EA047988B1 - HBV VACCINES AND HBV TREATMENT METHODS - Google Patents

Description

This application claims priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/908,494, filed September 30, 2019, the contents of which are incorporated herein by reference in their entirety for all purposes.

List of sequences

This application contains a sequence listing that was filed electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the sequence listing in ASCII format created on September 4, 2020 is named 1324_PF_SL.txt and is 296,675 bytes in size.

State of the art

Numerous attempts have been made to use vaccination to treat patients with chronic hepatitis B virus (HBV) infection to increase the rate of loss of HBV surface antigen (sAg), the main marker of functional therapy. Such attempts include vaccination with recombinant proteins (Dikici, et al., J Gastroenterol Hepatol. (2003) 18(2):218–22; Pol, et al., J Hepatol. (2001) 34(6):917–21; Vandepapeliere, et al., Vaccine (2007) 25(51):8585–97; Yalcin, et al., J Clin Gastroenterol. (2003) 37(4):330–5; Al-Mahtab, Hepatol Int. (2013) 7(4):981–9; Hoa, et al., Antimicrob Agents Chemother. (2009) 53(12):5134–40; and Yalcin, et al., Infection. (2003) 31(4):221-5), recombinant DNA (Mancini-Bourgine, et al., Hepatology. (2004) 40(4):874-82; Yang, et al., World J Gastroenterol. (2017) 23(2):306-17; Yang, et al., J Viral Hepat. (2012) 19(8):581-93; Yoon, et al., Liver Int. (2015) 35(3):805-15; Cavenaugh, et al., PLoS One. (2011) 6(2):e14626 and Godon, et al., Mol Ther. (2014) 22(3):675-84), dendritic cells (Luo, et al., Vaccine. (2010) 28(13):2497–504 and Wei et al., Int Immunopharmacol. (2015) 27(2):238–43), yeast vector (Gane. et al., J Hepatol. (2019) Epub 2019/07/16. doi: 10.1016/jjhep.2019.06.028. PubMed PMID: 31306680), and some viral vectors (Cavenaugh et al. and Zoulim et al., Hum Vaccin Immunother. (2019) Epub 2019/08/03. doi: 10.1080/21645515.2019.1651141. PubMed PMID: 31373537). Despite these numerous attempts, no vaccine therapy approach has yet demonstrated consistent benefit in chronic HBV infection (CHI). Failures in previous vaccine therapy approaches may explain the poor outcomes of previous vaccine therapy approaches.

Such constraints include limitations in the antigen configuration and the vaccine technology employed. An optimal antigen will contain highly conserved regions of HBV proteins and exclude low-conserved regions, since highly conserved regions may induce responses against epitopes that are identical to the vaccine antigen and the virus present in the treated patient, whereas low-conserved regions may elicit immunizing T cell responses against epitopes that are not present in the infecting patient's virus strain (Swadling, et al., Vaccines (Basel). (2016) 4(3). Epub 2016/08/05. doi: 10.3390/vaccines4030027. PubMed PMID: 27490575). However, some previous vaccines use antigen configurations that do not meet these criteria (Yalcin, et al., J Clin Gastroenterol. (2003) 37(4):330-5; Hoa, et al.; Yalcin, et al., Infection. (2003) 31(4):221-5; Mancini-Bourgine et al., Yang et al., J Viral Hepat. (2012) 19(8):581-93; Cavenaugh et al.; Godon, et al.; Gane et al.; and Obeng-Adjei et al., Cancer Gene Ther. (2013) 20(12):652-62). Furthermore, many previous vaccines did not induce the full combination of virus-specific CD4 ⁺ T cells, CD8 ⁺ T cells, and antibody responses (Dikici et al.; Pol, et al.; Vandepapeliere, et al.; Yalcin et al., J Clin Gastroenterol. (2003) 37(4):330–5; Al-Mahtab; Hoa et al.; Yalcin, et al., Infection. (2003) 31(4):221–5; Mancini-Bourgine, et al.; Yang, et al., J Viral Hepat. (2012) 19(8):581–93; Gane et al., and Zoulim et al.,). These immune components are particularly important for the treatment of chronic HBV infection, as CD8 ⁺ T cells have been shown to be the major effector cells responsible for viral clearance during acute HBV infection in chimpanzees (Thimme, et al., J Virol. (2003) 77(1):6876). In addition, antibodies that bind to HBV surface antigen (HBsAg) facilitate HBsAg clearance and prevent dissemination of residual HBV. Moreover, a high level of immune response is likely necessary to achieve therapeutic effect, but many previous HBV vaccines do not induce such a robust response (Mancini-Bourgine, et al, above; Yang, et al., J Viral Hepat. (2012) 19(8):581–93; Cavenaugh, et al.; Gane et al.; and Zoulim et al.). Finally, some previous SNV vaccine antigens were not stable enough in delivery vectors to support industrial vaccine production.

The essence of the invention

In one aspect, truncated hepatitis B virus (HBV) polymerase polypeptides are provided, e.g., capable of inducing or eliciting an immune response in a human when administered. In some embodiments, the truncated HBV polymerase polypeptide comprises an inactivated reverse transcriptase domain and an inactivated RNase H and lacks all or a portion of the terminal protein (TP) domain and all or a portion of the spacer domain. In some embodiments, the polypeptide is no more than 600 amino acids in length, such as no more than 595, 590, 585, 580, 575, 570, 565, 560, 555, 550, 545, 540, or 535 amino acids in length. In some embodiments, the reverse transcriptase domain does not comprise a YMDD motif (SEQ ID NO: 97) and the RNase H domain does not comprise an AELL motif (SEQ ID NO: 98). In some embodiments, the YMDD motif (SEQ ID NO: 97) in the reverse transcriptase domain is mutated to YMHD (SEQ ID NO: 99) and wherein the AELL motif (SEQ ID NO: 98) in the RNase H domain is mutated to

- 1 047988

AHLL (SEQ ID NO: 100). In some embodiments, the polypeptide is of HBV genotype A, B, C, or D. In some embodiments, (a) the polypeptide is of HBV genotype B and does not comprise the polypeptide sequence (e.g., the sequence is excluded, deleted, or not included) of SEQ ID NO: 50, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 50; or (b) the polypeptide is of HBV genotype D and does not comprise the polypeptide sequence of SEQ ID NO: 51, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 51. In some embodiments, the truncated HBV polymerase polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 13-14, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 13-14.

In another aspect, mutant HBV polymerase polypeptides with a deletion are provided.

In some embodiments, a deletion mutant HBV polymerase polypeptide comprises, in sequence from N-terminus to C-terminus, a terminal protein (TP) domain, an inactivated reverse transcriptase domain, and an inactivated RNase H, wherein the mutant polypeptide lacks all or a portion of a spacer domain. In some embodiments, the polypeptide is no more than 800 amino acids in length, such as no more than 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710, or 705 amino acids in length. In some embodiments, the reverse transcriptase domain lacks a YMDD motif (SEQ ID NO: 97) and the RNase H domain lacks an AELL motif (SEQ ID NO: 98). In some embodiments, the YMDD motif (SEQ ID NO: 97) in the reverse transcriptase domain is mutated to YMHD (SEQ ID NO: 99) and wherein the AELL motif (SEQ ID NO: 98) in the RNase H domain is mutated to AHLL (SEQ ID NO: 100). In some embodiments, the polypeptide is of HBV genotype A, B, C, or D. In some embodiments, (a) the polypeptide is of HBV genotype A and does not comprise the polypeptide of SEQ ID NO: 42 or 46 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 42 or 46; (b) the polypeptide belongs to genotype B of HBV and does not comprise the polypeptide of SEQ ID NO: 43 or 47, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 43 or 47; (c) the polypeptide belongs to genotype C of HBV and does not comprise the polypeptide of SEQ ID NO: 44 or 48, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 44 or 48; or (d) the polypeptide is of HBV genotype D and does not comprise the polypeptide of SEQ ID NO: 45 or 49, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 45 or 49. In some embodiments, the deletion mutant HBV polymerase polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 5-12, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 5-12. In some embodiments, the deletion mutant HBV polymerase polypeptide further comprises (e.g., is a fusion protein comprising) an HBV capsid polypeptide. In some embodiments, the deletion mutant HBV polymerase polypeptide comprises, sequentially from N-terminus to C-terminus, an HBV capsid polypeptide and a deletion mutant HBV polymerase polypeptide as described herein. In some embodiments, the deletion mutant HBV polymerase polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 19-26 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 19-26.

In a further aspect, a capsid-sAg HBV fusion protein is provided. In some embodiments, the capsid-sAg fusion protein comprises, sequentially from N-terminus to C-terminus, a capsid polypeptide of HBV and a small polypeptide of a surface antigen (sAg) of HBV. In various embodiments, the capsid polypeptide is of genotype B or C of HBV, and the sAg polypeptide is of genotype C of HBV. In some embodiments, the capsid polypeptide is of genotype D of HBV, and the sAg polypeptide is of genotype D of HBV. In some embodiments, the capsid-sAg fusion protein comprises: (a) a capsid polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 65 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65, and an sAg polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3; or (b) a capsid polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 66 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 66, and an sAg polypeptide comprising or consisting of the amino acid sequence

- 2 047988 of SEQ ID NO: 4 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4. In some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at an amino acid position, corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a leucine (L) residue at an amino acid position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In various embodiments, the sAg polypeptide lacks a pre-S1 polypeptide. In various embodiments, the sAg polypeptide lacks a pre-S2 polypeptide. In some embodiments, the sAg polypeptide lacks an HBV pre-S2 polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 79-83 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to any of SEQ ID NOs: 79-83. In some embodiments, the sAg polypeptide does not comprise an HBV pre-S1 polypeptide or an HBV pre-S2 polypeptide. In some embodiments, the sAg polypeptide does not comprise an HBV pre-S1-pre-S2 polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 84-88 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 84-88. In various embodiments, the capsid-sAg fusion protein comprises a cleavable linker operably linked to and positioned between the HBV polypeptide and the HBV sAg polypeptide. In some embodiments, the cleavable linker is a cleavable 2A peptide. In some embodiments, the cleavable linker is a cleavable 2A peptide selected from foot-and-mouth disease virus (F2A), equine rhinitis virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A). In some embodiments, the cleavable linker is a porcine teschovirus-1 (P2A) linker. In some embodiments, the cleavable linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 57), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 58), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 59) or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 57), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 58) or EGRGSLLTCGDVEENPGP (SEQ ID NO: 59). In some embodiments, the cleavable linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56). In some embodiments, the capsid-sAg fusion protein comprises a flexible linker and/or a furin recognition/cleavage site operably linked to and N-terminally to the cleavable linker and an HBV capsid polypeptide and C-terminally to the HBV capsid polypeptide. In some embodiments, the furin recognition/cleavage region comprises or consists of an amino acid sequence selected from RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62). In some embodiments, the flexible linker comprises a polyglycine or polyalanine sequence. In some embodiments, the flexible linker comprises or consists of a polyglycine or polyalanine sequence selected from AA, AAA, AAY, GG, GGG, GGS, GSG, and GGGS (SEQ ID NO: 63). In some embodiments, the capsid-sAg fusion protein is no more than 450 amino acids in length, such as no more than 445, 440, 435, 430, 425, 420, 415, or 410 amino acids in length. In some embodiments, the capsid-sAg fusion protein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 38-41, such as SEQ ID NO: 41, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41, such as SEQ ID NO: 41. In some embodiments, the fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position

- 3 047988 acidic position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein the position numbers have a reference to SEQ ID NO:41. In various embodiments, the capsid-sAg fusion polypeptide does not comprise an amino sequence or fragment thereof from an HBV protein selected from the group consisting of X, precapsid, preS1, and pre-S2.

With respect to immunogenic HBV polypeptides, in some embodiments, the truncated HBV polymerase polypeptide, the deletion mutant HBV polymerase polypeptide, or the capsid-sAg fusion protein as described herein further comprises an N-terminal signal peptide or leader sequence. In various embodiments, the signal peptide or leader sequence belongs to a parent protein selected from a serum protein, a cytokine, a chemokine, a chaperone protein, an invariant protein, and a protein that targets proteins to a lysosomal compartment. In various embodiments, the signal peptide or leader sequence belongs to a parent protein selected from colony stimulating factor 2 (CSF2, GM-CSF), tissue plasminogen activator (PLAT, t-PA), C-C motif chemokine ligand 7 (CCL7, MCP-3), C-X-C motif chemokine ligand 10 (CXCL10, IP-10), catenin beta-1 (CTNNB1), CD74 (p33; DHLAG; HLADG; Ia-GAMMA, invariant chain), serum albumin (ALB), polyubiquitin B/C (UBB/UBC), calreticulin (CALR), vesicular stomatitis virus G protein (VSV-G), lysosome-associated membrane protein 1 (LAMP-1), and lysosome-associated membrane protein 2 (LAMP-2). In some embodiments, the signal peptide or leader sequence is selected from the amino acid sequence of any one of SEQ ID NOs: 67-78 or a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 67-78. In various embodiments, the truncated polymerase polypeptide, HBV polypeptide, deletion mutant HBV polymerase polypeptide, and/or capsid-sAg fusion protein as described herein can be recombinantly produced or chemically synthesized. In various embodiments, a truncated HBV polymerase polypeptide, a deletion mutant HBV polymerase polypeptide, and/or a capsid-sAg fusion protein as described herein are capable of inducing, activating, or promoting an immune response (e.g., expanding and/or activating CD8+ and/or CD4+ T cells; producing antibodies that bind to and/or neutralize one or more of HBV polymerase, HBV capsid, and HBV sAg) in a human. In various embodiments, a truncated HBV polymerase polypeptide, a deletion mutant HBV polymerase polypeptide, and/or a capsid-sAg fusion protein as described herein are capable of inducing, activating, or promoting an immune response against HBV (e.g., that prevents, delays the progression of, inhibits, and/or reverses HBV infection) in a human. In various embodiments, a truncated HBV polymerase polypeptide, a deletion mutant HBV polymerase polypeptide, and/or a capsid-sAg fusion protein as described herein are capable of inducing, activating, or promoting the proliferation and/or activation of one or more cell types selected from monocytic dendritic cells (DCs), CD8+ T cells, and CD4+ T cells.

In a further aspect, polynucleotides encoding immunogenic HBV polypeptides as described herein are provided. For example, polynucleotides encoding one or more of a truncated HBV polymerase polypeptide, a deletion mutant HBV polymerase polypeptide, or a capsid-sAg fusion protein as described herein are provided. In some embodiments, the polynucleotide comprises cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). In some embodiments, the polynucleotide comprises self-replicating or self-amplifying alphaviral replicons. In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence of any of SEQ ID NOs: 27-37, such as SEQ ID NOs: 37 and 89-94, such as SEQ ID NOs: 29, 89, 90 or 92, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NOs: 27-37, such as SEQ ID NOs: 37 and 89-94, such as SEQ ID NOs: 29, 89, 90 or 92.

In another aspect, a lipid nanoparticle (LNP) is provided comprising one or more polynucleotides encoding an immunogenic HBV polypeptide as described herein.

In another aspect, expression cassettes are provided comprising one or more polynucleotides encoding an immunogenic HBV polypeptide as described herein operably linked to one or more regulatory sequences. In some embodiments, the polynucleotide is operably linked to and controlled by a constitutive promoter. In some embodiments, the promoter is selected from the cytomegalovirus essential immediate early

- 4 047988 ca (CMV), the CMV enhancer fused to the promoter of chicken beta-actin (CAG), human elongation factor la (HEF-la), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1a (CHEF1a), and phosphoglycerate kinase (PGK).

In another aspect, one or more polynucleotides encoding an immunogenic HBV polypeptide as described herein, or one or more expression cassettes comprising such polynucleotides are provided. In various embodiments, the vector is a plasmid vector, a bacterial vector, or a viral vector. In some embodiments, the vector is a viral vector. In various embodiments, the viral vector is a DNA virus or an RNA virus. In some embodiments, the viral vector is a virus selected from adenovirus, adeno-associated virus, arenavirus, alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, Maraba virus, and vaccinia virus. In some embodiments, the viral vector is derived from a virus from a taxonomic family selected from adenoviruses, arenaviruses, herpes viruses (e.g., cytomegalovirus), poxviruses (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA), flaviviruses (e.g., yellow fever virus), rhabdoviruses (e.g., vesiculovirus, e.g., Maraba vesiculovirus), togaviruses (e.g., alphavirus). In some embodiments, the viral vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Kali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV) and Whitewater Arroyo mammarenavirus (WWAV). In some embodiments, the viral vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Cali mammarenavirus (LCMV) (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)). In some embodiments, the viral vector is a human adenovirus or a simian adenovirus (e.g., a chimpanzee adenovirus, a gorilla adenovirus, or a rhesus macaque adenovirus). In some embodiments, the viral vector is an adenovirus vector selected from adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (e.g., ChAdOx1, ChAdOx2, ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 (AdC9), ChAd10 (AdC10), ChAd11 (AdC11), ChAd17 (AdC17), ChAd16 (AdC16), ChAd19 (AdC19), ChAd20 (AdC20), ChAd22 (AdC22),

ChAd24 (AdC24), ChAdY25, ChAd26 (AdC26), ChAd28 (AdC28), ChAd30 (AdC30), ChAd31 (AdC31),

ChAd37 (AdC37), ChAd38 (AdC38), ChAd43 (AdC43), ChAd44 (AdC44), ChAd55 (AdC55), ChAd63 (AdC63), ChAdV63, ChAd68 (AdC68), ChAd73 (AdC73), ChAd82 (AdC82), ChAd83 (AdC83), ChAdl43 (AdC143), ChAdl44 (AdC144), ChAdl45 (AdC145), ChAdl47 (AdC147)), gorilla adenovirus (e.g. GC44, GC45, GC46) and rhesus macaque adenovirus (e.g. RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66). In some embodiments, the viral vector is replication defective, replication deficient, replication attenuated, or replication competent. In some embodiments, the viral vector is a replication defective arenavirus having a two-segment genome. In some embodiments, the viral vector is a replication attenuated arenavirus having a three-segment genome.

In a further aspect, arenavirus vectors are provided. In one embodiment, an arenavirus vector is provided comprising a polynucleotide encoding an HBV capsid sAg fusion polypeptide comprising or consisting of the amino acid sequence of any of SEQ ID NO: 3841, such as SEQ ID NO: 41, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NOs: 38-41, such as SEQ ID NO: 41, and wherein the sAg polypeptide does not comprise an HBV pre-S1 polypeptide and/or an HBV pre-S2 polypeptide. In some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO:65 or SEQ ID NO:66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at an amino acid position corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a leucine (L) residue at an amino acid position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. ...

- 5 047988 the capsid-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a a tyrosine (Y) at an amino acid position corresponding to position 338, a glycine (G) residue at an amino acid position corresponding to position 363, and an alanine (A) residue at an amino acid position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO: 41. In some embodiments, the polynucleotide comprises or consists of a nucleic acid sequence of any of SEQ ID NOs: 33-37, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 33-37. In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 37, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments, the arenavirus vector has a two-segment genome and further comprises a polynucleotide encoding a truncated HBV polymerase comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 13-14, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 13-14, and wherein the truncated HBV polymerase does not comprise all of the HBV polymerase terminal protein (TP) domain and does not comprise all or a portion of the HBV polymerase spacer domain. In some embodiments, the truncated HBV polymerase does not comprise the polypeptide sequence of SEQ ID NO: 50 or SEQ ID NO: 51, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 50 or SEQ ID NO: 51. In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence of any of SEQ ID NOs: 29 and 89-94, such as SEQ ID NOs: 29, 89, 90, or 92, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 29 and 89-94, such as SEQ ID NOs: 29, 89, 90, or 92. In some embodiments, the arenavirus vector is a lymphocytic choriomeningitis mammarenavirus (LCMV) vector and the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 29, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 29. In some embodiments, the arenavirus vector is a Kali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)) vector and the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90.

Additionally provided is an arenavirus vector comprising a polynucleotide encoding a truncated HBV polymerase comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 13-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NOs: 13-14, and wherein the truncated HBV polymerase does not comprise the entire terminal protein (TP) domain of the HBV polymerase and does not comprise the entire spacer domain of the HBV polymerase or a portion thereof. In some embodiments, the truncated HBV polymerase does not comprise the polypeptide sequence of SEQ ID NO: 50 or SEQ ID NO: 51, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 50 or SEQ ID NO: 51. In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence of any of SEQ ID NOs: 29 and 89-94, such as SEQ ID NOs: 29, 89, 90, or 92, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 29 and 89-94, such as SEQ ID NOs: 29, 89, 90, or 92. In some embodiments, the arenavirus vector is a mammarenavirus lymphocytic choriomeningitis virus (LCMV) vector and the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 29, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 29. In some embodiments, the arenavirus vector is a Kali mammarenavirus vector and the polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 90, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

- 6 047988

95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90. In some embodiments, the arenavirus vector is replication defective, replication deficient, or replication incompetent.

In a further aspect, host cells are provided comprising one or more polynucleotides encoding one or more immunogenic HBV polypeptides as described herein, or one or more vectors comprising such polynucleotides. In some embodiments, the one or more polynucleotides encoding one or more immunogenic HBV polypeptides as described herein are not integrated into the genome of the host cell, such as being episomal. In some embodiments, the one or more polynucleotides are integrated into the genome of the host cell. In some embodiments, the host cell is a mammalian cell, such as a human cell. In various embodiments, the host cell can be in vitro or in vivo.

In another aspect, immunogenic compositions are provided comprising one or more immunogenic HBV polypeptides as described herein. In some embodiments, the immunogenic composition comprises one or more, such as two or more, truncated HBV polymerase polypeptides, one or more, such as two or more, deletion mutant HBV polymerase polypeptides, and/or one or more, such as two or more, capsid-sAg fusion proteins as described herein. In some embodiments, the immunogenic composition comprises one or more, such as two or more, polynucleotides encoding one or more, such as two or more, truncated HBV polymerase polypeptides, one or more, such as two or more, deletion mutant HBV polymerase polypeptides, and/or one or more, such as two or more, capsid-sAg fusion proteins as described herein. In some embodiments, the immunogenic composition comprises one or more, such as two or more, one or more, such as two or more, vectors comprising one or more, such as two or more, polynucleotides encoding one or more, such as two or more, truncated HBV polymerase polypeptides, one or more, such as two or more, deletion mutant HBV polymerase polypeptides, and/or one or more, such as two or more, capsid-sAg fusion proteins as described herein, the immunogenic compositions further comprising a pharmaceutically acceptable carrier. In some embodiments, the immunogenic composition comprises one or more polynucleotides in the form of DNA, cDNA, mRNA, or self-replicating RNA. In various embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide or a deletion mutant HBV polymerase polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein as described. In some embodiments, an immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-14; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41. In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 13-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-14; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41. In some embodiments, the immunogenic compositions comprise a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 41

- 7,047,988 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at an amino acid position corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a leucine (L) residue at an amino acid position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the capsid-sAg fusion polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 12, an asparagine (N) residue at an amino acid position corresponding to position 67, a valine (V) residue at an amino acid position corresponding to position 74, a phenylalanine (F) residue at an amino acid position corresponding to position 97, a threonine (T) residue at an amino acid position corresponding to position 249, a threonine (T) residue at an amino acid position corresponding to position 250, a serine (S) residue at an amino acid position corresponding to position 317, a serine (S) residue at an amino acid position corresponding to position 318, an arginine (R) residue at an amino acid position corresponding to position 326, a tyrosine (Y) at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO: 41. In some embodiments, the immunogenic compositions comprise a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90, or 92 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 33-37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 33-37. In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 90; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 37. In various embodiments, the first viral expression vector and the second viral expression vector are independently derived from a taxonomic family selected from adenoviruses, arenaviruses, herpes viruses (e.g., cytomegalovirus), poxviruses (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA)), flaviviruses (e.g., yellow fever virus), rhabdoviruses (e.g., vesiculovirus, e.g., Maraba vesiculovirus), togaviruses (e.g., alphavirus). In various embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition can be from the same taxonomic family or different taxonomic families. In some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are from arenaviruses. In some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are independently derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). ...

- 8 047988 the vector and the second viral expression vector are independently derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Kali mammarenavirus (LCMV) (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)). In some embodiments, the first viral expression vector and the second viral expression vector are replication defective or replication deficient. In some embodiments, the first viral expression vector and the second viral expression vector are replication attenuated. According to some embodiments, the immunogenic composition comprises a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 37. In some embodiments, the immunogenic composition comprises a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 90 or a sequence that is at least at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 37. In various embodiments, the first viral expression vector and the second viral expression vector are provided in the immunogenic composition in a ratio in the range of 1:10 to 10:1, such as 1:9 to 9:1, 1:8 to 8:1, from 1:7 to 7:1, from 1:6 to 6:1, from 1:5 to 5:1, from 1:4 to 4:1, from 1:3 to 3:1, from 1:2 to 2:1, or 1:1. In some embodiments, the immunogenic composition comprises in the range of from about 10 ³ to about 10 ¹² viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (VP) per milliliter, such as from about 10 ⁴ to about 10 ⁷ viral FFU or PFU or IU or IU per milliliter, such as from about 10 ³ to about 10 ⁴ , 10 ⁵ , 10 6, 10 ⁷ , 10 ⁸ , 109, ¹⁰¹⁰ , 1011, or ¹⁰¹² viral FFU, or PFU, or IE, or VP per milliliter, of each of the first viral expression vector and the second viral expression vector. In some embodiments, the immunogenic composition further comprises one or more of an adjuvant, a detergent, a micelle former, and an oil. In various embodiments, the immunogenic composition is formulated for administration by a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., buccal, intranasal, intrarectal, intravaginal). In some embodiments, the immunogenic composition is an aqueous solution or suspension, e.g., formulated as a liquid. In some embodiments, the immunogenic composition is lyophilized.

In a further aspect, kits are provided. In various embodiments, a kit comprises one or more, such as two or more, unit doses of one or more, such as two or more, truncated HBV polymerase polypeptides, one or more, such as two or more, deletion mutant HBV polymerase polypeptides, and/or one or more, such as two or more, capsid-sAg fusion proteins as described herein. In some embodiments, a kit comprises one or more, such as two or more, unit doses of one or more, such as two or more, polynucleotides encoding one or more, such as two or more, truncated HBV polymerase polypeptides, one or more, such as two or more, deletion mutant HBV polymerase polypeptides, and/or one or more, such as two or more, capsid-sAg fusion proteins as described herein. In some embodiments, a kit comprises one or more, such as two or more unit doses of one or more, such as two or more, vectors comprising one or more, such as two or more, polynucleotides encoding one or more, such as two or more, truncated HBV polymerase polypeptides, one or more, such as two or more, deletion mutant HBV polymerase polypeptides, and/or one or more, such as two or more, capsid-sAg fusion proteins as described herein. In various embodiments, a kit comprises one or more, such as two or more, unit doses of one or more, such as two or more, immunogenic compositions as described herein. In some embodiments, the one or more unit doses in the kit are in a single container. In some embodiments, the one or more unit doses in the kit are in two or more separate containers. In some embodiments, a kit comprises one or more containers selected from vials, ampoules, and prefilled syringes. In some embodiments, a kit

- 9 047988 contains one or more containers containing one or more polypeptides, one or more polynucleotides, one or more vectors, or one or more immunogenic compositions in an aqueous solution or suspension, or as a lyophilized preparation. In various embodiments, one or more unit doses may be the same or different. In some embodiments, the kit comprises one or more unit doses of one or more viral vectors as described herein, wherein the unit doses are in the range of from about 10 ³ to about 10 ¹² viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (VP), such as from about 10 ⁴ to about 10 ⁷ viral FFU, or PFU, or IU, or IU per milliliter, such as from about 10 ³ to about 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 9, 10 ¹⁰ , 10 ¹¹ or 10 ¹² viral FFU, or PFU, or IU, or IU. In some embodiments, the kit comprises one or more polynucleotides encoding, or one or more expression vectors, or an immunogenic composition comprising at least two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-14; and (b) an HBV capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41. In some embodiments, the kit comprises one or more polynucleotides encoding, or one or more expression vectors, or an immunogenic composition comprising at least two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 13-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 13-14; and (b) an HBV capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41. In some embodiments, the kit comprises one or more polynucleotides encoding, or one or more expression vectors, or an immunogenic composition comprising at least two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; and (b) an HBV capsid-sAg fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the polypeptide sAg comprises an isoleucine (I) residue at an amino acid position corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a leucine (L) residue at an amino acid position corresponding to position a position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the capsid-sAg fusion polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 12, an asparagine (N) residue at an amino acid position corresponding to position 67, a valine (V) residue at an amino acid position corresponding to position 74, a phenylalanine (F) residue at an amino acid position corresponding to position 97, a threonine (T) residue at an amino acid position corresponding to position 249, a threonine (T) residue at an amino acid position corresponding to position 250, a serine (S) residue at an amino acid position corresponding to position 317, a serine (S) residue at an amino acid position corresponding to position 318, a arginine (R) at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position

- 10 047988 a position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO: 41. In some embodiments, the kit comprises first and second vectors encoding first and second immunogenic polypeptides, respectively, wherein the first and second immunogenic polypeptides respectively comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; and (b) an HBV capsid-sAg fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the kit comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90 or 92 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90 or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 33-37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 33-37. In some embodiments, the kit comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 90; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments, the kit comprises one or more unit doses of an immunogenic composition comprising first and second viral expression vectors as described herein, wherein the first and second viral expression vectors comprise a replication-deficient or replication-defective Kali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)). In some embodiments, the kit comprises one or more unit doses of an immunogenic composition comprising first and second viral expression vectors as described herein, wherein the first and second viral expression vectors comprise a replication-deficient, replication-defective lymphocytic choriomeningitis mammarenavirus (LCMV). In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the adenovirus family; and (b) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the poxvirus family (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA)). In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the arenavirus family; and (b) one or more unit doses of an immunogenic composition as described herein, wherein the first and second viral expression vectors are from the adenovirus family; In some embodiments, the kit comprises a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments, the kit comprises a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that

- 11 047988 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37.

In various embodiments, the kit further comprises one or more unit doses of one or more additional therapeutic agents. In some embodiments, the kit further comprises one or more agonists or activators of one or more Toll-like receptors (TLRs). In some embodiments, the kit further comprises one or more TLR agonists or activators selected from a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the kit further comprises a TLR7 agonist selected from GS 9620 (vesatolimod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod). In some embodiments, the kit further comprises a TLR8 agonist selected from GS-9688, R848 (resiquimod), and NKTR-262 (a dual TLR7/TLR8 agonist). In some embodiments, the kit further comprises one or more interleukin receptor agonists of an interleukin receptor selected from IL-2, IL-7, IL-12, and IL15. In some embodiments, the kit further comprises one or more cytokines selected from IL-2, IL-7, IL-12, IL-15, and variants thereof. In some embodiments, the kit further comprises one or more innate immune activators. In some embodiments, the kit further comprises one or more innate immune activators comprising a receptor agonist selected from tyrosine kinase 3 (FLT3), stimulator of interferon genes receptor (STING), interferon receptor, DExD/H-box helicase 58 (DDX58; also known as RIG-I), nucleotide-binding oligomerization domain containing protein 2 (NOD2). In some embodiments, the kit further comprises one or more unit doses of GS-3583 and/or GS-9992. In some embodiments, the kit further comprises one or more antagonists or inhibitors of an inhibitory immune checkpoint protein or receptor and/or one or more activators or agonists of a mimetic immune checkpoint protein or receptor. In some embodiments, the kit further comprises one or more immune checkpoint proteins or receptors selected from CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); variable immunoglobulin-like domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4); Vset domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated protein 2 (HHLA2, B7H7); inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death protein 1 (PDCD1, PD1, PD-1); cytotoxic T-lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B71), CD28; nectin cell adhesion molecule-2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); T-cell immunoglobulin and mucin domain-containing protein 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation gene protein 3 (LAG3, CD223); signaling lymphocyte-activating molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); U116-binding protein 1 (ULBP1); CD3-binding protein 2 (ULBP2); Cytotoxicity-binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); lymphocyte activation gene protein 3 (CD223); immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); lectin-like killer cell receptor C1 (KLRC1, NKG2A, CD159A); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); lectin-like killer cell receptor C2 (KLRC2, CD159c, NKG2C); lectin-like killer cell receptor C3 (KLRC3, NKG2E); lectin-like killer cell receptor C4 (KLRC4, NKG2F); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1); lectin-like cell receptor

- 12 047988 killer D1 (KLRD1); and SLAM family member 7 (SLAMF7). In some embodiments, the kit further comprises one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more T cell inhibitory immune checkpoint proteins or receptors selected from CD274 (CD274, PDL1, PD-L1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 protein (PDCD1, PD1, PD-1); cytotoxic T lymphocyte associated protein 4 (CTLA4, CD152); CD276 (B7H3); T-cell activation inhibitor with variable immunoglobulin-like domain-containing 1 (VTCN1, B7H4); V-set domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation gene protein 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); and immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1). In some embodiments, the kit further comprises one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more T cell stimulatory immune checkpoint proteins or receptors selected from CD27, CD70; CD40, CD40LG; inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; nectin cell adhesion molecule-2 (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the kit further comprises one or more unit doses of AGEN-2373 and/or AGEN-1223. In some embodiments, the kit further comprises one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more NK cell inhibitory immune checkpoint proteins or receptors selected from: killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1); lectin-like killer cell receptor C1 (KLRC1, NKG2A, CD159A); and lectin-like killer cell receptor D1 (KLRD1, CD94). In some embodiments, the kit further comprises one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the kit further comprises one or more NK cell stimulatory immune checkpoint proteins or receptors selected from CD16, CD226 (DNAM-1); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7). In some embodiments, the kit further comprises one or more protein inhibitors of PD-L1 (CD274), PD-1 (PDCD1), and/or CTLA4. In some embodiments, the kit further comprises one or more CTLA4 protein inhibitors selected from ipilimumab, tremelimumab, BMS986218, AGEN1181, AGEN1884, BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, FPT-155 (CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4), KN-046 (PD1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717 (PD-1/CTLA4) and AK-104 (CTLA4/PD-1). In some embodiments, the kit further comprises one or more PD-L1 (CD274) or PD-1 (PDCD1) protein inhibitors selected from zimberelimab (AB122), pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, ASC22, durvalumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP3055), AK-103 (HX-008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genolimzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306, (MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI

- 13 047988

308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4/PD- L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-013 (PD-1/LAG-3), FS118 (LAG-3/PD-L1) MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), RO7121661 (PD-1/TIM-3), XmAb-20717 (PD-1/CTLA4), AK-104 (CTLA4/PD-1), М7824 (PD-L1/TGFe-EC domain), CA-170 (PD-L1/VISTA), CDX-527 (CD27/PD-L1), LY-3415244 (TIM3 /PDL1) and INBRX-105 (41BB/PDL1). In some embodiments, the kit further comprises one or more small molecule inhibitors of CD274 (PDL1, PD-L1), programmed cell death protein 1 (PDCD1, PD1, PD-1) and/ or CTLA4. In some embodiments, the kit further comprises one or more small molecule inhibitors of CD274 or PDCD1 selected from GS-4224, GS4416, INCB086550, and MAX10181. In some embodiments, the kit further comprises a small molecule CTLA4 inhibitor, BPI-002. In some embodiments, the kit further comprises one or more antiviral agents. In some embodiments, the kit further comprises one or more antiviral agents selected from lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF or VEMLIDY®), and ledipasvir + sofosbuvir (HARVONI®). In some embodiments, the kit further comprises one or more therapeutic agents selected from HBV antigen inhibitors (e.g., HBV human antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), antigen antibodies to HBV, HBV-targeted inhibitory nucleic acids (e.g. antisense oligonucleotide, short interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi), HBV-targeted gene editors (e.g. CRISPRCas (e.g. Cas9, Cas12, Cascade, Cas13) , zinc finger nucleases, homing endonucleases, homing meganucleases (e.g., aRCUS), synthetic nucleases, TALENs), covalently closed circular DNA (cccDNA) inhibitors, and inhibitors secretion or assembly of HBsAg and inhibitors of HBV virus entry.

In a further aspect, methods are provided for eliciting an immune response to human hepatitis B virus (HBV) in a subject in need thereof. Methods for treating or preventing human hepatitis B virus (HBV) in a subject in need thereof are also provided. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of one or more immunogenic compositions as described herein. In some embodiments, the methods comprise administering one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide or a deletion mutant HBV polymerase polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein as described herein. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-14; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 13-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 13-14; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector

- 14 047988 contains a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41. In some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO: 65 or SEQ ID NO: 66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at an amino acid position corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a a leucine (L) at an amino acid position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the capsid-sAg fusion polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 12, an asparagine (N) residue at an amino acid position corresponding to position 67, a valine (V) residue at an amino acid position corresponding to position 74, a phenylalanine (F) residue at an amino acid position corresponding to position 97, a threonine (T) residue at an amino acid position corresponding to position 249, a threonine (T) residue at an amino acid position corresponding to position 250, a serine (S) residue at an amino acid position corresponding to position 317, a serine (S) residue at an amino acid position, corresponding to position 318, an arginine residue (R) at the amino acid position corresponding to position 326, a tyrosine residue (Y) at the amino acid position corresponding to position 338, a glycine residue (G) at the amino acid position corresponding to position 363, and an alanine residue (A) at the amino acid position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO:41. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90, or 92 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 27-32, and 89-94, such as SEQ ID NO: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NO: 33-37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 33-37. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 90; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments of the methods, the first viral expression vector and the second viral expression vector are from the arenavirus family. In some embodiments of the methods, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments of the methods, the first viral expression vector and the second viral expression vector

- 15 047988 are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Kali mammarenavirus (LCMV) (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)). In some embodiments of the methods, the first viral expression vector and the second viral expression vector are replication defective or replication deficient. In some embodiments of the methods, the first viral expression vector and the second viral expression vector are replication attenuated. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of one or more immunogenic compositions comprising a mixture comprising a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of a ... the sequence of SEQ ID NO: 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In some embodiments of the methods, the subject is infected with HBV, is suspected of being infected with HBV, or is at risk of being infected with HBV. In some embodiments of the methods, the subject is asymptomatic. In some embodiments of the methods, the subject is chronically infected with HBV. In some embodiments of the methods, the subject exhibits or experiences one or more symptoms selected from liver failure, liver cancer, liver fibrosis, and liver cirrhosis. In some embodiments of the methods, the subject has an acute HBV infection. In some embodiments of the methods, the subject exhibits or experiences one or more symptoms selected from jaundice, visible networks of swollen blood vessels in the skin, dark colored (e.g., orange or brown) urine, discolored stool, fever, persistent fatigue, discomfort, abdominal pain, abdominal fluid, loss of appetite, nausea, and vomiting. In some embodiments of the methods, the subject is co-infected with hepatitis D virus (HDV). In some embodiments of the methods, the composition is administered by a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., buccal, intranasal, intrarectal, intravaginal). In some embodiments, administering to the subject from about 10 ³ to about 10 ¹² viral focus-forming units (FFU), or plaque-forming units (PFU), or infectious units (IU), or viral particles (VP), such as from about 10 ⁴ to about 10 ⁷ viral FFU, or PFU, or IU, or IVP, such as from about 10 ³ to about 10 ⁴ , 10 ⁵ , 10 6, 10 ⁷ , 10 ⁸ , 10 9, 10 ¹⁰ , 10 11 or 10 ¹² viral FFU, or PFU, or IU, or IVP per administration. In some embodiments of the methods, one or more compositions are administered multiple times. In some embodiments, the methods comprise administering intravenously or intramuscularly from about 106 to about ¹⁰⁸ viral FFU, or PFU, or IE, or VP per administration every two weeks (Q2W) or monthly (Q4W). In some embodiments, the methods comprise multiple administrations of one or more immunogenic compositions over a period of at least about 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or more, or until sAg is not detectable in the serum or plasma of the subject. In some embodiments, the methods comprise a prime/boost cycle comprising administering a priming composition at a first time point and administering one or more boosting compositions at one or more subsequent time points. Depending on the circumstances, the methods may comprise repeating the priming/boost cycle for one or more cycles. In some embodiments of the methods, the administrations of the priming composition and the one or more boosting compositions are spaced apart by at least 1 week and up to at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments of the methods, the priming composition and the boosting composition may comprise the same immunogenic composition or may comprise different

- 16 047988 personal immunogenic compositions. In some embodiments of the methods, the priming composition and the boosting composition comprise one or more polypeptides and the same viral expression vector. In some embodiments of the methods, the priming composition and the boosting composition comprise different polypeptides and/or different viral expression vectors. In some embodiments, the methods comprise priming with a priming composition comprising one or more (e.g., first and second) viral expression vectors and boosting with a boosting composition comprising one or more (e.g., third and fourth) viral expression vectors. In various embodiments, the priming/boosting cycle comprises: (a) priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more polynucleotides, wherein the one or more polynucleotides comprise DNA, cDNA, mRNA, or self-replicating RNA; (b) priming with a priming composition comprising one or more polynucleotides, wherein the one or more polynucleotides comprise DNA, cDNA, mRNA or self-replicating RNA, and amplification with an enhancing composition comprising one or more viral expression vectors, (c) priming with a priming composition comprising one or more viral expression vectors and amplification with an enhancing composition comprising one or more viral expression vectors, wherein the one or more viral expression vectors in the priming composition and the one or more viral expression vectors in the enhancing composition are from identical, related or unrelated taxonomic families; (d) priming with a priming composition comprising one or more replication-deficient viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors, wherein the one or more replication-deficient viral expression vectors in the priming composition and the one or more replication-deficient viral expression vectors in the boosting composition are from identical, related, or unrelated taxonomic families; (e) priming with a priming composition comprising one or more replication-attenuated viral expression vectors, and amplifying with a boosting composition comprising one or more replication-attenuated viral expression vectors, wherein the one or more replication-attenuated viral expression vectors in the priming composition and the one or more replication-attenuated viral expression vectors in the boosting composition are from identical, related, or unrelated taxonomic families; (f) priming with a priming composition comprising one or more replication-deficient viral expression vectors, and amplifying with a boosting composition comprising one or more replication-attenuated viral expression vectors; (g) priming with a priming composition comprising one or more replication-attenuated viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors; (h) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more Pichinde mammarenavirus (PICV) viral expression vectors; (i) priming with a priming composition comprising one or more Pichinde mammarenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors; (j) priming with a priming composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors; (k) priming with a priming composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) viral expression vectors; (l) priming with a priming composition comprising one or more arenavirus viral expression vectors and boosting with a boosting composition comprising one or more adenovirus viral expression vectors; (m) priming with a priming composition comprising one or more adenovirus viral expression vectors and boosting with a boosting composition comprising one or more arenavirus viral expression vectors; (n) priming with a priming composition comprising one or more poxvirus viral expression vectors and boosting with a boosting composition comprising one or more arenavirus viral expression vectors; (o) priming with a priming composition comprising one or more arenavirus viral expression vectors and boosting with a boosting composition comprising one or more

- 17 047 988 more poxvirus viral expression vectors; (p) priming with a priming composition comprising one or more poxvirus viral expression vectors and boosting with a boosting composition comprising one or more adenovirus viral expression vectors; or (q) priming with a priming composition comprising one or more adenovirus viral expression vectors and boosting with a boosting composition comprising one or more poxvirus viral expression vectors. In some embodiments, the methods comprise a priming/boosting cycle that comprises: (a) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more Pichinde mammarenavirus (PICV) viral expression vectors; (b) priming with a priming composition comprising one or more Pichinde mammaryenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammaryenavirus (LCMV) viral expression vectors; (c) priming with a priming composition comprising one or more replication-deficient Pichinde mammaryenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient lymphocytic choriomeningitis mammaryenavirus (LCMV) viral expression vectors; or (d) priming with a priming composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) viral expression vectors. In some embodiments, the priming composition and the boosting composition comprise an immunogenic composition as described herein. In some embodiments, the subject does not receive antiviral therapy or the antiviral therapy is discontinued prior to administration of the one or more immunogenic compositions. In some embodiments of the methods, the antiviral therapy is discontinued after one or more administrations of the one or more immunogenic compositions. In some embodiments, the methods further comprise administering to the subject one or more additional therapeutic agents, such as two, three, four or more additional therapeutic agents. In some embodiments, the methods comprise co-administering one or more agonists or activators of one or more Toll-like receptors (TLRs). In some embodiments, the methods comprise co-administering one or more TLR agonists or activators selected from: a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the methods comprise co-administering a TLR7 agonist selected from GS-9620 (vesatolimod), R848 (resiquimod), DS-0509, LHC-165, and TMX-101 (imiquimod). In some embodiments, the methods comprise co-administering a TLR8 agonist selected from GS-9688, R848 (resiquimod), and NKTR-262 (a dual TLR7/TLR8 agonist). In some embodiments, the methods comprise co-administering one or more interleukin receptor agonists of an interleukin receptor selected from IL-2, IL-7, IL-12, and IL-15. In some embodiments, the methods comprise co-administering one or more cytokines selected from IL-2, IL-7, IL-12, IL-15, and variants thereof. In some embodiments, the methods comprise co-administering one or more innate immune activators. In some embodiments, the methods comprise co-administering one or more innate immune activators comprising a receptor agonist selected from tyrosine kinase 3 (FLT3), stimulator of interferon genes receptor (STING), interferon receptor, DExD/H-box helicase 58 (DDX58; also known as RIG-I), nucleotide-binding oligomerization domain containing protein 2 (NOD2). In some embodiments, the methods comprise co-administering GS-3583 and/or GS-9992. In some embodiments, the methods comprise co-administering one or more antagonists or inhibitors of an inhibitory immune checkpoint protein or receptor and/or one or more activators or agonists of a stimulatory immune checkpoint protein or receptor. In some embodiments, the methods comprise co-administering one or more immune checkpoint proteins or receptors selected from the following: CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain containing protein 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); variable immunoglobulin-like domain-containing inhibitor of T-cell activation 1 (VTCN1, B7H4); Vset domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated protein 2 (HHLA2, B7H7); inducible T-cell costimulator (ICOS, CD278); inducible T-cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270),

- 18 047988

TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); programmed cell death protein 1 (PDCD1, PD1, PD-1); cytotoxic T-lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B71), CD28; nectin cell adhesion molecule-2 (NECTIN2, CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); T-cell immunoglobulin and mucin domain-containing protein 4 (TIMD4; TIM4); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); lymphocyte activation gene protein 3 (LAG3, CD223); signaling lymphocyte-activating molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); ^16-binding protein 1 (ULBP1); ^16-binding protein 2 (ULBP2); ^16-binding protein 3 (ULBP3); retinoic acid early transcript 1E (RAET1E; ULBP4); retinoic acid early transcript 1G (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); lymphocyte activation gene protein 3 (CD223); immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); lectin-like killer cell receptor C1 (KLRC1, NKG2A, CD159A); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); lectin-like killer cell receptor C2 (KLRC2, CD159c, NKG2C); lectin-like killer cell receptor C3 (KLRC3, NKG2E); lectin-like killer cell receptor C4 (KLRC4, NKG2F); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2);

immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor D1 (KLRD1); and SLAM family member 7 (SLAMF7). In some embodiments, the methods comprise co-administering one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the methods comprise co-administering one or more T cell inhibitory immune checkpoint proteins or receptors selected from CD274 (CD274, PDL1, PD-L1); programmed cell death ligand 2 1 (PDCD1LG2, PD-L2, CD273); programmed cell death protein 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); variable immunoglobulin-like domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation gene protein 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR3DL1). In some embodiments, the methods comprise co-administering one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the methods comprise co-administering one or more T cell stimulatory immune checkpoint proteins or receptors selected from CD27, CD70; CD40, CD40LG; inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; cell adhesion molecule-2 nectin (NECTIN2, CD112); CD226 (DNAM-1); poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). In some embodiments, the methods involve co-administering AGEN-2373 and/or AGEN-1223. In some embodiments, the methods involve co-administering one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In some embodiments, the methods involve co-administering one or more NK cell inhibitory immune checkpoint proteins or receptors selected from a killer cell immunoglobulin-like receptor, three Ig domains

- 19 047988 and long cytoplasmic tail 1 (KIR, CD158E1); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); lectin-like killer cell receptor C1 (KLRC1, NKG2A, CD159A); and lectin-like killer cell receptor D1 (KLRD1, CD94). In some embodiments, the methods comprise co-administering one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. In some embodiments, the methods comprise co-administering one or more NK cell stimulatory immune checkpoint proteins or receptors selected from CD16, CD226 (DNAM-1);

lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7). In some embodiments, the methods comprise co-administering one or more protein inhibitors of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the methods comprise co-administering one or more CTLA4 protein inhibitors selected from ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884, BMS986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, FPT-155 (CTLA4/PD-L1/CD28), PF06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717 (PD-1/CTLA4) and AK-104 (CTLA4/PD-1). In some embodiments, the methods comprise co-administering one or more PD-L1 (CD274) or PD-1 (PDCD1) protein inhibitors selected from zimberelimab (AB122), pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, ASC22, durvalumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab), JNJ63723283, genolimzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK, BAT-1306, (MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintillimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-013 (PD-l/LAG-3), FS-118 ( LAG-3/PDL1) MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), RO-7121661 (PD1/TGM-3), XmAb-20717 (PD-1/CTLA4), AK-104 (CTLA4/PD-1), M7824 (domain PD-L1/TGFe-EC), CA-170 (PD-L1/VISTA), CDX-527 (CD27/PD-L1), LY-3415244 (TM3/PDL1), and INBRX-105 (4-1BB/PDL1). In some embodiments, the methods comprise co-administering one or more small molecule inhibitors of CD274 (PDL1, PD-L1), programmed cell death protein 1 (PDCD1, PD1, PD-1), or CTLA4. In some embodiments, the methods comprise co-administering one or more small molecule inhibitors of CD274 or PDCD1 selected from GS-4224, GS-4416, INCB086550, and MAX10181. In some embodiments, the methods comprise co-administering BPI-002 (a small molecule inhibitor of CTLA4). In some embodiments, the methods comprise co-administering to the subject one or more antiviral agents. In some embodiments, the methods comprise co-administering one or more antiviral agents selected from lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF or VEMLIDY®), and ledipasvir + sofosbuvir (HARVONI®). In some embodiments, the methods comprise co-administering to the subject one or more therapeutic agents selected from HBV antigen inhibitors (e.g., HBV human antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), HBV antigen antibodies, inhibitory nucleic acids targeting HBV (e.g., antisense oligonucleotide, short interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi), gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing meganucleases (e.g., ARCUS), synthetic nucleases, TALEN), covalently closed circular DNA inhibitors (ccDNA) and inhibitors of HBsAg secretion or assembly and inhibitors of HBV viral entry. In some embodiments, the method is activated in CD8 T cells and/or CD4+ T cells of the subject targeting one or more HBV polypeptide epitopes. In some embodiments, the method causes the subject to produce antibodies that bind one or more HBV polypeptides.

Brief description of graphic materials

Figure 1 shows the immunogenicity of adenovirus vectors expressing HBsAg from genotypes (GT) A, B, C, and D in outbred mice. Five- to seven-week-old outbred mice (n=8 per group) were injected intramuscularly with ^1x108 viral particles (VP) of adenovirus encoding con

- 20 047988 HBsAg sense sequences of HBV genotypes (GT)-A, B, C, D (SEQ ID NOs: 1-4, respectively). On day 14 after administration, splenocytes were collected for analysis, and T-cell immune responses were assessed using interferon (ZFN)-y ELISPOT (BD mouse IFN-γ ELISPOT kit, Cat. No. 551083). Each symbol corresponds to an individual mouse that was assessed for responses to overlapping peptide pools corresponding to HBsAg GT-A, B, C, and D.

Fig. 2 shows schematic representations of each Pol-containing antigen configuration. Each Pol domain is labeled separately (TR, terminal protein; RT, reverse transcriptase; RNH, RNase H). The approximate locations of the D-H mutation in the YMDD motif (SEQ ID NO: 97) in RT and the EH mutation in the AELL motif (SEQ ID NO: 98) in RNH are indicated below the RT and RNH domains. The designation of each construct is shown on the left, and the amino acid size range of the GT-A, B, C, and D constructs is shown on the right. YMHD and AHL are described in SEQ ID NOs: 99 and 100, respectively.

Figure 3 shows the immunogenicity of adenovirus vectors expressing the capsid-Pol fusion protein in C57BL/6 mice. Six- to eight-week-old C57BL/6 mice (n=5 per group) were injected with 1x108 adenovirus viral particles (VPs) encoding the capsid-Pol fusion variants of SEQ ID NOs: 15-26. The genotype of each antigen is shown above each graph, and the antigen designations are shown on the horizontal axis (mutation: capsid-Pol ^mut , ^Δ1 : capsid-Pol ^Δ1 , Δ3: capsid-Pol ^Δ3 ). At day 14 post-administration, splenocytes were collected for analysis and T cell immune responses were assessed by IFN-γ ELISPOT (BD mouse IFN-γ ELISPOT kit, Cat# 551083) using overlapping pools of peptides corresponding to GT-D capsid and Pol. Bars show summed geometric mean responses for each group. SFU: halo forming units.

Figures 4A–4B show the immunogenicity of adenovirus vectors expressing capsid-Pol fusion proteins in outbred mice. Five- to seven-week-old outbred mice (n=8 per group) were injected intramuscularly with 1x108 viral particles (VP) of adenovirus encoding GT-A ^{capsidΡο1Δ3} or GT-B, C, or D capsid- ^PolΔ1 . At day 14 after injection, splenocytes were collected for analysis, and T cell immune responses were assessed by IFN-γ ELISPOT responses (BD mouse IFN-γ ELISPOT kit, Cat# 551083) to overlapping pools of peptides corresponding to GT-A and D capsid and Pol. Statistical comparisons between responses to peptides of different genotypes in mice receiving the same vaccine were assessed using Wilcoxon signed-rank tests. Statistical comparisons between mice receiving different vaccines were assessed using Mann-Whitney tests. (A) Responses to Pol peptides. (B) Responses to capsid peptides. SFU: halo-forming cells.

Figure 5 shows the immunogenicity of Pol-expressing adenovirus vectors. Six- to eight-week-old C57BL/6 mice (n=5 per group) were injected with 1x108 adenovirus viral particles (VPs) expressing Pol antigen variants SEQ ID NOs: 8, 12, 13, 14 or the full-length unmodified GT-D Pol sequence (GT-D Pol ^control ). Splenocytes were collected at day 14 after injection and T cell immune responses were assessed by IFN-γ ELISPOT (BD mouse IFN-γ ELISPOT kit, Cat# 551083) using overlapping pools of GT-D Pol-specific peptides. SFU: halo forming cells.

Figure 6 shows a schematic diagram of a study evaluating the efficacy of Ad5 vectors expressing HBV and vaccinia vectors in a mouse model of AAV CHB (AAV-HBV). Six- to eight-week-old C57BL/6 mice were transduced with ¹⁰¹² copies of the AAV-HBV genome on day -35. Mice were randomized to treatment groups based on serum HBsAg levels on day -7. Adenovirus type 5 primer vaccines expressing HBV antigens were administered intramuscularly (IM) in a volume of 50 μL on day 0, and booster vaccinia vaccines expressing the same HBV antigens were administered IM in a volume of 50 μL on day 32. Beginning on days 46–67, mice were injected with either the anti-PD-1 (anti-CD279) RMP 1-14 monoclonal antibody or an isotype control mAb. Blood samples for viral antigen testing were collected on days -7, 14, 27, 46, 60, 67, and 88. Splenocytes were collected for testing on day 88 and assessed for IFN-γ ELISPOT.

Figure 7 shows the immunogenicity of booster vaccination with Ad5 priming vaccine in AAV-HBV mice. Splenocytes were collected on day 88 in the study shown in Figure 6. T cell immune responses to HBsAg and Pol were assessed by IFN-γ ELISPOT (BD mouse IFN-γ ELISPOT kit, Cat# 551083) using overlapping pools of peptides corresponding to the GT-D sAg capsid and Pol. The dotted line indicates the highest signal in the HBsAg ELISPOT observed in mice receiving the control vaccine. mAb: monoclonal antibody injected. Iso: isotype control. aPD-1: anti-PD-L Vax: indicates whether the vaccine contained HBV antigens or control (Ctrl) antigens. SFU: halo forming cells.

Figure 8 shows the effects of booster vaccination with an Ad5 priming vaccine expressing HBV in combination with PD-1 blockade in AAV-HBV mice. Serum HBeAg levels in the study shown in Figure 6 were determined by ELISA (International Immunodicics) at the indicated time points. The dotted line indicates the lower limit of detection. Ad: adenovirus 5 vector. Vac: vaccinia vector. Ctrl: control antigen. Isotype: isotype control antibody.

- 21 047988 lo. aPD-1: anti-mouse PD-1 antibody.

Figures 9A-9C provide an overview of the arenavirus vector platforms demonstrated in the examples provided herein. (A) Schematic diagram of the phylogenetic tree of the arenavirus family (Arenaviridae). In the examples provided herein, lymphocytic choriomeningitis mammarenavirus (LCMV) (NCBI:txid1 1623) from the Old World (OW) clade and Kali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)) (NCBI:xid2169993) from the New World (NW) clade were selected to generate HBV antigen-encoding vectors. See, e.g., Buchmeier et al., 2001, Arenaviridae: The Viruses and Their Replication, Fields Virology Vol 2, 1635-1668. Arenavirus taxonomy has been reviewed in more detail recently, for example in Radoshitzky, et al., Arch Virol. (2015) 160(7):1851–74. Phylogenetic information on the Arenaviridae family is also available on the Viral Pathogen Resource website at viprbrc.org. (B) Schematic representation of replication-deficient arenavirus vectors having a two-segment genome as described in WO200908310 and (C) replication-attenuated arenavirus vectors having a three-segment genome as described in WO2016075250 and WO2017198726. The replication-deficient arenavirus vectors having a two-segment genome as described in WO2009083210 and used in the examples provided herein encode three of the four viral proteins (L, Z, and NP) and an open reading frame for insertion of a heterologous polynucleotide, such as encoding an antigen. The replication-deficient arenavirus vectors having a two-segment genome can only be propagated by delivery of the viral GP in trans. The replication-attenuated arenavirus vectors with a three-segment genome described in WO 2016075250 and WO 2017198726 have an artificial duplication of the genomic S segment, encode all four viral proteins (L, Z, NP and GP) and have two open reading frames for insertion of one or two heterologous polynucleotides, for example encoding one or two antigens.

Figure 10 shows the immunogenicity of Pol antigens in replication-incompetent mammarena lymphocytic choriomeningitis virus (LCMV) vectors. Six- to eight-week-old C57BL/6 mice (n=6 per group) were injected intravenously with 1x106 focus-forming units (FFU) of replication-incompetent LCMV vectors expressing the Pol antigen variants GT-D and GT-B Ρο1 ^δ1 (SEQ ID NOS: 6 and 8), Ρο1 ^Δ3 (SEQ ID NOS: 10 and 12), and Pol ³⁰⁰ (SEQ ID NOS: 13 and 14), or with medium as a negative control. On day 7 post-administration, splenocytes were collected for analysis and T cell immune responses were assessed by IFN-γ ELISPOT (BD mouse IFN-γ ELISPOT kit, Cat. No. 551083) using overlapping pools of Pol peptides corresponding to the genotype of the immunization antigen in each group. SFU: halo-forming units.

Figure 11 shows the immunogenicity of LCMV vectors expressing the capsid-HBsAg fusion protein in C57BL/6 mice. Six- to eight-week-old C57BL/6 mice (n=6 per group) were inoculated with 1x106 focus-forming units (FFU) of replication-incompetent LCMV vectors expressing the capsid-HBsAg fusion variants of SEQ ID NOs: 38-41 or mock-vaccinated as a negative control. At day 7 after administration, splenocytes were collected for analysis and T cell immune responses were assessed by IFN-γ ELISPOT (BD mouse IFN-γ ELISPOT kit, Cat# 551083) using overlapping pools of capsid and HBsAg peptides corresponding to the genotype of the immunization antigen in each group. SFU: halo-forming cells.

Figure 12 shows the antibody response to HBsAg obtained from mice injected with replication-incompetent LCMV vectors expressing the capsid-sAg fusion protein. Six- to eight-week-old C57BL/6 (left) or Balb/c (right) mice (n=5 per group) were injected with 1x106 focus-forming units (FFU) of replication-incompetent LCMV vectors expressing the capsid-sAg fusion variants of SEQ ID NOs: 38-41 or with medium as a negative control. On day 17 after injection, sera were collected and tested for anti-HBsAg antibody by ELISA (International Immunodeiagics). The dotted line indicates the lower limit of detection of 11 mIU/mL. *p<0.05 by Mann-Whitney test.

Fig. 13 shows the effect of nucleotide sequence modification on the T cell immunogenicity of capsid-P2A-sAg fusion proteins. Six- to eight-week-old C57BL/6 mice (n=6 per group) were injected with 1x106 focus-forming units (FFU) of replication-incompetent LCMV vectors with GT-D capsid-P2A-sAg (SEQ ID NO:36) or GT-D iCore-P2A-sAg (SEQ ID NO:37) or mock-vaccinated as a negative control. On day 7 post-administration, splenocytes were collected for analysis and T-cell immune responses were assessed by IFN-γ ELISPOT (BD mouse IFN-γ ELISPOT kit, Cat. #551083) using overlapping pools of capsid and sAg peptides. Statistical analysis was performed using Mann-Whitney tests.

Figures 14A–14B show the immunogenicity of prime/boost vaccination with replication-incompetent LCMV (VV1) vectors encoding GT-B/C capsid-P2A-sAg or GT-D iCore-P2A-sAg (Figure 14A) and GT-B Ρο1 ^Δ3 or GT-B Pol ³⁰⁰ (Figure 14B) in outbred mice. Animals were given 2 doses of each vaccine on days 0 and 28 as described in Table 9. Splenocytes were collected for study on day 42, and T cell immune responses to HBV antigens were measured by

- 22 047988

IFN-γ ELISPOT using pools of sAg, capsid and polymerase peptides from different viral genotypes as indicated. Data are expressed as background-subtracted values (without peptide). Statistical analysis was performed using Mann-Whitney tests. ns: not statistically significant; *p < 0.0332.

Figures 15A–15B show the coverage of HBV-specific T cell responses generated by priming/boosting vaccination with replication-incompetent LCMV (VV1) vectors encoding GT-D iCore-P2A-sAg (Figure 15A) or GT-B Pol ³⁰⁰ (Figure 15B) in outbred mice. IFN-γ ELISPOT was performed with peptides from the same viral genotype (filled rings) or a different viral genotype (open rings).

Figures 16A–16B show the immunogenicity of priming/boosting vaccination with replication-incompetent LCMV (VV1) vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ delivered either as individual vectors or as a combination cocktail in C57BL/6 mice. Animals were treated with 2 doses of vectors on days 0 and 21 as described in Table 10. Splenocytes were collected for study on day 28, and HBV-specific T cell responses were measured by FIN-γ ELISPOT using pools of capsid (16A), sAg (16B), and Pol (16C) peptides.

Figures 17A–17F show the immunogenicity of booster vaccinations with replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ in cynomolgus monkeys. A group of animals was also vaccinated with Ad5 and vaccinia vectors encoding the same HBV antigens. Animals were treated with vectors as described in Table 11. 17A: Group 1; 17B: Group 2; 17C: Group 3; 17D; Group 4; 17E: Group 5; 17F: Group 6. T cell responses to HBV antigens were assessed by performing IFN-γ ELISPOT using sAg, capsid, and Pol peptide pools at the indicated time points. Data are expressed as overall HBV-specific T cell responses defined as the sum of IFN-γ ELISPOT values obtained after stimulation with sAg, capsid and polymerase peptide pools.

Figures 18A-18F show the immunogenicity of booster vaccinations with replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Po1300 in cynomolgus monkeys as described in Figure 17 and Table 11. Figures 18A-18F focus on IFN-γ ELISPOT obtained after stimulation with capsid peptide pools. 18A: Group 1; 18B: Group 2; 18C: Group 3; 18D; Group 4; 18E; Group 5; 18F; Group 6.

Figures 19A-19F show the immunogenicity of booster vaccinations with replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ in cynomolgus monkeys as described in Figure 17 and Table 11. Figures 19A-19F focus on IFN-γ ELISPOT obtained after stimulation with sAg peptide pools. 19A: Group 1; 19B: Group 2; 19C: Group 3; 19D; Group 4; 19E: Group 5; 19F: Group 6.

Figures 20A-20F show the immunogenicity of booster vaccinations with replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Po1300 in cynomolgus monkeys as described in Figure 17 and Table 11. Figures 20A-20F focus on IFN-γ ELISPOT obtained after stimulation with Pol peptide pools. 20A: Group 1; 20B: Group 2; 20C: Group 3; 20D; Group 4; 20E: Group 5; 20F: Group 6.

Figures 21A–21B show the frequency of peripheral HBV-specific CD8+ T cells (A) and CD4+ T cells (B) at week 14 in cynomolgus monkeys from groups 1, 2, and 6 as described in Table 11. Data were obtained from PBMCs collected for studies at week 14 and restimulated with pools of sAg, capsid, and HBV polymerase peptides. CD4+ and CD8+ T subsets were then analyzed for intracellular IFN-γ by flow cytometry.

Figures 22A-22C show the HBsAg antibody response in cynomolgus monkeys from Group 1 (22A), Group 2 (22B), and Group 6 (22C) as described in Table 11. Serum samples were collected at the indicated time points and quantified for HBsAg antibodies by ELISA. The dotted line indicates the lower limit of quantification of the assay (5 mIU/mL).

Figure 23 shows a schematic diagram of a study evaluating the immunogenicity of replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ (HBV vaccine) alone or in combination with the immunomodulators anti-PD1, anti-CTLA4, anti-CD137, and FLT3LFc fusion in a mouse model of AAV-HBV. Six- to ten-week-old C57BL/6 mice were transduced with 10 ¹¹ copies of the AAV-HBV genome on day -35. Mice were randomized to treatment groups based on serum HBsAg levels on day 11. Replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Po1300 were administered intravenously (i.v.) in a volume of 200 μl on days 0, 21, and 42. Mice were injected intraperitoneally with 200 μl of (i) saline on days 0, 7, 14, 21, 28, 35, 42, 49, and 56; (ii) anti-PD-1 monoclonal antibody RMP1-14 on days 42, 46, 49, 53, 56, and 60; iii) anti-CTLA-4 monoclonal antibody clone 9D9 on days 0, 4, 7, 11, 14, 18, 21, 25, 28, 32, 35, 39, 42, 46, 49, and 53; iv) anti-CD137 monoclonal antibody clone mAb8 (IgG2b) on days 0, 21, and 42; v) FLT3L-Fc fusion protein on days -7, 14, and 35. Asterisks indicate the doses of each immunomodulator. Splenocytes were collected for

- 23 047988 studies on day 105 and assessed for IFN-γ ELISPOT using pools of sAg peptide, capsid and Pol. A group of C57BL/6 mice that did not receive AAV-HBV but were injected with replication-incompetent LCMV vectors separately were used as a positive control for IFN-γ ELISPOT.

Figures 24A–24C show the immunogenicity of booster vaccinations with replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Po1300 in AAVHBV mice as described in Table 12 and Figure 23. Splenocytes were collected at day 105 and assessed for IFN-γ ELISPOT using pools of sAg peptide (24A), capsid (24B), and polymerase (24C). Statistical analysis was performed using the Mann-Whitney tests. ns: not statistically significant; *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001.

Figure 25 shows the immunogenicity of priming/boosting vaccination with replication-incompetent PICV (VV2) vectors encoding GT-B Pol ³⁰⁰ ori or GT-B Pol ³⁰⁰ dint in C57BL/6 mice. Animals received 2 doses of vaccine on days 0 and 21. Splenocytes were collected on day 28, and HBV polymerase-specific T cell immune responses were measured by FIN-γ ELISPOT using Pol peptide pools. Data are expressed as background-subtracted values (without peptide). Statistical analysis was performed using Mann-Whitney tests. **p < 0.0021.

Figures 26A–26C show the immunogenicity of homologous and heterologous prime/boost vaccination with replication-incompetent LCMV (VV1) and PICV (VV2) vectors encoding GT-D iCore-P2A-sAg or GT-B Pol ³⁰⁰ in C57BL/6 mice. Animals were treated with 2 doses of vector on days 0 and 21 as described in Table 15. Splenocytes were collected on day 28, and HBV-specific T cell responses were measured by FIN-γ ELISPOT using sAg (26A), capsid (26B), and polymerase (26C) peptide pools. Data are expressed as background-subtracted values (no peptide).

Figure 27 shows the HBsAg antibody response in C57BL/6 mice administered replication-incompetent LCMV and PICV vectors encoding GT-D iCore-P2A-sAg using homologous (VV1/VV1) or heterologous (VV2/VV1) prime/boost vaccination on days 0 and 21. Serum samples were collected on day 28 and quantified for HBsAg antibodies by ELISA. The dotted line indicates the lower limit of quantification of the assay (20 mIU/mL). Statistical analysis was performed using the Mann-Whitney tests. **p < 0.0021.

Figures 28A–28C show the immunogenicity of homologous and heterologous prime/boost vaccination with replication-attenuated LCMV (TT1) and PICV (TT2) vectors encoding GT-D cascade-P2A-sAg or GT-B Pol ³⁰⁰ in C57BL/6 mice. Animals were treated with 2 doses of vectors on day 0 and day 21 as described in Table 16. Splenocytes were collected for study on day 28, and HBV-specific T cell responses were measured by FIN-γ ELISPOT using sAg (28A), capsid (28B), and polymerase (28C) peptide pools.

Figure 29 shows the immunogenicity of homologous and heterologous prime/boost vaccination with replication-deficient LCMV (VV1) and PICV (VV2) vectors encoding GT-D capsid-P2A-sAg and GT-B Pol ³⁰⁰ in cynomolgus monkeys. Animals were treated with 2 doses of vectors, one at week 0 and one at week 4. PBMCs were collected for study at week 6, and HBV-specific T cell immune responses were measured by FIN-γ ELISPOT using sAg, capsid, and polymerase peptide pools. Data are expressed as total HBV-specific T cell responses, defined as the sum of IFN-γ ELISPOT values obtained after stimulation with sAg, capsid, and polymerase peptide pools. The lower limit of quantification (LLOQ) of ELISPOT (dashed line) was defined as 200 IFN-y+ SFU/10 ⁶ PBMC. Statistical analysis was performed using the Mann-Whitney test.

Figure 30 shows the immunogenicity of homologous and heterologous prime/boost vaccination with replication-deficient LCMV (VV1) and PICV (VV2) vectors encoding GT-D capsid-P2A-sAg and GT-B Pol ³⁰⁰ administered weekly to cynomolgus monkeys. Animals were injected with 4 doses of vectors at weeks 0, 1, 2, and 3. PBMCs were collected for study at week 4, and HBV-specific T cell responses were measured by FIN-γ ELISPOT using sAg, capsid, and polymerase peptide pools. Data are expressed as total HBV-specific T cell responses defined as the sum of IFN-γ ELISPOT values obtained after stimulation with sAg, capsid and polymerase peptide pools. The lower limit of quantification (LLOQ) of ELISPOT (dashed line) was defined as 200 ZFN-y+ SFU/10 ⁶ PBMC.

Detailed description of the essence of the invention

1. Introduction

Polypeptides are provided that are used to provide protective immune responses to one or more hepatitis B virus (HBV) antigens in humans, the immunogenic polypeptides described herein are capable of inducing prophylactic and/or therapeutic immune responses in

- 24 047 988 humans to one or more hepatitis B virus (HBV) antigens. Typically, the immunogenic polypeptides described herein comprise highly conserved regions of HBV proteins to induce responses to epitopes that are identical between the vaccine antigen and the infecting HBV present in the patient, while also excluding lowly conserved regions, thereby preventing the emergence of immunodominant T cell responses targeting epitopes absent from the infecting HBV strain of the patient. The immunogenic polypeptides described herein additionally induce CD4+ and CD8+ T cell immune responses to facilitate the killing of infected cells, as well as additionally humoral immune responses to sAg antibodies that facilitate the clearance of sAg, thereby reducing or eliminating the dissemination of residual virus if absolute viral clearance is not achieved. Moreover, the immunogenic polypeptides described herein have demonstrated immunogenic activity when produced using vaccine technologies that are capable of inducing desired responses in humans and are stable in delivery vectors through a sufficient number of vector replication cycles to enable industrial production of the vaccine, the immunogenic polypeptides can be used in a variety of vector systems that induce CD4+ and CD8+ T cells and provide antibody responses in humans and other primates. In certain embodiments, the immunogenic polypeptides are expressed from arenavirus vectors that can be dosed multiple times without inducing antibodies to the vector, thereby overcoming the limitation of many previous viral vector technologies and providing the potential for enhanced therapeutic effect with multiple dosing.

2. Polypeptides used to stimulate the immune response to hepatitis B virus (HBV)

Immunogenic polypeptides used to stimulate, induce, and/or elicit an immunogenic response to one or more hepatitis B virus (HBV) antigens are provided. In various embodiments, the immunogenic polypeptides comprise variants and/or fragments of polypeptides encoded by the HBV polymerase (Pol) gene, and fusion polypeptides having, in sequence, from N-terminus to C-terminus, a variant and/or fragment of a polypeptide encoded by the HBV capsid gene and a variant and/or fragment of a polypeptide encoded by the surface antigen (sAg) gene. The immunogenic polypeptides may comprise amino acid sequences based on consensus or near-consensus sequences of HBV genotypes A, B, C, or D, and combinations thereof. Typically, the immunogenic polypeptides described herein do not comprise a sequence of HBV protein X (HBx), precapsid, pre-S1, pre-S2, or fragments thereof.

In various embodiments, the immunogenic polypeptides described herein and/or polynucleotides encoding such polypeptides are in isolated form. This means that such a polypeptide or polynucleotide is at least 50% by weight pure of interfering proteins, cellular and other contaminants arising from its production or purification, but does not exclude the possibility of combining the agent with excess pharmaceutically acceptable carrier(s) or other carrier medium designed to facilitate its use. The term isolated when applied to a polypeptide or polynucleotide as described herein means that the polypeptide or polynucleotide is substantially free of cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and can be in a dry or aqueous solution. Purity and homogeneity can be determined using known methods, for example, analytical chemistry techniques such as polyacrylamide gel electrophoresis, column chromatography, thin layer chromatography or high performance liquid chromatography (HPLC). The protein, which is the predominant species present in the preparation, is substantially purified. The isolated or purified polypeptide or polynucleotide is substantially free of other cellular material or culture medium when produced by recombinant techniques or chemical precursors or other chemicals when chemically synthesized. In various embodiments, the purified polypeptides and/or polynucleotides are at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (w/w) separated from, cleared of, or free of interfering proteins and contaminants from production or purification. Often, the agent is the predominant macromolecular species remaining after its purification.

HBV polymerase polypeptide variants

In various embodiments, truncated and/or mutant polypeptides with an internal deletion of hepatitis B virus (HBV) polymerase are provided.

Wild-type HBV has four domains arranged in tandem in a single polypeptide from N-terminus to C-terminus: a protein terminal (TP) domain conserved among the hepadnavirus family (aa residues 1–177), a spacer region (aa residues 178–335), a reverse transcriptase (RT) terminal protein binding domain (aa residues 336–678; containing the conserved NCBI pfam00078 or cd01645 domain), and a C-terminal RNase H (RH) domain (aa residues 679–832). See, e.g., Lanford, et al, J. Virol. (1999) 73(3): 1885–93; Voros, et al., J Virol. (2014) 88(5):2584–99 and Jones, et al., J Virol. (2014) 88(3):1564–72. In the HBV polymerase variants described herein, all or part of the spacer region has been deleted or eliminated. In the truncated polymerase mutants,

- 25 047988 zy VGV the entire TR domain was removed or excluded.

Typically, the enzymatic domains, i.e., the reverse transcriptase and RNase H domains, are inactivated in the HBV polymerase protein mutants described herein. In various embodiments, the reverse transcriptase domain does not comprise a YMDD motif (SEQ ID NO: 97). In some embodiments, the YMDD motif (SEQ ID NO: 97) in the reverse transcriptase domain is altered to YMHD (SEQ ID NO: 99). In some embodiments, the RNase H domain does not comprise an AELL motif (SEQ ID NO: 98). In some embodiments, the AELL motif (SEQ ID NO: 98) in the RNase H domain is altered to AHLL (SEQ ID NO: 100).

Truncated polymerase mutants

In some embodiments, the truncated HBV polymerase polypeptides comprise an inactivated reverse transcriptase domain and an inactivated RNase H, wherein the polypeptide does not comprise all of a terminal protein (TP) domain and does not comprise all or a portion of a spacer domain (i.e., the terminal protein (TP) domain and all or a portion of the spacer domain are removed, isolated, or excluded). In the truncated HBV polymerase polypeptides described herein, all of the TP domain and all or a portion or region of the spacer domain are removed or excluded. For example, in some embodiments, the N-terminal 300 amino acids of native or wild-type HBV polymerase are removed or excluded from the truncated HBV polymerase polypeptides described herein. In various embodiments, the inactivated reverse transcriptase domain and the inactivated RNase H can be directly fused or operably linked or connected via a linker as described herein. In some embodiments, the truncated HBV polymerase polypeptide is no more than 600 amino acids in length, such as no more than 595, 590, 585, 580, 575, 570, 565, 560, 555, 550, 545, 540, or 535 amino acids in length. In some embodiments, the truncated HBV polymerase polypeptides comprise 528, 529, 530, 531, 532, 533, 534, or 535 C-terminal amino acids of native or wild-type HBV polymerase.

In some embodiments, the truncated HBV polymerase polypeptides comprise an amino acid sequence corresponding to amino acid residues 300-832, 301-832, 302-832, 303-832, 304-832, 305-832, 306-832, 307-832, 308-832, 309-832, 310-832, 311-832, 312-832, 313-832, 314-832, 315-832, 316-832, 317-832, 318-832, 319-832, 320-832, 325-832, 326-832, 327-832, 328-832, 329-832, 330832, 331-832, 332-832, 333-832, 334-832, 335-832 or 336-832 of a native or wild-type HBV polymerase. As used herein, the numbering of a given amino acid polymer or nucleic acid polymer corresponds to, is consistent with, or is relative to the numbering of a selected or reference amino acid polymer or nucleic acid polymer when the position of any given polymer component (e.g., amino acid, nucleotide, also referred to generically as a residue) is designated by reference to the same or equivalent position (e.g., based on an optimal alignment or consensus sequence) in the selected amino acid polymer or nucleic acid polymer, rather than by the actual numerical position of the component in the polymer. In various embodiments, the truncated HBV polymerase polypeptides comprise an amino acid sequence corresponding to amino acid residues 300-832. In such embodiments, the N-terminus corresponds to amino acid position 300 of the prototype genotype D pol protein. The N-terminal 6 amino acid residues of this sequence are SARSQS (SEQ ID NO: 95) in the genotype D Pol antigen and SSRSQS (SEQ ID NO: 96) in the genotype B Pol antigen. Literature data have shown that this N-terminal initial region provides for RT domain function (see, e.g., Lanford, et al., supra) and expression of the truncated protein in vitro (see, e.g., Voros, et al., supra).

In some embodiments, the truncated HBV polymerase polypeptide is of HBV genotype B and comprises or consists of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In some embodiments, the truncated HBV polymerase polypeptide is of HBV genotype B and does not comprise (i.e., the sequence is excluded, isolated, or deleted; the sequence is not included) the polypeptide sequence of SEQ ID NO: 50, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 50.

In some embodiments, the truncated HBV polymerase polypeptide is of HBV genotype D and comprises or consists of the amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14. In some embodiments, the truncated HBV polymerase polypeptide is of HBV genotype D and does not comprise the polypeptide sequence of SEQ ID NO: 51 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 51.

Modifications can be made to the structure of the polypeptides and polynucleotides encoding such polypeptides described herein and still produce a functional molecule that encodes a variant or derivative of the polypeptide with desired (e.g., immunogenic) properties.

- 26 047988 ristics. When it is desired to change the amino acid sequence of a polypeptide to create an equivalent or even improved version or portion of the polypeptide described herein, one skilled in the art typically changes one or more codons of the coding DNA sequence.

For example, some amino acids can be replaced by other amino acids in the structure of a protein without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells. Since it is the binding ability and nature of a protein that determine its biological functional activity, certain amino acid sequence substitutions can be made in the protein sequence and, of course, in its coding DNA sequence, and still obtain a protein with similar properties. Thus, it is expected that various changes can be made in the polypeptide sequences of the described polypeptides or the corresponding DNA sequences that code for such polypeptides without appreciable loss of their biological effectiveness or activity.

As used herein, the term "substitution" means the replacement of one or more amino acids or nucleotides with other amino acids or nucleotides, respectively.

In many cases, a polypeptide variant will contain one or more conservative substitutions. A conservative substitution is one in which an amino acid is replaced by another amino acid that has similar properties, such that a peptide chemist would expect the secondary structure and hydropathicity profile of the polypeptide to be substantially unchanged.

As used herein, the term "identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned for maximum sequence matching, i.e., taking into account gaps and insertions. The sequences are substantially aligned for maximum matching over a designated region, for example, a region at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more amino acids or nucleotides in length and may be up to the full length of a reference polypeptide or polynucleotide sequence. For sequence comparison, typically one sequence is used as a reference sequence against which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer program, subsequence coordinates are designated if necessary, and the parameters of the sequence algorithm program are designated. Otherwise, standard parameters can be used. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence based on the specified program parameters.

When comparing polynucleotide and polypeptide sequences, two sequences are said to be identical if the nucleotide or amino acid sequence in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons of two sequences are typically performed by comparing the sequences in a comparison window to identify and compare local regions of sequence similarity. A comparison window, as used herein, refers to a segment of at least about 20 contiguous positions, typically from 30 to about 75, from 40 to about 50, or the entire length of the sequence, in which the sequence can be compared to a reference sequence with the same number of contiguous positions after the two sequences have been optimally aligned.

Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, WI) using default parameters. Several alignment schemes are implemented in this program, described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein, J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5: 151-153; Myers, E.W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E.D. (1971) Comb. Theor77: 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R. (1973) Numerical Taxonomy - the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

Alternatively, optimal alignment of sequences for comparison can be performed by the local alignment algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the similarity search method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual inspection.

- 27 047988

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein, to determine percent sequence identity of the polynucleotides and polypeptides described herein. Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information (ncbi. nlm.nih.gov).

In one illustrative example, cumulative scores can be calculated using the nucleotide sequence parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0). Expansion of word scores in each direction ceases when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score approaches zero or below due to the accumulation of one or more negative residue alignment scores; or at the end of each sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11 and an expectation (E) of 10, and a BLOSUM62 weight matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) of alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

For amino acid sequences, a scoring matrix can be used to calculate a cumulative score. Expansion of word scores in each direction ceases when: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score approaches zero or below due to accumulation of one or more negative residue alignment scores; or at the end of each sequence. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment.

In one approach, the percent sequence identity is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, such as at least 50 positions, at least 100 positions, or the entire length of a reference sequence, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may have additions or deletions (i.e., gaps) of 20 percent or less, typically 5 to 15% or 10 to 12%, compared to the reference sequences (which have no additions or deletions) for an optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, by dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the results by 100 to obtain the percent sequence identity.

A polypeptide variant, as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically described herein by one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more of the above polypeptide sequences described herein and assessing one or more biological activities of the polypeptide as described herein and/or using any of a number of methods well known in the art. The term variant may also refer to any natural or engineered molecule containing one or more nucleotide or amino acid mutations.

Illustrative truncated mutants of HBV polymerase for use in activating, inducing or eliciting an immunogenic response, such as with respect to a polymerase antigen expressed by HBV, are provided in Table A. Illustrative N-terminal sequence segments removed or excluded, and therefore not contained therein, of the truncated mutants of HBV polymerase described herein are provided in Table B.

- 28 047988

Table A. Pol ³⁰⁰ mutants - motifs containing inactivating mutations are underlined (YMDD mutated to YMHD, AELL mutated to AHLL)

SEQ ID NO: HBV genotype Length (number of amino acids) Polypeptide sequence 13 IN 534 MSSRSQSQGPVLSCWWLQFRNSEPCSEYCLCHIVNLIEDWGPCTEHGE HRIRTPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNTRVSWP KFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSG LSRYVARLSSNSRIINNQHRTMQNLHDSCSRNLYVSLMLLYKTYGRK LHLYSHPIILGFRK IPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYM HDVVLGAKSVQHLESLYAAVTNFLLSLGIHLNPHKTKRWGYSLNFMG YVIGSWGTLPQEHIVQKIKMCFRKLPVNRPIDWKVCQRIVGLLGFAAP FTQCGYPALMPLYACIQAKQAFTFSPTYKAFLSKQYLHLYPVARQRPG LCQVFADATPTGWGLAIGHQRMRGAFVSPLPIHTAHLLAACFARSRS GAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPAD DPSRGRLGLYRPLLRLLYRPTTGRTSLYADSPSVPSHLPDRVHFASPLH VAWRPP 14 D 534 MSARSQSERPVFPCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCAEHG EHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGNYRVSW PKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSS GLSRYVARLSSNSRIFNYQHGTMQNLHDSCSRNLYVSLMLLYQTFGR KLHLYSHPIIL GFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSY MHDVVLGAKSVQHLESLFTAVTNFLLSLGIHLNPNKTKRWGYSLHFM GYVIGCYGSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRIVGLLGFAAP FTQCGYPALMPLYACIQSKQAFTFSPTYKAFLCKQYLNLYPVARQRPG LCQVFADATPTGWGLVMGHQRMRGTFKAPLPIHTAHLLAACFARSRS GANILGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPAD DPSRGRLGLYRPLLRLPFRPTTGRTSLYADSPSVPSHLPDRVHFASPLH VAWRPP

Table B. N-terminal polypeptide sequence deleted from Pol ³⁰⁰ truncation mutants

SEQ ID NO: HBV genotype Polypeptide sequence 50 IN PLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKVG NFTGLYSSTVPVFNPEWQTPSFPHIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPARF YPNLTKYLPLDKGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRESTRSASFCGS PYSWEQDLQHGRLVFQ TSKRHGDKSFCPQSPGILPRSSVGPCIQNQLRKSRLGPQPA QGQLAGRQQGGSGSIRARVHPSPWGTVGVEPSGSGHIHNCASNSSSCLHQSAVRKA AYSHISTSKGHSSSGHAVELHHFPPS 51 D PLSYQHFRRLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLNLNVSIPWTHKVG NFTGLYSSTVPVFNPHWKTPSFPNIHLHQDIIKKCEQFVGPLTVNEKRRLQLIMPARF YPNVTKYLPLDKGIKPYYPEHLVNHYFQTRHYLHTLWKAGILYKRETTHSASFCGS PYSWEQELQHGAESFH QQSSGILSRPPVGSSLQSKHRKSRLGLQSQQGHLARRQQG RGWSIRAGIHPTARRPFGVEPSGSGHTANLASKSASCLYQSAVRKAAYPVVSTFKK HSSSGHAVELHNLPPN

In some embodiments, the truncated HBV polymerase polypeptide does not comprise an amino sequence or fragment thereof from another HBV protein. In some embodiments, the truncated HBV polymerase polypeptide does not comprise an amino sequence or fragment thereof from an HBV protein selected from the group consisting of precapsid, capsid, X, and envelope (e.g., small, medium, or large surface antigen (sAg)).

Internal deletion mutant polypeptides

Additionally provided are internal deletion mutant HBV polymerase polypeptides. In some embodiments, the internal deletion mutant HBV polymerase polypeptides comprise, in sequence from N-terminus to C-terminus, a terminal protein (TP) domain, an inactivated reverse transcriptase domain, and an inactivated RNase H, wherein the mutant polypeptide does not comprise all or a portion of a spacer domain (i.e., all or a portion of a spacer domain or region is removed or eliminated). In various embodiments, the deletion mutant HBV polymerase polypeptide is no more than 800 amino acids in length, such as no more than 795, 790, 785, 780, 775, 770, 765, 760, 755, 750, 745, 740, 735, 730, 725, 720, 715, 710, or 705 amino acids in length. In some embodiments, the internal deletion mutant HBV polymerase polypeptides comprise, in sequence from N-terminus to C-terminus, a terminal protein (TP) domain and an amino acid sequence corresponding to amino acid residues 300-832, 301-832, 302-832, 303-832, 304-832, 305-832, 306-832, 307-832, 308-832, 309-832, 310-832, 311-832, 312-832, 313-832, 314-832, 315-832, 316-832, 317-832, 318-832,319-832,

320-832,325-832,326-832, 327-832,328-832, 329-832, 330-832, 331-832, 332-832, 333-832, 334-832, 335-832, or 336-832 of a native or wild-type HBV polymerase. In various embodiments, the tail protein (TP) domain, the inactivated reverse transcriptase domain, and the inactivated RNase H can independently be directly fused or operably linked or connected via a linker, e.g., as described herein, e.g., as set forth in Table J.

In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of HBV genotype A and comprises or consists of the amino acid sequence

- 29 047988 any of SEQ ID NOs: 5 and 9, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 5 and 9. In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of HBV genotype A and does not comprise the polypeptide of SEQ ID NOs: 42 or 46, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 42 or 46.

In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of genotype B of HBV and comprises or consists of the amino acid sequence of any of SEQ ID NOs: 6 and 10, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 6 and 10. In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of genotype B of HBV and does not comprise the polypeptide of SEQ ID NO: 43 or 47, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 43 or 47.

In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of HBV genotype C and comprises or consists of the amino acid sequence of any of SEQ ID NOs: 8 and 11, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 8 and 11. In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of HBV genotype C and does not comprise the polypeptide of SEQ ID NO: 44 or 48, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 44 or 48.

In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of HBV genotype D and comprises or consists of the amino acid sequence of any of SEQ ID NOs: 9 and 12, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 9 and 12. In some embodiments, the internal deletion mutant HBV polymerase polypeptide is of HBV genotype D and does not comprise the polypeptide of SEQ ID NO: 45 or 49, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 45 or 49.

In some embodiments, the internal deletion mutant HBV polymerase polypeptide does not comprise an amino sequence or fragment thereof from another HBV protein. In some embodiments, the internal deletion mutant HBV polymerase polypeptide does not comprise an amino sequence or fragment thereof from an HBV protein selected from the group consisting of precapsid, capsid, X, and envelope (e.g., small, medium, or large surface antigen (sAg)).

Exemplary internal deletion mutant HBV polymerase polypeptides for use in activating, inducing, or eliciting an immunogenic response, e.g., to a polymerase antigen expressed by HBV, are provided in Tables C and E. Exemplary internal amino acid sequence segments deleted or excluded, and therefore not contained therein, of the internal deletion mutant HBV polymerase polypeptides described herein, e.g., corresponding to all or a portion of the spacer region of HBV polymerase, are provided in Tables D and F.

Capsid polymerase fusion polypeptides

In various embodiments, the internal deletion variants of the HBV polymerase polypeptide described herein are fused with an HBV capsid polypeptide. The capsid polypeptide can be positioned either N-terminal or C-terminal to the HBV polymerase. Also provided are fusion polypeptides comprising, sequentially from N-terminus to C-terminus, an HBV capsid polypeptide and a truncated or internal deletion mutant HBV polymerase polypeptide as described herein. In some embodiments, a capsid-Pol fusion polypeptide comprises a deletion mutant HBV polymerase polypeptide described herein, comprises, sequentially from N-terminus to C-terminus, an HBV capsid polypeptide and an internal deletion mutant HBV polymerase polypeptide as described herein.

In some embodiments, the capsid-Pol fusion polypeptide comprises or consists of an amino acid sequence of any of SEQ ID NOs: 19-26 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 19-26.

In some embodiments, the internal deletion mutant HBV capsid polymerase fusion protein does not comprise an amino sequence or fragment thereof from an HBV protein selected from the group consisting of X, precapsid, and envelope (e.g., small, medium, or large surface antigen (sAg)).

Illustrative capsid polymerase fusion proteins for use in activation, induction

- 30 047988 or inducing an immunogenic response, for example, against a capsid antigen and/or polymerase expressed by HBV, are presented in Table G.

Table C. Pol ^A1 mutants: Motifs containing inactivating mutations are underlined (YMDD mutated to YMHD, AELL mutated to AHLL). Amino acids in bold + underlined + italics at the site of deletion (last amino acid before the deleted region and first amino acid after the deleted region).

SEQ ID NO: HBV genotype Length (number of amino acids) Polypeptide sequence 5 A 755 MPLSYQHFRKLLLLLDDETEAGPLEEELPRLADEDLNRRVAEDLNL GNLNVSIPWTHKVGNFTGLYSSTVPIFNPEWQTPSFPKIHLHEDIAN RCQQFVGPLTVNEKRRLRLIMPARFYPNSTKYLPLDKGIKPYYPDH VVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELHH GRLVIKTSQRHGDEPFCSQPSGILSRSSVGPEFHSFPPSSARSQSQGP VFSCWWLQFRNTQPCSKYCLSHLVNLLEDWGPCDEHGEHHIRIPR TPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGITRVSWPKFAVP NLQ SLTNLLSSNLSWLSLDVSAAFYHIPLHPAAMPHLLVGSSGLSR YVARLSSNSRIHNNQHGTLQNLHDSCSRQLYVSLMLLYKTYGRKL HLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSY MHDVVLGAKSVQHLESLYTAVTNFLLSLGIHLNPNKTKRWGYSLN FMGYVIGSWGTLPQDHIVQKIKHCFRKLPINRPIDWKVCQRIVGLL GFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYKAFLSKQYLNLYP VARQRPGLCQVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAHLL AACFARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCTANWILRGTSF VYVPSALNP ADDPSRGRLGLYRPLLLRLPYRPTTGRTSLYAVSPSVPS HLPVRVHFASPLHVAWRPP 6 IN 749 MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGN LNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPHIHLQEDIINRC QQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLDKGIKPYYPEHVV NHYFQTRHYLHTLWKAGILYKRESTRSASFCGSPYSWEQDLQHGR LVFQ TSKRHGDKSFCPQSPGILPRNELHHFPPSSSRSQSQGPVLSCW WLQFRNSEPCSEYCLCHIVNLIEDWGPCTEHGEHRIRTPRTPARVT GGVFLVDKNPHNTTESRLVVDFSQFSRGNTRVSWPKFAVPNLQSL TNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRYVARL SSNSRIINNQHRTMQNLHDSCSRNLYVSLMLLYKTYGRKLHLYSHP IILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMHDW LGAKSVQHLESLYAAVTNFLLSLGIHLNPHKTKRWGYSLNFMGYV IGSWGTLPQEHIVQKIKMCFRKLPVNRPIDWKVCQRIVGLLGFAAP FTQCGYP ALMPLYACIQAKQAFTFSPTYKAFLSKQYLHLYPVARQ RPGLCQVFADATPTGWGLAIGHQRMRGAFVSPLPIHTAHLLAACF ARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVP SALNPADDPPSRGRLGLYRPLLRLLYRPTTGRTSLYADSPSVPSHLPD RVHFASPLHVAWRPP 7 WITH 753 MPLSYQHFRKLLLLLDDEAGPLEEELPRLADEDLNRRVAEDLNLGN LNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPHIHLQEDIINRC QQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLDKGIKPYYPEFITV NH YFKTRHYLHTLWKAGILYKRETTRSASFCGSPYSWEQELQHGRLV FQTSTRHGDESFCSQSSGILSRSPVGPELHNFPPSSARSQSEGPLLSC WWLQFRNSKPCSDYCLSFIIVNLLEDWGPCTEFIGEFINIRIPRTPARV TGGVFLVDKNPHNTTESRLVVDFSQFSRGSTHVSWPKFAVPNLQSL TNLLSSNLSWLSLD VSAAFYHLPLHPAAMPHLLVGSSGLSRYVARL SSTSRNINYQHGAMQDLHDSCSRNLYVSLLLLYKTFGRKLHLYSHP IILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMHDW LGAKSVQHLESLFTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVI GSWGTLPQEHIVLKIKQCFRKLPVNRPIDWKVCQRIVGLLGFAAPF TQCGYPALMPLYACIQAKQAFTFSPTYKAFLCKQYLNLYPVARQR SGLCQVFADATPTGWGLAVGHQRMRGTFVSPLPIHTAHLLAACFA RSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPS ALNP ADDPSRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPVR VHFASPLHVAWRPP 8 D 742 MPLSYQHFRRLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGN LNVSIPWTHKVGNFTGLYSSTVPVFNPHWKTPSFPNIHLHQDIIKKC KNP HNTAESRLVVDFSQFSRGNYRVSWPKFAVPNLQSLTNLLSSNL SWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRYVARLSSNSRIFN YQHGTMQNLHDSCSRNLYVSLMLLYQTFGRKLHLYSHPIILGFRKI PMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMHDVVLGAKSV QHLESLFTAVTNFLLSLGIHLNPNKTKRWGYSLHFMGYVIGCYGSL PQDFHIQKIKECFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPA LMPLYACIQSKQAFTFSPTYKAFLCKQYLNLYPVARQRPGLCQVFA DATPTGWGLVMGHQRMRGTFKAPLPIHTAHLLAACFARSRSGANI LGTDNSWLSRKY TSFPWLLGCAANWTLRGTSFVYVPSALNPADD PSRGRLGLYRPLLRLPFRPTTGRTSLYADSPSVPSHLPDRVHFASPL HVAWRPP

- 31 047988

Table D. Polypeptide sequences with an internal spencer deleted from Ρο1 ^Δ1 mutants and Ρο1 ^δ1 capsid-fusion proteins

SEQ ID NO: HBV genotype Polypeptide sequence 42 A CIRSQFKQSRLGLQPHQGPLATSQSGRSGSIRARVHSPTRRCFGVEPSGSGHIGHSA SSSSSCLHQSAVRKAAYSHLSTSKRQSSSGHAV 43 IN SVGPCIQNQLRKSRLGPQPAQGQLAGRQQGGSGSIRARVHPSPWGTVGVEPSGSG HIHNCASNSSSCLHQSAVRKAAYSHISTSKGHSSSGHAV 44 WITH CIRSQLKQSRLGLQPQQGSLARSKSGRSGSIRARVHPTTRQSFGVEPSGSGHIDNSA SSASSCLHQSAVRKTAYSHLSTSKRQSSSGHAV 45 D SLQSKHRKSRLGLQSQQGHLARRQQGRGWSIRAGIHPTARRPFGVEPSGSGHTAN LASKSASCLYQSAVRKAAYPVVSTFKKHSSSGHAV

Table E. Pol ^Δ3 mutants - motifs containing inactivating mutations are underlined (YMDD mutated to YMHD, AELL mutated to AHLL). Amino acids in bold + underlined + italicized at the site of deletion (last amino acid before the deleted region and first amino acid after the deleted region).

SEQ ID NO: HBV genotype Length (number of amino acids) Polypeptide sequence 9 A 705 MPLSYQHFRKLLLLDDETEAGPLEEELPRLADEDLNRRVAEDL NLGNLNVSIPWTHKVGNFTGLYSSTVPIFNPEWQTPSFPKIHLHE DIANRCQQFVGPLTVNEKRRLRLIMPARFYPNSTKYLPLDKGIK PYYPDHVVNHYFQTRHYLHTLWKAGILYKRETTRSASFCGSPY SWEQELHHGCW WLQFRNTQPCSKYCLSHLVNLLEDWGPCDE HGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRG ITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLHPA AMPHLLVGSSGLSRYVARLSSNSRIHNNQHGTLQNLHDSCSRQ LYVSLMLLYKTYGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQF TSAICSVVRRAFPHCLAFSYMHDVVLGAKSVQHLESLYTAVTN

- 32 047988

FLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQDHIVQKI KHCFRKLPINRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYA CIQAKQAFTFSPIYKAFLSKQYLNLYPVARQRPGLCQVFADAT PTGWGLAIGHQRMRGTFVAPLPIHTAHLLAACFARSRSGAKLIG TDNSVVLSRKYTSFPW LLGCTANWILRGTSFVYVPSALNPADD PSRGRLGLYRPLLLRLPYRPTTGRTSLYAVSPSVPSHLPVRVHFAS PLHVAWRPP 10 IN 703 MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNL GNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPHIHLQED IINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLDKGIKP YYPEHVVNHYFQTRHYLHTLWKAGILYKRESTRSASFCGSPYS WEQDLQHGCWWLQFR NSEPCSEYCLCHIVNLIEDWGPCTEHG EHRIRTPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNT RVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAA MPHLLVGSSGLSRYVARLSSNSRII NNQHRTMQNLHDSCSRNLYVSLMLLYKTYGRKLHLYSHPIILG FRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMHDWL GAKSVQHLESLYAAVTNFLLSLGIHLNPHKTKRWGYSLNFMG YVIGSWGTLPQEHIVQKIKMCFRKLPVNRPIDWKVCQRIVGLLG FAAPFTQCGYPALMPLYAC IQAKQAFTFSPTYKAFLSKQYLHL YPVARQRPGLCQVFADATPTGWGLAIGHQRMRGAFVSPLPIHT AHLLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWLLGCAANW ILRGTSFVYVPSALNPADDPPSRGRLGLYRPLLRLLYRPTTGRTSL YADSPSVPSHLPDRVHFASPLHVAWRPP 11 WITH 703 MPLSYQHFRKLLLLLDDEAGPLEEELPRLADEDLNRRVAEDLNL GNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFPHIHLQED IINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLDKGIKP YYPEHTVNHYFKTRHYLHTLWKAGILYKRETTRSASFCGSPYS WEQELQHfiCWWLQ FRNSKPCSDYCLSHIVNLLEDWGPCTEHG EHNIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGST HVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAA MPHLLVGSSGLSRYVARLSSTSRNINYQHGAMQDLHDSCSRNL YVSLLLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTS AICSVVRRAFPHCLAFSYMHDVVLGAKSVQHLESLFTAVTNFL LSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQEHIVLKIKQ CFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACI QAKQAFTFSPTYKAFL CKQYLNLYPVARQRSGLCQVFADATPT GWGLAVGHQRMRGTFVSPLPIHTAHLLAACFARSRSGAKLIGT DNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDP SRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPVRVHFASP LHVAWRPP 12 D 703 MPLSYQHFRRLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNL GNLNVSIPWTHKVGNFTGLYSSTVPVFNPHWKTPSFPNIHLHQD IIKKCEQFVGPLTVNEKRRLQLIMPARFYPNVTKYLPLDKGIKP YYPEHLVNHYFQTRHYLHTLWKAGILYKRETTHSASFCGSPYS WEQELQHfiGWWL QFRNSKPCSDYCLSHIVNLLEDWGPCAEHG EHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGNY RVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAA MPHLLVGSSGLSRYVARLSSNSRIFNYQHGTMQNLHDSCSRNL YVSLMLLYQTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTS AICSVVRRAFPHCLAFSYMHDVVLGAKSVQHLESLFTAVTNFL LSLGIHLNPNKTKRWGYSLHFMGYVIGCYGSLPQDHIIQKIKEC FRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQ SKQAFTFSPTYKAFLCKQYLNLYPVARQRPGLCQVFADATPTG WGLVMGHQRMRGTFKAPLPIHTAHLLAACFARSRSGANILGTD NSWLSRKYTSFPWLLGCAANWIL RGTSFVYVPSALNPADDPS RGRLGLYRPLLRLPFRPTTGRTSLYADSPSVPSHLPDRVHFASPL HVAWRPP

Table F. Polypeptide sequences with internal spacer deleted from Ρο1 ^Δ3 mutants and ρ-ρsid-RoP ³ fusion proteins

SEQ ID NO: HBV genotype Polypeptide sequence 46 A RLVIKTSQRHGDEPFCSQPSGILSRSSVGPCIRSQFKQSRLGLQPHQGPLATSQSG RSGSIRARVHSPTRRCFGVEPSGSGHIGHSASSSSSCLHQSAVRKAAYSHLSTSKR QSSSGHAVEFHSFPPSSARSQSQGPVFS 47 IN RLVFQTSKRHGDKSFCPQSPGILPRSSVGPCIQNQLRKSRLGPQPAQGQLAGRQQ GGSGSIRARVHPSPWGTVGVEPSGSGHIHNCASNSSSCLHQSAVRKAAYSHISTS KGHSSSGHAVELHHFPPSSRSQSQGPVLS 48 c RLVFQTSTRHGDESFCSQSSGILSRSPVGPCIRSQLKQSRLGLQPQQGSLARSKSG RSGSIRARVHPTTRQSFGVEPSGSGHIDNSASSASSCLHQSAVRKTAYSHLSTSKR QSSSGHAVELHNFPPSSARSQSEGPLLS 49 D AESFHQQSSGILSRPPVGSSLQSKHRKSRLGLQSQQGHLARRQQGRGWSIRAGIH PTARRPFGVEPSGSGHTANLASKSASCLYQSAVRKAAYPVVSTFKKHSSSGHAV ELHNLPPNSARSQSERPVFP

- 33 047988

Table G. Capsid-Pol fusion proteins

SEQ ID NO: HBV genotype Length (number of amino acids) Polypeptide sequence Capsid-Po! ^mutated fusion proteins - Capsid sequences are shown in bold + underlined. Motifs containing inactivating mutations in Pol are underlined (YMDD mutated to YMHD, AELL mutated to AHLL). 15 A 1030 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMTLATWVGNNLEDPASRDLVV NYVNTNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSOSP RRRRSOSRESOCMPLSYOHFRKLLLLLDDETEAGPLEEELPRLADE DLNRRVAEDLNLNLNVSIPWTHKVGNFTGLYSSTVPIFNPEWQT PSFPKIHLHEDIANRCQQFVGPLTVNEKRRLRLIMPARFYPNSTKY LPLDKGIKPYYPDHVVNHYFQTRHYLHTLWKAGILYKRETTRSAS FCGSPYSWEQEL HHGRLVIKTSQRHGDEPFCSQPSGILSRSSVGPCI RSQFKQSRLGLQPHQGPLATSQSGRSGSIRARVHSPTRRCFGVEPS GSGHIGHSASSSSSCLHQSAVRKAAYSHLSTSKRQSSSGHAVEFHS FPPSARSQSQGPVFSCWWLQFRNTQPCSKYCLSHLVNLLEDWGP CDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSR GITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLHPAA MPHLLVGSSGLSRYVARLSSNSRIHNNQHGTLQNLHDSCSRQLYV SLMLLYKTYGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICS VVRRAFPHCLAFSYMHDVVL GAKSVQHLESLYTAVTNFLLSLGIH LNPNKTKRWGYSLNFMGYVIGSWGTLPQDHIVQKIKHCFRKLPIN RPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSP TYKAFLSKQYLNLYPVARQRPGLCQVFADATPTGWGLAIGHQRM RGTFVAPLPIHTAHLLAACFARSRSGAKLIGTDNSVVLSRKYTSFP WLLGCTANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLLRLPY RPTTGRTSLYAVSPSVPSHLPVRVHFASPLHVAWRPP 16 IN 1026 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRELVV SYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRKLLLLLDDEAGPLEEELPRLADEGLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFP HIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLD KGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRESTRSASFCGS PYSWEQDLQH GRLVFQTSKRHGDKSFCPQSPGILPRSSVGPCIQNQ LRKSRLGPQPAQGQLAGRQQGGSGSIRARVHPSPWGTVGVEPSGS GHIHNCASNSSSCLHQSAVRKAAYSHISTSKGHSSSGHAVELHHFP PSSSRSQSQGPVLSCWWLQFRNSEPCSEYCLCHIVNLIEDWGPCTE HGEHRIRTPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNT RVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMP HLLVGSSGLSRYVARLSSNSRIINNQHRTMQNLHDSCSRNLYVSL MLLYKTYGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVV RRAFPHCLAFSYMHDVVLGAKSVQ HLESLYAAVTNFLLSLGIHLN PHKTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKMCFRKLPVNRP IDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTSPT YKAFLSKQYLHLYPVARQRPGLCQVFADATPTGWGLAIGHQRMR GAFVSPLPIHTAHLLAACFARSRSGAKLIGTDNSVVLSRKYTSFPW LLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLLYR PTTGRTSLYADSPSVPSHLPDRVHFASPLHVAWRPP 17 WITH 1026 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRELVV SYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSOSPR

- 34 047988

RRRSOSRESOCMPLSYOHFRKLLLLLDDEAGPLEEELPRLADEDLN RRVAEDLNLNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSF PHIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPL DKGIKPYYPEHTVNHYFKTRHYLHTLWKAGILYKRETTRSASFCG SPYSWEQELQ HGRLVFQTSTRHGDESFCSQSSGILSRSPVGPCIRSQ LKQSRLGLQPQQGSLARSKSSGRSGSIRARVHPTTRQSFGVEPSGSG HIDNSASSASSCLHQSAVRKTAYSHLSTSKRQSSSGHAVELHNFPP SSARSQSEGPLLSCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCTE HGEHNIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGST HVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMP HLLVGSSGLSRYVARLSSTSRNINYQHGAMQDLHDSCSRNLYVSL LLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVV QHLESLFTAVTNFLLSLGIHLN PNKTKRWGYSLNFMGYVIGSWGTLPQEHIVLKIKQCFRKLPVNRP IDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPT YKAFLCKQYLNLYPVARQRSGLCQVFADATPTGWGLAVGHQRM RGTFVSPLPIHTAHLLAACFARSRSGAKLIGTDNSVVLSRKYTSFP WLLGCAANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPF RPTTGRTSLYAVSPSVPSHLPVRVHFASPLHVAWRPP 18 D 1015 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGVNLEDPASRDLVV SYVNTNMGLKFROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRRLLLLDDEAGPLEEELPRLADEGLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPHWKTPSFP NIHLHQDIIKKCEQFVGPLTVNEKRRLQLIMPARFYPNVTKYLPLD KGIKPYYPEHLVNHYFQTRHYLHTLWKAGILYKRETTHSASFCGS PYSWEQELQH GAESFHQQSSGILSRPPVGSSLQSKHRKSRLGLQSQ QGHLARRQQGRGWSIRAGIHPTARRPFGVEPSGSGHTANLASKSA SCLYQSAVRKAAYPVVSTFKKHSSSGHAVELHNLPPNSARSQSER PVFPCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCAEHGEHHIRIPR TPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGNYRVSWPKFA VPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSG LSRYVARLSSNSRIFNYQHGTMQNLHDSCSRNLYVSLMLLYQTFG RKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCL AFSYMHDVVLGAKSVQHLESLF TAVTNFLLSLGIHLNPNKTKRWG YSLHFMGYVIGCYGSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRI VGLLGFAAPFTQCGYPALMPLYACIQSKQAFTFSPTYKAFLCKQY LNLYPVARQRPGLCQVFADATPTGWGLVMGHQRMRGTFKAPLPI HTAHLLAACFARSRSGANILGTDNSVVLSRKYTSFPWLLGCA AN WILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPFRPTTGRTSL YADSPSVPSHLPDRVHFASPLHVAWRPP Capsid-Po1 ^L1 fusion proteins — Capsid sequences are shown in bold + underline. Motifs containing inactivating mutations in Pol are underlined (YMDD mutated to YMHD, AELL mutated to AHLL). Amino acids in bold + italics are at the deletion site (last amino acid before the deleted region and first amino acid after the deleted region). 19 A 940 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMTLATWVGNNLEDPASRDLVV NYVNTNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPS

- 35 047988

PRRRRSOSPRRRRSOSRESOCMPLSYOHFRKLLLLDDETEAGPLE EELPRLADEDLNRRVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTV PIFNPEWQTPSFPKIHLHEDIANRCQQFVGPLTVNEKRRLRLIMPAR FYPNSTKYLPLDKGIKPYYPDHVVNHYFQTRHYLHTLWKAGILYK RETTRSASFCGSPYSWE QELHHGRLVIKTSQRHGDEPFCSQPSGILS RSSVGPEFHSFPPSSARSQSQGPVFSCWWLQFRNTQPCSKYCLSHL VNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESR LVVDFSQFSRGITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAA FYHIPLHPAAMPHLLVGSSGLSRYVARLSSNSRIHNNQHGTLQNLH DSCSRQLYVSLMLLYKTYGRKLHLYSHPIILGFRKIPMGVGLSPFL LAQFTSAICSVVRRAFPHCLAFSYMHDVVLGAKSVQHLESLYTAV TNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQDHIVQKI KHCFRKLPINRPIDWKVCQ RIVGLLGFAAPFTQCGYPALMPLYACI QAKQAFTFSPTYKAFLSKQYLNLYPVARQRPGLCQVFADATPTG WGLAIGHQRMRGTFVAPLPIHTAHLLAACFARSRSGAKLIGTDNS VVLSRKYTSFPWLLGCTANWILRGTSFVYVPSALNPADDPSRGRL GLYRPLLLRLPYRPTTGRTSLYAVSPSVPSHLPVRVHFASPLH VAWR RR 20 IN 932 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRELVV SYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRKLLLLLDDEAGPLEEELPRLADEGLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFP HIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLD KGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRESTRSASFCGS PYSWEQDLQH GRLVFQTSKRHGDKSFCPQSPGILPRAELHHFPPSS SRSQSQGPVLSCWWLQFRNSEPCSEYCLCHIVNLIEDWGPCTEHG EHRIRTPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNTRV SWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHL LVGSSGLSRYVARLSSNSRIINNQHRTMQNLHDSCSRNLYVSLML LYKTYGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRR AFPHCLAFSYNfflDVVLGAKSVQHLESLYAAVTNFLLSLGIHLNPH KTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKMCFRKLPVNRPID WKVCQRIVG LLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYK AFLSKQYLHLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGA FVSPLPIHTAHLLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWLL GCAANWILRGTSFVYVPSALNPADDPPSRGRLGLYRPLLRLLYRPT TGRTSLYADSPSVPSHLPDRVHFASPLH VAWRPP 21 WITH 936 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRELVV SYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRKLLLLLDDEAGPLEEELPRLADEDLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFP HIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLD KGIKPYYPEHTVNHYFKTRHYLHTLWKAGILYKRETTRSASFCGS PYSWEQELQHGR LVFQTSTRHGDESFCSQSSGILSRSPVGPELHNF PPSSARSQSEGPLLSCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCT EHGEHNIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGS THVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAM PHLLVGSSGLSRYVARLSSTSRNINYQHGAMQDLHDSCSRNLYVS LLLLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSV VRRAFPHCLAFSYMHDVVLGAKSVOHL

- 36 047988

ESLFTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQ EFIIVLKIKQCFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPAL MPLYACIQAKQAFTFSPTYKAFLCKQYLNLYPVARQRSGLCQVFA DATPTGWGLAVGHQRMRGTFVSPLPIHTAHLLAACFARSRSGAKL IGTDNSV VLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADD PSRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPVRVHFASPL HVAWRPP 22 D 925 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGVNLEDPASRDLW SYVNTNMGLKFROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTWRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRRLLLLDDEAGPLEEELPRLADEGLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPHWKTPSFP NIHLHQDIIKKCEQFVGPLTVNEKRRLQLIMPARFYPNVTKYLPLD KGIKPYYPEHLVNHYFQTRHYLHTLWKAGILYKRETTHSASFCGS PYSWEQELQH GAESFHQQSSGILSRPPVGyELHNLPPNSARSQSER PVFPCWWLQFRNSKPCSDYCLSFIIVNLLEDWGPCAEFIGEFIFIIRIPR TPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGNYRVSWPKFA VPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSG LSRYVARLSSNSRIFNYQHGTMQNLHDSCSRNLYVSLMLLYQTFG RKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCL AFSYMHDVVLGAKSVQHLESLFTAVTNFLLSLGIHLNPNKTKRWG YSLHFMGYVIGCYGSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRI VGLLGFAAPFTQCGY PALMPLYACIQSKQAFTFSPTYKAFLCKQY LNLYPVARQRPGLCQVFADATPTGWGLVMGHQRMRGTFKAPLPI htahllaacfarsrsganilgtdnsvvlsrkytsfpwllgcaan WILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPFRPTTGRTSL YADSPSVPSHLPDRVHFASPLHVAWRPP Capsid-Po1 ^L3 fusion proteins — Capsid sequences are shown in bold + underlined. Motifs containing inactivating mutations in Pol are underlined (YMDD mutated to YMHD, AELL mutated to AHLL). Amino acids in bold + underlined + italicized are at the deletion site (last amino acid before the deleted region and first amino acid after the deleted region). 23 A 890 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMTLATWVGNNLEDPASRDLW NYVNTNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTWRRRDRGRSPRRRTPSPRRRRSOSP RRRRSOSRESOCMPLSYOHFRKLLLLLDDETEAGPLEEELPRLADE DLNRRVAEDLNLNLNVSIPWTHKVGNFTGLYSSTVPIFNPEWQT PSFPKIFILFIEDIANRCQQFVGPLTVNEKRRLRLIMPARFYPNSTKY LPLDKGIKPYYPDHVVNHYFQTRHYLHTLWKAGILYKRETTRSAS fcgspysweq elhhgcwwlqfrntqpcskyclshlvnlledwgp CDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSR GITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLHPAA MPHLLVGSSGLSRYVARLSSNSRIHNNQHGTLQNLHDSCSRQLYV SLMLLYKTYGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICS VVRRAFPHCLAFSYMHDVVLGAKSVQHLESLYTAVTNFLLSLGIH LNPNKTKRWGYSLNFMGYVIGSWGTLPQDHIVQKIKHCFRKLPIN RPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSP TYKAFLSKQ YLNLYPVARQR PGLCQVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAHLLAACFA RSRSGAKLIGTDNSVVLSRKYTSFPWLLGCTANWILRGTSFVYVPS ALNPADDPPSRGRLGLYRPLLLRLPYRPTTGRTSLYAVSPSVPSHLPV RVHFASPLHVAWRPP

- 37 047988

24 IN 886 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRELW SYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTWRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRKLLLLLDDEAGPLEEELPRLADEGLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFP HIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLD KGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRESTRSASFCGS PYSWEQDLQH GCWWLQFRNSEPCSEYCLCHIVNLIEDWGPCTEH GEHRIRTPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGNTR VSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPH LLVGSSGLSRYVARLSSNSRIINNQHRTMQNLHDSCSRNLYVSLM LLYKTYGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVR RAFPHCLAFSYMHDVVLGAKSVQHLESLYAAVTNFLLSLGIHLNP HKTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKMCFRKLPVNRPI DWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTY KAFLSKQYL HLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRG AFVSPLPIHTAHLLAACFARSRSGAKLIGTDNSVVLSRKYTSFPWL LGCAANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLLYRP TTGRTSLYADSPSVPSHLPDRVHFASPLHVAWRPP 25 WITH 886 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRELVV SYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTWRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRKLLLLLDDEAGPLEEELPRLADEDLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPEWQTPSFP HIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPARFYPNLTKYLPLD KGIKPYYPEHTVNHYFKTRHYLHTLWKAGILYKRETTRSASFCGS PYSWEQELQHGC WWLQFRNSKPCSDYCLSHIVNLLEDWGPCTEH GEHNIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGSTH VSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPH LLVGSSGLSRYVARLSSTSRNINYQHGAMQDLHDSCSRNLYVSLL LLYKTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVR RAFPHCLAFSYMHDVVLGAKSVQHLESLFTAVTNFLLSLGIHLNP NKTKRWGYSLNFMGYVIGSWGTLPQEHIVLKIKQCFRKLPVNRPI DWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTY KAFLCKQY LNLYPVARQRSGLCQVFADATPTGWGLAVGHQRMR GTFVSPLPIHTAHLLAACFARSRSGAKLIGTDNSVVLSRKYTSFPW LLGCAANWILRGTSFVYVPSALNPADDPPSRGRLGLYRPLLRLPFRP TTGRTSLYAVSPSVPSHLPVRVHFASPLHVAWRPP 26 D 886 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALES PEHCSPHHTALROAILCWGELMNLATWVGVNLEDPASRDLVV SYVNTNMGLKFROLLWFHISCLTFGRETVLEYLVSFGVWIRTP PAYRPPNAPILSTLPETTWRRRGRSPRRRTPSPRRRRSOSPRR RRSOSRESOCMPLSYOHFRRLLLLDDEAGPLEEELPRLADEGLNR RVAEDLNLGNLNVSIPWTHKVGNFTGLYSSTVPVFNPHWKTPSFP NIHLHQDIIKKCEQFVGPLTVNEKRRLQLIMPARFYPNVTKYLPLD KGIKPYYPEHLVNHYFQTRHYLHTLWKAGILYKRETTHSASFCGS PYSWEQELQH GCWWLQFRNSKPCSDYCLSHIVNLLEDWGPCAEH GEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLWDFSQFSRGNYR VSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPH LLV GSSGLSRYVARLSSNSRIFNYQHGTMQNLHDSCSRNLYVSLMLLY QTFGRKLHLYSHPIILGFRKIPMGVGLSPFLLAQFTSAICSVVRRAF PHCLAFSYMHDVVLGAKSVQHLESLFTAVTNFLLSLGIHLNPNKT KRWGYSLHFMGYVIGCYGSLPQDHIIQKIKECFRKLPVNRPIDWK VCQRIVGLLGFAAPFTQ CGYPALMPLYACIQSKQAFTFSPTYKAFL CKQYLNLYPVARQRPGLCQVFADATPTGWGLVMGHQRMRGTFK APLPIHTAHLLAACFARSRSGANILGTDNSVVLSRKYTSFPWLLGC AANWILRGTSFVYVPSALNPADDPSRGRLGLYRPLLRLPFRPTTGR TSLYADSPSVPSHLPDRVHFASPLHVAWRPP

Capsid-sAg fusion proteins

Fusion proteins are further provided, consisting of an N-terminal portion comprising a HBV capsid polypeptide or an immunogenic fragment thereof, and a C-terminal portion comprising a small HBV surface antigen or an immunogenic fragment thereof. In various embodiments, the HBV capsid polypeptide or a fragment thereof and the HBV small surface antigen (sAg) or a fragment thereof are directly fused or adjacent. In some embodiments, the HBV capsid polypeptide or a fragment thereof and the HBV small surface antigen or a fragment thereof are connected via a linker.

HBV capsid polypeptide or its immunogenic fragment

In various embodiments, the HBV capsid polypeptide or immunogenic fragment thereof from the capsid-sAg fusion protein may independently be derived from HBV genotype A, B, C, or D. Exemplary amino acid sequences of the HBV capsid polypeptide that may be used in the capsid-sAg fusion proteins described herein are provided in Table H.

- 38 047988

Table H. Illustrative sequences of HBV capsid polypeptides

SEQ ID NO: HBV genotype Polypeptide sequence 64 A MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETV LEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPR RRRSQSRESQC 65 B/S MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETV LEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRR RSQSRESQC 66 D MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMNLATWVGVNLEDPASRDLVVSYVNTNMGLKFRQLLWFHISCLTFGRET VLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRR RRSQSRESQC

In some embodiments, the capsid polypeptide in the capsid-sAg fusion polypeptide comprises or consists of an amino acid sequence of any of SEQ ID NOs: 64-66 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 64-66. In some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO: 65 or SEQ ID NO: 66.

HBV small surface antigen or its immunogenic fragment

In various embodiments, the HBV sAg polypeptide or immunogenic fragment thereof from the capsid-sAg fusion protein can independently be derived from HBV genotype A, B, C, or D. Exemplary amino acid sequences of the HBV sAg polypeptide that can be used in the capsid-sAg fusion proteins described herein are provided in Table 1 in Example 1 below.

In some embodiments, the sAg polypeptide in the capsid-sAg fusion polypeptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-4 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-4, such as comprising one or more serine (S) residues at the amino acid position corresponding to position 53, isoleucine (I) residues at the amino acid position corresponding to position 68, threonine (T) residues at the amino acid position corresponding to position 125, proline (P) at the amino acid position corresponding to position 127, phenylalanine residues (F) at the amino acid position corresponding to position 161, tyrosine residues (Y) at the amino acid position corresponding to position 200, serine residues (S) at the amino acid position corresponding to position 210, and leucine residues (L) at the amino acid position corresponding to position 213.

With respect to fusion proteins, the capsid-sAg, the HBV capsid polypeptide, and the HBV sAg polypeptide may be derived from the same or different HBV genotypes. In some embodiments, the capsid-sAg fusion protein comprises, in sequence from N-terminus to C-terminus, an HBV capsid polypeptide and a small HBV surface antigen (sAg) polypeptide, wherein the capsid polypeptide belongs to HBV genotype A, and the sAg polypeptide belongs to HBV genotype A;

the capsid polypeptide belongs to the genotype B or C of HBV, and the sAg polypeptide belongs to the genotype B of HBV;

the capsid polypeptide belongs to the genotype B or C of HBV, and the sAg polypeptide belongs to the genotype C of HBV;

the capsid polypeptide belongs to the HBV genotype D, and the sAg polypeptide belongs to the HBV genotype D;

the capsid polypeptide belongs to the HBV genotype A, and the sAg polypeptide belongs to the HBV genotype B;

The capsid polypeptide belongs to the HBV genotype A, and the sAg polypeptide belongs to the HBV genotype C;

the capsid polypeptide belongs to the HBV genotype A, and the sAg polypeptide belongs to the HBV genotype D;

the capsid polypeptide belongs to genotype B or C of HBV, and the sAg polypeptide belongs to genotype A of HBV;

the capsid polypeptide belongs to genotype B or C of HBV, and the sAg polypeptide belongs to genotype D of HBV;

The capsid polypeptide belongs to the genotype D of HBV, and the sAg polypeptide belongs to genotype A

- 39 047988

VGV;

the capsid polypeptide belongs to genotype D of HBV and the sAg polypeptide belongs to genotype B of HBV. or the capsid polypeptide belongs to genotype D of HBV and the sAg polypeptide belongs to genotype C of HBV.

In some embodiments, the capsid-sAg fusion protein comprises, sequentially from N-terminus to C-terminus, an HBV capsid polypeptide and a small HBV surface antigen (sAg) polypeptide, wherein:

the capsid polypeptide belongs to genotype B or C of HBV, and the sAg polypeptide belongs to genotype C of HBV; or the capsid polypeptide belongs to genotype D of HBV, and the sAg polypeptide belongs to genotype D of HBV.

In some embodiments, the capsid-sAg fusion protein comprises, sequentially from N-terminus to C-terminus, (i) an HBV capsid polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 65 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 65; and (ii) an HBV small surface antigen (sAg) polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

In some embodiments, the capsid-sAg fusion protein comprises, sequentially from N-terminus to C-terminus, (i) an HBV capsid polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 66 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 66; and (ii) an HBV small surface antigen (sAg) polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 4 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.

In various embodiments, the capsid-sAg fusion proteins described herein comprise an HBV small surface antigen isoform, but do not comprise an HBV medium surface antigen isoform or an HBV large surface antigen isoform. Accordingly, in some embodiments, the capsid-sAg fusion proteins described herein do not comprise an HBV pre-S1 polypeptide. In some embodiments, the capsid-sAg fusion proteins described herein do not comprise an HBV pre-S2 polypeptide. In some embodiments, the capsid-sAg fusion proteins described herein do not comprise both an HBV pre-S1 polypeptide and an HBV pre-S2 polypeptide.

An exemplary HBV pre-S2 polypeptide not included in the capsid-sAg fusion protein described herein is shown below:

MQWNST[A/T]FHQ[T/A]LQDPRVR[A/G]LYFP[A/G]GGSS[L/S]G[A/T][V/I]NPV[L/P]TT[A /V]S[P/H]

[L/I]SSIF[S/A]RIGDP[A/V][L/M/P/T]N (SEQ ID NO: 79).

An illustrative HBV pre-S2 consensus polypeptide from HBV genotype A, not included in the capsid-sAg fusion protein described herein, is shown below:

MQWNSTAFHQALQDPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN (SEQ ID NO: 80).

An illustrative HBV pre-S2 consensus polypeptide from HBV genotype B, not included in the capsid-sAg fusion protein described herein, is shown below:

MQWNSTTFHQTLQDPRVRALYFPAGGSSSGTVSPAQNTVSAISSILSKTGDPVPN (SEQ ID NO: 81).

An illustrative HBV pre-S2 consensus polypeptide from HBV genotype C, not included in the capsid-sAg fusion protein described herein, is shown below:

MOWNSTTFHQALLDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPN (SEQ ID NO: 82).

An illustrative HBV pre-S2 consensus polypeptide from HBV genotype D, not included in the capsid-sAg fusion protein described herein, is shown below:

MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRIGDPALN (SEQ ID NO: 83).

In some embodiments, the capsid-sAg fusion proteins described herein do not comprise an HBV pre-S2 polypeptide comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 79-83 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to any of SEQ ID NOs: 79-83.

An exemplary HBV pre-S1-pre-S2 polypeptide not included in the capsid-sAg fusion protein described herein is shown below:

- 40 047988

MGQNLSTSNPLGFFPDHQL[D/A]PAFRANT[A/G/R]NPDWDFNPNKDTWDANKVGAGAFGLGFTP PHGGLLGWSPQAQGI[I/L]QT[L/V]PANPPPAS[T/A]NRQ[S/T]GRQPTPLSPPLR[N/D]THPQAMQWN ST[A/ T]FHQ[T/A]LQDPRVR[A/G]LYFP [A/G] GGS S [L/S] G[A/T] [V/I]NP V[L/P] TT[A/V ] S [P/H] [L/I] SSI F[S/A]RIGDP[A/V][L/M/P/T]N (SEQ ID NO: 84).

An illustrative HBV pre-S1-pre-S2 consensus polypeptide from genotype ABHBV, not included in the capsid-sAg fusion protein described herein, is shown below:

MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPAANQVGVGAFG PGLTPPHGGILGWSPQAQGILTTVSTIPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTAFHQALQ DPRVRGLYFPAGGSSSGTVNPAPNIASHISSISARTGDPVTN (SEQ ID NO: 85).

An illustrative HBV pre-S1-pre-S2 consensus polypeptide from HBV genotype B, not included in the capsid-sAg fusion protein described herein, is shown below:

MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDANKVGVGAFG PGFTPPHGGLLGWSPQAQGLLTTVPAAPPPASTNRQSGRQPTPLSPPLRDTHPQAMQWNSTTFHQTL QDPRVRALYFPAGGSSSGTVSPAQNTVSAISSILSKTGDPVPN (SEQ ID NO: 86).

An illustrative HBV pre-S1-pre-S2 consensus polypeptide from HBV genotype C, not included in the capsid-sAg fusion protein described herein, is shown below:

MGGWSSKPRQGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANQVGAGAFG PGFTPPHGGLLGWSPQAQGILTTVPAAPPPASTNRQSGRQPTPISPPLRDSHPQAMQWNSTTFHQALL DPRVRGLYFPAGGSSSGTVNPVPTTASPISSIFSRTGDPAPN (SEQ ID NO: 87).

An illustrative HBV pre-S1-pre-S2 consensus polypeptide from HBV genotype D, not included in the capsid-sAg fusion protein described herein, is shown below:

MGQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLG WSPQAQGILQTLPANPPPASTNRQSGRQPTPLSPPLRNTHPQAMQWNSTTFHQTLQDPRVRGLYFPA GGSSSGTVNPVPTTASPISSIFSRIGDPALN (SEQ ID NO: 88).

In some embodiments, the capsid-sAg fusion proteins described herein do not comprise an HBV pre-S1-pre-S2 polypeptide comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 84-88 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 84-88.

Optional polypeptide linker

Where appropriate, the HBV capsid polypeptide and the HBV sAg polypeptide in the capsid-sAg fusion protein may be directly adjacent or fused, or may be connected, joined, or linked by one or more peptide linkers. In various embodiments, the one or more peptide linkers are selected from one or more of a polyalanine linker, a polyglycine linker, a cleavable linker, a flexible linker, a rigid linker, and combinations thereof, such as within a linker or within a full-length fusion polypeptide. Exemplary fusion protein linkers that may be used in the present fusion polypeptides to connect the HBV capsid polypeptide and the HBV sAg polypeptide are described, for example, in Chen, et al., Adv Drug Deliv Rev. (2013) 65(10): 1357-1369. In some embodiments, the polyalanine linker comprises or consists of 2 or 3 contiguous alanine residues, e.g., AA, AAA, AAY, or AAX, wherein X is any amino acid (e.g., A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Y). In some embodiments, a polyglycine linker is used, e.g., GG, GGG, GGS, GSG, or GGGS (SEQ ID NO: 63). In some embodiments, the cleavable linker is selected from a 2A cleavable peptide. Exemplary 2A cleavable peptides that can be used to link an HBV capsid polypeptide and an HBV sAg polypeptide are described in, e.g., Donnelly, et al., J. Gen. Virol (2001), 82, 1027-1041 and Chng, et al., mAbs (2015) 7:2, 403-412. Exemplary 2A cleavage peptides that can be used to link an HBV capsid polypeptide and an HBV sAg polypeptide include, but are not limited to, 2A cleavage sequences (e.g., foot-and-mouth disease virus (F2A), equine rhinitis virus (E2A), porcine teschovirus-1 (P2A), and Thosea aigna virus (T2A)), optionally in combination with furan recognition/cleavage sequences (e.g., RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62)). In certain embodiments, a furin recognition/cleavage sequence (e.g., RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62)) is combined or fused with a 2A cleavable peptide (e.g., foot-and-mouth disease virus (F2A), equine rhinitis virus (E2A), porcine teschovirus-1 (P2A), and Thosea aigna virus (T2A)) in a single linker. See, e.g., Chng, et al., mAbs (2015) 7:2, 403-412. In some embodiments, the linker comprises a porcine teschovirus-1 (P2A) linker. In various embodiments, the cleavable 2A linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 57),

- 41 047988

QCTNYALLKLAGDVESNPGP (SEQ ID NO: 58) or EGRGSLLTCGDVEENPGP (SEQ ID NO: 59) or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 57), QCTNYALLKLAGDVESNPGP (SEQ ID NO: 58) or EGRGSLLTCGDVEENPGP (SEQ ID NO: 59). In various embodiments, the cleavable 2A linker comprises or consists of the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56), or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to ATNFSLLKQAGDVEENPGP (SEQ ID NO: 56). Where appropriate, in certain embodiments, the furin recognition/cleavage sequence can be located at either the N-terminus or the C-terminus of the 2A linker. In some embodiments, the cleavable linker comprises or consists of a furin recognition/cleavage region selected from RAKR (SEQ ID NO: 60), REKR (SEQ ID NO: 61), and RRKR (SEQ ID NO: 62). Illustrative linkers that can be used to link or connect the HBV capsid polypeptide and the HBV sAg polypeptide are shown in Table J.

Table J. Illustrative linkers for joining HBV capsid and HBV sAg polypeptides _______________________in a capsid-sAg fusion protein_______________________

SEQ ID NO: NAME poly-alanine (2) poly-alanine (3) poly-alanine-Tug poly-alanine-XXX poly-glycine (2) poly-glycine (3) poly-glycine/serine (3) SEQUENCE AA AAA AAY AAX (X=any amino acid) GG GGG GGS poly-glycine/serine (3) GSG 63 Gly3Ser GGGS 60 furin recognition site RAKR 61 furin recognition site REKR 62 furin recognition site RRKR 56 P2A ATNFSLLKQAGDVEENPGP 57 F2A APVKQTLNFDLLKLAGDVESNPGP 58 E2A QCTNYALLKLAGDVESNPGP 59 T2A EGRGSLLTCGDVEENPGP

In some embodiments, the capsid-sAg fusion protein is no more than 450 amino acids in length, such as no more than 445, 440, 435, 430, 425, 420, 415, or 410 amino acids in length.

In some embodiments, the capsid-sAg fusion protein does not comprise an amino sequence or fragment thereof from an HBV protein selected from the group consisting of X, precapsid, pre-S1, pre-S2, and polymerase.

In some embodiments, the capsid-sAg fusion protein comprises or consists of the amino acid sequence of any of SEQ ID NOs: 38-41, e.g., SEQ ID NO: 41 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 38-41, SEQ ID NO: 41. In some embodiments, the fusion polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 12, an asparagine (N) residue at an amino acid position corresponding to position 67, a valine (V) residue at an amino acid position corresponding to position 74, a phenylalanine (F) residue at an amino acid position corresponding to position 97, a threonine (T) at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO:41.

Exemplary capsid-sAg fusion proteins, for example, for use in activating, inducing or eliciting an immune response, for example, against capsid and/or small surface antigens expressed by HBV, are provided in Table K.

- 42 047988

Table K. Capsid-sAg fusion proteins

SEQ ID NO: HBV genotype Length (number of amino acids) Polypeptide sequence Capsid-sAg fusion proteins — Capsid sequences are shown in bold + underline. Flexible GSG linker is shown in italics. Cleavable P2A linker is shown underlined. 38 Capsid: B/C sAg: C 409 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREAL ESPEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRE LVVSYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGV WIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRR RSOSPRRRRSOSRESOCMESTTSGFLGPLLVLQAGFFLLTRILTI PQSLDSWWTSLNFLGGAPTCPGQNSQSPTSNHSPTSCPPICPGYR WMCLRRFIIFLCILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTG PCKTCTTPAQGTSMFPSCCCTKPTDGNCTCIPIPSSWAFARFLWE WASVRFSWLSLLVPFVQWFVGLSPTVWLS VIWMMWYWGPSL YNILSPFLPLLPIFFCLWVYI 39 Capsid: B/C sAg: C 430 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREAL ESPEHCSPHHTALROAILCWGELMNLATWVGSNLEDPASRE LVVSYVNVNMGLKIROLLWFHISCLTFGRETVLEYLVSFGV WIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRR RSOSPRRRRSOSRESOCG.SGATNFSLLKQAGDVEENPGPESTT SGFLGPLLVLQAGFFLTRILTIPQSLDSWWTSLNFLGGAPTCPG QNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLCILLLCLIFLLVL LDYQGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKP TDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVG LSPTVWLSVIWMMWYWGPSLY NILSPFLPLLPIFFCLWVYI 40 Capsid: D sAg: D 409 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREAL ESPEHCSPHHTALROAILCWGELMNLATWVGVNLEDPASR DLVVSYVNTNMGLKFROLLWFHISCLTFGRETVLEYLVSFG VWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRR RRSOSPRRRRSOSRESOCMENITSGFLGPLLVLOAGFFLLTRIL TIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPICPG YRWMCLRRFIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTS TGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSWAFGKFL WEWASARFSWLSLLVPFVQWFVGLSPTVWLSVI WMMWYWGP SLYSILSPFLPLLPIFFCLWVYI 41 Capsid: D sAg: D 430 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYREAL ESPEHCSPHHTALROAILCWGELMNLATWVGVNLEDPASR DLVVSYVNTNMGLKFROLLWFHISCLTFGRETVLEYLVSFG VWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRR RRSOSPRRRRSOSRESOCGSGATNFSLLKQ 41 Capsid: D sAg: D 430 AGDVEENPGPENITSGFLGPLLVLOAGFFLTRILTIPQSLDSWW TSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFI IFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCTTPA QGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFS WLSLLVPFVQWFVGLSPTVWLSVIWMM WYWGPSLYSILSPFLP LLPIFFCLWVYI

Signal or leader sequences

In various embodiments, the immunogenic polypeptides described herein comprise a signal sequence or a signal peptide, such as direct intracellular trafficking of the polypeptide to the proteasomal or lysosomal compartment. In various embodiments, the immunogenic polypeptide comprises a signal sequence at the N-terminus and/or C-terminus. In some embodiments, the immunogenic polypeptide comprises an N-terminal signal peptide or leader sequence. In various embodiments, the signal peptide or leader sequence belongs to a parent protein selected from a serum protein, a cytokine, a chemokine, a chaperone protein, an invariant protein, and a protein that targets proteins to a lysosomal compartment. In some embodiments, the signal peptide or leader sequence is derived from a parent protein selected from colony stimulating factor 2 (CSF2, GM-CSF), tissue plasminogen activator (PLAT, t-PA), C-C motif chemokine ligand 7 (CCL7, MCP-3), C-X-C motif chemokine ligand 10 (CxCl10, IP-10), catenin beta 1 (CTNNB1), CD74 (p33; DHLAG; HLADG; Ia-GAMMA, invariant chain), serum albumin (ALB), polyubiquitin B/C (UBB/UBC), calreticulin (CALR), vesicular gelatin virus protein G (VSV-G), lysosome-associated membrane protein-1 (LAMP-1), and lysosome-associated membrane protein-2 (LAMP-2). In various embodiments, the signal peptide or leader sequence is selected from the amino acid sequence of any of SEQ ID NOs: 67-76 or a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 67-76. In certain embodiments, the immunogenic polypeptide comprises N-terminal and C-terminal sequences from LAMP-1, such as SEQ ID NOs: 77 and 78, respectively. Exemplary signal sequences that can be used in the present immunogenic polypeptides are provided in Table L.

- 43 047988

Table L. Illustrative signal sequences

SEQ ID NO: name of the original protein SUBSEQUENCE 67 CSF2, GM-CSF MWLQSLLLLGTVACSISV 68 PLAT, t-PA MDAMKRGLCCVLLLCGAVFVSAR 69 CD74 MHRRRSRSCREDQKPV 70 albumin KWVTFISLLFLFS S AYS 71 β-catenin MRKAAVSHWQQQSYLDSGIHSGATTTAPSLS 72 CCL7, MCP-3 MNPSAAVIFCLILLGLSGTQGILDMAQPVGINTSTTCCYRFINKKIPKQ RLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKK TQTPKLASAGA 73 ubiquitin MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQ LEDGRTLSDYNIQKESTLHLVLRLRGG 74 calreticulin MLLSVPLLLGLLGLAVA 75 VSV-G MKCLLYLAFLFIGVNC 76 CXCL10, IP-10 MNQTAILICCLIFLTLSGIQG 77 LAMP-1 N-terminal MAPRSARRPLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFS AAFSVNYDTKSGPKNMTLDLPSDATVVLNRSSCGKENTSDPSLVIAF GRGHTLTLNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESI TDIRADIDKKYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGET RCEQDRPSPTTAPPAPPSSPSPVP KSPSVDKYNVSGTNGTCLLASMG LQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTV LLFQFGMNASSSRFFLQGIQLNT1LPDARDPAFKAANGSLRALQATVG NSYKCNAEEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDE NSLEDI 78 LAMP-1 C-terminal GSEFTLIPIAVGGALAGLVIVLIAYLVGRKRSHAGYQTI

Additionally provided are methods for producing immunogenic polypeptides as described herein. In some embodiments, the methods comprise constructing immunogenic polypeptides using peptide synthesis. In some embodiments, the methods comprise constructing, using synthetic or recombinant DNA technology, polynucleotides encoding each of the polypeptides of a bivalent antigen and expressing the polypeptides from an expression vector. In some embodiments, the methods may further comprise introducing the polynucleotides into one or more vectors and expressing the encoded polypeptides in a cell. This may be accomplished using known recombinant techniques.

3. Polynucleotides encoding immunogenic polypeptides

Provided are polynucleotides encoding the immunogenic polypeptides described herein, vectors comprising such polynucleotides, and host cells (e.g., human cells, mammalian cells, yeast cells, plant cells, insect cells, bacterial cells, e.g., E. coli) comprising such polynucleotides or expression vectors. Provided herein are polynucleotides comprising nucleotide sequence(s) encoding any of the immunogenic polypeptides provided herein, as well as expression cassettes and vector(s) comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells. In various embodiments, the polynucleotide is DNA, cDNA, mRNA, self-amplifying RNA (SAM), self-replicating RNA, or self-amplifying replicon RNA (RepRNA). In some embodiments, the polynucleotide comprises or is expressed from an alphaviral self-replicating or self-amplifying replicon RNA (RepRNA). Self-replicating RNA and self-amplifying replicon RNA have been described as methods for delivering a vaccine, for example, in Tews, et al., Methods Mol Biol (2017) 1499:15-35; Demoulins, et al., Methods MolBiol. (2017) 1499:3775; Englezou, et al., Mol Ther Nucleic Acids. (2018) 12:118-134; McCollough, et al., Vaccines (Basel). (2014) 2(4):735-54; and McCollough, et al., Mol Ther Nucleic Acids. (2014)3:e173.

The terms polynucleotide and nucleic acid molecule refer interchangeably to the polymeric form of nucleotides and include both sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers of the foregoing. As used herein, the term nucleic acid molecule may be interchangeable with the term polynucleotide. In some embodiments, nucleotide refers to a ribonucleotide, a deoxynucleotide, or a modified form of any type of nucleotide, and combinations thereof. The terms also include, without limitation, single- and double-stranded forms of DNA. In addition, a polynucleotide, such as cDNA or mRNA, may include both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, tags, methylation, replacement of one or more naturally occurring nucleotides with an analogue, internucleotide

- 44 047988 modifications such as uncharged linkages (e.g. methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g. polypeptides), intercalators (e.g. acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g. alpha-anomeric nucleic acids, etc.). The above term is also intended to include any topological conformation, including single-stranded, double-stranded, partially duplex, triplex, hairpin, circular and closed conformations. Reference to a nucleic acid sequence includes its complement unless otherwise specified. Thus, it should be understood that reference to a nucleic acid molecule having a particular sequence includes its complementary strand with its complementary sequence. The term also includes codon-biased polynucleotides for improved expression in a desired viral expression vector or host cell.

As used herein, the term "substitution" means the replacement of one or more amino acids or nucleotides with other amino acids or nucleotides, respectively.

An isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present outside of a chromosome or in a chromosomal location that differs from its natural chromosomal location. An isolated nucleic acid encoding an immunogenic polypeptide refers to one or more nucleic acid molecules encoding such immunogenic polypeptides, including such nucleic acid molecule(s) in a single vector or a plurality of separate vectors, and such nucleic acid molecule(s) are present in one or more locations in a host cell.

As used herein, the term "polynucleotide variant" is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein by one or more substitutions, deletions, additions, and/or insertions. Such variants may be naturally occurring or may be produced synthetically, such as by modifying one or more of the above polynucleotide sequences described herein and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of methods well known in the art.

In some embodiments, the nucleic acid molecule is codon biased to enhance expression in a desired host cell, e.g., human cells, mammalian cells, yeast cells, plant cells, insect cells, or bacterial cells, e.g., E. coli cells. Accordingly, provided are polynucleotides encoding an immunogenic polypeptide as described herein, wherein the polynucleotides are codon biased, contain substituted heterologous signal sequences, and/or have mRNA instability elements removed. Methods for creating codon biased nucleic acids can be implemented by adapting methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6794498. Preferred codon usage frequencies for expressing immunogenic polypeptides from desired viral expression vectors and/or in desired host cells are provided, for example, at kazusa.or.jp/codon/; and genscript.com/tools/codon-frequency-table.

In some embodiments, a polynucleotide encoding an immunogenic polypeptide as described herein is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 27-37 and 89-94, as set forth in Table M.

Accordingly, in some embodiments, the 3' end of a polynucleotide encoding one or more immunogenic polypeptides described herein comprises one or more tandem stop codons, such as two or more tandem TAG (amber), TAA (ochre), or TGA (opal or umber) stop codons. Multiple tandem stop codons may be the same or different.

Additionally provided are expression cassettes comprising a polynucleotide encoding an immunogenic polypeptide as described herein operably linked to one or more regulatory sequences. In some embodiments, the polynucleotide is operably linked to and controlled by a constitutive promoter. In some embodiments, the promoter is selected from cytomegalovirus (CMV) major immediate early, a CMV enhancer fused to a chicken beta-actin (CAG) promoter, human elongation factor-1a (HEF-1a), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1a (CHEF-1a), and phosphoglycerate kinase (PGK).

- 45 047988

Table M. Polynucleotides encoding immunogenic polypeptides

SEQ ID NO: HBV genotype Name Polynucleotide sequence 27 IN Ρο1 ^Δ1 ATGCCCCTGAGCTACCAGCACTTCAGGAAGCTGCTGCTGCTGGATG ATGAGGCTGGCCCTCTGGAGGAGGAGCTGCCCAGGCTGGCAGATG AGGGCCTCAACAGGAGAGTGGCAGAGGACCTGAACCTGGGCAACC TGAATGTGAGCATCCCCTGGACCCAAAGTGGGGAACTTCACTGG CCTCTACAGCAGCACAGTGCCAGTGTTCAACCCTGAGTGGCAGACC CCCTCCTTCCCC CACATCCACCTCCAGGAGCATCATCAACAGAT GTCAGCAGTATGTGGGCCCTCTGACAGTCAATGAGAAGAGGAGGC TGAAGCTGATCATGCCTGCCAGGTTCTACCCCAACCTGACCAAGTA CCTCCCACTGGACAAGGGCATCAAGCCATACTATCCTGAGCATGTG GTGAACCACTACTTTCAGACCAGGCACTACCTGCACACACTGTGGA AGGCTGGCATCCTGTACAAGAGGGAGAGCACCAGATCAGCCTCTTT CTGTGGCTCCCCCTACAGCTGGGAGCAGGATCTCCAGCATGGCAGA CTGGTGTTCCAGACCTCCAAGAGGCATGGGGACAAGTCCTTTTGCC CCCAGAGCCCTGGCATCCTGCCCAGGAGCGAGCTCCACCACTTCCC CCCCTCCTCCAGCAGA AGCCAGTCCCAGGGACCTGTGCTGTCCTGC TGGTGGCTCCAGTTCAGGAACAGTGAGCCCTGCAGTGAGTACTGTC TGTGTCACATTGTGAACCTGATTGAGGACTGGGGGCCCTGCACTGA GCATGGAGAGCACAGGATCAGAACCCCCAGGACCCCAGCCAGAGT GACTGGAGGTGTGTTCCTGGTGGACAAGAACCCCCACAACACCAC AGAGGCAGACTGGTGGTGGACTTCTCCCAGTTTTCAAGGGGCAAC ACCAGAGTGTCCTGGCCCAAGTTTGCAGTGCCCAACCTCCAGAGCC TGACCAACCTGCTGTCATCAAACCTGAGCTGGCTGTCCCTGGATGT GTCTGCTGCCTTCTACCACCTGCCCCTGCACCCTGCAGCCATGCCTC ACCTCCT GGTGGGCAGCTCAGGCCTGAGCAGGTATGTGGCCAGGCT GTCAAGCAACTCCAGAATCATCAACAACCAGCACAGGACCATGCA GAACCTGCATGACTCTTGCAGCAGGAACCTGTATGTGAGCCTGATG CTGCTGTACAAGACCTATGGCAGGAAGCTGCACCTGTACTCCCACC CCATCATCCTGGGTTTCAGGAAGATCCCCATGGGAGTGGGACTGTC CCCCTTCCTGCTGGCCCAGTTCACCCTGCCATCTGCTCTGTGGTGA GGAGAGCCTTCCCCCACTGCCTGGCCTTCTCCTACATGCATGATGT GGTGCTGGGGGCCAAGTCAGTGCAGCACCTGGAGTCTCTGTATGCT GCAGTCACCAACTTCCTGCTCAGCCTGGGCATCCACCTGAACCCCC ACAAGACCAA GAGGTGGGGCTACTCTCTGAACTTCATGGGCTATGT GATAGGCAGCTGGGGCACCCTGCCACAGGAGCACATAGTGCAGAA GATCAAGATGTGCTTCAGGAAGCTGCCAGTGAACAGGCCCATTGAT TGGAAGGTGTGCCAGAGGATTGTGGGCCTGCTGGGCTTTGCAGCAC CCTTCACACAGTGTGGCTACCCAGCTCTGATGCCCCTGTATGCCTGC ATCCAGGCCAAGCAGGCCTTCACCTTCTCCCCCACTTACAAGGCCT TCCTGTCCAAGCAGTACCTGCACCTGTACCCTGTGGCAAGGCAGAG GCCAGGCCTCTGCCAGGTGTTTGCAGATGCCACCCACAGGCTGG GGCCTGGCCATTGGCCACCAGAGGATGAGAGGGGCCTTTGTGAGC GCCAATCCACACAGCCCACCTGCTGGCAGCATGCTTTGCCA GGTCCAGGTCTGGTGCAAAGCTGATTGGCACTGACAACAGTGTGGT GCTGTCCAGAAAGTACACCAGCTTCCCCTGGCTGCTGGGATGTGCT GCCAACTGGATTCTGAGGGGCACCAGCTTTGTCTATGTGCCCTCTG CACTGAACCCTGCAGATGACCCCTCCAGGGGCAGACTGGGGCTGTA CAGGCC ACTGCTCAGACTGCTGTACAGGCCCACCACTGGCAGAACCTCCCTG TATGCAGACAGCCCCTCAGTGCCCTCTCACCTGCCAGACAGAGTGC ACTTTGCCAGCCCCCTGCATGTTGCCTGGAGGCCCCCC 28 V Ρο1 ^Δ3 ATGCCCCTGAGCTACCAGCACTTCAGGAAGCTGCTGCTGCTGGATG ATGAGGCTGGCCCTCTGGAGGAGGAGCTGCCCAGGCTGGCAGATG AGGGCCTCAACAGGAGAGTGGCAGAGGACCTGAACCTGGGCAACC TGAATGTGAGCATCCCCTGGACCCAAAGTGGGGAACTTCACTGG CCTCTACAGCAGCACAGTGCCAGTGTTCAACCCTGAGTGGCAGACC CCCTCCTTCCCC CACATCCACCTCCAGGAGCATCATCAACAGAT GTCAGCAGTATGTGGGCCCTCTGACAGTCAATGAGAAGAGGAGGC TGAAGCTGATCATGCCTGCCAGGTTCTACCCCAACCTGACCAAGTA CCTCCCACTGGACAAGGGCATCAAGCCATACTATCCTGAGCATGTG GTGAACCACTACTTTCAGACCAGGCACTACCTGCACACACTGTGGA AGGCTGGCATCCTGTACAAGAGGGAGAGCACCAGATCAGCCTCTTT CTGTGGCTCCCCCTACAGCTGGGAGCAGGATCTCCAGCATGGCTGC TGGTGGCTCCAGTTCAGGAACAGTGAGCCCTGCAGTGAGTACTGTC TGTGTCACATTGTGAACCTGATTGAGGACTGGGGGCCCTGCACTGA GCATGGAGAGCA CAGGATCAGAACCCCCAGGACCCCAGCCAGAGT GACTGGAGGTGTGTTCCTGGTGGACAAGAACCCCCACAACACCAC AGAGAGCAGACTGGTGGTGGACTTCTCCCAGTTTTCAAGGGGCAAC ACCAGAGTGTCCTGGCCCAAGTTTGCAGTGCCCAACCTCCAGAGCC TGACCAACCTGCTGTCATCAAACCTGAGCTGGCTGTCCCTGGATGT GTCTGCTGCCTTCTACCACCTGCCCCTGCACCCTGCAGCCATGCCTC

- 46 047988

ACCTCCTGGTGGGCAGCTCAGGCCTGAGCAGGTATGTGGCCAGGCT GTCAAGCAACTCCAGAATCATCAACAACCAGCACAGGACCATGCA GAACCTGCATGACTCTTGCAGCAGGAACCTGTATGTGAGCCTGATG CTGCTGTACAAGACCTATGGCAGGAAGCTGCACCTGTACTCCCACC CCATCATCCTGGGTTTCAGGAAGATCCCCATGGGAGTGGGACTGTC CCCCTTCCTGCTG GCCCAGTTCACCCTGCCATCTGCTCTGTGGTGA GGAGAGCCTTCCCCCACTGCCTGGCCTTCTCCTACATGCATGATGT GGTGCTGGGGGCCAAGTCAGTGCAGCACCTGGAGTCTCTGTATGCT GCAGTCACCAACTTCCTGCTCAGCCTGGGCATCCACCTGAACCCCC ACAAGACCAAGAGGTGGGGCTACTCTCTGAACTTCATGGGCTATGT GATAGGCAGCTGGGGCACCCTGCCACAGGAGCACATAGTGCAGAA GATCAAGATGTGCTTCAGGAAGCTGCCAGTGAACAGGCCCATTGAT TGGAAGGTGTGCCAGAGGATTGTGGGCCTGCTGGGCTTTGCAGCAC CCTTCACACAGTGTGGCTACCCAGCTCTGATGCCCCTGTATGCCTGC CCAAGCAGGCCTTCACCTTCTCCCCCACTTACAAGGCCT TCCTGTCCAAGCAGTACCTGCACCTGTACCCTGTGGCAAGGCAGAG GCCAGGCCTCTGCCAGGTGTTTGCAGATGCCACCCCCACAGGCTGG GGCCTGGCCATTGGCCACCAGAGGATGAGAGGGGCCTTTGTGAGC CCACTGCCAATCCACACAGCCCACCTGCTGGCAGCATGCTTTGCCA GGTCCAGGTCTGGTGCAAAGCTGATTGGCACTGACAACAGTGTGGT GCTGTCCAGAAAGTACACCAGCTTCCCCTGGCTGCTGGGATGTGCT GCCAACTGGATTCTGAGGGGCACCAGCTTTGTCTATGTGCCCTCTG CACTGAACCCTGCAGATGACCCCTCCAGGGGCAGACTGGGGCTG TACAGGCCACTGCTCAGACTGCTGTACAGGCCCACCACTGGCAGAA CCTCCCTGTATGCAGACAGCCCCTCAGTGCCCTCTCACCTGCCAGA CAGAGTGCACTTTGCCAGCCCCCTGCATGTTGCCTGGAGGCCCCCC 29 IN Floor ³⁰⁰ AGC AGACTGGTGGTGGACTTCTCCCAGTTTTCAAGGGGCAACACCA GAGTGTCCTGGCCCAAGTTTGCAGTGCCCAACCTCCAGAGCCTGAC CAACCTGCTGTCATCAAACCTGAGCTGGCTGTCCCTGGATGTGTCT GCTGCCTTCTACCACCTGCCCCTGCACCCTGCAGCCATGCCTCACCT CCTGGTGGGCAGCTCAGGCCTGAGCAGGTATGTGGCCAGGCTGTCA AGCAACTCCAGAATCATCAACAACCAGCACAGGACCATGCAGAAC CTGCATGACTCTTGCAGCAGGAACCTGTATGTGAGCCTGATGCTGC TGTACAAGACCTATGGCAGGAAGCTGCACCTGTACTCCCACCCCAT CATCCTGGGTTTCAGGAAGATCCCCATGGGAGTGGGACTGTCCCCC GCTGGCCCAGTTCACCCTGCCATCTGCTCTGTGGTGAGGAG AGCCTTCCCCCACTGCCTGGCCTTCTCCTACATGCATGATGTGGTGC TGGGGGCCAAGTCAGTGCAGCACCTGGAGTCTCTGTATGCTGCAGT CACCAACTTCCTGCTCAGCCTGGGCATCCACCTGAACCCCCACAAG ACCAAGAGGTGGGGCTACTCTCTGAACTTCATGGGCTATGTGATAG GCAGCTGGGGCACCCTGCCACAGGAGCACATAGTGCAGAAGATCA AGATGTGCTTCAGGAAGCTGCCAGTGAACAGGCCCATTGATTGGAA GGTGTGCCAGAGGATTGTGGGCCTGCTGGGCTTTGCAGCACCCTTC ACACAGTGTGGCTACCCAGCTCTGATGCCCCTGTATGCCTGCATCC AGGCCAAGCAG GCCTTCACCTTCTCCCCCACTTACAAGGCCTTCCT GTCCAAGCAGTACCTGCACCTGTACCCTGTGGCAAGGCAGAGGCCA GGCCTCTGCCAGGTGTTTGCAGATGCCACCCCCACAGGCTGGGGCC TGGCCATTGGCCACCAGAGGATGAGAGGGGCCTTTGTGAGCCCACT GCCAATCCACACAGCCCACCTGCTGGCAGCATGCTTTGCCAGGTCC AGGTCTGGTGCAAAGCTGATTGGCACTGACAACAGTGTGGTGCTGT CCAGAAAGTACACCAGCTTCCCCTGGCTGCTGGGATGTGCTGCCAA CTGGATTCTGAGGGGCACCAGCTTTGTCTATGTGCCCTCTGCACTG

- 47 047988

AACCCTGCAGATGACCCCTCCAGGGGCAGACTGGGGCTGTACAGG CCACTGCTCAGACTGCTGTACAGGCCCACCACTGGCAGAACCTCCC TGTATGCAGACAGCCCCTCAGTGCCCTCTCACCTGCCAGACAGAGT GCACTTTGCCAGCCCCCTGCATGTTGCCTGGAGGCCCCCC 89 IN Pol ³⁰⁰ on ATGTCTTCAAGATCCCAGAGTCAGGGCCCTGTACTTTCCTGCTGGT GGCTCCAGTTCAGGAACAGTGAGCCCTGCTCCGAATACTGTCTCTG CCATATCGTCAATCTTATCGAAGACTGGGGACCCTGTACCGAACAT GGGAACATCGCATCAGGACTCCTAGGACCCCTGCTCGTGTTACAG GCGGGGTTTTTCTTGTTGACAAAAATCCCTCACAATACCACAGAGTC CGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCCGT GTGTCTTGGCCAAAATTCGCAGTCCCAAATCTCCAGTCACTCACCA ACCTGTTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTC TGCGGCGTTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATC TTCTTGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCCGTTTGTCC TCTAATTCCAGGATCATCAACAACCAGCACCGGACCATGCAAAACC TGCACGACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTG TACAAACCTACGGACGGAAACTGCACTTGTATTCCCATCCCATCA CTTTCGCAAAATTCCTATGGGAGTGGGCCTCAGTCCGTTT CTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGC TTTCCCCCACTGTCTGGCTTTCAGTTATATGCATGATGTGGTATTGG GGGCCAAGTCTGTACAACATCTTGAGTCCCTTTATGCCGCTGTTACC AATTTTCTTTTGTCTTTGGGTATACATTTAAACCCTCACAAAACAAA AAGATGGGGATATTCCCTTAACTTCATGGGATATGTAATTGGGAGT TGGGGCACATTGCCGCAGGAACATATTGTACAAAAAATCAAAATG TGTTTTAGGAAACTTCCTGTAAACCGGCCTATTGATTGGAAAGTAT GTCAACGAATTGTGGGTCTTTTGGGGTTTGCCGCCCCTTTCACGCAA TGTGGATATC CTGCTTTAATGCCTTTATATGCATGTATACAAGCAAA ACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTCCTAAGTAAAC AGTATCTGCACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGC CAAGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCTTGGCCATAG GCCATCAGCGCATGCGTGGAGCCTTCGTGTCTCCTCTGCCGATCCA TACTGCGCATCTCCTGGCCGCTTGTTTTGCTCGCAGCAGGTCTGGGG CAAAACTCATCGGGACTGACAATTCTGTCGTGCTCTCCCGCAAGTA TACATCCTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGC GCGGGACGTCCTTTGTTTACGTCCCGTCGGCGCTGAATCCCGCGGA CGACC CCTCCCGGGGCCGCTTGGGGCTCTACCGCCCGCTTCTCCGC TTGTTGTACCGACCGACTACGGGGCGCACCTCTCTCTACGCGACT CCCCGTCTGTGCCTTCTCATCTGCCGGACCGTGTGCACTTCGCTTCA CCTCTGCACGTCGCATGGGAGACCACCGT 90 IN Floor ³⁰⁰ dint ATGTCATCCAGATCCCAGAGTCAGGGCCCTGTCCTTTCCTGTTGGTG GCTCCAGTTCAGGAACAGTGAGCCCTGTTCTGAGTACTGTCTCTGC CACATTGTCAATCTGATTGAGGACTGGGGCCCCTGCACAGAGCATG GTGAACACAGGATCAGGACTCCCAGGACCCCTGCCAGGGTGACTG GTGGGGTTTTCCTTGTTGACAAAAATCCTCACAACACCACAGTC CTTGTGGTGGACTTCTCTCAATTTTCAAGGGGGAACACAAGG GTGTCTTGGCCCAAATTTGCAGTCCCAAATCTCCAGTCTCTGACCA ACCTGTTGTCCTCCAATTTGTCCTGGTTGTCTCTGGATGTCTCTGCT GCCTTTTATCATCTTCCTTCCATCCTGCTGCCATGCCTCATCTTCTT GTTGGTTCTTCTGGCCTCTCTAGGTATGTTGCCAGATTGTCCTCCAA TTCCAGGATCATCAACAACCAGCACAGGACCATGCAAAACCTGCAT GACTCCTGCTCCAGAAACCTCTATGTTTCTCTCATGTTGCTGTACAA AACCTATGGCAGGAAACTGCATTTGTATTCCCATCCCATCATCTTG GGCTTCAGGAAAATTCCCATGGGAGTGGGCCTCAGTCCCTTCCTCT TGGCTCAGTT CACCAGTGCCATTTGTTCTGTTGTCAGGAGGGCTTTC CCCCACTGTCTTGCTTTCAGTTACATGCATGATGTGGTCTTGGGGGC CAAGTCTGTCCAACATCTTGAGTCACTTTATGCTGCTGTGACCAACT

- 48 047988

TTCTTTTGTCTTTGGGCATCCATTTGAACCCTCACAAAACCAAAAGA TGGGGCTATTCCCTCAATTTCATGGGCTATGTCATTGGG AACAG TATCTCCACCTTTACCCTGTTGCAAGGCAGAGGCCTGGTCT GTGCCAAGTGTTTGCTGATGCAACCCCCACTGGTTGGGGCTTGGCC ATTGGCCATCAGAGAATGAGAGGTGCCTTTGTGTCTCCTCTCCCCA TCCACACTGCTCATCTCCTGGCAGCTTGCTTTGCAAGGAGCAGGTC TGGAGCCAAACTCATAGGGACTGACAATTCTGTGGTGCTCTCCAGA AAGTACACCTCCTTTCCTTGGCTGCTGGGCTGTGCAGCCAACTGGA TCCTGAGGGGGACTTCCTTTGTTTATGTCCCCTCTGCCCTGAATCCT GCAGATGACCCCTCCAGGGGCAGGTTGGGGCTCTACAGACCCCTTC TCAGGTTGTTGTACAGACCAACACAGGGAGGACCTCTCTATGC AGAT TCCCCCTCTGTTCCTTCTCATCTTCCAGACAGAGTGCACTTTG CTTCTCCTCTGCATGTGGCTTGGAGACCTCCC 91 IN Pol ³⁰⁰ huCo low GC ATGTCTAGCAGAAGCCAGTCCCAGGGACCTGTGCTGTCTTGTTGGT GGCTTCAGTTTCGGAATAGCGAGCCATGTAGCGAGTATTGCCTGTG TCACATCGTGAATCTGATTGAGGATTGGGGACCATGCACAGAGCAC GGAGAGCACCGGATCAGAACCCCTAGGACACCAGCCCGCGTGACA GGAGGCGTGTTCCTGGTGGATAAGAACCCCCATAATACAACAGAG AGC AGACTGGTGGTGGATTTTTCTCAGTTTTTCTCGGGGCAATACAA GAGTGTCCTGGCCAAAGTTTGCCGTGCCCAATCTCCAGAGCCTGAC AAACCTGCTGTCTTCTAATCTGAGCTGGCTGTCCCTGGACGTGTCCG CCGCCTTTTACCACCTGCCACTGCACCCTGCCGCCATGCCCCACCTG CTGGTGGGCAGCTCCGGACTGAGCAGATACGTGGCAAGGCTGTCTA GCAATTCTAGAATTATTAATAATCAGCACAGAACAATGCAGAATCT GCATGATTCTTGTAGCAGGAATCTGTACGTGAGCCTGATGCTGCTG TATAAGACATATGGACGCAAGCTGCACCTGTATTCTCACCCTATTA TTCTGGGCTTCCGGAAGATCCCTATGGGCGTGGGACTGTCCCCATT CCTGCTG GCCCAGTTTACCTCGCCATCTGCTCTGTGGTGCGGAGA GCCTTCCCACATTGTCTGGCCTTTTCTTACATGCACGATGTGGTGCT GGGCGCCAAATCCGTGCAGCACCTGGAGTCTCTGTATGCCGCCGTG ACAAACTTCCTGCTGAGCCTGGGCATCCACCTGAATCCACATAAGA CAAAGCGGTGGGGCTATTCTCTGAATTTTATGGGCTATGTGATCGG CAGCTGGGGAACCCTGCCACAGGAGCACATTGTGCAGAAGATCAA GATGTGCTTTCGCAAGCTGCCCGTGAATCGGCCTATCGATTGGAAG GTGTGCCAGAGGATCGTGGGACTGCTGGGATTCGCAGCACCCTTTA CCCAGTGCGGCTACCCAGCCCTGATGCCACTGTATGCCTGTATCCA GGCC AAACAGGCCTTCACCTTTTCCCCTACATATAAGGCTTTTCTGT CTAAGCAGTACCTGCATCTGTATCCAGTGGCAAGGCAGAGGCCAG GACTGTGCCAGGTGTTTGCAGATGCAACACCACAGGATGGGGAC TGGCAATCGGACACCAGAGGATGAGAGGAGCCTTCGTGAGCCCAC TGCCAATTCACACCGCCCACCTGCTGGCAGCATGCTTTGCAAGGTC CCGCTCTGGAGCAAAGCTGATTGGCACCGATAACAGCGTGGTGCTG TCCAGAAAATACACCAGCTTCCCCTGGCTGCTGGGATGTGCAGCAA ATTGGATTC TGAGGGGCACCAGCTTCGTGTATGTGCCTTCCGCCCTGAATCCTGC CGATGATCCATCTCGAGGCAGACTGGGACTGTATAGGCCACTGCTG AGACTGCTGTATAGGCCTACCACAGGCAGAACATCCCTGTATGCCG ACAGCCCATCCGTGCCCTCTCACCTGCCAGATAGAGTGCATTTCGC AAGCCCACTGCATGTGGCATGGAGGCCACCC 92 IN Pol ³⁰⁰ oridel CpG ATGTCTTCAAGATCCCAGAGTCAGGGCCCTGTACTTTCCTGCTGGT GGCTCCAGTTCAGGAACAGTGAGCCCTGCTCTGAATACTGTCTCTG CCATATTGTCAATCTTATAGAAGACTGGGGACCCTGTACTGAACAT

- 49 047988

GGAGAACATAGGATCAGGACTCCTAGGACCCCTGCTAGAGTTACA GGGGGGGTTTTTCTTGTTGACAAAAATCCCTCACAATACCACAGAGT CTAGACTTGTGGTGGACTTCTCTCAATTTTCTAGGGGGAACACCAG GGTGTCTTGGCCAAAATTTGCAGTCCCAAATCTCCAGTCACTCACC AACCTGTTGTCCTCCAATTTGTCCTGGTTATCCCTGGATGTGTCTGC AGCC TTTTATCATCTTCCTCTGCATCCTGCTGCTATGCCTCATCTTCT TGTTGGTTCTTCTGGACTATCAAGGTATGTTGCCAGGTTGTCCTCTA ATTCCAGGATCATCAACAACCAGCACAGGACCATGCAAAACCTGC ATGACTCCTGCTCAAGGAACCTCTATGTTTCCCTCATGTTGCTGTAC AAAACCTATGGAAGGAAACTGCACTTGTATTCCCATCCCATCATCT TGGGCTTTAGAAAAATTCCTATGGGAGTGGGCCTCAGTCCCTTTCT CTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTAGAAGGGCTT TCCCCCACTGTCTGGCTTTCAGTTATATGCATGATGTGGTATTGGGG GCCAAGTCTGTACAACATCTTGAGTCCCTTTATGCTGCTGTTACCAA TTTT CTTTTGTCTTTGGGTATACATTTAAACCCTCACAAAACAAAAA GATGGGGATATTCCCTTAACTTCATGGGATATGTAATTGGGAGTTG GGGCACATTGCCTCAGGAACATATTGTACAAAAAATCAAAATGTGT TTTAGGAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTC AAAGAATTGTGGGTCTTTTGGGGTTTGCAGCCCCTTTCACCCAATGT GGATATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAAAAC AGGCTTTTACTTTCTCCCCAACTTACAAGGCCTTCCTAAGTAAACAG TATCTGCACCTTTACCCTGTTGCTAGGCAAAGGCCTGGTCTGTGCCA AGTGTTTGCTGATGCAACCCCCACTGGTTGGGGCTTGGCCATAGGC CAT CAGAGGATGAGGGGAGCCTTTGTGTCTCCTCTGCCTATCCATA CTGCCCATCTCCTGGCAGCTTGTTTTGCTAGGAGCAGGTCTGGGGC AAAACTCATTGGGACTGACAATTCTGTTGTGCTCTCCAGAAAGTAT ACATCCTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGGATCCTGA GGGGGACATCCTTTGTTTATGTCCCTTCAGCACTGAATCCTGCTGAT GACCCCTCCAGGGGCAGATTGGGGCTCTACAGGCCCCTTCTCAGGT TGTTGTACAGACCCACTACTGGGAGAACCTCTCTATGCAGACTC CCCCTCTGTGCCTTCTCATCTGCCTGACAGGGTGCACTTTGCTTCAC CTCTGCATGTTGCATGGAGACCACCT 93 IN Pol ³⁰⁰ IDT GTA GATTGGTCGTTGATTTCAGTCAATTTTCAAGAGGCAATACACG AGTTTCTTGGCCGAAAT TCGCCGTACCGAATCTGCAATCCTTGACAAATTTGCTTAGTTCTAAT TTGTCTTGGCTTTCTCTCGATGTTTCCGCCGCTTTCTATCACTTGCCC CTTCACCCAGCCGCGATGCCGCATCTCTTGGTGGGCAGCTCTGGAC TTAGTAGATACGTAGCTAGACTCAGTTCTAACTCACGGATAATAAA TAACCAACATCGCACTATGCAGAACCTGCATGATTCTTGTTCCCGG AACT TGTATGTCTCCTTGATGTTGTTGTATAAAACTTATGGGCGAAA GCTTCATCTGTATAGCCATCCGATTATATTGGGTTTTAGGAAAATTC CTATGGGTGTTGGCTTGAGCCCTTTTCTGCTGGCGCAATTTACTTCA GCTATCTGCTCAGTAGTACGCCGGGCGTTTCCCCATTGTCTTGCTTT CTCATACATGCATGATGTAGTACTTGGGGCCAAGTCTGTACAACAC CTTGAGAGTTTGTATGCCGCCGTAACTAATTTCCTTCTCTCTCGG GATCCATCTTAACCCTCACAAAACGAAGAGGTGGGGTTATTCTCTG AATTTCATGGGATATGTTATCGGGTCTTGGGGAACGCTGCCTCAGG AACACATCGTCCAGAAAATCAAGATGTGTTTCAGAAAGTTGCCAGT CGATAGATTGGAAGGTTTGCCAAAGAATTGTTGGCTTG TTGGGATTCGCAGCCCCATTCACACAGTGCGGGTATCCGGCTTTGA TGCCCCTTTATGCTTGTATCCAGGCAAAACAGGCATTCACCTTTTCCA CCGACTTACAAAGCATTTCTTTCTAAGCAGTATCTCCATCTTTACCC

- 50 047988

TGTCGCTCGACAGCGGCCGGGGCTTTGCCAGGTTTTCGCAGACGCA ACCCCAACTGGTTGGGGTCTTGCGATCGGCCACCAGAGGATGCGCG GTGCATTCGTGTCCCCGCTCCCAATCCATACGGCCCACTTGCTGGC GGCGTGCTTCGCTCGAAGTAGAAGCGGGCTAAATTGATCGGCAC GGACAATTCAGTCGTGTTGTCACGCAAATATACCTCTTCCCTGGT TTGCGCAGCAAACTGGATACTTCGGGGAACTAGTTTCGT TTATGTGCCCTCTGCTCTCAACCCCGCCGACGATCCTTCACGAGGG AGGCTGGGTCTTTACCGCCCATTGCTCAGGCTGCTTTACCGGCCTAC CACTGGGAGAACAAGCTTGTACGCCGACAGCCCGAGCGTCCCGTCT CATCTGCCCGACAGAGTTCACTTTGCGAGTCCATTGCACGTCGCTT GGCGCCCGCCG 94 IN Pol ³⁰⁰ _ID TCpGdel ATGAGTTCCAGATCACAGAGTCAGGGGCCTGTCCTTTCATGTTGGT GGCTTCAGTTTAGAAACTCAGAGCCATGTTCTGAGTATTGTCTCTGC CACATTGTGAATCTTATTGAAGACTGGGGCCCCTGCACAGAGCATG GAGAGCACAGAATAAGGACACCTAGAACCCCAGCAAGAGTGACAG GTGGAGTGTTCCTGGTAGACAAGAATCCACACACAACTGAGA TTGGTGGTTGATTTCAGTCAATTTTCAAGAGGCAATACAAG AGTTTCTTGGCCAAAATTTGCTGTACCCAATCTGCAATCCTTGACAA ATTTGCTTAGTTCTAATTTGTCTTGGCTTTCTCTAGATGTTTCTGCAG CTTTCTATCACTTGCCCCTTCACCCAGCAGCTATGCCTCATCTCTTG GTGGGCAGCTCTGGACTTAGTAGATATGTAGCTAGACTCAGTTCTA ACTCAAGGATAATAAATAACCAACATAGGACTATGCAGAACCTGC ATGATTCTTGTTCCAGGAACTTGTATGTCTCCTTGATGTTGTTGTAT AAAACTTATGGGAGAAAGCTTCATCTGTATAGCCATCCTATTATAT TGGGTTTTAGGAAAATTCCTATGGGTGTTGGCTTGAGCCCTTTTCTG CTGGCCCAATTT ACTTCAGCTATCTGCTCAGTAGTAAGGAGGGCCT TTCCCCATTGTCTTGCTTTCTCATACATGCATGATGTAGTACTTGGG GCCAAGTCTGTACAACACCTTGAGAGTTTGTATGCAGCAGTAACTA ATTTCCTTCTCTCTCT TGGGATCCATCTTAACCCTCACAAAACCAAGAGGTGGGGTTATTCT CTGAATTTCATGGGATATGTTATAGGGTCTTGGGGAACCCTGCCTC AGGAACACATTGTCCAGAAAATCAAGATGTGTTTCAGAAAGTTGCC AGTGAACAGACCAATAGATTGGAAGGTTTGCCAAAGAATTGTTGG CTTGTTGGGATTTGCAGCCCCATTCACACAGTGTGGGTATCCTGCTT TGATGCCCC TTTATGCTTGTATCCAGGCAAAACAGGCATTCACCTTT TCACCCACTTACAAAGCATTTCTTTCTAAGCAGTATCTCCATCTTTA CCCTGTGGCTAGACAGAGGCCAGGGCTTTGCCAGGTTTTTGCAGAT GCAACCCCAACTGGTTGGGGTCTTGCAATTGGCCACCAGAGGATGA GAGGTGCATTTGTGTCCCCACTCCCAATCCATACTGCCCACTTGCTG GCAGCTTGCTTTGCTAGAAGTAGAAGTGGGGCTAAATTGATTGGCA CAGACAATTCAGTTGTGTTGTCAAGGAAATATACCTCCTTTCCCTG GTTGCTTGGTTGTGCAGCAAACTGGATACTTAGGGGAACTAGTTTT GTTTATGTGCCCTCTGCTCTCAACCCTGCAGATGATCCTTCAAGAGG GAGGCTG GGTCTTTACAGGCCATTGCTCAGGCTGCTTTACAGGCCT ACCACTGGGAGAACAAGCTTGTATGCAGACAGCCCCAGTGTCCCCT CTCATCTGCCTGACAGAGTTCACTTTGCAAGTCCATTGCATGTTGCT TGGAGACCTCCA 30 D Ρο1 ^Δ1 ATGCCCCTGAGCTACCAACACTTCAGGAGACTGCTGCTGCTGGATG ATGAGGCAGGCCCTCTGGAGGAGGAGCTGCCCAGGCTGGCAGATG AGGGCCTGAACAGGAGGTGGCTGAGGACCTGAACCTGGGCAACC TGAATGTGAGCATCCCTTGGACCCAAAGTGGGCAACTTCACAGG CCTGTACAGCAGCACTGTGCCTGTGTTCAACCCCCACTGGAAGACA CCCAGCTTCCCC AACATCCACCTGCACCAGGACATCATCAAGAAGT GTGAGCAGTTTGTGGGCCCCCTGACAGTCAATGAGAAGAGGAGGC TCCAGCTGATCATGCCAGCCAGGTTCTACCCCAATGTGACCAAGTA CCTCCCCCTGGACAAGGGCATCAAGCCTTACTATCCAGAGCACCTG GTGAACCACTACTTCCAGACCAGACACTACCTGCACACACTGTGGA

- 51 047988

AGGCAGGCATCCTGTACAAGAGGGAGACCACACACAGTGCCTCCT TCTGTGGCAGCCCCTACTCCTGGGAGCAGGAGCTGCAACATGGAGC TGAGTCCTTCCACCAGCAGTCCAGTGGCATCCTGAGCAGGCCCCCT GTGGGCAGCGAGCTGCACAACCTGCCCCCCAACTCTGCCAGATCCC AGTCTGAGAGGCCAGTGTTCCCTTGCTGGTGGCTCCAGTTCAGGAA AGCCCTGCTCAGACTACTGCCTGAGCCACATTGTGAACCTG CTGGAGGACTGGGGCCCCTGTGCAGAGCATGGGGAGCACCACATC AGAATCCCCAGGACCCCTGCCAGGGTGACAGGAGGGGTGTTCCTG GTGGACAAGAACCCCCACAACACTGCAGAGTCCAGGCTGGTGGTG GACTTCTCCCAGTTCAGCAGGGGCAACTACAGAGTCTCCTGGCCAA AGTTTGCTGTGCCCAACCTCCAGAGCCTGACAAACCTGCTGAGCAG CAACCTGTCCTGGCTCCCTGGATGTGAGTGCAGCCTTCTATCACC TGCCCCTGCACCCAGCAGCCATGCCACACCTGCTGGTGGGCTCCAG TGGCCTGTCCAGGTATGTGGCCAGGCTCTCCTCCAACTCCAGGATC TTCAACTAT CAGCATGGCACCATGCAGAACCTGCATGACAGCTGCT CCAGGAACCTGTATGTGTCCCTGATGCTGCTCTATCAGACCTTTGGC AGGAAGCTGCACCTGTACAGCCACCCCATCATCCTGGGGTTCAGGA AGATCCCCATGGGTGTGGGCCTGTCCCCCTTCCTGCTGGCCCAGTTC ACCAGTGCCATCTGCTCAGTGGTGAGGAGG GCCTTCCCACACTGCCTGGCCTTCTCTTACATGCATGATGTGGTCCT GGGTGCCAAGTCTGTGCAGCACCTGGAGAGCCTGTTCACAGCTGTG ACAAACTTTCTCCTGAGCCTGGGCATCCACCTGAACCCCAACAAGA CCAAGAGGTGGGGTTATTCACTGCACTTCATGGGCTATGTGATTGG CTGCTATGGCTCTCTGCCACAGGACCACATCATCCAGAAGATCAAG GAGTGCTTCAGAAA GCTGCCAGTGAACAGGCCAATTGACTGGAAG GTGTGCCAGAGGATTGTGGGCCTGCTGGGCTTTGCAGCCCCCTTCA CCCAGTGTGGCTACCCTGCCCTGATGCCCCTGTATGCCTGCATCCA GAGCAAGCAGGCCTTCACCTTTTCCCCCACTTACAAGGCCTTCCTGT GCAAGCAGTACCTGAACCTGTACCCTGTGGCCAGGCAGAGACCTG GGCTGTGCCAGGTGTTTGCAGATGCCACCCCCACAGGATGGGGACT GGTCATGGGACACCAGAGGATGAGGGGCACCTTCAAGGCACCCCT GCCCATCCACACAGCCCACCTGCTGGCTGCCTGCTTTGCCAGGAGC AGGAGTGGGCCAACATCCTGGGCACAGACAACTCTGTGGTGCTG AGCAGGA AGTACACATCCTTCCCCTGGCTGCTGGGATGTGCAGCCA ACTGGATCCTGAGGGGCACCAGCTTTGTGTATGTGCCCTCTGCCCT CAACCCTGCAGATGATCCAAGCAGGGGCAGGCTGGGACTGTACAG GCCACTGCTCAGACTGCCCTTCAGGCCCACCACTGGCAGGACCAGC CTGTATGCTGACTCCCCATCTGTGCCCTCCCACCTGCCTGACAGAGT GCACTTTGCCTCCCCACTGCATGTGGCCTGGAGGCCCCCA 31 D Ρο1 ^Δ3 ATGCCCCTGAGCTACCAACACTTCAGGAGACTGCTGCTGCTGGATG ATGAGGCAGGCCCTCTGGAGGAGGAGCTGCCCAGGCTGGCAGATG AGGGCCTGAACAGGAGGGTGGCTGAGGACCTGAACCTGGGCAACC TGAATGTGAGCATCCCTTGGACCCAAAGTGGGCAACTTCACAGG CCTGTACAGCAGCACTGTGCCTGTGTTCAACCCCCACTGGAAGACA CCCAGCTTCCCCAACATCCACCTGCACCAGGACATCATCAAGAAGT GTGAGCAGTTTGTGGGCCCCCTGACAGTCAATGAGAAGAGGAGGC TCCAGCTGATCATGCCAGCCAGGTTCTACCCCAATGTGACCAAGTA CCTCCCCCTGGACAAGGGCATCAAGCCTTACTATCCAGAGCACCTG GTGAACCACTACTTCCAGACCAGACACTACCTGCACACACTGTGGA AGGCAGGCATCCTGTACAAGAGGGAGACCACACACAGTGCCTCCT TCTGTGGCAGCCCCTACTCCTGGGAGCAGGAGCTGCAACATGGATG CTGGTGGCTCCAGTTCAGGAACAGCAAGCCCTGCTCAGACTACTGC CTGAGCCACATTGTGAACCTGCTGGAGGACTGGGGCCCCTGTGCAG AGCATGGGGAGCACCACATCAGAATCCCCAGGACCCCTGCCAGGG TGACAGGAGGGGTGTTCCTGGTGGACAAGAACCCCCACAACACTG CAGAGTCCAGGCTGGTGGTGGACTTCTCCCAGTTCAGCAGGGGCAA CTACAGAGTCTCCTGGCCAAAGTTTGCTGTGCCCAACCTCCAGAGC

- 52 047988

CTGACAAACCTGCTGAGCAGCAACCTGTCCTGGCTCTCCCTGGATG TGAGTGCAGCCTTCTATCACCTGCCCCTGCACCCAGCAGCCATGCC ACACCTGCTGGTGGGCTCCAGTGGCCTGTCCAGGTATGTGGCCAGG CTCTCCTCCAACTCCAGGATCTTCAACTATCAGCATGGCACCATGC AGAACCTGCATGACAGCTGCTCCAGGAACCTGTATGTGTCCCTGAT GCTGCTC TATCAGACCTTTGGCAGGAAGCTGCACCTGTACAGCCAC CCCATCATCCTGGGGTTCAGGAAGATCCCCATGGGTGTGGGCCTGT CCCCCTTCCTGCTGGCCCAGTTCA CCAGTGCCATCTGCTCAGTGGTGAGGAGGGCCTTCCCACACTGCCT GGCCTTCTCTTACATGCATGATGTGGTCCTGGGTGCCAAGTCTGTGC AGCACCTGGAGAGCCTGTTCACAGCTGTGACAAACTTTCTCCTGAG CCTGGGCATCCACCTGAACCCCAACAAGACCAAGAGGTGGGGTTA TTCACTGCACTTCATGGGCTATGTGATTGGCTGCTATGGCTCTGC CACAGGACCA CATCATCCAGAAGATCAAGGAGTGCTTCAGAAAGC TGCCAGTGAACAGGCCAATTGACTGGAAGGTGTGCCAGAGGATTG TGGGCCTGCTGGGCTTTGCAGCCCCCTTCACCCAGTGTGGCTACCCT GCCCTGATGCCCCTGTATGCCTGCATCCAGAGCAAGCAGGCCTTCA CCTTTTCCCCCACTTACAAGGCCTTCCTGTGCAAGCAGTACCTGAAC CTGTACCCTGTGGCCAGGCAGACCTGGGCTGTGCCAGGTGTTTG CAGATGCCACCCCCACAGGATGGGGACTGGTCATGGGACACCAGA GGATGAGGGGCACCTTCAAGGCACCCCTGCCCATCCACACAGCCCA CCTGCTGGCTGCCTGCTTTGCCAGGAGCAGGAGTGGGGCCAACATC CACAGACAACTCTGTGGTGCTGAGCAGGAAGTACACATCCT TCCCCTGGCTGCTGGGATGTGCAGCCAACTGGATCCTGAGGGGCAC CAGCTTTGTGTATGTGCCCTCTGCCCTCAACCCTGCAGATGATCCAA GCAGGGGCAGGCTGGGACTGTACAGGCCACTGCTCAGACTGCCCTT CAGGCCCACCACTGGCAGGACCAGCCTGTATGCTGACTCCCCATCT GTGCCCTCCCACCTGCCTGACAGAGTGCACTTTGCCTCCCCACTGC ATGTGGCCTGGAGGCCCCCA 32 D Floor ³⁰⁰ ATGTCTGCCAGATCCCAGTCTGAGAGGCCAGTGTTCCCTTGCTGGT GGCTCCAGTTCAGGAACAGCAAGCCCTGCTCAGACTACTGCCTGAG CCACATTGTGAACCTGCTGGAGGACTGGGGCCCCTGTGCAGAGCAT GGGGAGCACCACATCAGAATCCCCAGGACCCCTGCCAGGGTGACA GGAGGGGTGTTCCTGGTGGACAAGAACCCCCACAACACTGCAGAG TCCAGGCT GGTGGTGGACTTCTCCCAGTTCAGCAGGGGCAACTACA GAGTCTCCTGGCCAAAGTTTGCTGTGCCCAACCTCCAGAGCCTGAC AAACCTGCTGAGCAGCAACCTGTCCTGGCTCTCCCTGGATGTGAGT GCAGCCTTCTATCACCTGCCCCTGCACCCAGCAGCCATGCCACACC TGCTGGTGGGCTCCAGTGGCCTGTCCAGGTATGTGGCCAGGCTCTC CTCCAACTCCAGGATCTTCAACTATCAGCATGGCACCATGCAGAAC CTGCATGACAGCTGCTCCAGGAACCTGTATGTGTCCCTGATGCTGC TCTATCAGACCTTTGGCAGGAAGCTGCACCTGTACAGCCACCCCAT CATCCTGGGGTTCAGGAAGATCCCCATGGGTGTGGGCCTGTCCCCC TGGCCCAGTTCACCAGTGCCATCTGCTCAGTGGTGAGGA GGGCCTTCCCACACTGCCTGGCCTTCTCTTACATGCATGATGTGGTC CTGGGTGCCAAGTCTGTGCAGCACCTGGAGAGCCTGTTCACAGCTG TGACAAACTTTCTCCTGAGCCTGGGCATCCACCTGAACCCCAACAA GACCAAGAGGTGGGGTTATTCACTGCACTTCATGGGCTATGTGATT GGCTGCTATGGCTCTCTGCCACAGGACCACATCATCCAGAAGATCA AGGAGTGCTTCAGAAAGCTGCCAGTGAACAGGCCAATTGACTGGA AGGTGTGCCAGAGGATTGTGGGCCTGCTGGGCTTTGCAGCCCCCTT CACCCAGTGTGGCTACCCTGCCCTGATGCCCCTGTATGCCTGCATCC AGAGCAAGC AGGCCTTCACCTTTTCCCCCACTTACAAGGCCTTCCT GTGCAAGCAGTACCTGAACCTGTACCCTGTGGCCAGGCAGAGACCT GGGCTGTGCCAGGTGTTTG CAGATGCCACCCCCACAGGATGGGGACTGGTCATGGGACACCAGA GGATGAGGGGCACCTTCAAGGCACCCCTGCCCATCCACACAGCCCA

- 53 047988

CCTGCTGGCTGCCTGCTTTGCCAGGAGCAGGAGTGGGGCCAACATC CTGGGCACAGACAACTCTGTGGTGCTGAGCAGGAAGTACACATCCT TCCCCTGGCTGCTGGGATGTGCAGCCAACTGGATCCTGAGGGGCAC CAGCTTTGTGTATGTGCCCTCTGCCCTCAACCCTGCAGATGATCCAA GCAGGGGCAGGCTGGGACTGTACAGGCCACTGCTCAGACTGCCCTT CAGGCCCACC ACTGGCAGGACCAGCCTGTATGCTGACTCCCCATCT GTGCCCTCCCACCTGCCTGACAGAGTGCACTTTGCCTCCCCACTGC ATGTGGCCTGGGAGGCCCCCA 33 B/C CapsidsAg ATGGACATTGACCCCTACAAGGAGTTTGGGGCCAGTGTGGAGCTGC TGTCTTTTCTGCCATCTGACTTCTTCCCCAGTGTGAGGGACCTGCTG GACACTGCCTCAGCACTGTACAGAGAGGCCCTGGAGAGCCCAGAG CACTGCTCCCCCCACCACACAGCCCTGAGGCAGGCCATCCTCTGCT GGGGGGAGCTGATGAACCTGGCCACCTGGGTGGGCTCCAACCTGG AGGACCCT GCCTCAAGGGAGCTGGTGGTCAGCTATGTCAATGTGAA CATGGGCCTCAAGATCAGGCAGCTGCTGTGGTTCCACATCTCCTGC CTGACCTTTGGCAGGGAGACAGTCCTGGAGTACCTGGTGAGCTTTG GGGTGTGGATCAGGACCCCCCCTGCCTACAGGCCCCCCAATGCTCC CATCCTGTCCACCCTGCCAGAGACCACTGTGGTCAGGAAGGGGC AGGTCCCCCAGGAGGAGAACCCCCTCCCAGGAGGAGGAGAAGC CAGTCCCCCAGGAGGAGGAGGAGCCAGAGCAGAGAGTCTCAGTGC ATGGAGAGCACCACATCAGGCTTCCTGGGCCCCCTGCTGGTGCTCC AGGCAGGCTTCTTTCTGCTGACCAGGATTCTGACCATCCCCCAGTC CCTGGACAG CTGGTGGACCTCCCTGAATTTTCTGGGGGGGGCCCCT ACCTGTCCTGGCCAGAACTCTCAGTCTCCCACCTCGAATCACTCAC CAACCAGCTGTCCCCCCATCTGTCCTGGCTACAGGTGGATGTGCCT GAGGAGATTCATCATCTTCCTGTGCATCCTGCTGCTGTGCCTGATCT TTCTGCTGGTGCTGCTGGACTACCAGGGCATGCTGCCAGTGTGCCC TCTCATCCCAGGCAGCTCCACCACATCCACAGGACCTTGCAAGACA TGCACCACACCAGCCCAGGGCACCAGCATGTTCCCCTCCTGCTGTT GCACCAAGCCAACAGATGGCAACTGCACATGCATTCCCATCCCCTC CAGCTGGGCCTTTGCCAGGTTTCTGTGGGAGTGGGCCAGTGTGAGA CTGTCTCTTCTGGTGCCCTTTGTGCAGTGGTTTGTGGG CCTGTCCCCTACAGTGTGGCTGAGTGTCATCTGGATGATGTGGTAC TGGGGCCCCTCCCTGTACAACATCCTCTCTCCCTTTCTGCCTCTGCT GCCAATCTTCTTTTGCCTGTGGGTGTACATC 34 B/S Capsid- P2A-sAg ATGGACATTGACCCCTACAAGGAGTTTGGGGCCAGTGTGGAGCTGC TGTCTTTTCTGCCATCTGACTTCTTCCCCAGTGTGAGGGACCTGCTG GACACTGCCTCAGCACTGTACAGAGAGGCCCTGGAGAGCCCAGAG CACTGCTCCCCCCACCACACAGCCCTGAGGCAGGCCATCCTCTGCT GGGGGGAGCTGATGAACCTGGCCACCTGGGTGGGCTCCAACCTGG AGGACCCT GCCTCAAGGGAGCTGGTGGTCAGCTATGTCAATGTGAA CATGGGCCTCAAGATCAGGCAGCTGCTGTGGTTCCACATCTCCTGC CTGACCTTTGGCAGGGAGACAGTCCTGGAGTACCTGGTGAGCTTTG GGGTGTGGATCAGGACCCCCCCTGCCTACAGGCCCCCCAATGCTCC CATCCTGTCCACCCTGCCAGAGACC ACTGTGGTCAGGAGAAGGGGCAGGTCCCCCAGGAGGAGAACCCCC TCTCCCAGGAGGAGGAGAAGCCAGTCCCCCAGGAGGAGGAGGAGC CAGAGCAGAGAGTCTCAGTGCGGCAGTGGGGCAACCAACTTCAGC CTCCTGAAACAGGCAGGGGATGTGGAGGAAAACCCAGGCCCCGAG AGCACCACATCAGGCTTCCTGGGCCCCCTGCTGGTGCTCCAGGCAG GCTTCTTTCTG CTGACCAGGATTCTGACCATCCCCCAGTCCCTGGAC AGCTGGTGGACCTCCCTGAATTTTCTGGGGGGGGCCCCTACCTGTC CTGGCCAGAACTCTCAGTCTCCCACCTCGAATCACTCACCAACCAG CTGTCCCCCCATCTGTCCTGGCTACAGGTGGATGTGCCTGAGGAGA TTCATCATCTTCCTGTGCATCCTGCTGCTGTGCCTGATCTTTCTGCTG GTGCTGCTGGACTACCAGGGCATGCTGCCAGTGTGCCCTCTCATCC CAGGCAGCTCCACCACATCCACAGGACCTTGCAAGACATGCACCAC

- 54 047988

ACCAGCCCAGGGCACCAGCATGTTCCCCTCCTGCTGTTGCACCAAG CCAACAGATGGCAACTGCACATGCATTCCCATCCCCTCCAGCTGGG CCTTTGCCAGGTTTCTGTGGGAGTGGGCCAGTGTGAGATTTTCCTG GCTGTCTCTTCTGGTGCCCTTTGTGCAGTGGTTTGTGGGCCTGTCCC CTACAGTGTGGCTGAGTGTCATCTGGATGATGTGGTACTGGGGCCC CCTGTACAACATCCTCTCTCCCTTTCTGCCTCTGCTGCCAATCTT CTTTTGCCCTGTGGGTGTACATC 35 D/D CapsidsAg ATGGACATTGACCCCTACAAGGAGTTTGGGGCCAGTGTGGAGCTGC TCTCCTTCCTGCCCTCAGACTTCTTTCCCAGTGTGAGGGACCTGCTT GACACAGCCTCTGCCCTCTACAGAGAGGCCCTGGAGAGCCCAGAG CATTGCTCCCCCCACCACAGCACTGAGGCAGGCCATCCTGTGCT GGGGGGAGCTCATGAACCTGGCCACCTGGGTGGGTGTCAACCTGG GCTTCCAGGATCTGGTGGTCAGCTATGTGAACACAAA CATGGGCCTCAAGTTCAGGCAGCTGCTCTGGTTCCACATCTCCTGC CTGACCTTTGGCAGGGAGACTGTGCTGGAGTACCTGGTGAGCTTTG GAGTGTGGATCAGGACCCCACCTGCCTACAGGCCCCCCAATGCCCC CATCCTGTCCACCCTGCCTGAGACCACAGTGGTGAGGAGGAGGGG GAGGTCCCCAGAAGGAGGACCCCTTCTCCCAGGAGGAGGAGGAG TCAGTCTCCCAGGAGGAGGAGGAGCCAGAGCAGAGAGTCCCAGTG TATGGAGAACATCACCTCTGGCTTTCTGGGACCCCTGCTGGTGCTC CAGGCAGGCTTTTTCCTGCTGACCAGGATCCTGACCATCCCTCAGA ACTCCTGGTGGACATCTCTGAATTTTCTTGGGGGCACCAC TGTGTGCCTGGGACAGAACTCCCAGTCTCCCACCTCCAACCACAGC CCAACATCCTGTCCCCCCATCTGCCCAGGCTACAGGTGGATGTGCC TGAGGAGGTTCATCATCTTCCTGTTCATCCTGCTGCTGTGCCTGATC TTTCTGCTGGTGCTCCTGGACTATCAGGGCATGCTGCCAGTGTGCCC ACTGATCCCAGGCAGCTCCACCACAAGCACAGGACCTTGCAGGAC ATGCACCACACCTGCCCAGGGCACTTCCATGTACCCATCTTGCTGTT GCACCAAGCCATCTGATGGCAATTGCACCTGCATCCCCATCCCCTC AAGCTGGGCCTTTGGCAAGTTCCTGTGGGAGTGGGCAAGTGCCAGA TTCTCTTGG CTGAGCCTGCTGGTCCCTTTTGTGCAGTGGTTTGTGGG CCTGAGCCCCACTGTGTGGCTGTCTGTGATCTGGATGATGTGGTAC TGGGGCCCCTCCCTGTATTCAATCCTGAGCCCTTTTCTGCCACTGCT GCCCATCTTCTTTTGTCTGTGGGTGTACATC 36 D/D Capsid- P2A-sAg ATGGACATTGACCCCTACAAGGAGTTTGGGGCCAGTGTGGAGCTGC TCTCCTTCCTGCCCTCAGACTTCTTTCCCAGTGTGAGGGACCTGCTT GACACAGCCTCTGCCCTCTACAGAGAGGCCCTGGAGAGCCCAGAG CATTGCTCCCCCCACCACAGCACTGAGGCAGGCCATCCTGTGCT GGGGGGAGCTCATGAACCTGGCCACCTGGGTGGGTGTCAACCTGG AGGACCCAGCTTCCAGGGATCTGGTGGTCAGCTATGTGAACACAAA CATGGGCCTCAAGTTCAGGCAGCTGCTCTGGTTCCACATCTCCCTGC CTGACCTTTGGCAGGGAGACTGTGCTGGAGTACCTGGTGAGCTTTG GAGTGTGGATCAGGACCCCACCTGCCTACAGGCCCCCCAATGCCCC CATCCTGTCCACCCTGCCTGAGACCACAGTGGTGAGGAGGAGGGG GAGGTCCCCCAGAAGGAGGACCCCTTCTCCCAGGAGGAGGAGGAG TCAGTCTCCCAGGAGGAGGAGGAGCCAGAGCAGAGAGTCCCAGTG TGGCAGTGGGGCAACCAACTTCAGCCTCCTGAAACAGGCAGGGGA TGTGGAGGAAAACCCAGGCCCCGAGAACATCACCTCTGGCTTTCTG GGACCCCTGCTGGTGCTCCAGGCAGGCTTTTTCCTGCTGACCAGGA TCCTGACCATCCCTCAGAGCCTGGACTCCTGGTGGACATCTCTGAA TTTTCTTGGGGGCACCACTGTGTGCCTGGGACAGAACTCCCAGTCT CCCACCTCCAACCACAGCCCAACATCCTGTCCCCCCATCTGCCCAG GCTACAGGTGGATGTGCCTGAGGAGGTTCATCATCTTCCTGTTCAT CCTGCTGCTGTGCCTGATCTTTCTGCTGGTGCTCCTGGACTATCAGG GCATGCTGCCAGTGTGCCCACTGATCCCAGGCAGCTCCACCACAAG CACAGGACCTTGCAGGACATGCACCACACCTGCCCAGGGCACTTCC ATGTACCCATCTTGCTGTTGCACCAAGCCATCTGATGGCAATTGCA

4. Vectors and host cells

Additionally provided are vectors comprising one or more polynucleotides encoding one or more immunogenic polypeptides described herein, or an expression cassette comprising such polynucleotides. The vector may be of any type, for example, a recombinant vector, such as an expression vector. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), as well as vectors derived from plant or animal (including human) bacteriophages or viruses. Vectors may comprise an origin of replication recognized by the intended host cell, and in the case of expression vectors, a promoter and other regulatory regions recognized by the host cell. In further embodiments, the vector comprises one or more polynucleotides encoding one or more immunogenic polypeptides of the present invention, operably linked to a promoter and, optionally, additional regulatory elements. Some vectors are capable of autonomous replication in the host into which they are introduced (e.g., vectors having a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into the host genome upon introduction into the host and thus replicate along with the host genome. Vectors include, but are not limited to, vectors suitable for recombinant production of the immunogenic polypeptides described herein.

In the context of this document, the term vector refers to a nucleic acid molecule capable of

- 55 047988 capable of providing for the reproduction of another nucleic acid linked thereto. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector that is incorporated into the genome of a host cell into which it has been introduced. Some vectors are suitable for delivering the nucleic acid molecule or polynucleotide of the present application. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as expression vectors.

The term operably linked refers to two or more nucleic acid sequences or polypeptide sequence elements that are normally physically linked and in a functional relationship with one another. For example, in the context of nucleic acid sequence elements, a promoter is operably linked to a coding sequence if the promoter is capable of initiating or regulating transcription or expression of the coding sequence, in which case the coding sequence is to be understood as being under the control of the promoter.

The choice of vector depends on the recombinant procedures used and the host used. The introduction of vectors into host cells can be accomplished by, among other things, calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofectamine transfection, electroporation, viral infection, or administration to a subject as described herein. Vectors can replicate autonomously or can replicate together with the chromosome into which they have been integrated. In certain embodiments, the vectors comprise one or more selectable markers. The choice of markers can depend on the host cells selected. These include, among other things, a gene for resistance to kanamycin, neomycin, puromycin, hygromycin, zeocin, the herpes simplex virus thymidine kinase gene (HSV-TK), and the mouse dihydrofolate reductase gene (dhfr). Vectors comprising one or more nucleic acid molecules encoding the immunogenic polypeptides described herein, operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the immunogenic polypeptides, are also encompassed by the present invention. These proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase, and beta-galactosidase.

In other embodiments, the pcDNA™ 3.1+ vector (ThermoFisher, MA) is used.

In some embodiments, the vector is a viral vector.

Where appropriate, the viral vector may be a DNA virus or RNA virus, including a self-replicating RNA virus. Self-replicating RNA viruses include alphaviruses and are described, for example, in Lundstrom, Molecules. (2018) 23(12). pii: E3310 (PMID: 30551668); and Ljungberg, et al., Expert Rev Vaccines. (2015) 14(2): 177-94). In various embodiments, the viral vector is derived from a virus selected from the group consisting of adenovirus, adeno-associated virus, arenavirus, alphavirus, self-replicating alphavirus, poxvirus, cytomegalovirus, rhabdovirus, vesicular stomatitis virus, flavivirus, Maraba virus, and vaccinia virus. In some embodiments, the viral vector is derived from a virus family selected from the group consisting of the following: Adenoviruses (e.g., adenoviruses, adeno-associated virus), arenaviruses (e.g., lymphocytic choriomeningitis mammarenavirus, Kali mammarenavirus (also known as Pichinde mammarenavirus (PICV)), poxviruses (e.g., vaccinia virus), herpes viruses (e.g., cytomegalovirus, herpes virus, e.g., HSV-1), parvoviruses (e.g., parvovirus H1), poxviruses (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA), flaviviruses (e.g., yellow fever virus), reoviruses (e.g., reovirus), retroviruses (e.g., lentivirus), picornaviruses (e.g., coxsackievirus, Seneca Valley virus, poliovirus), paramyxoviruses (e.g. measles virus, Newcastle disease virus (NDV)), rhabdoviruses (e.g. vesiculovirus, including Maraba vesiculovirus and vesicular stomatitis virus (VSV), togaviruses (e.g. alphavirus, e.g. self-replicating alphavirus; Sindbis virus), enteroviruses (e.g. echovirus). Exemplary modified vaccinia virus vectors used to express the present immunogenic polypeptides are described, for example, in WO 2019/134049.

In some embodiments, the viral expression vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) (NCBI:txid11623), Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus) (NCBI:txid2169993), Guanarito virus (GTOV) (NCBI:txid45219), Argentine mammarenavirus (also known as Junin virus (JUNV)) (NCBI:xid2169991), Lassa virus (LASV) (NCBI:xid11620), Luyo virus (LUJV) (NCBI:txid649188), Machupo virus (MACV) (NCBI:xid11628), Brazilian mammarenavirus (also known as Sabia virus (SABV)) (NCBI:xid2169992) and Whitewater Arroyo virus (WWAV) (NCBI:xid46919). In some embodiments, the viral expression vector is an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Cali mammarenavirus (LCMV) (also known as mamma

- 56 047988 Pichinde renavirus or Pichinde arenavirus (PICV)). Exemplary arenavirus vectors that can be used as delivery and expression vehicles for the immunogenic polypeptides described herein are described, for example, in WO 2009/083210; WO 2015/183895; WO 2016/075250; WO 2017/198726; and U.S. Patent Nos. 9,943,585 and 10,342,861, which are incorporated herein by reference in their entirety for all purposes.

In some embodiments, the viral expression vector is an adenovirus vector, such as a human adenovirus or a simian adenovirus (e.g., a chimpanzee adenovirus, a gorilla adenovirus, or a rhesus macaque adenovirus). In various embodiments, the adenovirus vector is selected from adenovirus serotype 5 (Ad5), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), chimpanzee adenovirus (e.g., ChAdOx1, ChAdOx2, ChAd3 (AdC3), ChAd5 (AdC5), ChAd6 (AdC6), ChAd7 (AdC7), ChAd8 (AdC8), ChAd9 (AdC9), ChAd10 (AdC10), ChAd11 (AdC11), ChAd17 (AdC17), ChAd16 (AdC16), ChAd19 (AdC19), ChAd20 (AdC20), ChAd22 (AdC22), ChAd24 (AdC24), ChAdY25, ChAd26 (AdC26), ChAd28 (AdC28), ChAd30 (AdC30), ChAd31 (AdC31), ChAd37 (AdC37), ChAd38 (AdC38), ChAd43 (AdC43), ChAd44 (AdC44), ChAd55 55), ChAd63 (AdC63), ChAdV63, ChAd68 (AdC68), ChAd73 (AdC73), ChAd82 (AdC82), ChAd83 (AdC83), ChAd143 (AdC143), ChAd144 (AdC144), ChAd145 (AdC145), ChAd147 (AdC147)), gorilla adenovirus (e.g. GC44, GC45, GC46) and rhesus macaque adenovirus (e.g. RhAd51, RhAd52, RhAd53, RhAd54, RhAd55, RhAd56, RhAd57, RhAd58, RhAd59, RhAd60, RhAd61, RhAd62, RhAd63, RhAd64, RhAd65, RhAd66). Illustrative chimpanzee, gorilla, and rhesus macaque adenovirus vectors that can be used as delivery and expression vehicles for the immunogenic polypeptides described herein are described, for example, in WO 2012/172277 (ChAdOx1), WO 2017/221031 (ChAdOx2), WO 2019/076880; WO 2019/076877; Andrabi et al., (2019) Cell Reports 27:2426-2441 Guo, et al., Hum Vaccin Immunother. (2018) 14(7):1679-1685; Abbink, et al., J Virol. (2015) 89(3):1512-22; and Abbink, et al., J Virol. (2018) 92(6). pii:e01924-17.

In various embodiments, the viral expression vector is incapable of replication (i.e., is replication-defective or replication-deficient), has a reduced or limited replication capacity, e.g., compared to a wild-type viral vector (i.e., replication-attenuated), or is replication-competent. In various embodiments, the viral expression vector is a replication-defective or replication-deficient arenavirus vector having a two-segment genome, e.g., as described in WO 2009/083210 and WO 2017/076988. In various embodiments, the viral expression vector is a replication-attenuated arenavirus vector having a two-segment, three-segment genome, such as described in WO 2016/075250, WO 2017/076988, and WO 2017/198726.

Additionally provided are host cells comprising one or more polynucleotides encoding one or more immunogenic polypeptides or one or more vectors expressing immunogenic polypeptides as described herein. Any of a variety of host cells can be used. In one embodiment, the host cell is a prokaryotic cell, such as E. coli. In another embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, a mammalian cell such as a Chinese hamster ovary (CHO) cell line or a CHO-derived cell line (e.g., CHO-S, CHO DG44, ExpiCHO™, ZFN-modified GS-/-CHO cell line CHOZN®, CHO-Ki, CHO-KY), COS cells, BHK cells, NSO cells, or Bowes melanoma cells. Examples of human host cells include, but are not limited to, HeLa, 911, AT1080, A549, and HEK293 (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293™). Additionally, immunogenic polypeptides can be expressed in yeast cells such as Pichia (see, e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces.

The terms host cell, host cell line, and host cell culture are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include initially transformed cells and progeny derived therefrom, regardless of passage number. Progeny may not be completely identical to the original cell in nucleic acid content, and may contain mutations. Mutant progeny that have the same function or biological activity that is the subject of study or selection in the initially transformed cell are included herein.

If desired, host cells can be stably or transiently transfected with one or more polynucleotides encoding one or more immunogenic polypeptides as described herein. In appropriate cases, host cells can be infected with one or more vectors expressing one or more immunogenic polypeptides as described herein. In some embodiments, host cells are capable of being infected and expanded with one or more replication-attenuated or replication-

- 57 047 988 competent vectors expressing one or more immunogenic polypeptides as described herein. Exemplary cells suitable for infection and/or propagation of viral vectors include, but are not limited to, BHK-21, A549, Vero, and HEK293 cells (e.g., HEK293E, HEK293F, HEK293H, HEK293T, Expi293™). In certain embodiments, the host cells express a coxsackievirus and an adenovirus receptor (CAR), such as MDCK, Caco-2, or Calu-3 host cells. In certain embodiments, the polynucleotides are integrated into the host cell genome.

5. Pharmaceutical compositions/immunogenic compositions

Provided are pharmaceutical compositions or immunogenic compositions comprising one or more immunogenic HBV polypeptides as described herein, or a polynucleotide encoding one or more immunogenic HBV polypeptides as described herein, or a viral expression vector comprising one or more such polynucleotides, and a pharmaceutically acceptable diluent, carrier, or excipient. A pharmaceutically acceptable excipient includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

Typically, the pharmaceutical compositions described herein are immunogenic. In certain embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more (e.g., two or more, three or more) immunogenic HBV polypeptides, or one or more (e.g., two or more, three or more) polynucleotides encoding one or more (e.g., two or more, three or more) immunogenic HBV polypeptides, or one or more (e.g., two or more, three or more) viral expression vectors comprising one or more (e.g., two or more, three or more) polynucleotides encoding one or more immunogenic HBV polypeptides.

Various pharmaceutically acceptable diluents, carriers, and excipients, as well as methods for preparing and using the pharmaceutical compositions, will be known to those skilled in the art in light of the present invention. Illustrative pharmaceutical compositions and pharmaceutically acceptable diluents, carriers, and excipients are also described in, for example, Loyd V. Allen Jr (Editor), Remington: The Science and Practice of Pharmacy, 22 ^nd Edition, 2012, Pharmaceutical Press; Brunton, Knollman and Hilal-Dandan, Goodman and Gilman's The Pharmacological Basis of Therapeutics, 13 th Edition, 2017, McGraw-Hill Education / Medical; McNally and Hastedt (Editors), Protein Formulation and Delivery, 2nd Edition, 2007, CRC Press; Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, 3rd Edition, 2015, CRC Press; Lars Hovgaard, Frokjaer and van de Weert (Editors), Pharmaceutical Formulation Development of Peptides and Proteins, 2nd Edition, 2012, CRC Press; Carpenter and Manning (Editors), Rational Design of Stable Protein Formulations: Theory and Practice, 2002, Springer (Pharmaceutical Biotechnology (Book 13)); Meyer (Editor), Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, 2012, Woodhead Publishing.

In certain embodiments, the polynucleotides or vectors are formulated into lipid nanoparticles. For example, in some embodiments in which the immunogenic HBV polypeptides are expressed from self-replicating or self-amplifying RNA molecules, the self-replicating or self-amplifying RNA can be formulated into lipid nanoparticles (LNPs). As used herein, the term lipid nanoparticle refers to one or more spherical nanoparticles with an average diameter of about 10 to about 1000 nm that contain a solid lipid core matrix that can solubilize lipophilic molecules. In some embodiments, the lipid core is stabilized with surfactants (e.g., emulsifiers) and can comprise one or more of triglycerides (e.g., tristearin), diglycerides (e.g., glycerol bahenate), monoglycerides (e.g., glycerol monostearate), fatty acids (e.g., stearic acid), steroids (e.g., cholesterol), and waxes (e.g., cetyl palmitate), including combinations thereof. Lipid nanoparticles are described in, e.g., Petrilli et al., Curr Pharm Biotechnol. 15:847-55, 2014; and U.S. Patent Nos. 6,217,912; 6,881,421; 7,402,573; 7,404,969; 7,550,441; 7,727,969; 8,003,621; 8,691,750; 8871509; 9017726; 9173853; 9220779; 9227917 and 9278130, each of which is incorporated herein by reference in its entirety. In one embodiment, a self-replicating or self-amplifying RNA molecule encoding one or more immunogenic HBV polypeptides described herein is formulated or condensed into polyethyleneimine (PEI) polyplex carriers, such as described in Demoulins, et al., Nanomedicine. (2016) Apr; 12(3):711-722 and Demoulins, et al., J Control Release. (2017) Nov 28; 266:256-271, which can be nanoparticles.

In embodiments in which the immunogenic HBV polypeptides are expressed from a viral expression vector, the viral expression vector can be formulated to require

- 58 047988 any route of administration, such as in the form of an isotopically pharmaceutically acceptable aqueous solution or suspension suitable for intravenous, intramuscular, subcutaneous, or intradermal administration. In some embodiments, the viral expression vector may be formulated for mucosal, e.g., buccal, intranasal, intravaginal, or intrarectal administration. Exemplary formulations for viral expression vectors that may be used in the pharmaceutical compositions and methods described herein are described, for example, in Manfredsson and Benskey, editors, Viral Vectors for Gene Therapy: Methods and Protocols (Methods in Molecular Biology), 2019, Book 1937 in Methods in Molecular Biology Series, Humana Press; WO 2017/013169 (formulation of adenovirus vectors in an aqueous mixture or freeze-dried composition in the presence of amorphous sugar and a low salt concentration); and Kumru, et al., J Pharm Sci. (2018) Nov; 107(11):2764-2774 (aqueous formulations buffered in trometamol and containing proline, lactose and mannitol as stabilizing additives). Formulation of arenavirus vectors is described, for example, in WO 2009/083210; WO 2016075250; and WO 2017/198726. In certain embodiments, viral expression vectors are delivered via microneedle-mediated delivery, for example, as described in Zaric, et al., Expert Opin Drug Deliv. (2017) Oct; 14(10): 1177-1187.

In some embodiments, each carrier, diluent, or excipient is acceptable in the sense that it is compatible with the other ingredients of the pharmaceutical composition and is not deleterious to the subject. Often, a pharmaceutically acceptable carrier is an aqueous pH buffer solution. Some examples of materials that can serve as pharmaceutically acceptable carriers, diluents, or excipients include the following: water; buffers, such as a buffer having a pKa in the range of from about 6.0 to about 8.0, such as a physiologically acceptable buffer, such as selected from phosphate, carbonate, bicarbonate, citrate, maleate, glycineglycine, HEPES, HEPpSo, HEPPS, imidazole, BICINE, TRICINE, trometamol, and BIS-trometamol; sugars such as lactose, trehalose, glucose, and sucrose; starches such as corn starch and potato starch; Cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Hank's solution, Ringer's solution; ethyl alcohol; phosphate buffer solutions; amino acids (e.g., charged amino acids, including, but not limited to, aspartate, asparagine, glutamate, glutamine, histidine, arginine, lysine); and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, anti-adhesive agents, coatings, sweeteners, flavors, and fragrances, preservatives, and antioxidants may also be present in the compositions.

In one particular formulation, an arenavirus vector (e.g., an LCMV or Pichinde mammaryenavirus (PICV) vector) described herein is formulated in an isotonic aqueous solution containing a biocompatible buffer having a pKa in the range of about 6.0 to about 8.0 (e.g., HEPES and NaCl) at neutral or near neutral pH and a nonionic surfactant (e.g., PLURONIC® F68 (also referred to as poloxamer 188)). In one particular formulation, an arenavirus vector (e.g., an LCMV or Pichinde mammaryenavirus vector) described herein is contained in an isotonic aqueous solution containing HEPES buffer at pH 7.4, NaCl, and PLURONIC® F68 (also referred to as poloxamer 188). Schleiss, et al. (Clin Vaccine Immunol. 2017 Jan 5;24(1):e00300-16) describes LCMV formulated from LCMV vectors in a diluent of 25 mM HEPES, 150 mM NaCl, 0.01% PLURONIC® F68; pH 7.4) that can be used to formulate the arenavirus vectors described herein. A final concentration of 10% sorbitol was added before freezing below -60 °C.

The composition and methods of delivery of pharmaceutical compositions will generally be tailored to the site and disorder to be treated. Examples of compositions include, but are not limited to, those suitable for parenteral administration, such as intravenous, intraarterial, intramuscular, or subcutaneous administration, including compositions encapsulated in micelles, liposomes, or capsules with controlled drug release (active agents included in a biocompatible coating designed to provide slow release); compositions for oral administration; compositions for topical use, such as creams, ointments, and gels; and other compositions such as inhalants, aerosols, and sprays. In some embodiments, the pharmaceutical compositions are formulated for parenteral, such as intravenous, subcutaneous, or oral administration. In some embodiments, the pharmaceutical compositions are formulated for mucosal administration, such as buccal, intranasal, intrarectal, and/or intravaginal administration.

- 59 047988

In certain embodiments, the pharmaceutical compositions are sterile. In some embodiments, the pharmaceutical composition has a pH in the range of 4.5 to 8.5, 4.5 to 6.5, 6.5 to 8.5, 6.0 to 8.0, 6.5 to 8.5, or a pH of about 5.0, about 5.5, about 6.0, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.5, about 8.0, or about 8.5. In one embodiment, the pharmaceutical composition has an osmolarity in the range of 240-260 or 250-330 mOsmol/L. In certain embodiments, the pharmaceutical composition is isotonic or nearly isotonic.

In some embodiments, the pharmaceutical compositions are liquids or solids. In some embodiments, the pharmaceutical composition comprises an aqueous solution or suspension. In some embodiments, the pharmaceutical composition is lyophilized or is a frozen liquid.

In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, such as a second therapeutic agent or a second and third therapeutic agents, for use in combination therapies as described herein.

In certain embodiments, the pharmaceutical composition further comprises an adjuvant. Exemplary adjuvants that can be co-formulated or co-administered with the immunogenic HBV polypeptides, polynucleotides encoding such immunogenic HBV polypeptides, and vectors expressing such immunogenic HBV polypeptides described herein include, but are not limited to, cytokines, chemokines, immune costimulatory molecules, Toll-like receptor agonists, or inhibitors of immune suppressive pathways, as described herein and in Li, et al., Curr Issues Mol Biol. (2017) 22:17-40. Other adjuvants that can be co-formulated or co-administered with the immunogenic HBV polypeptides, polynucleotides encoding such immunogenic HBV polypeptides, and vectors expressing such immunogenic HBV polypeptides described herein include, but are not limited to, mineral salts (e.g., aluminum salts (e.g., aluminum alum), calcium phosphate, incomplete Freund's adjuvant), lipid particles (e.g., MF59, cochleates, virus-like particles), microparticles (e.g., virosomes, polylactic acid (PLA), poly[lactide-coglycolide] (PLG)), immunostimulants (e.g., dsRNA:Poly(I:C), Poly-IC:LC, monophosphoryl lipid A (MPL), LPS, flagellin, imidazoquinolines: imiquimod (R837), resiquimod (848), CpG oligodeoxynucleotides (ODN), muramyl dipeptide (MDP), saponins (QS-21), and mucosal adjuvants (e.g., cholera toxin (CT), heat-labile enterotoxin (LTK3 and LTR72), chitosan). Adjuvants that can be co-formulated or co-administered with the immunogenic HBV polypeptides described herein, polynucleotides encoding such immunogenic HBV polypeptides, and vectors expressing such immunogenic HBV polypeptides are summarized in Apostolico, et al., J Immunol Res. (2016) 2016:1459394.

In some embodiments, the pharmaceutical compositions or immunogenic compositions comprise mixtures of two or more immunogenic HBV polypeptides, two or more polynucleotides encoding such immunogenic HBV polypeptides, or two or more vectors expressing such immunogenic HBV polypeptides. In some embodiments, the pharmaceutical composition comprises two or more immunogenic HBV polypeptides, two or more polynucleotides encoding such immunogenic HBV polypeptides, or two or more vectors expressing such immunogenic HBV polypeptides.

In various embodiments, the immunogenic composition comprises one or more polynucleotides encoding, or one or more vectors capable of expressing, two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-14; and (b) an HBV capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41.

In various embodiments, the immunogenic composition comprises one or more polynucleotides encoding, or one or more vectors capable of expressing, two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 13-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-14; and (b) an HBV capsid-sAg fusion protein comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any

- 60 047988 fight from SEQ ID NO: 38-41.

In various embodiments, the immunogenic composition comprises one or more polynucleotides encoding, or one or more vectors capable of expressing, two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; and (b) a HBV capsid-sAg fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41.

With respect to the capsid-sAg fusion polypeptide in the immunogenic composition, in some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO:65 or SEQ ID NO:66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at an amino acid position corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at an amino acid position corresponding to position 127, a phenylalanine (F) residue at an amino acid position corresponding to position 161, a tyrosine (Y) residue at an amino acid position corresponding to position 200, a serine (S) residue at an amino acid position corresponding to position 210, and a a leucine (L) at an amino acid position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the capsid-sAg fusion polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 12, an asparagine (N) residue at an amino acid position corresponding to position 67, a valine (V) residue at an amino acid position corresponding to position 74, a phenylalanine (F) residue at an amino acid position corresponding to position 97, a threonine (T) residue at an amino acid position corresponding to position 249, a threonine (T) residue at an amino acid position corresponding to position 250, a serine (S) residue at an amino acid position corresponding to position 317, a serine (S) residue at an amino acid position, corresponding to position 318, an arginine residue (R) at the amino acid position corresponding to position 326, a tyrosine residue (Y) at the amino acid position corresponding to position 338, a glycine residue (G) at the amino acid position corresponding to position 363, and an alanine residue (A) at the amino acid position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO: 41.

In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90, and 92 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90 and 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 33-37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 33-37.

In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 37.

In some embodiments, the immunogenic composition comprises a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second arenavirus expression vector

- 61 047988 sa LCMV comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 37.

According to some embodiments, the immunogenic composition comprises a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 37.

If necessary or desired, the HBV polymerase polypeptide and the HBV capsid-sAg fusion protein can be provided in the immunogenic composition in a ratio in the range of 1:10 to 10:1, such as in the range of 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1. In various embodiments, the ratios can be measured in plaque forming units (PFU), focus forming units (FFU), infectious units (IU), or viral particles (VP).

In various embodiments, one or more polynucleotides are DNA, cDNA, mRNA, or self-replicating RNA.

In some embodiments, the immunogenic composition comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated HBV polymerase polypeptide or a deletion mutant HBV polymerase polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein as described herein. If necessary or desired, the first viral expression vector and the second viral expression vector can be provided in a ratio in the range of 1:10 to 10:1, such as in the range of 1:9 to 9:1, 1:8 to 8:1, 1:7 to 7:1, 1:6 to 6:1, 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.

In some embodiments, the immunogenic composition comprises in the range of about 10 ³ to about 10 ¹² viral focus-forming units (FFU), or plaque-forming units (PFU), or infectious units (IU), or viral particles (VP), such as about 10 ⁴ to about 10 ⁷ viral FFU or IU, such as about 10 ³ to about 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 9, 10 ¹⁰ , 10 11 or 10 ¹² viral FFU or PFU or IU or IU per milliliter, of each of the first viral expression vector and the second viral expression vector.

In various embodiments, the first viral expression vector and the second viral expression vector of the immunogenic composition are independently derived from a taxonomic family selected from adenoviruses, arenaviruses, herpes viruses (e.g., cytomegalovirus), poxviruses (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA), flaviviruses (e.g., yellow fever virus), rhabdoviruses (e.g., vesiculovirus, e.g., Maraba vesiculovirus), togaviruses (e.g., alphavirus). In various embodiments, the first viral expression vector and the second viral expression vector can be derived from the same taxonomic family or different taxonomic families. For example, in some embodiments, both the first viral expression vector and the second viral expression vector in the immunogenic composition are from the adenovirus, arenavirus, or poxvirus family (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA) smallpox vaccine Ankara (MVA)).

In some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are derived from arenaviruses. In some embodiments, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments, the first viral expression vector and the second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Kali mammarenavirus (LCMV) (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)).

In various embodiments, the first viral expression vector and the second viral expression vector of the immunogenic composition are replication defective or rep

- 62 047988 replication-deficient. In some embodiments, the first viral expression vector and the second viral expression vector in the immunogenic composition are replication-attenuated.

6. Treatment methods

Additionally, methods for eliciting an immune response to human hepatitis B virus (HBV) in a subject in need thereof are provided. Methods for treating or preventing human hepatitis B virus (HBV) in a subject in need thereof are also provided. Methods for inhibiting HBV replication in an infected individual are also provided.

Additionally provided are methods for reducing the viral load associated with HBV infection. In various embodiments, the methods comprise administering to a subject an effective amount of an immunogenic composition as described herein. In various embodiments, the subject or individual is a human, a marmot, a Pekin duck, a mouse, or a non-human primate (e.g., a chimpanzee).

As used herein, the term "treating" or "treating" refers to an approach to obtain beneficial or desired results. For purposes of the present disclosure, beneficial or desired results include, but are not limited to, alleviating a symptom and/or reducing the severity of a symptom, delaying the progression of, and/or preventing the worsening of a symptom associated with a disease or condition. The term "treating" may include one or more of the following: a) inhibiting a disease or condition (e.g., alleviating one or more symptoms resulting from a disease or condition and/or reducing the severity of a disease or condition); b) slowing or stopping the progression of one or more symptoms associated with a disease or condition (e.g., stabilizing a disease or condition, delaying the worsening or progression of a disease or condition); and c) alleviating a disease or condition, such as causing regression of clinical symptoms, alleviating a painful condition, delaying the progression of a disease, improving quality of life, and/or prolonging survival.

As used herein, the term "delay" refers to the development of a disease or condition, meaning delaying, slowing, inhibiting, holding back, stabilizing and/or postponing the development of a disease or condition. This delay may be of varying length, depending on the history of the disease and/or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay may, in fact, include prevention, since the individual does not develop the disease or condition.

As used herein, the terms "prevent" or "prevention" refer to a regimen that prevents the manifestation of a disease or disorder such that clinical symptoms of the disease do not develop. Thus, prophylaxis refers to the administration of a therapy (e.g., administration of a therapeutic substance) to a subject before the subject exhibits signs of a disease (e.g., administration of a therapeutic substance to a subject in the absence of a detectable infectious agent (e.g., a virus) in the subject). The subject may be an individual at risk of developing the disease or disorder, such as an individual who has one or more risk factors that are known to be associated with the development or manifestation of the disease or disorder. Thus, in some embodiments, the term "prevention of HBV infection" refers to the administration of an HBV therapeutic substance to a subject who does not have a detectable HBV infection. It is understood that the subject of the HBV prophylactic therapy may be an individual at risk of infection with the HBV virus. It is also understood that prophylaxis does not require a 100% success rate. In some cases, prevention may involve reducing the risk of infection, but not completely eliminating the occurrence of infection.

The term "therapeutically effective amount" or "effective amount" as used herein refers to an amount that is effective to elicit a desired biological or medical response, including an amount of an immunogenic composition that, when administered to a subject for treating a disease, is sufficient to effect such treatment of the disease. An effective amount will vary depending on the immunogenic composition, the disease and its severity, and the age, weight, etc. of the subject being treated. An effective amount may include a range of amounts. An effective amount may be in one or more doses, i.e., a single dose or multiple doses may be required to achieve the desired treatment endpoint. An effective amount may be considered in the context of administering one or more therapeutic agents, and it may be considered that one agent may be considered to be given in an effective amount if, in combination with one or more other agents, a desired or beneficial result can be achieved or is achieved. Suitable doses of any co-administered compounds may optionally be reduced due to the combined effects (e.g., additive or synergistic effects) of the compounds.

In various embodiments, the administered immunogenic composition comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a truncated

- 63 047988 an HBV polymerase polypeptide as described herein or a deletion mutant HBV polymerase polypeptide as described herein; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein as described herein.

In various embodiments, the immunogenic composition to be administered comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 5-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 5-14; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 38-41. Such an immunogenic composition can be introduced into a priming composition and/or a stimulatory composition.

In various embodiments, the immunogenic composition to be administered comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 13-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 13-14; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of the amino acid sequence of any of SEQ ID NO: 3841 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of SEQ ID NO: 3841. Such an immunogenic composition can be administered in a priming composition and/or a boosting composition.

With respect to the capsid-sAg fusion polypeptide in the administered immunogenic composition, in some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO:65 or SEQ ID NO:66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at an amino acid position corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position corresponding to position 125, a proline (P) residue at the amino acid position corresponding to position 127, a phenylalanine (F) residue at the amino acid position corresponding to position 161, a tyrosine (Y) residue at the amino acid position corresponding to position 200, a serine (S) residue at the amino acid position corresponding to position 210, and a leucine (L) residue at the amino acid position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the capsid-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) residue at the amino acid position corresponding to position a position corresponding to position 97, a threonine (T) residue at an amino acid position corresponding to position 249, a threonine (T) residue at an amino acid position corresponding to position 250, a serine (S) residue at an amino acid position corresponding to position 317, a serine (S) residue at an amino acid position corresponding to position 318, an arginine (R) residue at an amino acid position corresponding to position 326, a tyrosine (Y) residue at an amino acid position corresponding to position 338, a glycine (G) residue at an amino acid position corresponding to position 363, and an alanine (A) residue at an amino acid position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO:41.

In various embodiments, the administered immunogenic composition comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; and (b) the second viral expression vector comprises a polynucleotide encoding a capsid fusion protein

- 64 047988 sAg comprising or consisting of the amino acid sequence of SEQ ID NO: 41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41. Such an immunogenic composition can be introduced into a priming composition and/or a stimulating composition.

In various embodiments, the immunogenic composition to be administered comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90, and 92, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90 and 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 33-37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 33-37. Such an immunogenic composition can be administered in a priming composition and/or a boosting composition.

In various embodiments, the administered immunogenic composition comprises a mixture comprising a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29, 89, 90, or 92 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37. Such an immunogenic composition can be administered in a priming composition and/or a boosting composition.

In various embodiments, the first viral expression vector and the second viral expression vector of the administered immunogenic composition are independently derived from a taxonomic family selected from adenoviruses, arenaviruses, herpes viruses (e.g., cytomegalovirus), poxviruses (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA), flaviviruses (e.g., yellow fever virus), rhabdoviruses (e.g., vesiculovirus, e.g., Maraba vesiculovirus), togaviruses (e.g., alphavirus), as described above and herein. In various embodiments, the first viral expression vector and the second viral expression vector can be from the same taxonomic family or different taxonomic families. For example, in some embodiments, both the first viral expression vector and the second viral expression vector of the administered immunogenic composition are from an adenovirus, arenavirus, or poxvirus family (e.g., virus vaccinia, such as modified vaccinia virus Ankara (MVA).

In some embodiments, the first viral expression vector and the second viral expression vector are derived from the arenavirus family. In some embodiments, the first viral expression vector and the second viral expression vector of the administered immunogenic composition are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Cali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV), and Whitewater Arroyo virus (WWAV). In some embodiments, the first viral expression vector and the second viral expression vector of the administered immunogenic composition are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Kali mammarenavirus (LCMV) (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)).

In various embodiments, the first viral expression vector and the second viral expression vector of the administered immunogenic composition are replication-defective or replication-deficient. In some embodiments, the first viral expression vector and the second viral expression vector of the administered immunogenic composition are replication-attenuated.

In various embodiments, the administered immunogenic composition comprises a mixture comprising a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second ...

- 65 047988 leic acid of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37. Such an immunogenic composition can be administered in a priming composition and/or in a boosting composition.

In various embodiments, the administered immunogenic composition comprises a mixture comprising a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37. Such an immunogenic composition can be administered in a priming composition and/or a boosting composition.

In various embodiments, the subject is infected with HBV, suspected of being infected with HBV, or at risk of being infected with HBV. As used herein, the term "at-risk individual" refers to an individual who is at risk of developing the pathological condition being treated. An at-risk individual may or may not have a detectable disease or condition, and may or may not have exhibited a detectable disease prior to the use of the treatment methods described herein. At-risk means that the individual has one or more so-called risk factors, which are measurable parameters that correlate with the development of the disease or pathological condition and are known in the art. An individual who has one or more of these risk factors has a higher probability of developing the disease or condition than an individual without the risk factor(s). In various embodiments, the subject is chronically infected with HBV, such as infected with HBV for more than 6 months. Typically, the subject suffers from chronic hepatitis B infection, although it is within the scope of the present invention to treat people who have acute HBV infection. Accordingly, in some embodiments, the subject has acute HBV infection. In some embodiments, the subject is co-infected with hepatitis D virus (HDV).

In various embodiments, the subject may be asymptomatic. In some embodiments, the subject experiences or exhibits symptoms associated with HBV infection. Symptoms of HBV may include, for example, jaundice, visible networks of distended blood vessels, dark-colored (e.g., orange or brown) urine, discolored stool, fever, persistent fatigue, discomfort, abdominal pain, abdominal fluid, loss of appetite, nausea, and vomiting. Chronic HBV infection may result in one or more symptoms, including, for example, liver failure, liver cancer, liver fibrosis, and liver cirrhosis. One or more administrations of immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein can prevent, delay, alleviate, reduce, suppress, reverse, or eliminate one or more symptoms associated with or caused by HBV infection.

In some embodiments, the immunogenic composition is administered by a route selected from intravenous, intramuscular, intradermal, subcutaneous, and mucosal (e.g., buccal, intranasal, intrarectal, intravaginal).

In some embodiments, the administered dose of the immunogenic composition comprises in the range of about 10 ³ to about 10 ¹² viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (VP), such as about 10 4 to about 10 ⁷ viral FFU or IU, such as about 10 ³ to about 10 4, 10 5, 10 ⁶ , 10 7, 10 ⁸ , 10 9, 10 ¹⁰ , 10 ¹¹ or 10 ¹² viral FFU or PFU or IU or IU per milliliter, of each of the first viral expression vector and the second viral expression vector. In some embodiments, the methods comprise administering intravenously or intramuscularly from about 106 to about ¹⁰⁸ viral FFU or PFU or IE or VP per administration every two weeks (Q2W) or monthly (Q4W).

In various embodiments, the methods comprise a prime/boost cycle. In some embodiments, the prime/boost cycle comprises administering a priming composition at a first time point and administering one or more boosting compositions at one or more subsequent time points. If desired, the methods may comprise repeating the priming/boost cycle for one or more cycles. In various embodiments, the administration of the priming composition and the one or more boosting compositions are spaced apart by at least 1 week and up to at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. Where appropriate, the dosage or dosing frequency of the immunogenic composition

- 66 047988 may be adjusted during the course of treatment based on the judgment of the prescribing physician. In appropriate cases, the subject may be treated with multiple administrations over a period of at least about 2 weeks to 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or longer, or until sAg is no longer detectable in the subject's serum or plasma.

In some embodiments, following one or more administrations of one or more immunogenic polypeptides as described herein, or one or more polynucleotides encoding one or more immunogenic polypeptides as described herein, or one or more vectors expressing one or more immunogenic polypeptides as described herein, optionally with one or more additional therapeutic agents as described herein, the subject is free of HBV symptoms in the absence of antiviral therapy for at least 6 months, at least 1 year, at least 2 years, at least 3 years, or more. In some embodiments, following one or more administrations of one or more immunogenic polypeptides as described herein, or one or more polynucleotides encoding one or more immunogenic polypeptides as described herein, or one or more vectors expressing one or more immunogenic polypeptides as described herein, optionally with one or more additional therapeutic agents described herein, sAg is no longer detectable in the serum or plasma of the subject, in the absence of antiviral therapy for at least 6 months, such as at least 1 year, at least 2 years, at least 3 years, or more.

If necessary or desired, the priming composition and the boosting composition may comprise the same immunogenic composition or different immunogenic compositions. In various embodiments, the priming composition and the boosting composition comprise one or more polypeptides and the same expression vector (e.g., a viral expression vector). In some embodiments, the priming composition and the boosting composition comprise different polypeptides and/or different viral expression vectors (e.g., viral expression vectors). For example, in some embodiments, the priming composition and the boosting composition comprise one or more polypeptides and different expression vectors (e.g., viral vectors from different viral species in a taxonomic family, viral vectors from different taxonomic families, viral vectors with different replication competencies). In some embodiments, the priming composition and the boosting composition comprise different immunogenic polypeptides and the same expression vector (e.g., a viral expression vector).

In some embodiments, the methods comprise priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more viral expression vectors. In some embodiments, the priming/boosting cycle comprises:

a) priming with a priming composition comprising one or more viral expression vectors and amplifying with an amplifying composition comprising one or more polynucleotides, wherein the one or more polynucleotides comprise DNA, cDNA, mRNA or self-replicating RNA;

b) priming with a priming composition comprising one or more polynucleotides, wherein the one or more polynucleotides comprise DNA, cDNA, mRNA or self-replicating RNA, and amplification with an enhancing composition comprising one or more viral expression vectors,

c) priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more viral expression vectors, wherein the one or more viral expression vectors in the priming composition and the one or more viral expression vectors in the boosting composition are from identical, related or unrelated taxonomic families;

d) priming with a priming composition comprising one or more replication-deficient viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors, wherein the one or more replication-deficient viral expression vectors in the priming composition and the one or more replication-deficient viral expression vectors in the boosting composition are from identical, related, or unrelated taxonomic families;

e) priming with a priming composition comprising one or more replication-attenuated viral expression vectors and amplification with a boosting composition comprising one or more replication-attenuated viral expression vectors

- 67 047988 tori, wherein one or more replication-attenuated viral expression vectors in the priming composition and one or more replication-attenuated viral expression vectors in the booster composition are from identical, related or unrelated taxonomic families;

f) priming with a priming composition comprising one or more replication-deficient viral expression vectors and amplification with a boosting composition comprising one or more replication-attenuated viral expression vectors;

g) priming with a priming composition comprising one or more replication-attenuated viral expression vectors and enhancing with a boosting composition comprising one or more replication-deficient viral expression vectors;

h) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more Pichinde mammarenavirus (PICV) viral expression vectors;

i) priming with a priming composition comprising one or more Pichinde mammaryenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammaryenavirus (LCMV) viral expression vectors;

j) priming with a priming composition comprising one or more replication-deficient viral expression vectors of Pichinde mammaryenavirus (PICV) and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors of lymphocytic choriomeningitis mammaryenavirus (LCMV);

k) priming with a priming composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient Pichinde mammarenavirus (PICV) viral expression vectors;

l) priming with a priming composition comprising one or more arenavirus viral expression vectors and enhancing with a boosting composition comprising one or more adenovirus viral expression vectors;

m) priming with a priming composition comprising one or more adenovirus viral expression vectors and enhancing with a boosting composition comprising one or more arenavirus viral expression vectors;

n) priming with a priming composition comprising one or more poxvirus viral expression vectors and enhancing with a boosting composition comprising one or more arenavirus viral expression vectors;

o) priming with a priming composition comprising one or more arenavirus viral expression vectors and enhancing with a boosting composition comprising one or more poxvirus viral expression vectors;

p) priming with a priming composition comprising one or more poxvirus viral expression vectors and enhancing with a boosting composition comprising one or more adenovirus viral expression vectors; or

q) priming with a priming composition comprising one or more adenovirus viral expression vectors and enhancing with a boosting composition comprising one or more poxvirus viral expression vectors.

In some embodiments, the methods comprise priming with a priming composition comprising one or more viral expression vectors and boosting with a boosting composition comprising one or more viral expression vectors. In some embodiments, the priming/boosting cycle comprises:

a) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more Pichinde mammarenavirus (PICV) viral expression vectors;

b) priming with a priming composition comprising one or more Pichinde mammaryenavirus (PICV) viral expression vectors and boosting with a boosting composition comprising one or more lymphocytic choriomeningitis mammaryenavirus (LCMV) viral expression vectors;

c) priming with a priming composition comprising one or more replication-deficient viral expression vectors of Pichinde mammaryenavirus (PICV) and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors of lymphocytic choriomeningitis mammaryenavirus (LCMV); or

d) priming with a priming composition comprising one or more replication-deficient viral expression vectors of lymphocytic choriomeningitis mammaryenavirus

- 68 047988 gita (LCMV) and amplification with an enhancing composition comprising one or more replication-deficient viral expression vectors of Pichinde mammarenavirus (PICV).

In various embodiments, the priming composition and the boosting composition comprise an immunogenic composition as described herein.

In some embodiments, the subject does not receive antiviral therapy or the antiviral therapy is discontinued prior to administration of one or more immunogenic compositions. In some embodiments, the antiviral therapy is discontinued after one or more administrations of the compositions.

In some embodiments, the treatment methods are activated in CD8+ T cells of the subject targeting one or more HBV polypeptide epitopes. In some embodiments, the treatment methods cause the subject to produce antibodies that bind one or more HBV polypeptides.

7. Combination therapies

In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one, two, three, or four additional therapeutic agents. In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with two additional therapeutic agents. In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with three additional therapeutic agents. In certain embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with four additional therapeutic agents. The one, two, three, four, or more additional therapeutic agents may be different therapeutic agents selected from the same class of therapeutic agents, and/or they may be selected from different classes of therapeutic agents.

As used herein, the term "co-administration" refers to the administration of single doses of immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein, prior to or subsequent to the administration of single doses of one or more additional therapeutic agents. For example, administering an immunogenic composition described herein within seconds, minutes, or hours of the administration of one or more additional therapeutic agents. For example, in some embodiments, a single dose of an immunogenic composition of the present invention is administered first, followed within seconds or minutes by the administration of a single dose of one or more additional therapeutic agents. Alternatively, in other embodiments, a single dose of one or more additional therapeutic agents is administered, followed by the administration of a single dose of an immunogenic composition of the present invention within seconds or minutes. In some embodiments, a single dose of immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein is administered first, then after a period of several hours (e.g., 1-12 hours), by administering a single dose of one or more additional therapeutic agents. In other embodiments, a single dose of one or more additional therapeutic agents is administered first, then after a period of several hours (e.g., 1-12 hours), by administering a unit dose of immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein.

Co-administration of immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein with one or more additional therapeutic agents generally refers to the simultaneous or sequential administration of an immunogenic composition described herein and one or more additional therapeutic agents such that therapeutically effective amounts of each agent are present in the patient's body.

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more additional therapeutic agents as described herein, the components of the composition being administered in a simultaneous or sequential cycle. When administered sequentially

- 69 047988 the combination can be administered in two or more doses.

In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one, two, three, four or more additional therapeutic agents selected from HBV combination therapies, HBV vaccines, HBV DNA polymerase inhibitors, immunomodulators, Toll-like receptor (TLR) modulators, interferon alpha receptor ligands, hyaluronidase inhibitors, HBV antigen inhibitors (e.g., HBV capsid antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), anti-HBV antibodies that inhibit nucleic acids targeting HBV (e.g., an antisense oligonucleotide, short interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi), inhibitors of HBsAg secretion or assembly, inhibitors of HBV entry, immune checkpoint inhibitor, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor, cyclophilin inhibitor, endonuclease modulators, ribonucleotide reductase inhibitor, covalently closed circular DNA (cccDNA) inhibitor, farnesoid X receptor (FXR) agonist, STING agonist, anti-HBV antibodies, CCR2 chemokine antagonists, thymosin agonists, cytokines, nucleoprotein modulators, retinoic acid-inducible gene 1 stimulators, NOD2 stimulators, phosphatidylinositol 3-kinase (PI3K) inhibitors, pathway inhibitors indoleamine 2,3-dioxygenase (IDO) inhibitors, ZCCHC14 inhibitors, tertiary lymphoid aggregate inhibitors, nucleic acid polymers (eg, NAP and STOPS), PD-1 inhibitors, PD-L1 inhibitors, recombinant Bruton tyrosine kinase (BTK) inhibitors, lysine demethylase inhibitors (KDM), HBV replication inhibitors, arginase inhibitors, gene therapy and cell therapy, gene editors, cell therapy, TCR-T cell therapy, and other anti-HBV drugs.

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein can be combined or co-administered with one or more of a chemotherapeutic agent, an immunomodulator, an immunotherapeutic agent, a therapeutic antibody, a therapeutic vaccine, a bispecific antibody, and an antibody-like therapeutic protein (e.g., DARPins®, TCR-like antibodies to pMHC, DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs®, Fab derivatives), an antibody drug conjugate (ADC), gene modifiers or gene editors targeting HBV (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Casl3), zinc finger nucleases, homing endonucleases, homing meganucleases (e.g. ARCUS), synthetic nucleases, TALEN), cell therapy (e.g. T cells, NK cells, chimeric antigen receptor (CAR) macrophages), TCR-T (engineered T cell receptor), or any combination thereof.

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one, two, three, four or more additional therapeutic agents, such as 3-dioxygenase inhibitors (IDO), apolipoprotein A1 modulator, arginase inhibitors, B and T lymphocyte attenuator inhibitors, Bruton tyrosine kinase (BTK) inhibitors, a CCR2 chemokine antagonist, CD137 inhibitors, CD160 inhibitors, CD305 inhibitors, a CD4 agonist and modulator, hepatitis B capsid antigen (HBcAg) targeting compounds, capsid protein allosteric modulators, covalently closed circular DNA inhibitors (cccDNA), cyclophilin inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors, DNA polymerase inhibitors, endonuclease modulators, epigenetic modifiers, farnesoid X receptor (FXR) agonists, HBV DNA polymerase inhibitors, HBV replication inhibitors, HBV RNase inhibitors, HBV viral entry inhibitors, HBx inhibitors, hepatitis B major envelope protein modulator, hepatitis B major envelope protein stimulator, hepatitis B structural protein modulator, hepatitis B surface antigen (HBsAg) inhibitors, hepatitis B surface antigen (HBsAg) secretion or assembly inhibitors, hepatitis B antigen E inhibitors, hepatitis B virus replication inhibitors, hepatitis virus structural protein inhibitor, HIV-1 reverse transcriptase inhibitor, hyaluronidase inhibitor, protein inhibitor inhibitors of apoptosis proteins (IAP) family, IL-2 agonist, IL-7 agonist, immunomodulators, indoleamine-2 inhibitors, ribonucleotide reductase inhibitors, interleukin-2 ligand, ipi4 inhibitors, lysine demethylase inhibitors, histone demethylase inhibitors, KDM1 inhibitors, KDM5 inhibitors, lectin-like killer cell receptor subfamily G member 1 inhibitors, lymphocyte activating gene 3 inhibitors, lymphotoxin beta receptor activators, B7-H3 modulators, B7-H4 modulators, CD160 modulators, CD161 modulators, CD27 modulators, CD47 modulators, CD70 modulators, GITR modulators, HEVEM modulators, ICOS modulators, Mer modulators, NKG2A modulators, modulators

- 70 047988

NKG2D, OX40 modulators, SIRPalpha modulators, TIGIT modulators, Tim-4 modulators, Tyro modulators, Na+-taurocholate cotransporting polypeptide (NTCP) inhibitors, natural killer cell receptor 2B4 inhibitors, NOD2 gene stimulator, nucleoprotein inhibitor, nucleoprotein modulators, OX-40 receptor agonist, PD-1 inhibitors, PD-L1 inhibitors, peptidyl prolyl isomerase inhibitors, phosphatidylinositol-3 kinase (PI3K) inhibitors, retinoic acid-inducible gene 1 stimulator, reverse transcriptase inhibitor, ribonuclease inhibitor, RNA-DNA polymerase inhibitor, SLC10A1 gene inhibitor, SMAC mimetics, Src tyrosine kinase, interferon stimulating gene agonists (STING), NOD1 stimulators, CD28 T-cell surface glycoprotein inhibitor, CD8 T-cell surface glycoprotein modulator, thymosin agonist, thymosin alpha-1 ligand, Tim-3 inhibitors, TLR-3 agonists, TLR-7 agonists, TLR-9 agonists, TLR9 gene agonists or stimulator, Toll-like receptor (TLR) modulators, viral ribonucleotide reductase inhibitors, and combinations thereof.

Antiviral drugs that inhibit HBV

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more antiviral agents. In some embodiments, the one or more antiviral agents are selected from the group consisting of lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir disoproxil fumarate and emtricitabine (TRUVADA®), tenofovir alafenamide (TAF or VEMLIDY®), and ledipasvir and sofosbuvir (HARVONI®).

Other anti-HBV drugs

Examples of other drugs used to treat HBV that can be combined or co-administered include alpha-hydroxytropolone, amdoxovir, anthroquinolone, beta-hydroxycytosine nucleosides, ARB-199, CCC-0975, ccc-R08, elvucitabine, ezetimibe, cyclosporine A, gentiopicrin (gentiopicroside), HH-003, hepalatide, JNJ-56136379, nitazoxanide, birinapant, NJK14047, NOV-205 (molixan, BAM-205), oligotide, mivotilate, feron, GST-HG-131, levamisole, Ka Shu Ning, alloferon, WS-007, Y-101 (Ti Fen Tai), rSIFN-co, PEG-IIFNm, KW-3, BP-Inter-014, oleanolic acid, HepB-nRNA, cTP-5 (rTP-5), HSK-II-2, HEISCO-106-1, HEISCO-106, Hepbarna, IBPB-006IA, Hepuyinfen, DasKloster 0014-01, ISA-204, Jiangantai (Ganxikang), MIV-210, OB-AI-004, PF-06, picroside, DasKloster-0039, hepulantai, IMB-2613, NCO-48 fumarate, XTYW-001, SFA-001, TCM-800B, reduced glutathione, RO-6864018, ENOB-HB-01, RG-7834, QL-007 sofosbuvir, ledipasvir, UB-551, PA-1010, HPN-BV1, STSG-0002 and ZH-2N, and compounds described in US 20150210682, (Roche), US 2016/0122344 (Roche), WO 2015173164, WO 2016023877, US 2015252057A (Roche), WO 16128335A1 (Roche), WO 16120186A1 (Roche), US 2016237090A (Roche), WO 16107833A1 (Roche), WO 16107832A1 (Roche), US 2016176899A (Roche), WO 16102438A1 (Roche), WO 16012470A1 (Roche), US 2016220586A (Roche) and US 2015031687A (Roche).

Examples of combination drugs for the treatment of HBV that can be combined or co-administered include tenofovir disoproxil fumarate and emtriptabin (TRUVADA®), ledipasvir and sofosbuvir (HARVONI®); ABX-203 (NASVAC), lamivudine and PEG-IFNa; adefovir and PEG-IFNa; and INO-1800 (INO-9112 and RG7944).

HBV vaccines

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more HBV vaccines. HBV vaccines that can be combined or co-administered (e.g., in a prime/boost cycle) include both prophylactic and therapeutic vaccines. Examples of prophylactic HBV vaccines include Vaxelis, Hexaxim, Heplisav, Mosquirix, DTwP-HBV vaccine, Bio-Hep-B, D/T/P/HBV/M (LBVP-0101; LBVW-0101), DTwP-HepBHib-IPV vaccine, Heberpenta L, DTwP-HepB-Hib, V-419, CVI-HBV-001, Tetrabhay, hepatitis B prophylactic vaccine (Advax Super D), Hepatrol-07, GSK-223192A, ENGERIX B®, recombinant hepatitis B vaccine (intramuscular, Kangtai Biological Products), recombinant hepatitis B vaccine (Hansenual polymorpha yeast, intramuscular, Hualan Biological Engineering), recombinant surface vaccine hepatitis B antigen, Bimmugen, CARG-101, Euforavac, Eutravac, anrix-DTaPIPV-Hep B, HBAI-20, Infanrix-DTaP-IPV-Hep B-Hib, Pentabio Vaksin DTP-HB-Hib, Comvac 4, Twinrix, Euvax-B, Tritanrix HB, Infanrix Hep B, Comvax, DTP-Hib-HBV vaccine, DTP-HBV vaccine, Yi Tai, Heberbiovac HB, Trivac HB, GerVax, DTwP-Hep B-Hib vaccine, Bilive, Hepavax-Gene, SUPERVAX, Comvac5, Shanvac-B, Hebsulin, Recombivax HB, Revac B mcf, Revac B+, Fendrix, DTwP-HepB-Hib, DNA-001, Shan5, Shan6, rhHBsAG vaccine, HBI pentavacine, LBVD, Infanrix HeXa, YS-HBV-001, IR-101H, TVAX-008 and DTaP-rHB-Hib vaccine.

Examples of HBV vaccines that can be combined or co-administered (e.g.,

- 71 047988 in the priming/boosting cycle) include the HBsAG-HBIG complex, ARB-1598, Bio-Hep-B, abi-HB (intravenous), ABX-203 (NASVAC), Tetrabhay, GX-110E, GS-4774, peptide vaccine (epsilon-PA44), Hepatrol-07, NASVAC (NASTERAP), IMP-321, BEVAC, Revac B mcf, Revac B+, MGN-1333, KW-2, CVI-HBV-002, AltraHepB, VGX-6200, FP-02, FP-02.2 (HepTcell), NU-500, HBVax, im/TriGrid/antigen vaccine, adjuvant vaccine based on MEGA-CD40L, HepB-v, RG7944 (INO-1800), therapeutic vaccine based on recombinant virus-like particle (HBV infection, VLP Biotech), AdTG17909, AdTG-17910 AdTG-18202, ChronVac-B, TG-1050, WX-001, GSK-3528869A (ChAd155-hli-HBV + MVA-HBV +Hbc-HBs/AS01B-4), VBI-2601, VTP-300 (prime ChAdOx1-SIi-HBV-CPmut-TPA-Ssh and boost MVA-SIi-HBV-CPmut-TPA-Ssh), Lm HBV and BM32 (Tulaeva, et al., EBioMedicine (2020) 102953). Vaccines against the HBV arenavirus are described, for example, in WO2017076988 and WO2017198726.

HBV DNA polymerase inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more polymerase inhibitors. Examples of HBV DNA polymerase inhibitors that may be combined or co-administered include adefovir (HEPSERA®), emtricitabine (EMTRIVA®), tenofovir disoproxil fumarate (VIREAD®), tenofovir alafenamide, tenofovir, tenofovir disoproxil, tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, tenofovir dipivoxil, tenofovir dipivoxil fumarate, tenofovir octadecyloxyethyl ether, CMX-157, tenofovir exalidex, besifovir, entecavir (BARACLUDE®), entecavir maleate, telbivudine (TYZEKA®), filocilivir, pradefovir, clevudine, ribavirin, lamivudine (EPIVIR-HBV®), phosphazide, famciclovir, fuzolin, metacavir, SNC-019754, FMCA, aGx-1009, AR-II-04-26, HIP-1302, tenofovir disoproxil aspartate, tenofovir disoproxil orotate, AiB-001, and HS-10234.

Immunomodulators

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more immunomodulators (e.g., an immune checkpoint inhibitor, a tumor necrosis factor (TNF) receptor superfamily (TNFRSF) agonist, an immunostimulant, e.g., a TLR agonist). Examples of immunomodulators that can be combined or co-administered include rintatolimod, imidol hydrochloride, ingaron, dermavir, plaquenil (hydroxychloroquine), proleukin, hydroxyurea, mycophenolate mofetil (MPA) and its ester derivative mycophenolate mofetil (MMF), JNJ-440, WF-10, AB-452, ribavirin, IL-12, INO-9112, polyethyleneimine polymer (PEI), Gepon, VGV-1, MOR-22, CRV-431, JNJ-0535, TG-1050, ABI-H2158, BMS-936559, GS-9688, RO-7011785 and the corresponding prodrug RO-702053, RG-7854, RO-6871765, AIC-649, and IR-103.

To11-podo6 receptor (TLR) agonists

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more Toll-like receptor (TLR) agonists or stimulators. In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with a TLR agonist, such as a TLR1 (NCBI Gene ID: 7096), TLR2 (NCBI Gene ID: 7097), TLR3 (NCBI Gene ID: 7098), TLR4 (NCBI Gene ID: 7099), TLR5 (NCBI Gene ID: 7100), TLR6 (NCBI Gene ID: 10333), TLR7 (NCBI Gene ID: 51284), TLR8 (NCBI gene accession number: 51311), TLR9 (NCBI gene accession number: 54106), and/or TLR10 (NCBI gene accession number: 81793), TLR11, TLR12, and TLR13.

Examples of TLR3 agonists that can be combined or co-administered include rintatolimod, poly-ICLC, RIBOXXON®, Apoxxim, RIBOXXIM®, IPH-33, MCT-465, MCT-475, and ND-1.1.

Examples of TLR4 agonists that can be combined or co-administered include G-100 and GSK-1795091.

Examples of TLR7 agonists that can be combined or co-administered include, but are not limited to, AL-034, DSP-0509, GS-9620 (vesatolimod), LHC-165, TMX-101 (imiquimod), GSK2245035, resiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, telratolimod (MEDI-9197), 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7854, RG-7795, RO-7011785 and the corresponding prodrug RO-702053, and the compounds described in US 20100143301 (Gilead Sciences) O 2014/128189 (Janssen),

- 72 047988

US 20140350031 (Janssen), WO 2014/023813 (Janssen), US 20080234251 (Array Biopharma), US 20080306050 (Array Biopharma), US 20100029585 (Ventirx Pharma), US 20110092485 (Ventirx Pharma), US 20110118235 (Ventirx Pharma), US 20120082658 (Ventirx Pharma), US 20120219615 (Ventirx Pharma), US 20140066432 (Ventirx Pharma), US 20140088085 (Ventirx Pharma), US 20140275167 (Novira Therapeutics) and US 20130251673 (Novira Therapeutics).

Examples of dual TLR7/TLR8 agonists that can be combined or co-administered include NKTR-262, telratolimod, and BDB-001.

Examples of TLR8 agonists that can be co-administered include, but are not limited to, E-6887, IMO-4200, IMO-8400, IMO-9200, MCT-465, telratolimod (MEDI-9197), motolimod, resiquimod, selgantolimod (GS-9688), HRS-9950, VTX-1463, VTX-763, 3M-051, 3M-052, SBT6050, and the compounds described in US 2016289229 (Gilead Sciences), US 20140045849 (Janssen), US 20140073642 (Janssen), WO 2014/056953 (Janssen), WO 2014/076221 (Janssen), WO 2014/128189 (Janssen), US 20140350031 (Janssen), WO 2014/023813 (Janssen), US 20080234251 (Array Biopharma), US 20080306050 (Array Biopharma), US 20100029585 (Ventirx Pharma), US 201100924 85 (Ventirx Pharma) (Novira Therapeutics) and US 20130251673 (Novira Therapeutics), US patent No. 9670205 (Gilead Sciences, Inc.), US 20160289229 (Gilead Sciences, Inc.), WO 2017/048727 (Gilead Sciences, Inc.), US 20180065938 (Gilead Sciences, Inc.) and US 20180086755 (Gilead Sciences, Inc.).

Examples of TLR9 agonists that can be combined or co-administered include, but are not limited to, AST-008, cobitolimod, CMP-001, IMO-2055, IMO-2125, S-540956, litenimob, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, agatolimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, lefitolimod (MGN-1703), CYT-003, CYT-003-QbG10, tilsotolimod, and PUL-042.

Further examples of TLR7, TLR8 and TLR9 modulators that can be combined or co-administered include the compounds described in WO2017047769 (Teika Seiyaku), WO2015014815 (Janssen), WO2018045150 (Gilead Sciences Inc), WO2018045144 (Gilead Sciences Inc), WO2015162075 (Roche), WO2017034986 (University of Kansas), WO2018095426 (Jiangsu Hengrai Medicine Co Ltd), WO2016091698 (Roche), WO2016075661 (GlaxoSmithKline Biologicals), WO2016180743 (Roche), WO 2018089695 (Dynavax Technologies), WO 2016055553 (Roche), WO 2015168279 (Novartis), WO 2016107536 (Medshine Discovery), WO 2018086593 (Livo (Shanghai) Pharmaceutical), WO 2017106607 (Merck), WO 20170 61532 (Sumitomo Dainippon Pharma), WO 2016023511 (Chia Tai Tianqing Pharmaceutical), WO 2017076346 (Chia Tai Tianqing Pharmaceutical), WO 2017046112 (Roche), WO 2018078149 (Roche), WO 2017040233 (3M Co), WO 2016141092 (Gilead Sciences), WO 2018049089 (BristolMyers Squibb), WO 2015057655 (Eisai Co Ltd), WO 2017001307 (Roche), WO 2018005586 (BristolMyers Squibb), WO 201704023 (3M Co), O 2017163264 (Council of Scientific and Industrial Research (India)), WO 2018046460 (GlaxoSmithKline Biologicals), WO 2018047081 (Novartis), WO 2016142250 (Roche), WO 2015168269 (Novartis), WO 201804163 (Roche), WO 2018038877 (3M Co), WO 2015057659 (Eisai Co Ltd), WO 2017202704 (Roche), WO 2018026620 (BristolMyers Squibb), WO 2016029077 (Janus Biotherapeutics), WO 201803143 (Merck), WO 2016 096778 (Roche), WO 2017190669 (Shanghai De Novo Pharmatech), US 09884866 (University of Minnesota), WO 2017219931 (Sichuan KelunBiotech Biopharmaceutical), WO 2018002319 (Janssen Sciences), WO 2017216054 (Roche), WO 2017202703 (Roche), WO 2017184735 (IFM Therapeutics), WO 2017184746 (IFM Therapeutics), WO 2015088045 (Takeda Pharmaceutical), WO 2017038909 (Takeda Pharmaceutical), WO 2015095780 (University of Kansas), WO 2015 023958 (University of Kansas).

In certain embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with a TLR7, TLR8, or TLR9 agonist.

Interferon-alpha receptor ligands

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more interferon-alpha receptor ligands. Examples of interferon alpha receptor ligands that can be combined or co-administered include interferon alpha-1 (INTRON A®), pegylated interferon alpha-2a (PEGASYS®), pegylated interferon alpha-3 (HAPGEN®), Veldona, Infradure, Roferon-A, YPEG-interferon alpha-2a (YPEGrhIFNalpha-2a), P-1101, Algeron, Alpharona, Ingaron (interferon gamma), rSIFN-co (recombinant super interferon), YPEG-interferon alpha-2b (YPEG-rhIFNalpha-2b), MOR-22, peginterferon alpha-2b (PEG-INTRON®), Bioferon, Novaferon, Inmutag (Inferon), MULTIFERON®, interferon alpha-n1 (HUMOFERON®), interferon beta-N (AVONEX®), schaferon, interferon alpha^ (Axxo), Alfaferon, interferon alpha^ (BioGeneric Pharma), interferon-alpha 2 (CJ), laferon, VIPEG, BLAUFERON-A, m-B, Intermax Alpha, Realdiron, Lanstion, Pegaferon, PDferon-B, interferon alpha^ (IFN,

- 73 047988

Laboratories Bioprofarma), alphainterferon-2b, Calferon, Pegnano, Feronsur, PegiHep, interferon alpha-2b (Zydus-Cadila), interferon alpha-2a, Optipeg A, Realfa 2B, Reliferon, interferon alpha-2b (Amega), interferon alpha-2b (Virchow), ropeginterferon alpha-2b, rHSA-IFN alpha-2a (fusion protein of recombinant human serum albumin and interferon alpha 2a), peginterferonalfa, rHSA-IFN alpha 2b, recombinant human interferon alpha-(1b, 2a, 2b), peginterferon alpha-2b (Amega), peginterferon alpha-2a, Reaferon-EC, Proquiferon, Uniferon, Urifron, interferon alpha-2b (Changchun Institute of Biological Products), anterferon, shanferon, lifeferon, Shang Sheng Lei Tai, INTEFEN, SINOGEN, Fukangtai, pegstat, rHSA-IFN alpha-2b, SFR-9216 and Interapo (Interapa).

Hyaluronidase inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more hyaluronidase inhibitors. Examples of hyaluronidase inhibitors that can be combined or co-administered include astodrimer.

Hepatitis B surface antigen (HBsAg) inhibitors

In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more HBsAg inhibitors. Examples of HBsAg inhibitors that can be combined or co-administered include AK-074, HBF-0259, GP-605, PBHBV-001, PBHBV-2-15, PBHBV-2-1, REP9AC, REP-9C, REP-9, REP-2139, REP-2139-Ca, REP-2055, REP-2163, REP-2165, REP-2053, REP-2031, and REP-006 and REP-9AC. Examples of HBsAg secretion inhibitors that can be combined or co-administered include BM601, GST-HG-131, AB-452, and ALG-010093.

Cyclophilin inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more cyclophilin inhibitors. Examples of cyclophilin secretion inhibitors that can be combined or co-administered include CPI-431-32, EDP-494, OCB-030, SCY-635, NVP-015, NVP-018, NVP-019, STG-175, and the compounds described in US 8,513,184 (Gilead Sciences), US 20140030221 (Gilead Sciences), US 20130344030 (Gilead Sciences), and US 20130344029 (Gilead Sciences).

HBV entry inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more HBV entry inhibitors. Examples of HBV entry inhibitors that can be combined or co-administered include bulevirtide (Hepcludex; Myrcludex B).

Inhibitory nucleic acids

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more inhibitory nucleic acids (e.g., an antisense oligonucleotide, short interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi) that specifically target an HBV polynucleotide. In some embodiments, the HBV polynucleotide encodes an HBV protein (i.e., is located in a coding region in the HBV genome).

Antisense oligonucleotide targeting viral mRNA

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more antisense oligonucleotides. Examples of an antisense oligonucleotide targeting viral mRNA that can be combined or co-administered include ISIS-HBVRx, IONIS-HBVRx, IONIS-HBV-LRx, IONIS-GSK6-LRx, GSK-3389404, BNC-1701, and RG-6004.

Short interfering RNA (siRNA)

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more siRNAs specifically targeting an HBV polynucleotide. Examples of siRNAs specifically targeting the HBV polynucleotide that can be combined or co-administered include TKM-HBV (TKM-NerB), ALN-HBV, SR-008, HepB-nRNA, ARC-520, ARC-521, ARB-1740, ARB-1467, AB-729, DCR-HBVS, RG-6084 (PD-L1), RG-6217, ALN-HBV-02, JNJ-3989 (ARO-HBV), STSG-0002, LUNAR-HBV, and DCR-HBVS (DCR-S219).

- 74 047988

DNA-directed RNA interference (ddRNAi)

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more ddRNAi specifically targeting an HBV polynucleotide. Examples of ddRNAi specifically targeting an HBV polynucleotide that can be combined or co-administered include BB-HB-331.

Endonuclease modulators

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more endonuclease modulators. Examples of endonuclease modulators that can be combined or co-administered include PGN-514.

Ribonucleotide reductase inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more ribonucleotide reductase inhibitors. Examples of ribonucleotide reductase inhibitors that can be combined or co-administered include Trimidox.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs)

In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more NNRTIs. Examples of NNRTIs that can be combined or co-administered include the compounds described in WO2018118826 (Merck), WO2018080903 (Merck), WO2018119013 (Merck), WO2017100108 (Idenix), WO2017027434 (Merck), WO2017007701 (Merck), WO2008005555 (Gilead).

Inhibitors of HBV replication

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more HBV replication inhibitors. Examples of HBV replication inhibitors that can be combined or co-administered include GP-31502, isothiafludine, IQP-HBV, RM-5038, and Xingantie.

Inhibitors of covalently closed circular DNA (cccDNA)

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more cccDNA inhibitors. Examples of cccDNA inhibitors that can be combined or co-administered include BSBI-25, ccc-R08, and CHR-101.

Farnesoid X receptor (FXR) agonists

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more FXR agonists. Examples of FXR agonists that can be combined or co-administered include EYP-001, silofexor (GS-9674), EDP-305, MET-409, Tropifexor, AKN-083, RDX-023, BWD-100, LMB-763, INV-3, NTX-023-1, EP-024297, and GS-8670.

Antibodies to HBV

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more antibodies that specifically bind to an HBV antigen, including an HBV peptide present in a major histocompatibility molecule (MHC) (pMHC). Examples of HBV antibodies targeting hepatitis B virus surface antigens that can be combined or co-administered include lenvervimab (GC-1102), XTL-17, XTL-19, KN-003, IV Hepabulin SN, and fully human monoclonal antibody (hepatitis B virus, Humabs BioMed). Antibodies targeting HBV protein X (HBx) that can be combined or co-administered are described, for example, in Kornyeyev, et al., J Virol. 2019 Jul 30; 93(16). pii: e00248-19.

Examples of HBV antibodies, including monoclonal antibodies and polyclonal antibodies, that can be combined or co-administered include Zutectra, Shang Sheng Gan Di, Uman Big (hyperimmune hepatitis B), Omri-Hep-B, Nabi-HB, Hepatect CP, HepaGamB, Igantibe, Niuliva, CT-P24, Hepatitis B Immunoglobulin (intravenous, pH4, HBV infection, Shanghai RAAS Blood Products), and Fovepta (BT-088).

Examples of fully human HBV monoclonal antibodies that can be combined

- 75 047988 or enter together, include NVS-34.

Antibodies to HBV viral peptide/major histocompatible complex (MHC) complexes (rMHC) that can be combined or co-administered are described, for example, in Sastry, et al., J Virol. 2011 Mar; 85(5):1935-42 and in WO 2011062562.

CCR2 chemokine antagonists

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more CCR2 chemokine antagonists. Examples of CCR2 chemokine antagonists that can be combined or co-administered include propagermanium.

Thymosin agonists

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more thymosin agonists, such as recombinant thymosin alpha-1. Examples of thymosin agonists that can be combined or co-administered include thymalfasin and recombinant thymosin alpha-1 (GeneScience). Examples of recombinant thymosin alpha-1 include NL-004 and peoptilized thymosin alpha-1.

Interleukin receptor agonists (eg, cytokines)

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more interleukin receptor agonists selected from IL-2, IL-7, IL-12, and IL-15. In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-21, IL-24, and variants thereof. Examples of IL-2 receptor agonists that can be combined or co-administered include proleukin (aldesleukin, IL-2); celloleukin; pegylated IL-2 (eg, NKTR-214); modified IL-2 variants (eg, THOR-707), bempegaldesleukin, AIC-284, ALKS-4230, CUI-101, and Neo-2/15. Examples of IL-15 receptor agonists that may be combined or co-administered include ALT803, NKTR-255 and hetIL-15, interleukin-15/Fc fusion protein, AM-0015, NIZ-985, SO-d01, syntorin IL-15, (pegylated IL-15), P-22339, and IL-15-PD-1 fusion protein N-809. Examples of IL-7 receptor agonists that may be combined or co-administered include CYT-107.

Nucleoprotein modulators

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more nucleoprotein modulators. Nucleoprotein modifiers can be inhibitors of the HBV capsid or capsid protein. Examples of nucleoprotein modulators that can be combined or co-administered include GS-4882, AB-423, AB-836, AT-130, ALG-001075, ALG001024, ALG-000184, EDP-514, GLS4, NVR-1221, NVR-3778, AL-3778, BAY 41-4109, morphothiadine mesylate, ARB-168786, ARB-880, ARB-1820, GST-HG-141, JNJ-379, JNJ-632, RG-7907, GST-HG-141, HEC72702, KL-060332, AB-506, ABI-H0731, ABI-H3733, JNJ-440, AK-0605, HRS-5091, VNRX-9945, ABIH2158, CB-HBV-001, AK-0605, SOC-10, SOC-11 and DVR-23.

Examples of capsid inhibitors that can be combined or co-administered include ALG-000184, ABI-H0731, NVR 3-778, and the compounds described in US 2018161307 (Gilead Sciences), US 20140275167 (Novira Therapeutics), US 20130251673 (Novira Therapeutics), US 20140343032 (Roche), WO 2014037480 (Roche), US 20130267517 (Roche), WO 2014131847 (Janssen), WO 2014033176 (Janssen), WO 2014033170 (Janssen), WO 2014033167 (Janssen), WO 2015/059212 (Janssen), WO 2015118057 (Janssen), WO 2015011281 (Janssen), WO 2014184365 (Janssen), WO 2014184350 (Janssen), WO 2014161888 (Janssen), WO 2013096744 (Novir a), US 20150225355 (Novira), US 20140178337 (Novira), US 20150315159 (Novira), US 20150197533 (Novira), US 20150274652 (Novira), US 20150259324, (Novira), US 20150132258 (Novira), US 9181288 (Novira), WO 2014184350 (Janssen), WO 2013144129 (Roche), WO 2017198744 (Roche), US 20170334882 (Novira), US 20170334898 (Roche), WO 20 17202798 (Roche), WO 2017214395 (Enanta), WO 2018001944 (Roche), WO 2018001952 (Roche), WO 2018005881 (Novira), WO 2018005883 (Novira), WO 2018011100 (Roche), WO 2018011160 (Roche), WO 2018011162 (Roche), WO 2018011163 (Roche), WO 2018036941 (Roche), WO 2018043747 (Kyoto Univ), US 20180065929 (Janssen), WO 2016168619 (Indiana University), WO 2016195982 (The Penn State Foundation), WO 2017001655 (Janssen), WO 2017048950 (Assembly Biosciences), WO 2017048954 (Assembly Biosciences), WO 2017048962 (Assembly Biosciences), US 20170121328 (Novira), US 20170121329 (Novira).

Examples of transcript inhibitors that can be combined or co-administered include

- 76 047988 compounds described in WO 2017013046 (Roche), WO 2017016960 (Roche), WO 2017017042 (Roche), WO 2017017043 (Roche), WO 2017061466 (Toyoma chemicals), WO 2016177655 (Roche), WO 2016161268 (Enanta) are included. WO 2017001853 (Redex Pharma), WO 2017211791 (Roche), WO 2017216685 (Novartis), WO 2017216686 (Novartis), WO 2018019297 (Ginkgo Pharma), WO 2018022282 (Newave Pharma), US 20180030053 ( Novartis), WO 2018045911 (Zhejiang Pharma).

Innate immune activators

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more innate immune activators. In various embodiments, the one or more innate immune activators comprise a receptor agonist selected from the group consisting of fms-like tyrosine kinase 3 (FLT3), the receptor stimulator of interferon genes (STING), an interferon receptor, DExD/H-box helicase 58 (DDX58; also known as RIG-I), nucleotide-binding oligomerization domain containing protein 2 (NOD2). In some embodiments, the methods comprise co-administering GS-3583 and/or GS-9992. In some embodiments, the methods include combining or co-administering a FLT3 agonist, such as GS3583 or CDX-301.

STING agonists, RIG-I and NOD2 modulators

In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with an agonist of cGAMP interferon response stimulator 1 interactor 1 (STING or STING1; NCBI Gene ID: 340061). In some embodiments, the STING/STING1 agonist or activator is selected from the group consisting of ADU-S100 (MIW-815), SB-11285, MK-1454, SR8291, AdVCA0848, STINGVAX, GSK-532, SYN-STING, MSA-1, SR-8291, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), cyclic GMP-AMP (cGAMP), and cyclic di-AMP. Examples of STING agonists that can be combined or co-administered include the compounds described in WO 2018065360 (Biolog Life Science Institute Forschungslabor und Biochemica-Vertrieb GmbH, Germany), WO 2018009466 (Aduro Biotech), WO 2017186711 (InvivoGen), WO 2017161349 (Immune Sensor), WO 2017106740 (Aduro Biotech), US 20170158724 (Glaxo Smithkline), WO 2017075477 (Aduro Biotech), US 20170044206 (Merck), WO 2014179760 (University of California), WO 2018098203 (Janssen), WO 2018118665 (Merck), WO 2018118664 (Merck), WO 2018100558 (Takeda), WO 2018067423 (Merck), WO 2018060323 (Boehringer).

In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with DExD/H-box helicase 58 (DDX58; also known as retinoic acid-inducible gene 1 (RIG-I), RIG1, RIGI, RLR-1, SGMRT2; NCBI Gene Accession Number: 23586). Exemplary RIG-I agonists that can be combined or co-administered include inarigivir soproxil (SB-9200; GS-9992); SB-40, SB-44, ORI-7246, ORI-9350, ORI-7537, ORI-9020, ORI-9198, ORI-7170 and RGT-100.

In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with a nucleotide-binding oligomerization domain containing protein 2 (NOD2; NCBI Gene Accession Number: 64127), such as inarigivir soproxil (SB-9200; GS-9992) and IR-103.

Phosphatidylinositol 3-kinase (PI3K) inhibitors

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with an inhibitor of a phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit, such as a phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA, CLAPO, CLOVE, CWS5, MCAP, MCM, MCMTC, PI3K, PI3K-alpha, p110-alpha; NCBI Gene Accession Number: 5290); phosphatidylinositol 4,5-bisphosphate 3-kinase, beta catalytic subunit (PIK3CB, P110-beta, PI3K, PI3K-beta, PIK3C1; NCBI gene accession number: 5291); phosphatidylinositol 4,5-bisphosphate 3-kinase, gamma catalytic subunit (PIK3CG, PI3CG, PI3K, PI3K-gamma, PIK3, p110-gamma, p120-PI3K; gene accession number: 5494); and/or phosphatidylinositol 4,5-bisphosphate 3-kinase delta catalytic subunit (PIK3CD, APDS, IMD14, P110-delta, PI3K, p110D, NCBI gene accession number: 5293). In some embodiments, the PI3K inhibitor is a pan-PI3K inhibitor.

Examples of PI3K inhibitors include, but are not limited to, ACP-319, AEZA-129, AMG-319, AS252424, AZD8186, BAY 1082439, BEZ235, bimiralisib (PQR309), buparlisib (BKM120), BYL719

- 77 047988 (alpelisib), carboxyamidotriazole orotate (CTO), CH5132799, CLR-457, CLR-1401, copanlisib (BAY 80-6946), DS-7423, duvelisib (IPI-145), fimepinostat (CUDC-907), gedatolisib (PF-05212384), GDC0032, GDC-0084 (RG7666), GDC-0077, пистилисиб (GDC-0941), GDC-0980, GSK2636771, GSK2269577, идеалалисиб (Zydelig®), INCB040093, INCB50465, IPI-443, IPI- 549, KAR4141, LY294002, LY3023414, NERLYNX® (neratinib), nemiralisib (GSK2269557), omipalisib (GSK2126458, GSK458), OXY111A, panulisib (P7170, AK151761), PA799, perifosine (KRX-040 1), pilaralisib (SAR245408; XL147), pucutinitb mesylate (XC-302), SAR260301, seletalisib (UCB-5857), serabelisib (INK-1117, MLN-1117, TAK-117), SF1126, sonolisib (PX-866), RG7604, sodium rigosertib (ON-01910 sodium), RP5090, tenalisib (RP6530), RV-1729, SRX3177, taselisib, TG100115, umbralisib (TGR-1202), TGX221, voxtalisib (SAR245409), VS-5584, WX-037, X-339, X-414, XL499, XL756, wortmannin, ZSTK474 and compounds described in WO 2005/113556 (ICOS), WO 2013/052699 (Gilead Calistoga), WO 2013/116562 (Gilead Calistoga), WO 2014/100765 (Gilead Calistoga) , W.O. 2014/100767 (Gilead Calistoga) and WO 2014/201409 (Gilead Sciences).

Immune checkpoint modulators

In some embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more blockers or inhibitors of inhibitory immune checkpoint proteins or receptors and/or one or more stimulators, activators, or agonists of one or more stimulatory immune checkpoint proteins or receptors. Blockade or inhibition of inhibitory immune checkpoints can positively regulate T cell or NK cell activation and prevent immune evasion of infected cells. Activation or stimulation of stimulatory immune checkpoints can enhance the effect of immune checkpoint inhibitors in infectious therapy. In various embodiments, immune checkpoint proteins or receptors regulate T cell responses (e.g., discussed in Xu, et al., J Exp Clin Cancer Res.(2018) 37:110). In various embodiments, immune checkpoint proteins or receptors regulate NK cell responses (e.g., discussed in Davis, et al., Semin Immunol. (2017) 31:64-75 and Chiossone, et al., Nat Rev Immunol. (2018) 18(11):671-688).

Examples of immune checkpoint proteins or receptors include, but are not limited to, CD27 (NCBI Accession Number: 939); CD70 (NCBI Accession Number: 970); CD40 (NCBI Accession Number: 958); CD40LG (NCBI Accession Number: 959); CD47 (NCBI Accession Number: 961); CD48 (SLAMF2; NCBI Accession Number: 962); transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2, CD28H; NCBI Accession Number: 126259); CD84 (LY9B, SLAMF5; NCBI Accession Number: 8832); CD96 (NCBI Accession Number: 10225); CD160 (NCBI Accession Number: 11126); MS4A1 (CD20; NCBI Accession Number: 931); CD244 (SLAMF4; NCBI Accession Number: 51744); CD276 (B7H3; NCBI Accession Number: 80381); variable immunoglobulin-like domain-containing T cell activation inhibitor 1 (VTCN1, B7H4; NCBI Accession Number: 79679); V-set domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA; NCBI Accession Number: 64115); immunoglobulin superfamily member 11 (IGSF11, VSIG3; NCBI Accession Number: 152404); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6; NCBI Accession Number: 374383); HERV-H LTR-associated protein 2 (HHLA2, B7H7; NCBI Accession Number: 11148); inducible T cell costimulator (ICOS, CD278; NCBI Accession Number: 29851); inducible T cell costimulator ligand (ICOSLG, B7H2; NCBI Accession Number: 23308); TNF receptor superfamily member 4 (TNFRSF4, OX40; NCBI Accession Number: 7293); TNF superfamily member 4 (TNFSF4, OX40L; NCBI Accession Number: 7292); TNFRSF8 (CD30; NCBI Accession Number: 943); TNFSF8 (CD30L; NCBI Accession Number: 944); TNFRSF10A (CD261, DR4, TRAILR1; NCBI Accession Number: 8797); TNFRSF9 (CD137; NCBI Gene ID: 3604); TNFSF9 (CD137L; NCBI Gene ID: 8744); TNFRSF10B (CD262, DR5, TRAILR2; NCBI Gene ID: 8795); TNFRSF10 (TRAIL; NCBI Gene ID: 8743); TNFRSF14 (HVEM, CD270; NCBI Gene ID: 8764); TNFSF14 (HVEML; NCBI Gene ID: 8740); CD272 (B- and T-lymphocyte-associated protein (BTLA); NCBI Gene ID: 151888); TNFRSF17 (BCMA, CD269; NCBI Gene ID: 608); TNFSF13B (BAFF; NCBI Gene ID: 10673); TNFRSF18 (GITR;

- 78 047988 NCBI Gene Accession Number: 8784); TNFSF18 (GITRL; NCBI Gene Accession Number: 8995); MHC class I polypeptide-related sequence A (MICA; NCBI Gene Accession Number: 100507436); MHC class I polypeptide-related sequence B (MICB; NCBI Gene Accession Number: 4277); CD274 (CD274, PDL1, PD-L1; NCBI Gene Accession Number: 29126); programmed cell death protein 1 (PDCD1, PD1, PD-1; NCBI Gene Accession Number: 5133); Cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152; NCBI Accession Number: 1493); CD80 (B7-1; NCBI Accession Number: 941); CD28 (NCBI Accession Number: 940); Nectin cell adhesion molecule-2 (NECTIN2, CD112; NCBI Accession Number: 5819); CD226 (DNAM-1; NCBI Accession Number: 10666); Poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155; NCBI Accession Number: 5817); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R; NCBI Gene ID: 79037); T cell immunoreceptor with Ig and ITIM domains (TIGIT; NCBI Gene ID: 201633); T cell immunoglobulin and mucin domain-containing protein 4 (TIMD4; TIM4; NCBI Gene ID: 91937); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3; NCBI Gene ID: 84868); galectin 9 (LGALS9; NCBI Gene ID: 3965); lymphocyte activation gene protein 3 (LAG3, CD223; NCBI Gene Accession Number: 3902); signaling lymphocyte activating molecule family member 1 (SLAMF1, SLAM, CD150; NCBI Gene Accession Number: 6504); lymphocyte antigen 9 (LY9, CD229, SLAMF3; NCBI Gene Accession Number: 4063); SLAM family member 6 (SLAMF6, CD352; NCBI Gene Accession Number: 114836); SLAM family member 7 (SLAMF7, CD319; NCBI Gene Accession Number: 57823); Ul16 binding protein 1 (ULBP1; NCBI Gene ID: 80329); UL16 binding protein 2 (ULBP2; NCBI Gene ID: 80328); UL16 binding protein 3 (ULBP3; NCBI Gene ID: 79465); retinoic acid early transcript 1E (RAET1E; ULBP4; NCBI Gene ID: 135250); retinoic acid early transcript 1G (RAET1G; ULBP5; NCBI Gene ID: 353091); retinoic acid early transcript 1L (RAET1L; ULBP6; NCBI Gene ID: 154064); lectin-like killer cell receptor C1 (KLRC1, NKG2A, CD159A; NCBI Gene ID: 3821); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314; NCBI Gene ID: 22914); lectin-like killer cell receptor C2 (KLRC2, CD159c, NKG2C; NCBI Gene ID: 3822); lectin-like killer cell receptor C3 (KLRC3, NKG2E; NCBI Gene ID: 3823); lectin-like killer cell receptor C4 (KLRC4, NKG2F; NCBI Gene ID: 8302); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1; NCBI Gene ID: 3802); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2; NCBI Gene ID: 3803); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3; NCBI Gene ID: 3804); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1, KIR, CD158E1; NCBI Gene ID: 3811) (e.g., lirilumab (IPH2102/BMS-986015), IPH-4102); and killer cell lectin-like receptor D1 (KLRD1; NCBI Gene ID: 3824).

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. Exemplary T cell inhibitory immune checkpoint proteins or receptors include, but are not limited to, CD274 (CD274, PDL1, PDL1); programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death 1 protein (PDCD1, PD1, PD-1); cytotoxic T lymphocyte associated protein 4 (CTLA4, CD152); CD276 (B7H3); variable immunoglobulin-like domain-containing inhibitor of T cell activation 1 (VTCN1, B7H4); V-set domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation gene protein 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); ha

- 79 047988 lectin 9 (LGALS9); immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and immunoglobulin-like killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1). In various embodiments, the agents as described herein are combined with one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. Exemplary T cell stimulatory immune checkpoint proteins or receptors include, but are not limited to, CD27, CD70; CD40, CD40LG; inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; cell adhesion molecule-2 nectin (NECTIN2, CD112); CD226 (DNAM-1); CD244 (2B4, SLAMF4), poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155). See, e.g., Xu, et al., J Exp Clin Cancer Res. (2018) 37:110.

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. Exemplary NK cell inhibitory immune checkpoint proteins or receptors include, but are not limited to, killer cell immunoglobulin-like receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin-like receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A) and killer cell lectin-like receptor D1 (KLRD1, CD94).

In some embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. Exemplary NK cell stimulatory immune checkpoint proteins or receptors include, but are not limited to, CD16, CD226 (DNAM1); CD244 (2B4, SLAMF4); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); SLAM family member 7 (SLAMF7). See, e.g., Davis, et al., Semin Immunol. (2017) 31:64-75; Fang, et al., Semin Immunol. (2017) 31:37–54 and Chiossone, et al., Nat Rev Immunol. (2018) 18(11):671–688).

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more cytotoxic T lymphocyte-associated protein 4 (CTLA4) inhibitors (CD152; NCBI Gene Accession Number: 1493). Examples of CTLA4 inhibitors that can be co-administered include, but are not limited to, ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884, AGEN2041, BMS-986249, MK-1308, REGN-4659, ADU-1604, CS-1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, JHL-1155, KN-044, CG-0161, ATOR-1144, PBI-5D3H5, BPI-002, betalasept, PSI-001, PRS-010, JHL-1155, and the polyspecific FPT-155 inhibitors (CTLA4/PDL1/CD28), PF-06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717 (PD-1/CTLA4) and AK-104 (CTLA4/PD-1).

PD-L1 (CD274) or PD-1 (PDCD1: CD279) inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more inhibitors of programmed cell death ligand 1 1 (PD-L1; CD274; NCBI Gene Accession Number: 29126) inhibitor of programmed cell death protein 1 (PD-1; PDCD1; CD279; NCBI Gene Accession Number: 5133). Examples of PD-L1 (CD274) or PD-1 (PDCD1) inhibitors that can be combined or co-administered include, but are not limited to, zimberelimab (AB122), pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, (MSB0010718C), ASC22, durvalumab, ALN-PDL, BMS-936559, CK-301, PF-06801591, BGB-108, BGB-A317 (tislelizumab), GLS-010

- 80 047988 (WBP-3055), AK-103 (HX-008), GB-226, AK-105, CS-1003, HLX-10, MGA-012, BI-754091, PDR-001, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genolimzumab (SVT-501), LZM-009, BCD-100, LY3300054, SHR-1201, SHR-1210 (camrelizumab), Sym-021, ABBV-181, PD1-PIK , BAT-1306, RO-6084 (PD-L1 antisense oligonucleotide), STI-1110, GX-P2, RG-7446, mDX-400, CX-072, CBT-502, TSR-042 (dostarlimab), MSB-2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155 ), MEDI-0680, envafolimab (KN-035), KD-033, KY-1003, IBI-308 (sintilimab), HLX-20, KL-A167, STI-A1014, STI-A1015 (IMC-001), BCD -135, FAZ-053, TQB-2450, MDX1105-01, MSB-0010718C, GS-4224, GS-4416, INCB086550, MAX10181, as well as polyspecific inhibitors FPT-155 (CTLA4/PD-L1/CD28), PF06936308 (PD-1/CTLA4), MGD-013 (PD-1/LAG-3), FS-118 (LAG-3 /PD-L1) MGD-019 (PD-1/CTLA4), KN046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), RO-7121661 (PD-1/TIM3), XmAb-20717 (PD-1/CTLA4), AK-104 (CTLA4/PD-1), M7824 (PD-L1/TGFe-EC domain), CA-170 (PD-L1/VISTA), CDX-527 (CD27/PDL1) , LY-3415244 (TIM3/PDL1), GNS-1480 (epidermal growth factor receptor antagonist; programmed cell death inhibitor 1), M-7824 (PD-L1/TGF-e bifunctional fusion protein), and INBRX-105 (4 -1BB/PDL1).

Examples of PD-1 inhibitors that can be combined or co-administered additionally include the compounds described in WO2017112730 (Incyte Corp), WO2017087777 (Incyte Corp), WO2017017624, WO2014151634 (BristolMyers Squibb Co), WO201317322 (BristolMyers Squibb Co), WO2018119286 (Incyte Corp), WO2018119266 (Incyte Corp), WO2018119263 (Incyte Corp), WO2018119236 (Incyte Corp), WO2018119221 (Incyte Corp), WO2018118848 (BristolMyers Squibb Co), WO 20161266460 (BristolMyers Squibb Co), WO 2017087678 (BristolMyers Squibb Co), WO 2016149351 (BristolMyers Squibb Co), WO 2015033299 (Aurigene Discovery Technologies Ltd), WO 2015179615 (Eisai Co Ltd; Eisai Research Institute), WO 2017066227 (BristolMyers Squibb Co), WO 2016142886 (Aurigene Discovery Technologies Ltd), WO 2016142852 (Aurigene Discovery Technologies Ltd), WO 2016142835 (Aurigene Discovery Technologies Ltd; Individual), WO 2016142833 (Aurigene Discovery Technologies Ltd), WO 2018085750 (BristolMyers Squibb Co), WO 2015033303 (Aurigene Discovery Technologies Ltd), WO 2017205464 (Incyte Corp), WO 2016019232 (3M Co; Individual; Texas A&M University System) 16077518 (BristolMyers Squibb Co), WO 2016057624 (BristolMyers Squibb Co), WO 2018044783 (Incyte Corp), WO 2016100608 (BristolMyers Squibb Co), WO 2016100285 (BristolMyers Squibb Co), WO 2016039749 (BristolMyers Squibb Co), WO 2015019284 (Cambridge Enterprise Ltd), WO 2016142894 (Aurigene Discovery Technologies Ltd), WO 2015134605 (BristolMyers Squibb Co), WO 2018051255 (Aurigene Discovery Technologies Ltd), WO 2018051254 (Aurigene Discovery Technologies Ltd) 2017176608 (BristolMyers Squibb Co), WO 2018009505 (BristolMyers Squibb Co), WO 2011161699 (Aurigene Discovery Technologies Ltd), WO 2015119944 (Incyte Corp; Merck Sharp & Dohme Corp), WO 2017192961 (Incyte Corp), WO 2017106634 (Incyte Corp), WO 2013132317 (Aurigene Discovery Technologies Ltd), WO 2012168944 (Aurigene Discovery Technologies Ltd), WO 2015036927 (Aurigene Discovery Technologies Ltd), WO 201504 4900 (Aurigene Discovery Technologies Ltd), WO 2018026971 (Arising International).

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more protein inhibitors (e.g., an antibody or fragment thereof or antibody mimetics) of PDL1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the one or more immune checkpoint inhibitors comprise small organic molecule inhibitors of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550, and MAX10181. Additional examples of small molecule PD-L1 inhibitors include those described in US Publication Nos. US2018305315 (Gilead Sciences), US2020017471 (Gilead Sciences), and US2019270727 (Gilead Sciences). In some embodiments, the small molecule CTLA4 inhibitor comprises BPI002.

T cell immunoreceptor inhibitors with Ig and ITIM domains (TIGIT)

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more T-cell immunoreceptor inhibitors with Ig domains and ITIM (TIGIT) (NCBI Gene Accession Number: 201633). Examples of TIGIT antibodies that can be combined or co-administered include ethylimab, BMS-986207, tiragolumab (also known as MTIG-7192A; RG-6058; RO 7092284), AGEN1307, AGEN1327, AGEN1777, COM-902, IBI-939, AB154, MG1131, and EOS884448 (EOS-448).

- 81 047988

Agonists or activators of members of the TNF receptor superfamily (TNFRSF)

In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more agonists of one or more members of the TNF receptor superfamily (TNFRSF), such as an agonist of one or more of TNFRSF1A (NCBI Gene ID: 7132), TNFRSF1B (NCBI Gene ID: 7133), TNFRSF4 (OX40, CD134; NCBI Gene ID: 7293), TNFRSF5 (CD40; NCBI Gene ID: 958), TNFRSF6 (FAS; NCBI Gene ID: 355), TNFRSF7 (CD27, NCBI Gene ID: 939), TNFRSF8 (CD30, NCBI Gene ID: 943), TNFRSF9 (4-1BB, CD137, NCBI Gene ID: 3604), TNFRSF10A (CD261, DR4, TRAILR1, NCBI Gene ID: 8797), TNFRSF 10B (CD262, DR5, TRAILR2, NCBI Gene ID: 8795), TNFRSF10C (CD263, TRAILR3, NCBI Gene ID: 8794), TNFRSF 10D (CD264, TRAILR4 (NCBI Gene ID: 8793), TNFRSF11A (CD265, RANK, NCBI Gene ID: 8792), TNFRSF11B (NCBI Gene ID: 4982), TNFRSF12A (CD266, NCBI Gene ID: 51330), TNFRSF13B (CD267, NCBI Gene ID: 23495), TNFRSF13C (CD268, NCBI Gene ID: 115650), TNFRSF16 (NGFR, CD271, NCBI Gene ID: 4804), TNFRSF17 (BCMA, CD269, NCBI Gene ID: 608), TNFRSF18 (GITR, CD357, NCBI Gene ID: 8784), TNFRSF19 (NCBI Gene ID: 55504), TNFRSF21 (CD358, DR6, NCBI Gene ID: 27242), and TNFRSF25 (DR3, NCBI Gene ID: 8718).

Examples of TNFRSF4 (OX40) antibodies that can be combined or co-administered include, but are not limited to, MEDI6469, MEDI6383, MEDI0562 (tavolyxizumab), MOXR0916, PF04518600, RG-7888, GSK-3174998, INCAGN1949, BMS-986178, GBR-8383, ABBV-368, and those described in WO2016179517, WO2017096179, WO2017096182, WO2017096281, and WO2018089628.

Examples of anti-TNFRSF5 (CD40) antibodies that can be combined or co-administered include, but are not limited to, RG7876, SEA-CD40, APX-005M, and ABBV-428.

In some embodiments, the anti-TNFRSF7 (CD27) antibody varlilumab (CDX-1127) is combined or co-administered.

Examples of anti-TNFRSF9 (4-1BB, CD137) antibodies that may be combined or co-administered include, but are not limited to, urelumab, utomilumab (PF-05082566), AGEN-2373, and ADG-106.

Examples of TNFRSF18 (GITR) antibodies that can be combined or co-administered include, but are not limited to, MEDI1873, FPA-154, INCAGN-1876, TRX-518, BMS-986156, MK-1248, GWN-323, and the antibodies described in WO2017096179, WO2017096276, WO2017096189, and WO2018089628. In some embodiments, an antibody or fragment thereof targeting TNFRSF4 (OX40) and TNFRSF18 (GITR) is co-administered. Such antibodies are described, for example, in WO2017096179 and WO2018089628.

Indoleamine pyrrole-2.3-dioxygenase (IDO1) inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with one or more indoleamine 2,3-dioxygenase 1 (IDO1; NCBI Gene Identification Number: 3620) inhibitors. Examples of IDO1 inhibitors that can be combined or co-administered include, but are not limited to, BLV-0801, epacadostat, resminostat, F-001287, GBV-1012, GBV-1028, GDC-0919, indoximod, NKTR-218, NLG-919 vaccine, PF-06840003, pyranonaphthoquinone derivatives (SN-35837), SBLK-200802, BMS-986205 and shIDO-ST, EOS-200271, KHK-2455, LY-3381916, and the compounds described in US 20100015178 (Incyte), US 2016137652 (Flexus Biosciences, Inc.), WO 2014073738 (Flexus Biosciences, Inc.) and WO 2015188085 (Flexus Biosciences, Inc.).

LAG-3 and TIM-3 inhibitors

In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with an anti-TIM-3 antibody such as TSR-022, LY-3321367, MBG-453, INCAGN-2390. In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with an anti-LAG-3 (lymphocyte activation) antibody such as relatlimab (ONO-4482), LAG-525, MK-4280, REGN-3767, INCAGN2385.

- 82 047988

Inhibitors of the apoptosis protein (IAP) family of proteins

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with an inhibitor of a protein of the inhibitor of apoptosis protein (IAP) family. Examples of IAP inhibitors include APG-1387.

Bruton tyrosine kinase inhibitors (BTK)

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with a Bruton tyrosine kinase inhibitor (BTK, AGMX1, AT, ATK, BPK, IGHD3, IMD1, PSCTK1, XLA; NCBI Gene Accession Number: 695). Examples of BTK inhibitors include, but are not limited to, (B)-6-amino-9-(1-(but-2-inoyl)pyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7H-purin-8(9H)-one, ABBV105, acalabrutinib (ACP-196), AC-058, AC-0025, ARQ-531, BMS-986142, dasatinib, ibrutinib (PCI32765, CRA-032765), GDC-0853, PRN-1008, SNS-062, BGB-3111, CB988, HM71224, KBP-7536, M-2951 (evobrutinib), M7583, tirabrutinib (ONO-4059), ML-319, MSC-2364447, PRN-1008, RDX-022, RG7845, spebrutinib (CC-292), TAK-020, TAS-5315, TP-0158, TP-4207, vecabrutinib (SNS-062), ARQ-531, SHR-1459, DTRMWXHS-12 and compounds described in US 20140330015 (Ono Pharmaceutical), US 20130079327 (Ono Pharmaceutical) and US 20130217880 (Ono Pharmaceutical).

Lysine demethylase inhibitors (KDM)

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with a lysine demethylase inhibitor (KDM). Examples of KDM5 inhibitors that can be combined or co-administered include the compounds described in WO2016057924 (Genentech/Constellation Pharmaceuticals), US20140275092 (Genentech/Constellation Pharmaceuticals), US20140371195 (Epitherapeutics), US20140371214 (Epitherapeutics), US20160102096 (Epitherapeutics), US20140194469 (Quanticel), US20140171432, US20140213591 (Quanticel), US20160039808 (Quanticel), US20140275084 (Quanticel), and WO2014164708 (Quanticel).

Examples of KDM1 inhibitors that may be combined or co-administered include the compounds described in US 9186337B2 (Oryzon Genomics), GSK-2879552, RG-6016, and ORY-2001.

Arginase inhibitors

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with an arginase inhibitor. Examples of arginase inhibitors include CB-1158, C-201, and resminostat.

Natural Killer (NK) Cell Attracting Bispecific and Trispecific Activators

In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with a bispecific NK cell-attracting activator (BiKE) or a trispecific NK cell-attracting activator (TriKE) (e.g., lacking an Fc) or a bispecific antibody (e.g., having an Fc) against an NK cell activating receptor, e.g., CD16A, C-type lectin receptors (CD94/NKG2C, NKG2D, NKG2E/H, and NKG2F), natural cytotoxicity receptors (NKp30, NKp44, and NKp46), killer cell lectin-like receptor C-type (NKp65, NKp80), Fc receptor FcyR (which mediates antibody-dependent cellular cytotoxicity), SLAM family receptors (e.g., 2B4, SLAM6, and SLAM7), killer cell immunoglobulin-like receptors (KIRs) (KIR-2DS and KIR-3DS), DNAM-1, and CD137 (41BB). Accordingly, bispecific molecules that bind to CD16 may or may not have an Fc. Exemplary NK cell-attracting bispecific activators that can be co-administered with a CD16 target and one or more HBV-associated antigens are described herein. BiKE and TriKE are described, for example, in Felices, et al., Methods Mol Biol. (2016) 1441:333–346; Fang, et al., Semin Immunol. (2017) 31:37–54.

Long-acting therapies

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with long-acting therapy. Long-acting entecavir (subcutaneous depot), long-acting triofovir implants (TFD and TAF), or subcutaneous depot. An example of long-acting entecavir is described in Henry, et al., Eur JPharm Sci. (2019) 136:104958.

- 83 047988

Gene therapy and cell therapy

In various embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered using a gene or cell therapy regimen. Gene therapy and cell therapy include, but are not limited to, genetic modification to suppress a gene; genetic approaches to directly kill infected cells; infusion of immune cells designed to replace a large portion of the patient's own immune system to enhance the immune response to infected cells, or activate the patient's own immune system to kill infected cells, or seek out and kill infected cells; genetic approaches to modify cellular activity to further alter the endogenous immune response against infection.

Gene editors

The genome editing system may be selected from the group consisting of the CRISPR/Cas9 system, the zinc finger nuclease system, the TALEN system, the homing endonuclease system, and the homing meganuclease system (e.g., the ARCUS system); for example, the removal of cccDNA by targeted cleavage and the alteration of one or more viral genes of the hepatitis B virus (HBV). An alteration (e.g., knockout and/or knockdown) of a PreC, C, X, PreSI, PreS2, S, P, or SP gene refers to (1) a decrease in or abolition of PreC, C, X, PreSI, PreS2, S, P, or SP gene expression, (2) an overlap of the precapsid, capsid, X protein, large surface protein, midsurface protein, S protein (also known as HBs antigen and HBsAg), polymerase protein, and/or hepatitis B spliced protein function (HBe, HBc, HBx, PreS1, PreS2, S, Pol, and/or HBSP), or (3) a decrease in or abolition of intracellular, serum, and/or intraparenchymal levels of HBe, HBc, HBx, LHB, MHB, SHB, Pol, and/or HBSP proteins. Knockdown of one or more of the PreC, C, X, PreSI, PreS2, S, P and/or SP genes is accomplished by targeting the gene(s) within the HBV cccDNA and/or the HBV pooled DNA. Additional examples of genome editing systems include, but are not limited to, those described in US 2019284543 (Gilead Sciences) and US 2019338263 (Gilead Sciences).

Examples of gene therapy such as liver-targeted gene therapy against HBV (using ARCUS technology) or using CRISPR/Cas9 or EBT106 gene editing technology (CRISPR/CasX nuclease delivered by LNP).

CAR-T cell therapy

CAR-T cell therapy comprises a population of immune effector cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an HBV antigen-binding domain. In certain embodiments, the antigen-binding domain is a domain as described herein. In certain embodiments, the antigen-binding domain is different from a domain as described herein. In some embodiments, the antigen is HBsAg (i.e., HbsAg-CART). The immune effector cell is a T cell or an NK cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, an NK cell, or a combination thereof. The cells may be autologous or allogeneic. An example of CART targeting HBV is described in Kruse, et al., Cytotherapy. (2018) 20(5):697-705.

TCR T cell therapy

TCR-T cell therapy involves T cells expressing HBV-specific T cell receptors. TCR-T cells are engineered to target HBV-derived peptides presented on the surface of virus-infected cells. An example of a TCR targeting HBV is described in Wisskirchen, et al., J Clin Invest. (2019) 129(7):2932–2945.

TCR-T cell therapy involves T cells expressing HBV surface antigen (HBsAg)-specific TCR, such as IMC-I109 V.

TCR-T cell therapy includes TCR-T therapies targeting HBV, such as LTCR-H2-1.

In various embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with an HBV DNA polymerase inhibitor, one or two additional therapeutic agents selected from the group consisting of immunomodulators, TLR modulators, HBsAg inhibitors, inhibitors of HBsAg secretion or assembly, therapeutic HBV vaccines, anti-HBV antibodies including anti-HBV antibodies targeting hepatitis B virus surface antigens and bispecific antibodies and antibody-like therapeutic proteins (e.g., DARTs®, DUOBODIES®, BITES®, XmAbs®, TandAbs®, Fab derivatives, or TCR-like antibodies), cyclophilin inhibitors, retinoic acid inducible gene 1 stimulators, acid, RIG-I-like receptor stimulators, PD-1 inhibitors, PD-L1 inhibitors, arginase inhibitors, PI3K inhibitors, IDO inhibitors and NOD2 stimulators, and one or two additional therapeutic agents selected from the group consisting of inhibitors

- 84 047988 HBV penetration inhibitors, NTCP inhibitors, HBx inhibitors, cccDNA inhibitors, HBV antibodies targeting hepatitis B surface antigens, siRNA, miRNA-based gene therapy agents, shRNA, KDM5 inhibitors or nucleoprotein modulators (modulators of HBV core or capsid protein).

In another specific embodiment, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with at least a second additional therapeutic agent selected from the group consisting of HBV DNA polymerase inhibitors, immunomodulators, TLR modulators, HBsAg inhibitors, therapeutic HBV vaccines, HBV antibodies including HBV antibodies targeting hepatitis B virus surface antigens, bispecific antibodies and antibody-like therapeutic proteins (e.g., DARPin®, anti-pMHC TCR-like antibodies, DART®, DUOBODY®, BITE®, XmAb®, TandAb®, Fab derivatives, or TCR-like antibodies), cyclophilin inhibitors, stimulators of hepatitis B inducible gene 1 retinoic acid, RIG-I-like receptor stimulators, PD-1 inhibitors, PD-L1 inhibitors, arginase inhibitors, PI3K inhibitors, IDO inhibitors and NOD2 stimulators.

In another specific embodiment, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with at least a second additional therapeutic agent selected from the group consisting of HBV DNA polymerase inhibitors, HBV viral entry inhibitors, NTCP inhibitors, HBx inhibitors, cccDNA inhibitors, HBV antibodies targeting hepatitis B surface antigen, siRNA, miRNA, shRNA, KDM5 inhibitors, and nucleoprotein modulators (HBV core or capsid protein modulators).

In certain embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with the compounds described in U.S. Pub. No. 2010/0143301 (Gilead Sciences), U.S. Pub. No. 2011/0098248 (Gilead Sciences), U.S. Pub. No. 2009/0047249 (Gilead Sciences), U.S. Pub. No. 8,722,054 (Gilead Sciences), U.S. Pub. No. 2014/0045849 (Janssen), U.S. Pub. No. 2014/0073642 (Janssen), WO 2014/056953 (Janssen), WO 2014/076221 (Janssen), WO 2014/128189 (Janssen), US Publication No. 2014/0350031 (Janssen), WO 2014/023813 (Janssen), US Publication No. 2008/0234251 (Array Biopharma), US Publication No. 2008/0306050 (Array Biopharma), US Publication No. 2010/0029585 (Ventirx Pharma), US Publication No. 2011/0092485 (Ventirx Pharma), US 2011/0118235 (Ventirx Pharma), US Publication No. 2012/0082658 (Ventirx Pharma), US Publication No. 2012/0219615 (Ventirx Pharma), US Publication No. 2014/0066432 (Ventirx Pharma), US Publication No. 2014/0088085 (Ventirx Pharma), US Publication No. 2014/0275167 (Novira Therapeutics), US Publication No. 2013/0251673 (Novira Therapeutics), US Publication No. 8513184 (Gilead Sciences), US Publication No. 2014/0030221 (Gilead Sciences), US Publication No. 2013/0344030 (Gilead Sciences), CIHA Publication No. 2013/0344029 (Gilead Sciences), US 20140275167 (Novira Therapeutics), US 20130251673 (Novira Therapeutics), US Publication No. 2014/0343032 (Roche), WO 2014037480 (Roche), US Publication No. 2013/0267517 (Roche), WO 2014131847 (Janssen), WO 2014033176 (Janssen), WO 2014033170 (Janssen), WO 2014033167 (Janssen), WO 2015/059212 (Janssen), WO 2015118057 (Janssen), WO 2015011281 (Janssen), WO 2014184365 (Janssen), WO 2014184350 (Janssen), WO 2014161888 (Janssen), WO 2013096744 (Novira), US 20150225355 (Novira), US 20140178337 (Novira), US 20150315159 (Novira), US 20150197533 (Novira), US 20150274652 (Novira), US 20150259 324, (Novira), US 20150132258 (Novira), US 9181288 (Novira), WO 2014184350 (Janssen), WO 2013144129 (Roche), US 20100015178 (Incyte), US 2016137652 (Flexus Biosciences, Inc.), WO 2014073738 (Flexus Biosciences, Inc.), WO 2015188085 (Flexus Biosciences, Inc.), US Publication No. 2014/0330015 (Ono Pharmaceutical), US Publication No. 2013/0079327 (Ono Pharmaceutical), US Publication No. 2013/0217880 (Ono pharmaceutical), WO 2016057924 (Genentech/Constellation Pharmaceuticals), US 20140275092 (Genentech/ConstellationPharmaceuticals), US 20140371195 (Epitherapeutics) and US 20140371214 (Epitherapeutics), US 20160102096 (Epitherapeutics), US 20140194469 (Quanticel), US 20140171432, US 20140213591 (Quanticel), US 20160039808 (Quanticel), US 20140275084 (Quanticel), WO 2014164708 (Quanticel), US 9186337B2 (Oryzon Genomics, and other drugs for the treatment of HBV, as well as their combinations.

In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with 5-30 mg of tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, or tenofovir alafenamide. In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with 510; 5-15; 5-20; 5-25; 25-30; 20-30; 15-30; or 10-30 mg of tenofovir alafenamide fumarate, tenofovir

- 85 047988 alafenamide hemifumarate or tenofovir alafenamide. In certain embodiments, the immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with 10 mg of tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, or tenofovir alafenamide. In certain embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with 25 mg of tenofovir alafenamide fumarate, tenofovir alafenamide hemifumarate, or tenofovir alafenamide. The immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein can be combined with the agents provided herein in any dosage of the compound (e.g., 50 to 500 mg of the compound) just as if each dosage combination were specifically and individually recited.

In certain embodiments, immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein are combined or co-administered with 100-400 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate, or tenofovir disoproxil. In some embodiments, an agent described herein or a pharmaceutically acceptable salt thereof is combined with 100-150;100-200, 100-250; 100-300; 100-350; 150-200; 150-250; 150-300; 150-350; 150-400; 200-250;200-300;200-350;200-400; 250-350; 250-400; 350-400 or 300-400 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate or tenofovir disoproxil. In certain embodiments, the agent described herein or a pharmaceutically acceptable salt thereof is combined with 300 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate or tenofovir disoproxil. In certain embodiments, an agent as described herein, or a pharmaceutically acceptable salt thereof, is combined with 250 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate, or tenofovir disoproxil. In certain embodiments, an agent as described herein, or a pharmaceutically acceptable salt thereof, is combined with 150 mg of tenofovir disoproxil fumarate, tenofovir disoproxil hemifumarate, or tenofovir disoproxil. Immunogenic polypeptides, polynucleotides encoding such polypeptides, vectors, LNPs, and immunogenic compositions comprising such polypeptides or polynucleotides as described herein can be combined with the agents provided herein at any dosage of the compound (e.g., 50 to 500 mg of the compound) just as if each dosage combination were specifically and individually recited.

8. Sets

Additionally provided is a kit comprising one or more unit doses of one or more of a truncated HBV polymerase polypeptide, one or more deletion mutant HBV polymerase polypeptide, one or more capsid-sAg fusion protein, one or more polynucleotides, one or more vectors, or one or more immunogenic compositions as described herein. In some embodiments, the kit comprises one or more unit doses of two or more truncated HBV polymerase polypeptides, deletion mutant HBV polymerase polypeptides, capsid-sAg fusion proteins, polynucleotides, vectors, or immunogenic compositions as described herein.

In various embodiments, if necessary or desired, one or more unit doses may be in a single container or in two or more separate containers. In various embodiments, one or more containers may be selected from the group consisting of vials, ampoules, and pre-filled syringes.

In some embodiments, one or more containers contain one or more polypeptides, one or more polynucleotides, one or more vectors, or one or more immunogenic compositions in an aqueous solution. In some embodiments, one or more containers contain one or more polypeptides, one or more polynucleotides, one or more vectors, or one or more immunogenic compositions in the form of a lyophilized preparation.

If necessary or desired, the one or more unit doses can be the same or different. In some embodiments, the kit comprises one or more unit doses of one or more viral vectors capable of expressing immunogenic polypeptides. In kits comprising viral vectors, the unit doses can range from about 10 ³ to about 10 ¹² viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (VP), such as from about 10 4 to about 10 ⁷ viral FFU or PFU, such as from about 10 ³ to about 10 4, 10 ⁵ , 10 ⁶ , 10 7 , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 11 or 10 ¹² viral FFU or PFU or IU or IU.

In various embodiments, the kit comprises one or more polynucleotides encoding

- 86 047988 vectors, or one or more vectors capable of expression, or immunogenic compositions comprising two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 5-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 5-14; and (b) an HBV capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41.

In various embodiments, the kit comprises one or more polynucleotides encoding, or one or more vectors capable of expression, or an immunogenic composition comprising two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 13-14 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 13-14; and (b) an HBV capsid-sAg fusion protein comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 38-41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 38-41.

In various embodiments, the kit comprises one or more polynucleotides encoding, or one or more vectors capable of expression, or an immunogenic composition comprising two immunogenic polypeptides, wherein the immunogenic polypeptides comprise: (a) a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; and (b) an HBV capsid-sAg fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 41 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41.

With respect to a capsid-sAg fusion polypeptide in a kit (e.g., expressed from a vector; in an immunogenic composition), in some embodiments, the capsid polypeptide comprises a serine (S) residue at an amino acid position corresponding to position 12 and an asparagine (N) residue at an amino acid position corresponding to position 67, wherein the position numbers are referenced to SEQ ID NO:65 or SEQ ID NO:66. In some embodiments, the sAg polypeptide comprises an isoleucine (I) residue at an amino acid position corresponding to position 68, wherein the position numbers are referenced to SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the sAg polypeptide comprises one or more of a serine (S) residue at an amino acid position corresponding to position 53, an isoleucine (I) residue at an amino acid position corresponding to position 68, a threonine (T) residue at an amino acid position, corresponding to position 125, a proline (P) residue at the amino acid position corresponding to position 127, a phenylalanine (F) residue at the amino acid position corresponding to position 161, a tyrosine (Y) residue at the amino acid position corresponding to position 200, a serine (S) residue at the amino acid position corresponding to position 210, and a leucine (L) residue at the amino acid position corresponding to position 213, wherein the position numbers are referenced to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the capsid-sAg fusion polypeptide comprises one or more of a serine (S) residue at the amino acid position corresponding to position 12, an asparagine (N) residue at the amino acid position corresponding to position 67, a valine (V) residue at the amino acid position corresponding to position 74, a phenylalanine (F) at the amino acid position corresponding to position 97, a threonine (T) residue at the amino acid position corresponding to position 249, a threonine (T) residue at the amino acid position corresponding to position 250, a serine (S) residue at the amino acid position corresponding to position 317, a serine (S) residue at the amino acid position corresponding to position 318, an arginine (R) residue at the amino acid position corresponding to position 326, a tyrosine (Y) residue at the amino acid position corresponding to position 338, a glycine (G) residue at the amino acid position corresponding to position 363, and an alanine (A) residue at the amino acid position corresponding to position 372, wherein the position numbers are referenced to SEQ ID NO: 41.

In some embodiments, the kit comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90, and 92, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 27-32 and 89-94, such as SEQ ID NOs: 29, 89, 90

- 87 047988 and 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 33-37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 33-37.

In some embodiments, the kit comprises a first viral expression vector and a second viral expression vector, wherein: (a) the first viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29, 89, 90, or 92 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, 89, 90, or 92; and (b) the second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37.

In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described above and herein, wherein the first and second viral expression vectors comprise a replication-deficient or replication-defective Kali mammarenavirus (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)); and (b) one or more unit doses of an immunogenic composition as described above and herein, wherein the first and second viral expression vectors comprise a replication-deficient or replication-defective lymphocytic choriomeningitis mammarenavirus (LCMV).

In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described above and herein, wherein the first and second viral expression vectors are from the adenovirus family; and (b) one or more unit doses of an immunogenic composition as described herein and above, wherein the first and second viral expression vectors are from the poxvirus family (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA)).

In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described above and herein, wherein the first and second viral expression vectors are from the arenavirus family; and (b) one or more unit doses of an immunogenic composition as described above and herein, wherein the first and second viral expression vectors are from the adenovirus family.

In some embodiments, the kit comprises: (a) one or more unit doses of an immunogenic composition as described above and herein, wherein the first and second viral expression vectors are from the arenavirus family; and (b) one or more unit doses of an immunogenic composition as described herein and above, wherein the first and second viral expression vectors are from the poxvirus family (e.g., vaccinia virus, e.g., modified vaccinia virus Ankara (MVA)).

In some embodiments, the kit comprises a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: (a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29; and (b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37.

In some embodiments, the kit comprises a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: (a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 90 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90; and (b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37.

In some embodiments, the kits comprise one or more unit doses of one or more additional therapeutic agents.

For example, in some embodiments, the kit comprises one or more agonists or activators of one or more Toll-like receptors (TLRs). In various embodiments, the TLR agonist or activator is selected from the group consisting of a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist. In some embodiments, the TLR7 agonist is selected from the group consisting of GS 9620 (vesatolimod), R848 (resiquimod), DS

- 88 047988

0509, LHC-165 and TMX-101 (imiquimod), and/or wherein the TLR8 agonist is selected from the group consisting of GS-9688, R848 (resiquimod) and NKTR-262 (a dual TLR7/TLR8 agonist).

In some embodiments, the kit comprises one or more interleukin receptor agonists of an interleukin receptor selected from IL-2, IL-7, IL-12, and IL-15. In some embodiments, the kit comprises one or more cytokines selected from the group consisting of IL-2, IL-7, IL12, IL-15, and variants thereof.

In some embodiments, the kit comprises one or more innate immune activators. In various embodiments, the one or more innate immune activators comprise a receptor agonist selected from the group consisting of fms-like tyrosine kinase 3 (FLT3), the receptor stimulator of interferon genes (STING), DExD/H-box helicase 58 (DDX58; also known as RIG-I), nucleotide-binding oligomerization domain containing protein 2 (NOD2). In some embodiments, the kit comprises one or more unit doses of GS-3583 and/or GS-9992.

In some embodiments, the kits comprise one or more antagonists or inhibitors of an inhibitory immune checkpoint protein or receptor and/or one or more activators or agonists of a stimulatory immune checkpoint protein or receptor. In various embodiments, the one or more immune checkpoint proteins or receptors are selected from the group consisting of: CD27, CD70; CD40, CD40LG; CD47, CD48 (SLAMF2), transmembrane and immunoglobulin domain-containing protein 2 (TMIGD2, CD28H), CD84 (LY9B, SLAMF5), CD96, CD160, MS4A1 (CD20), CD244 (SLAMF4); CD276 (B7H3); T cell activation inhibitor 1 containing variable immunoglobulin-like domain (VTCN1, B7H4); V-set domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); natural killer cell cytotoxicity receptor 3 ligand 1 (NCR3LG1, B7H6); HERV-H LTR-associated protein 2 (HHLA2, B7H7); inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF8 (CD30), TNFSF8 (CD30L); TNFRSF10A (CD261, DR4, TRAILR1), TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF10B (CD262, DR5, TRAILR2), TNFRSF10 (TRAIL); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); TNFRSF17 (BCMA, CD269), TNFSF13B (BAFF); TNFRSF18 (GITR), TNFSF18 (GITRL); MHC class I polypeptide-related sequence A (MICA); MHC class I polypeptide-related sequence B (MICB); CD274 (CD274, PDL1, PD-L1); Programmed cell death protein 1 (PDCD1, PD1, PD-1); Cytotoxic T-lymphocyte-associated protein 4 (CTLA4, CD152); CD80 (B71), CD28; Nectin cell adhesion molecule-2 (NECTIN2, CD112); CD226 (DNAM-1); Poliovirus receptor (PVR) cell adhesion molecule (PVR, CD155); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); T-cell immunoglobulin domain-containing protein-4 and mucin domain-containing protein-4 (TIMD4; TIM4); Hepatitis A virus cell receptor 2 (HAVCR2, TIMD3, TIM3); Galectin 9 (LGALS9); lymphocyte activation gene protein 3 (LAG3, CD223); signaling lymphocyte-activating molecule family member 1 (SLAMF1, SLAM, CD150); lymphocyte antigen 9 (LY9, CD229, SLAMF3); SLAM family member 6 (SLAMF6, CD352); SLAM family member 7 (SLAMF7, CD319); ULBP1; ULBP2; ULBP3; retinoic acid early transcript IE (RAET1E; ULBP4); retinoic acid early transcript IG (RAET1G; ULBP5); retinoic acid early transcript 1L (RAET1L; ULBP6); lymphocyte activation gene protein 3 (CD223); Immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); lectin-like killer cell receptor C1 (KLRC1, NKG2A, CD159A); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); lectin-like killer cell receptor C2 (KLRC2, CD159c, NKG2C); lectin-like killer cell receptor C3 (KLRC3, NKG2E); lectin-like killer cell receptor C4 (KLRC4, NKG2F); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); killer cell receptor immunoglobulin-like, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell receptor immunoglobulin-like, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); killer cell receptor immunoglobulin-like, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor D1 (KLRD1); and SLAM family member 7 (SLAMF7).

In some embodiments, the kits comprise one or more blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors. In various embodiments, the blockers or inhibitors of one or more T cell inhibitory immune checkpoint proteins or receptors are selected from the group consisting of: CD274 (CD274, PDL1, PD-L1); programmed cell death protein 1 ligand 2 (PDCD1LG2, PD-L2, CD273); programmed cell death protein 1 (PDCD1, PD1, PD-1); cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152); CD276 (B7H3); inhibitor 1

- 89 047988 T-cell activation protein containing variable immunoglobulin-like domain (VTCN1, B7H4); Vset domain-containing immunoregulatory receptor (VSIR, B7H5, VISTA); immunoglobulin superfamily member 11 (IGSF11, VSIG3); TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML); CD272 (B- and T-lymphocyte-associated protein (BTLA)); PVR-related immunoglobulin domain-containing protein (PVRIG, CD112R); T-cell immunoreceptor with Ig and ITIM domains (TIGIT); lymphocyte activation gene protein 3 (LAG3, CD223); hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3); galectin 9 (LGALS9); killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1); killer cell receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1).

In some embodiments, the kit comprises one or more agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors. In various embodiments, the agonists or activators of one or more T cell stimulatory immune checkpoint proteins or receptors are selected from the group consisting of CD27, CD70; CD40, CD40LG; inducible T cell costimulator (ICOS, CD278); inducible T cell costimulator ligand (ICOSLG, B7H2); TNF receptor superfamily member 4 (TNFRSF4, OX40); TNF superfamily member 4 (TNFSF4, OX40L); TNFRSF9 (CD137), TNFSF9 (CD137L); TNFRSF18 (GITR), TNFSF18 (GITRL); CD80 (B7-1), CD28; cell adhesion molecule-2 nectin (NECTIN2, CD112); CD226 (DNAM-1); cell adhesion molecule poliovirus receptor (PVR) (PVR, CD155). In some embodiments, the kit comprises one or more unit doses of AGEN-2373 and/or AGEN-1223.

In some embodiments, the kits comprise one or more blockers or inhibitors of one or more NK cell inhibitory immune checkpoint proteins or receptors. In various embodiments, the NK cell inhibitory immune checkpoint proteins or receptors are selected from the group consisting of: immunoglobulin-like killer cell receptor, three Ig domains and a long cytoplasmic tail 1 (KIR, CD158E1); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 1 (KIR2DL1); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 2 (KIR2DL2); immunoglobulin-like killer cell receptor, two Ig domains and a long cytoplasmic tail 3 (KIR2DL3); killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1); killer cell lectin-like receptor C1 (KLRC1, NKG2A, CD159A); and killer cell lectin-like receptor D1 (KLRD1, CD94).

In some embodiments, the kits comprise one or more agonists or activators of one or more NK cell stimulatory immune checkpoint proteins or receptors. In various embodiments, the NK cell stimulatory immune checkpoint proteins or receptors are selected from CD16, CD226 (DNAM-1); lectin-like killer cell receptor K1 (KLRK1, NKG2D, CD314); and SLAM family member 7 (SLAMF7).

In various embodiments of the kits, one or more immune checkpoint inhibitors comprise a protein inhibitor of PD-L1 (CD274), PD-1 (PDCD1), or CTLA4. In some embodiments, the CTLA4 protein inhibitor is selected from the group consisting of ipilimumab, tremelimumab, BMS-986218, AGEN1181, AGEN1884, BMS-986249, MK-1308, REGN-4659, ADU-1604, CS1002, BCD-145, APL-509, JS-007, BA-3071, ONC-392, AGEN-2041, JHL-1155, KN-044, CG-0161, ATOR1144, PBI-5D3H5, FPT-155 (CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), XmAb-20717 (PD-1/CTLA4) and AK-104 (CTLA4/PD-1). In some embodiments, the PD-L1 (CD274) or PD-1 (PDCD1) protein inhibitor is selected from the group consisting of zimberelimab (AB122), pembrolizumab, nivolumab, cemiplimab, pidilizumab, AMP-224, MEDI0680 (AMP-514), spartalizumab, atezolizumab, avelumab, ASC22, durvalumab, BMS-936559, CK-301, PF-06801591, BGB-A317 (tislelizumab), GLS-010 (WBP-3055), AK-103 (HX008), AK-105, CS-1003, HLX-10, MGA-012, BI-754091, AGEN-2034, JS-001 (toripalimab), JNJ-63723283, genolimzumab (CBT-501), LZM-009, BCD-100, LY-3300054, SHR-1201, SHR-1210 (camrelizumab), Sym021, ABBV-181, PD1-PIK, BAT-1306, (MSB0010718C), CX-072, CBT-502, TSR-042 (dostarlimab), MSB2311, JTX-4014, BGB-A333, SHR-1316, CS-1001 (WBP-3155, KN-035, IBI-308 (sintillimab), HLX-20, KLA167, STI-A1014, STI-A1015 (IMC-001), BCD-135, FAZ-053, TQB-2450, MDX1105-01, FPT-155 (CTLA4/PD-L1/CD28), PF-06936308 (PD-1/CTLA4), MGD-013 (PD-1/LAG-3), FS-118 (LAG-3/PD-L1) MGD-019 (PD-1/CTLA4), KN-046 (PD-1/CTLA4), MEDI-5752 (CTLA4/PD-1), RO-7121661 (PD-1/TIM-3), XmAb-20717 (PD-1/CTLA4), AK-104 (CTLA4/PD-1), M7824 (PD-L1/TGFe-EC domain), CA-170 (PDL1/VISTA), CDX-527 (CD27/PD-L1), LY-3415244 (TIM3/PDL1), and INBRX-105 (4-1BB/PDL1). In some embodiments, the one or more immune checkpoint inhibitors comprise a low

- 90 047988 a molecular inhibitor of CD274 (PDL1, PD-L1), programmed cell death protein 1 (PDCD1, PD1, PD-1) or CTLA4. In some embodiments, the small molecule inhibitor of CD274 or PDCD1 is selected from the group consisting of GS-4224, GS-4416, INCB086550 and MAX10181. In some embodiments, the small molecule inhibitor of CTLA4 comprises BPI-002.

In various embodiments, the kit comprises one or more antiviral agents. Exemplary antiviral agents that may be present in the kit include lamivudine (LAM), adefovir dipivoxil (ADV), entecavir (ETV), telbivudine (LdT), tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF or VEMLIDY®), and ledipasvir + sofosbuvir (HARVONI®). In some embodiments, the kit comprises one or more therapeutic agents selected from the group consisting of HBV antigen inhibitors (e.g., HBV human antigen (HBcAg) inhibitors, HBV surface antigen (HBsAg) inhibitors, HBx inhibitors, HBV E antigen inhibitors), HBV antigen antibodies, HBV-targeted inhibitory nucleic acids (e.g., antisense oligonucleotide, short interfering RNA (siRNA), DNA-directed RNA interference (ddRNAi), HBV-targeted gene editors (e.g., CRISPR-Cas (e.g., Cas9, Cas12, Cascade, Cas13), zinc finger nucleases, homing endonucleases, homing meganucleases (e.g., aRCUS), synthetic nucleases, TALEN), covalently closed circular DNA (cccDNA) inhibitors and inhibitors of HBsAg secretion or assembly and inhibitors of HBV virus entry.

If required, notice may be provided associated with such container(s) in a form prescribed by the governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products that reflects the agency's approval for manufacture, use, or sale for human administration.

Examples

The following examples are provided for illustrative purposes only and are not intended to limit the claimed invention.

Example 1. Identification of HBV sAg sequences that induce robust genotype-cross-reactive T cell responses

In this example, near-consensus, naturally occurring HBV sAg sequences in genotypes A, B, C, and D were identified, adenovirus type 5 vectors encoding each antigen were generated, and the intensity and cross-reactivity of T cells induced by each of these vectors in outbred mice were tested for genotype.

Selection of near-consensus, naturally occurring HBV sAg sequences

In selecting a specific HBV sAg amino acid sequence that could be used for therapeutic vaccination, attention was paid to an sAg sequence that was efficiently expressed and processed for antigen presentation while also eliciting T cell responses that are broadly responsive to a range of HBV genotypes. Although consensus sequences or mosaic antigens can be designed to attempt to improve T cell genotype reactivity, such sequences are not found in nature and are at risk of being inefficiently expressed or poorly processed into T cell epitopes. Therefore, near-consensus naturally occurring sAg sequences from HBV genotypes (GT) A, B, C, and D were identified. Using a database of sAg sequences from 14,207 patients infected with these HBV genotypes, consensus sequences were constructed for each genotype, and the naturally occurring sAg sequence closest to the consensus was then identified for each genotype. The naturally occurring near-consensus sAg sequences for HBV genotypes A, B, C, and D are presented in Table 1 as SEQ ID NOs: 1–4, respectively.

- 91 047988

Table 1. Naturally occurring, near-consensus sAg polypeptide sequences

SEQ ID NO: HBV genotype Polypeptide sequence 1 A MENITSGFLGPLLVLQAGFFLTRILTIPQSLDSWWTSLNFLGGTPVC LGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLV LLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTD GNCT CIPIPS SWAFAKYLWEWASVRF SWL S LLVP FVQW FVGL S PTVWL S VIWMMWYWGPSLYNILSPFIPLLPIFFCLWVYI 2 IN MESTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGAPTC PGQNLQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLV LLDYQGMLPVCPLIPGSSTTSTGPCRTCTTPAQGTSMFPSCCCTKPTD GNCT CIPIPS SWAFAKYLWEWASVRF SWL S LLVP FVQW FVGL S PTVWL WMMWYWGPSLYNILSPFMPLLPIFFCLWVYI 3 WITH MESTTSGFLGPLLVLQAGFFLTRILTIPQSLDSWWTSLNFLGGAPTC PGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLCILLLCLIFLLV LLDYQGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTD GNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFVGLSPTVWL SVIWMMWYWGPS LYNILSPFLPLLPIFFCLWVYI 4 D MENITSGFLGPLLVLQAGFFLTRILTIPQSLDSWWTSLNFLGGTTVC LGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLV LLDYQGMLPVCPLIPGSSTTSTGPCRTCTTPAQGTSMYPSCCCTKPSD GNCT CIPIPS SWAFGKFLWEWASARF SWL S LLVP FVQW FVGL S PTVWL MMWYWGPSLYSILSPFLPLLPIFFCLWVYI

Methods

To assess the immunogenicity of each antigen and to evaluate the genotype cross-reactivity of induced T cells to a wide range of epitopes in vivo, outbred mice from Jackson Laboratories were used for vaccination. Outbred mice were generated by random cross-mating of 160 recombinant inbred mouse strains and the colony is maintained by continuous random matings that prevent mating between siblings and mixtures. Outbred parental strains were developed by crossing eight unique and genetically diverse mouse strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Thus, outbred mice exhibit diversity in epitope selection and magnitude of T cell responses present in a highly diverse genetic population.

Results

All four naturally occurring, near-consensus HBV sAg sequences were robustly immunogenic in outbred mice (Fig. 1). Induced T cells responded to the GT-A, B, C, and D HBV sAg peptides approximately equally, demonstrating excellent genotype cross-reactivity of the T cell response. The geometric mean of the responses was greatest for GT-C and GT-D sAg.

Example 2. Identification of HBV capsid and Pol sequences that induce robust genotype-cross-reactive T cell responses

In this example, near-consensus, naturally occurring HBV capsid and HBV polymerase (Pol) sequences in genotypes A, B, C, and D were identified, adenovirus 5 expression vectors encoding Pol antigens or capsid-Pol fusion proteins were generated, and tested for intensity and cross-reactivity in genotype T cells induced in inbred and outbred animals.

Selection of near-consensus, naturally occurring HBV capsid and Pol sequences. In selecting a specific amino acid sequence of HBV capsid and Pol antigens that could be used for therapeutic vaccination, attention was paid to capsid and Pol sequences that were efficiently expressed and processed for antigen presentation while also inducing T cell responses that were broadly responsive to a range of HBV genotypes. Although consensus sequences or mosaic antigens could be designed to attempt to improve T cell genotype reactivity, such sequences are not naturally occurring and are at risk of being inefficiently expressed or poorly processed into T cell epitopes. Therefore, close-to-consensus naturally occurring capsid and Pol sequences of HBV from genotypes A, B, C, and D were identified. Using a database of core sequences from 5528 individuals infected with HBV genotypes A-D and Pol sequences from 4713 patients infected with HBV A-D, consensus sequences for capsid and Pol were constructed for each genotype, and then the naturally occurring capsid and Pol sequences closest to the consensus were identified for each genotype.

The GT-A, B, C, and D Pol sequences were then modified to improve the antigen characteristics. The enzymatic activity of polymerases can induce toxicity when overexpressed, so the enzymatic activity of the reverse transcriptase (RT) and RNase H (RNH) domains

- 92 047988 was deleted by mutations in the catalytic domains. The YMDD motif in RT was mutated to YMHD, and the AELL motif in RNH was mutated to AHLL (Radziwill, et al., j Virol. (1990) 64(2):613-20). The resulting Pol sequences are called Po1 ^mut . The Po1 ^mut sequences for HBV genotypes A, B, C, and D are presented in Table 2 as SEQ ID NOs: 52-55, respectively.

Table 2. Polypeptide sequence of Pol ^mut

SEQ ID NO: HBV genotype Polypeptide sequence - Motifs containing inactivating mutations in Pol are underlined (YMDD mutated to YMHD, AELL mutated to AHLL) 52 A MPLSYQHFRKLLLLDDETEAGPLEEELPRLADEDLNRRVAEDLNLGNLNVSIPWTH KVGNFTGLYSSTVPIFNPEWQTPSFPKIHLHEDIANRCQQFVGPLTVNEKRRLRLIMP ARFYPNSTKYLPLDKGIKPYYPDHVVNHYFQTRHYLHTLWKAGILYKRETTRSASF CGSPYSWEQELHHGRLVIK TSQRHGDEPFCSQPSGILSRSSVGPCIRSQFKQSRLGLQ PHQGPLATSQSGRSGSIRARVHSPTRRCFGVEPSGSGHIGHSASSSSSCLHQSAVRKA AYSHLSTSKRQSSSGHAVEFHSFPPSSARSQSQGPVFSCWWLQFRNTQPCSKYCLSH LVNLLEDWGPCDEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRG ITRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHIPLHPAAMPHLLVGSSGLS RYVARLSSNSRIHNNQHGTLQNLHDSCSRQLYVSLMLLYKTYGRKLHLYSHPIILGF RKIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFS YMHDVVLGAKSVQHLESLYTA VTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTLPQDHIVQKIKHCFRKLPINR PIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYKAFLSKQYLN LYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGTFVAPLPIHTAHLLAACFARSR SGAKLIGTDNSVVLSRKYTSFPWLLGCTANWILRGTSFVYVPSALNPADDPPSRGRLG LYRPLLLRLPYRPTTGRTSLYAVSPSVPSHLPVRVHFASPLHVAWRPP 53 IN MPLSYQHFRKLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLGNLNVSIPWTHKV GNFTGLYSSTVPVFNPEWQTPSFPHIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPAR FYPNLTKYLPLDKGIKPYYPEHVVNHYFQTRHYLHTLWKAGILYKRESTRSASFCGS PYSWEQDLQHGRLVFQTS KRHGDKSFCPQSPGILPRSSVGPCIQNQLRKSRLGPQPA QGQLAGRQQGGSGSIRARVHPSPWGTVGVEPSGSGHIHNCASNSSSCLHQSAVRKA AYSHISTSKGHSSSGHAVELHHFPPSSSRSQSQGPVLSCWWLQFRNSEPCSEYCLCHI VNLIEDWGPCTEHGEHRIRTPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGN TRVSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSR YVARLSSNSRIINNQHRTMQNLHDSCSRNLYVSLMLLYKTYGRKLHLYSHPIILGFR KIPMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYMHD WLGAKSVQHLESLYAA VTNFLLSLGIHLNPHKTKRWGYSLNFMGYVIGSWGTLPQEHIVQKIKMCFRKLPVN RPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSPTYKAFLSKQYL HLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGAFVSPLPIHTAHLLAACFARS RSGAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPSRGRL GLYRPLLRLLYRPTTGRTSLYADSPSVPSHLPDRVHFASPLHVAWRPP 54 WITH MPLSYQHFRKLLLLLDDEAGPLEEELPRLADEDLNRRVAEDLNLGNLNVSIPWTHKV GNFTGLYSSTVPVFNPEWQTPSFPHIHLQEDIINRCQQYVGPLTVNEKRRLKLIMPAR FYPNLTKYLPLDKGIKPYYPEHTVNHYFKTRHYLHTLWKAGILYKRETTRSASFCGS PYSWEQELQHGRLVFQTSTR HGDESFCSQSSGILSRSPVGPCIRSQLKQSRLGLQPQQ GSLARSKSGRSGSIRARVHPTTRQSFGVEPSGSGHIDNSASSASSCLHQSAVRKTAYS HLSTSKRQSSSGHAVELHNFPPSSARSQSEGPLLSCWWLQFRNSKPCSDYCLSHIVN LLEDWGPCTEHGEHNIRIPRTPARVTGGVFLVDKNPHNTTESRLVVDFSQFSRGSTH VSWPKFAVPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRY VARLSSTSRNINYQHGAMQDLHDSCSRNLYVSLLLLYKTFGRKLHLYSHPIILGFRKI PMGVGLSPFLLAQFTSAICSVVRRAFPHCLAFSYM HDVVLGAKSVQHLESLFTAVTNFLLSLGIHLNPNKTKRWGYSLNFMGYVIGSWGTL PQEHIVLKIKQCFRKLPVNRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQA KQAFTFSPTYKAFLCKQYLNLYPVARQRSGLCQVFADATPTGWGLAVGHQRMRGT FVSPLPIHTAHLLAACFARSRS GAKLIGTDNSVVLSRKYTSFPWLLGCAANWILRGT SFVYVPSALNPADDPPSRGRLGLYRPLLRLPFRPTTGRTSLYAVSPSVPSHLPVRVHFA SPLHVAWRPP 55 D MPLSYQHFRRLLLLDDEAGPLEEELPRLADEGLNRRVAEDLNLNLNVSIPWTHKV GNFTGLYSSTWVFNPHWKTPSFPNIHLHQDIIKKCEQFVGPLTVNEKRRLQLIMPAR FYPNVTKYLPLDKGIKPYYPEHLVNHYFQTRHYLHTLWKAGILYKRETTHSASFCG SPYSWEQELQHGAESFHQ QSSGILSRPPVGSSLQSKHRKSRLGLQSQQGHLARRQQG RGWSIRAGIHPTARRPFGVEPSGSGHTANLASKSASCLYQSAVRKAAYPVVSTFKKH SSSGHAVELHNLPPNSARSQSERPVFPCWWLQFRNSKPCSDYCLSHIVNLLEDWGPC AEHGEHHIRIPRTPARVTGGVFLVDKNPHNTAESRLVVDFSQFSRGNYRVSWPKFA VPNLQSLTNLLSSNLSWLSLDVSAAFYHLPLHPAAMPHLLVGSSGLSRYVARLSSNS RIFNYQHGTMQNLHDSCSRNLYVSLMLLYQTFGRKLHLYSHPIILGFRKIPMGVGLS PFLLAQFTSAICSVVRRAFPHCLAFSYMHDVVLGA KSVQHLESLFTAVTNFLLSLGIH LNPNKTKRWGYSLHFMGYVIGCYGSLPQDHIIQKIKECFRKLPVNRPIDWKVCQRIV GLLGFAAPFTQCGYPALMPLYACIQSKQAFTFSPTYKAFLCKQYLNLYPVARQRPGL CQVFADATPTGWGLVMGHQRMRGTFKAPLPIHTAHLLAACFARSRSGANILGTDNS VVLSRKYTSFPWLLGCAANWILRGTSFVYVPSALNPADDPPSRGRLGLYRPLLRLPFR PTTGRTSLYADSPSVPSHLPDRVHFASPLHVAWRPP

Pol ^mut sequences were then further modified to remove amino acid regions that are poorly conserved among HBV strains and genotypes, to produce Pol sequences of varying lengths to accommodate viral vectors with different constraints on the encoded antigen size, and to create capsid-Pol fusion proteins to encode two antigens with a single open reading frame. Pol consists of four functional domains, the terminal protein (TP), spacer, RT, and RNH. Of these three, TP, RT, and RNH are highly conserved.

- 93 047988 are common among HBV strains and genotypes and are therefore likely to induce strain- and genotype-cross-reactive T cells, while the spacer domain is highly variable. GT-A, B, C, and D Pol sequences with deletions in the spacer region were generated. In one set of sequences, designated Ρο1 ^δ1 , the deletion was based on a previously reported deletion mutant that retains enzymatic function in vitro, indicating that the deletion does not disrupt the expression, structure, or folding of the rest of the protein (Radziwill, et al., J Virol. (1990) 64(2):613–20). In a second set of vectors, designated Ρο1 ^Δ3 , the entire low-conserved region was identified by sequence alignment and removed. Capsid-Pol fusion proteins were generated by fusing near-consensus capsid sequences to Po1 ^mut , Pol , and Pol sequences for GT-A, B, C, and D. Finally, to accommodate viral vectors with less packaging constraints, shorter versions of each sequence of an inactivated near-consensus form of Pol, designated Pol ³⁰⁰ , were constructed. Pol ³⁰⁰ variants have large N-terminal deletions in which the entire TP and most of the spacer domain are removed but the RT and RNase domains are retained (Lanford et al., J Virol. (1999);73(3):1885–93). A sequence list of Pol-containing antigens tested in adenovirus or arenavirus vectors is presented in Table 3 and Fig. 2. The amino acid sequences deleted from each Pol deletion construct are presented in SEQ ID NOs: 42–51.

Table 3. Sequences of Pol-containing antigens

Polypeptide SEQ ID NO: Polypeptide 5-8 for genotype A-D respectively Ρο1 ^Δ1 9-12 for genotype A-D respectively Ρο1 ^Δ3 13-14 for genotype B and D respectively Floor ³⁰⁰ 15-18 for genotype A-D respectively Capsid-Ro1 ^mut 19-22 for genotype A-D respectively Capsid-Ro1 ^L1 23-26 for genotype A-D respectively Capsid-Ro1 ^lz

Methods

The immunogenicity of each GT-A, B, C, and D capsid-Pol fusion construct was initially tested in C57BL/6 mice to induce T cell responses reactive with GT-D capsid and Pol peptide pools to identify the variant in each genotype that induced the greatest immunogenic response (Figure 3). All genotypes elicited robust Pol responses, but capsid responses were weaker or absent. The weak or absent capsid response likely resulted from the fact that C57BL/6 mice are known to respond to only one peptide from the HBV GT-D capsid, namely MGLKFRQL (Chiale, et al., Antiviral Res. 2019 Aug; 168:156-167). Responses to this peptide in C57BL/6 mice are often weak or absent, and the peptide has an alternative sequence in the GT-A, B, and C capsid sequences of MGLKIRQL.

Results

All antigen genotypes showed little change in immunogenicity between capsid-Pol ^mut and capsid-Pol ^Δ1 . GT-A antigen had an increased response to capsid-Pol ^Δ3 compared to capsid-Pol ^mut and capsid-Pol , while GT-B, C and D all showed reduced immunogenicity with capsid-Pol .

T cell responses in inbred mouse strains are not ideal for comparing antigen immunogenicity across genotypes because responses may be characterized by one or a few epitopes that may vary in sequence among antigens. To better compare the immunogenicity of capsid-Pol antigens across genotypes, immunogenicity was tested in outbred mice to obtain responses across a broad range of epitopes. Outbred mice were immunized with GT-A capsid-Pol ^Δ3 or GT-B, C, or D capsid-Pol ^Δ1 and T cell responses were assessed for IFN-γ ELISPOT responses using the GT-A and GT-D peptide pools (Fig. 4). GT-B capsid-Pol gave the best overall Pol responses, with equally robust ELISPOT responses to the GT-A and GT-D peptide pools (Fig. 4A). Pol responses to GT-B capsid-Pol ^Δ1 were statistically significantly higher than those to GT-A capsid-Pol using GT-D peptides and to GT-C capsidΡο1 ^Δ1 using both peptide genotypes. The geometric mean of Pol ELISPOT responses to GTD capsid-Po1 ^m was numerically lower compared to GT-B capsid-Po1 ^m , but the difference was not statistically significant. Capsid responses were clearly detected in outbred mice for all four antigen genotypes (Fig. 4B). The pattern of capsid responses was similar to that of Pol responses with GT-B capsid-Po1 ^m , yielding the best results overall, although for capsid, comparisons between antigen genotypes reached statistical significance.

Example 3. Identification of smaller immunogenic Pol antigens

Different viral vectors have different limits on the maximum size of encoded antigens.

- 94 047988

Methods

To identify additional Pol variants that are smaller in size and thus usable in a wider range of vector systems, the immunogenicity of Pol variants expressed without capsid fusion was assessed. C57BL/6 mice were immunized with an adenovirus type 5 vector encoding GT-D Ρο1 ^δ1 , Ρο1 ^Δ3 and Pol ³⁰⁰ and GT-B Pol ³⁰⁰ and compared with a control vector encoding full-length unmodified GT-D polymerase (GT-D Pol ^Ctrl ) and mock vaccination with phosphate-buffered saline (PBS) as a negative control. IFN-γ ELISPOT responses were measured 14 days after immunization with GT-D Pol peptide pools (Fig. 5).

Results

All Pol antigen constructs tested were immunogenic with no statistically significant differences between groups.

Example 4. Efficacy of vaccination with close to consensus antigens in combination with an antibody to PD-1 in adeno-associated virus (AAV)-HBV

An adeno-associated virus (AAV)-HBV model (Dion, et al., J Virol. (2013) 87(10):5554-63; and Yang, et al., Cell Mol Immunol. (2014) 11(1):71-8) was used to determine whether the near-consensus antigen constructs described herein could have antiviral effects in a chronic HBV infection model.

Methods

In this model, C57BL/6 mice were transduced with AAV vectors encoding the 1.2x HBV GT-D gene, resulting in robust production of HBV protein and virions in hepatocytes, accompanied by serum antigenemia and viremia. Heterologous priming/boosting viral vectors consisting of a primary adenovirus (Ad) and a poxvirus boosting buffer have been shown to elicit potent T cell responses in humans (see, e.g., Barnes, et al., Sci Transl Med. (2012) 4(115):115ral; Ewer, et al., N Engl J Med. (2016) 374(17): 1635–46; Ewer, et al. Nat Commun. (2013) 4:2836; Green, et al., Sci Transl Med. (2015) 7(300):300ral26; Swadling, et al., Sci Transl Med. (2014) 6(261):261ral53), so vaccinia vectors based on the Western Reserve strain were generated (NCBI:xid696871) expressing GT-C sAg and GT-B capsid. Mice were vaccinated with AAV-HBV primary Ad5 and ^vaccinia boosting vector encoding GT-C sAg and GT-B capsid or irrelevant control antigens ^beta -galactosidase and green fluorescent protein. Mice were additionally treated with anti-PD-1 mAb or isotype control antibody after booster vaccination. The design of this AAV-HBV efficacy study is shown in Fig. 6 and the experimental groups are shown in Table 4. The control group received HBV vaccine but no AAV-HBV to determine whether vaccine responses were reduced in the presence of persistent HBV.

Table 4. Study groups in the AAV-HBV efficacy study

Group N AAV- HBV Priming Reinforcing Antibody 1 12 Y Αά-β-gal Vac-GFP Control isotype 2 12 Y Ad-sAg GT-C A0-capsid-po1 ^L1 GT-B Vac-sAg GT-C Vac-capsid-ρο1 ^Δ1 GT-B Control isotype 3 12 Y Αά-β-gal Vac-GFP anti-PD-l 4 12 Y Ad-sAg GT-C Ab-capsid-ro!' ¹ GT-B Vac-sAg GT-C Vac-capsid-ρο1 ^Δ1 GT-B anti-PD-l 5 12 N Ad-sAg GT-C Ad-Kancm-pol' ¹ GT-B Vac-sAg GT-C Vac-capsid-ρο1 ^Δ1 GT-B No

Ad: Adenovirus Vector 5

Vac: vaccinia vector β-gal: beta-galactosidase GFP: green fluorescent protein

Results

Figure 7 shows the IFN-γ ELISPOT responses in each group. It should be noted that responses were assessed using GT-D peptide pools corresponding to the HBV strain in the AAV-HBV vector, so T cell responses are detected only when exposed to virus present in the AAV-HBV mice. Capsid responses were tested, but none were detected in any group, consistent with the low immunogenicity of the capsid in C57BL/6 mice (Chiale, et al., supra). Robust Pol ELISPOT responses were detected in all groups primed with Ad and boosted with vaccinia vectors encoding HBV antigens. Pol ELISPOT magnitudes were similar in AAVHBV mice and control mice that did not receive AAV-HBV, indicating that AAV-HBV does not induce T cell tolerance to Pol. In contrast, sAg ELISPOT responses were significantly reduced in AAV-HBV mice compared with control mice, suggesting that AAV-HBV induces T cell tolerance to sAg. However, in mice that received AAV-HBV and were primed with adenovirus and boosted with vaccinia, 2-3 mice per group demonstrated that sAg ELISPOT responses were higher than those found in control mice vaccinated with control. ELISPOT response values were not altered by administration of anti-PD-1 antigen.

To assess any antiviral effects of HBV T cells induced by vaccination, serum HBeAg antigen was measured. Serum HBeAg is a better marker of T cell-mediated antiviral efficacy than serum HBsAg, as the latter can be reduced by vaccination-induced anti-HBsAg antibodies. Neither HBV vaccine alone nor anti-PD-1 antibody caused any reduction in serum HBeAg compared to mice receiving control vaccine and isotype control antibody. However, the combination of HBV vaccine and anti-PD-1 antibody resulted in a loss of detectable serum HBeAg in 4 of 12 mice (Fig. 8). These data demonstrate that vaccination with viral vectors encoding the improved antigen sequences described herein promotes HBV clearance as part of a combination therapy strategy.

Example 5. Immunogenicity of Pol antigens in arenavirus vectors

The HBV antigen constructs of the present invention have been further improved for use in arenavirus vectors. Unlike adenovirus vectors and most other viral vector systems, arenavirus vectors can be administered multiple times without inducing neutralizing antibodies to the vector. Furthermore, arenavirus vectors can be produced in several variants that differ with respect to the parent virus used to produce the vector, such as replication-incompetent with a two-segment genome (Flatz, et al., Nat Med. (2010) 16(3):339-45) or replication-attenuated with a three-segment (trisegmented) genome (Kallert, et al., Nat Commun. (2017) 8:15327) (Fig. 9). Specific HBV antigens were expressed in three-segmented replication-attenuated or two-segmented replication-defective arenavirus platforms with the lymphocytic choriomeningitis mammarenavirus (LCMV) or Kali mammarenavirus (LCMV) (also known as Pichinde mammarenavirus or Pichinde arenavirus (PICV)) backbone vector. The replication-defective arenavirus vectors used are described in WO 2009/083210. The replication-attenuated arenavirus vectors used are described in WO 2016075250 (LCMV) and WO 2017/198726 (Pichinde).

Arenaviral vectors can accept antigens of approximately 500-800 amino acids in the open reading frame. Therefore, GT-D and GT-B Pol ^A1 (SEQ ID NOs: 6 and 8), Ρο1 ^Δ3 (SEQ ID NOs: 10 and 12), and Pol ³⁰⁰ (SEQ ID NOs: 13 and 14) were tested for immunogenicity in replication-incompetent LCMV vectors. C57BL/6 mice were immunized intravenously with 106 focus-forming units (FFU) of replication-incompetent LCMV vectors, and IFN-γ ELISPOT responses were measured at day 7 post-immunization. All GT-B and GT-D Pol ³⁰⁰ antigens induced robust T cell responses, while GT-D Ρο1 ^Δ1 and Pol ^Δ3 induced reduced ELISPOT responses compared to the other antigen constructs (Fig. 10).

Example 6. Identification of genetically stable replication-incompetent LCMV receptors encoding immunogenic Pol antigens

The stability of various immunogenic Pol transgenes in replication-incompetent LCMV (VV1) vectors was assessed by polymerase chain reaction (PCR) after serial passaging of the vector containing the supernatant. Genetic stability was determined by the major band indicating the correct size of the full-length transgene (TG). The results are presented in Table 6.

Table 6. Comparative table for assessing the genetic stability of Pol transgenes

*VV1 denotes replication-incompetent LCMV vectors

P# stands for the number of passages (for example, P1 equals 1 passage)

Example 7. Immunogenicity of capsid-sAg fusion proteins in replication-incompetent LCMV vectors

Following the identification of stable immunogenic arenavirus vectors encoding HBV Pol, a series of capsid-sAg fusion proteins were further tested for immunogenicity in replication-incompetent LCMV vectors. Capsid-sAg fusions were generated by fusing a near-consensus

- 96 047988 a GT-B capsid and GT-C sAg or a GT-D capsid and GT-D sAg with the capsid at the N-terminus and sAg at the C-terminus. Direct fusions are expected to elicit T cell responses but may not induce antibodies to sAg since the fusion protein does not secrete sAg. Therefore, additional antigen constructs were tested with the capsid and sAg separated by a GSG linker and then with a porcine teschovirus-1 (P2A)-derived translational skip 2A site (Kim, et al., PLoS ONE. (2011) 6:e18556). This orientation will yield a 21-amino acid extension at the C-terminus of the capsid while allowing normal sAg secretion to elicit antibody responses. Sequence identification numbers for the amino acid sequences of antigens tested in arenavirus vectors and the nucleotide sequences used to encode antigens in arenavirus vectors are presented in Table 7.

Table 7. Sequences of vector antigens and antigen-encoding genes __________used in LCMV vectors__________

Polynucleotide SEQ ID NO: Polypeptide SEQ ID NO: Polypeptide 27 6 GT-B ROG ¹ 28 10 GT-B Ρο1 ^Δ3 29 13 GT-B Pol ³⁰⁰ 30 8 GT-D Ρο1 ^Δ1 31 12 GT-D Ρο1 ^Δ3 32 14 GT-D Pol ³⁰⁰ 33 38 GT-B/C capsid-sAg 34 39 GT-B/C Kancmi-P2A-sAg 35 40 GT-D capsid-sAg 36 41 GT-D Kancmi-P2A-sAg 37 41 GT-D iCore-P2A-sAg

Replication-incompetent LCMV vectors encoding capsid-sAg variants were tested for immunogenicity by immunization of C57BL/6 mice (Fig. 11). The overall abundance of HBV-specific IFN-γ ELISPOT responses was indistinguishable for all vectors tested, and inclusion of the P2A region did not affect ELISPOT responses to GT-B/C or GT-D antigens. Both capsid and sAg responses were observed for all vectors tested. Detection of capsid responses was notable since T cell-based responses are generally weak and difficult to detect in this mouse strain (Chiale, et al., supra). Similar results were observed in Balb/c mice immunized with the same vectors.

Because antibody responses develop more slowly than T cell responses following vaccination with a replication-incompetent LCMV vector, an additional set of C57BL/6 mice were immunized and antibody responses were measured at day 17 post-immunization (Fig. 12). As expected, direct capsid-sAg fusions did not elicit sAg antibody responses. Among the P2A-containing constructs, only the GT-D capsid-P2A-sAg vector consistently induced anti-sAg antibodies, whereas anti-sAg antibodies were observed in only one of five mice immunized with GT-B/C capsid-P2A-sAg. This result was unexpected, since Western blots demonstrated efficient separation of capsid and sAg in both the GT-D and GT-B/C P2A-sAg vectors. To confirm that the difference in sAg antibody responses was not an artifact of the mouse strain selected, the same experiment was performed in Balb/c mice. The results in Balb/c mice were similar to those in C57BL/6 mice: sAg antibodies were detected in 4 of 5 Balb/C mice immunized with GT-D capsid-P2A-sAg, but only 1 of 5 mice immunized with GT-B/C capsid-P2A-sAg (Fig. 12).

Example 8. Identification of genetically stable replication-incompetent LCMV receptors encoding immunogenic capsid-sAg fusion proteins

The stability of different immunogenic capsid-sAg fusion transgenes in replication-incompetent LCMV (VV1) vectors was assessed by PCR after serial passaging of the vector containing the supernatant. Genetic stability was determined by the major band indicating the correct size of the full-length transgene (TG). The results are presented in Table 8.

- 97 047988

Table 8. Comparative table for assessing the genetic stability of capsid-sAg transgenes

Genotype Vector Stable TG insertion up to GT-B/C W1-capsid-sAg P6 GT-B/C W1-capsid-P2 A-sAg P7 GT-D VV1-capsid-sAg P4 GT-D VV1 -capsid-P2 A-sAg P2 GT-D VVl-iCore-P2 A-sAg P6

*VV 1 denotes replication-incompetent LCMV vectors P# denotes the number of passages (e.g. P1 equals 1 passage)

GT-D Capsid-P2A sAg induced robust T cell responses and the highest anti-sAg antibody responses of the capsid-sAg fusion constructs tested, but did not have favorable genetic stability in this assay. However, the modified GT-D iCore-P2A-sAg transgene (polynucleotide SEQ ID NO:37 encoding the polypeptide SEQ ID NO:41) demonstrated improved genetic stability in the replication-incompetent LCMV vector (Table 8).

To confirm that the modified transgene does not compromise the immunogenicity of GT-D iCore-P2A-sAg T cells, C57BL/6 mice were immunized with replication-incompetent LCMV vectors carrying the GT-D capsid-P2A-sAg and GT-D iCore-P2A-sAg constructs, or mock immunized, and T cell responses were measured 7 days later by IFN-γ ELISPOT (Fig. 13). sAg ELISPOT responses were significantly higher with GT-D iCore-P2A-sAg, and capsid ELISPOT responses were also higher. Thus, the modified GT-D iCore-P2A-sAg transgene resulted in both improved genetic stability and improved immunogenicity.

Example 9.

Immunogenicity of replication-incompetent LCMV vectors in outbred mice The immunogenicity of replication-incompetent LCMV (VV1) vectors encoding various HBV antigens was assessed in outbred mice. These mice have more diverse MHC alleles than inbred C57BL/6 mice and are therefore better suited to assess genotype cross-reactivity of vaccination-induced T cell responses.

Methods

Outbred mice were immunized twice at day 0 and day 28 with replication-incompetent LCMV vectors as described in Table 9. HBV-specific T cell responses were measured at day 42 by IFN-γ ELISPOT using splenocytes.

Table 9. Study groups in the immunogenicity study

Group N Priming vector - day 0 Reinforcing vector - day 28 Selection day Dose/vector 1 8 Imitation Imitation 42 - 2 8 W1-GT-B/C capsid-P2 A-sAg VV1-GT-B/C Kanciu-P2A-sAg 42 10 ^b FUE 3 8 Wl-GT-D iCore-P2A-sAg Wl-GT-D iCore-P2A-sAg 42 10 ^b FUE 4 8 VV1-GT-B Ρο1 ^Δ3 Wl-GT-B Ρο1 ^Δ3 42 10 ^b FUE 5 8 Wl-GT-B Pol ³⁰⁰ Wl-GT-B Pol ³⁰⁰ 42 10 ^b FUE

Results

Replication-incompetent LCMV vectors encoding GT-B/C capsid-P2A-sAg and GT-D iCore-P2A-sAg induced comparable T cell responses specific for their respective capsid antigen (Fig. 14A). The vector encoding GT-D iCore-P2A-sAg induced a higher frequency of T cells specific for its respective sAg antigen compared to the vector encoding GT-B/C capsid-P2A-sAg (Fig. 14A). The vector encoding GT-B Pol ³⁰⁰ induced Pol antigen-specific T cell responses numerically superior to the vector encoding GT-B Pol ³³ (Fig. 14B). Thus, vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ are more immunogenic than vectors encoding GT-B/C capsid-P2A-sAg and GT-B Pol ³³ in outbred mice.

In addition to inducing T cell responses specific to their recognized antigens (i.e., GT-D capsid antigens, GT-D sAg, GT-B Pol), the GT-D iCore-sAg and GT-B Pol ³⁰⁰ vectors are also capable of generating T cell responses specific to antigens derived from different HBV viral genotypes (i.e., GT-B capsid antigens, GT-B sAg, GT-D Pol) (Fig. 15A and 15B). Thus, vectors encoding GT-D iCore-sAg and GT-B Pol ³⁰⁰ produce T cells that cross-react for different HBV genotypes.

- 98 047988

Example 10. Immunogenicity of replication-incompetent LCMV vectors administered as a single vector or co-formulated in C57BL/6 mice

Replication-incompetent LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Po1300 are immunogenic in mice. The immunogenicity of both vectors was then compared when delivered either as individual vectors or as a co-formulated mixture in C57BL/6 mice.

Methods

C57BL/6 mice were immunized twice at day 0 and day 21 with replication-incompetent LCMV vectors as described in Table 10. HBV-specific T cell responses were measured at day 28 by IFN-γ ELISPOT using splenocytes.

Table 10. Study groups in the immunogenicity study

Group N Vector format Priming vector - day 0 Reinforcing vector - day 21 Selection day Dose/vector 1 5 - Imitation Imitation 28 ΙΟ ⁶ FOB 2 5 One vector VV1-GT-D iCore_P2A_sAg VV1-GT-D iCore_P2A_sAg 28 ΙΟ ⁶ FOB 3 5 One vector VV1-GT-B Pol ³⁰⁰ VV1-GT-B Pol ³⁰⁰ 28 ΙΟ ⁶ FOB 4 5 Co-written VV1-GT-D iCore_P2A_sAg + VV1-GT-B Pol ³⁰⁰ VV1-GT-D iCore_P2A_sAg + VV1-GT-B Pol ³⁰⁰ 28 ΙΟ ⁶ FOB

Results

Consistent with the data described above, vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ induced sAg-, capsid-, and Pol-specific T cell responses when administered as vectors alone (Figs. 16A–16C). Administration of the same vectors as a co-formulated mixture induced comparable T cell responses (Figs. 16A–16C). Thus, the combination of LCMV vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ does not interfere with their immunogenicity in C57BL/6 mice.

Example 11. Immunogenicity of replication-incompetent LCMV vectors in cynomolgus macaques

The immunogenicity of replication-incompetent LCMV (VV1) vectors GT-D iCoreP2A-sAg and GT-B Pol ³⁰⁰ was assessed in cynomolgus macaques. Ad5 and vaccinia vectors encoding capsid antigens, sAg and Pol ³⁰⁰ were also tested.

Methods

Cynomolgus macaques were immunized using different routes, different doses, and different immunization schedules as listed in Table 11. HBV-specific T cell responses were measured in PBMCs every 2 weeks by IFN-γ ELISPOT. Intracellular cytokine staining was also performed on CD4+ and CD8+ T cells at week 14 by flow cytometry. Anti-sAg antibody responses were quantified every 4 weeks by ELISA.

Table 11. Study groups in the immunogenicity study

Group N Vaccine Dose Path Immunization schedule (week) 1 5 Wl-GT-D iCore-P2A-sAg + VV1-GT-B Pol ³⁰⁰ 5x10 ^b FOU/vector v/m Every 4 weeks: 0, 4, 8, 12, 16, 20 2 5 10 ⁸ FFU/vector v/m Every 4 weeks: 0, 4, 8, 12, 16, 20 3 5 5x10 ^b FOU/vector v/m Every 8 weeks: 0, 8, 16, 24 4 5 10 ⁸ FFU/vector v/m Every 8 weeks: 0, 8, 16, 24 5 5 10 ⁸ FFU/vector in/in Every 8 weeks: 0, 8, 16, 24 6 5 1. Ad5-GT-D capsid-sAg + Ad5-GT-B Pol ³⁰⁰ (days 0 and 5) 10 ¹¹ RF/vector v/m 0 (Ad5), 4 (Ad5), 8 (smallpox), 12 (smallpox) 2. Vaccinia vaccine GT-D capsid-sAg + vaccinia vaccine GT-B Pol ³⁰⁰ (days 8 and 12) 10 ⁸ PFU/vector

Results

The overall proportion of HBV-specific T cell responses (defined as the sum of capsid, sAg, and polymerase-specific responses shown in Figs. 18A–18F to 2OA–2OF) to the VV1 GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ vectors was highest when administered intramuscularly (IM) and every 4 weeks (Groups 1 and 2) (Fig. 17A–B). Ad5 and vaccinia vectors encoding the same antigens also

- 99 047988 induced comparable T cell responses. HBV-specific immune responses were detected after the first dose of VV1 GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ vectors, and doses two to four induced progressive increases in HBV-specific ELISPOT values. The fifth and sixth doses did not further increase the responses, indicating that the peak response to the vectors of the present invention was achieved after the fourth dose. The geometric mean response at week 14 was 1206-SFU/10 ⁶ PBMC in animals administered the full human dose (10 ⁸ FFU, Group 2), and approximately 2-fold lower at the lower dose of 5x10 ⁶ FFU (Group 1), indicating a dose-response capacity.

To quantify the contribution of CD4+ and CD8+ T cells to the overall T cell response, PBMCs from animals in groups 1, 2, and 6 were analyzed by intracellular cytokine staining (ICS) at week 14 of the study, when T cell responses were highest. Both groups 1 and 2 had elevated levels of IFN-Y+ CD8+ T cells in response to stimulation with HBV peptides. The background correction frequency of these cells ranged from 0.8 to 1.9% in group 1 and from 0.2 to 4% in group 2 (Fig. 21A). In contrast, IFN-Y+ CD4+ T cells specific for HBV were detectable but constituted less than 0.1% of total CD4+ T cells (Fig. 21B). Thus, the T cell response induced by the vectors of the present invention in non-human primates is predominantly composed of CD8+ T cells.

Anti-HBsAg antibodies were also induced by administration of the vectors of the present invention. Anti-sAg responses increased with dose level and with repeated administration of the vectors (Fig. 22).

Example 12. Immunogenicity of replication-incompetent LCMV vectors in combination with immunomodulators in C57BL/6 mice

The immunogenicity of replication-incompetent LCMV (VV1) vectors GT-D iCoreP2A-sAg and GT-B Po1300 alone or in combination with various immunomodulators (anti-PD-i, anti-CTLA-4 and anti-CD137 and FLT3 ligand) was assessed in a murine AAV-HBV model.

Methods

AAV-HBV C57BL/6 mice were treated with 3 doses of VV1-GT-D iCore-P2A-sAg and GT-B Po1300 vectors on days 0, 21, and 42. Mice also received saline, anti-mouse PD-1 inhibitory antibody, anti-mouse CTLA-4 inhibitory antibody, anti-mouse CD137 stimulatory antibody, and mouse FLT3-L as indicated in Table 12 and Fig. 23. Control mice received only HBV vaccine but no AAV-HBV to determine the impact of HBV vaccine immunogenicity in the context of chronic HBV infection. HBV-specific T cell responses were measured at 105 days post-primary vaccination using splenocyte-based IFN-γ ELISPOT. Data were expressed after subtraction of background signal in control wells without peptides. Serum HBeAg levels were measured at day -11 and day 105 by ELISA.

Table 12. Study groups in the AAV-HBV immunogenicity study

Group N AAV- HBV HBV vaccine immunomodulator Molecule and Dose 1 11 Yes Wl-GT-D iCore-P2A-sAg + Wl-GT-B Pol ³⁰⁰ Carrier Saline solution 2 12 Yes anti-PD-l CloneIMR 1-14 8 mg/kg/dose 3 12 Yes O-CTLA4 Clone 9D9 10 mg/kg/dose 4 12 Yes anti-CO137 Clone mAb8 2.5 mg/kg/dose 5 12 Yes FLT3L Mouse FLT3LFc 1 mg/kg/dose 6 5 No Carrier Saline solution

Results

Robust IFN-γ ELISPOT responses were observed for all 3 HBV antigens in mice in the absence of persistent HBV (Fig. 24). IFN-γ ELISPOT responses obtained in AAV-HBV mice that received only the HBV vaccine were reduced but still present, indicating that VV1 GT-D iCore-P2A-sAg and GT-B Po1300 were immunogenic even in the context of an immune system tolerized to HBV. Combination administration of VV1 GT-D iCore-P2A-sAg and GT-B Po1300 with anti-PD-I, anti-CTLA-4 or anti-CD137 further improved HBV-specific IFN-γ ELISPOT responses to capsid and sAg, while the combination of VV1 GT-D iCore-P2A-sAg and GT-B Po1300 with FLT3-L yielded the highest ELISPOT value for all 3 HBV antigens.

Furthermore, administration of VV1 GT-D iCore-P2A-sAg and GT-B Pol300 reduced serum HBeAg levels in those AAV-HBV mice as measured at control-day 11 and day 105 (Table 13). Importantly, combined administration of VV1 GT-D iCore-P2A-sAg and GT-B Pol300 vectors with antibodies to

- 100 047988

PD-1, CTLA-4, CD137 or FTL3-L further reduced serum HBeAg levels (Table 13). Thus, VV1 GT-D iCore-P2A-sAg and GT-B Po1300 vectors demonstrate antiviral efficacy in the AAV-HBV murine model, which can be enhanced in combination with certain immunomodulators.

Table 13. Control table of serum HBeAg levels in AAV-HB V mice

Group Serum HBeAg level (geometric mean, ng/ml) Animals with HBeAg in serum < 100 ng/ml on day 105 -11th day 105th day HBV vaccine + saline solution 868 528 0/11 HBV vaccines + α-PD-l 879 337 3/12 HBV vaccines + a-CTLA4 661 341 2/12 HBV vaccines + a-CD137 1069 500 1/12 HBV Vaccines + FLT3L-FC 773 315 3/12

Example 13. Identification of replication-incompetent Pichinde vectors (PICV) encoding immunogenic nucleotide-optimized HBV antigens

Replication-incompetent PICV (VV2) vectors encoding the GT-D capsid-P2A-sAg antigen (SEQ ID NO: 41) and the GT-B Pol ³⁰⁰ antigen (SEQ ID NO: 13) were generated initially using the same nucleotide sequences that were identified as stable and immunogenic in the replication-incompetent LCMV (VV1) vectors. The stability of the iCore-P2AsAg transgene in the VV2 vectors (SEQ ID NO: 37) was assessed by PCR after serial passaging of the vector containing the supernatant and found to be sufficiently stable for production (Table 13). Genetic stability was determined by a major band indicating the correct size of the full-length transgene (TG).

In contrast, when the same GT-B Pol ³⁰⁰ transgene used in VV1 vectors (SEQ ID NO: 29) was used in VV2 vectors, the transgene rapidly became unstable during serial passage (Table 14). To identify VV2 vectors with sufficient genetic stability for production, three additional VV2 vectors encoding the same GT-B Pol ³⁰⁰ antigen were generated using different nucleotide sequences, designated VV2-Pol300_IDT_CpGdel (SEQ ID NO: 94), Pol ³⁰⁰ ori (SEQ ID NO: 89) and Pol ³⁰⁰ dint (SEQ ID NO: 90), Pol300 huCo low GC (SEQ ID NO: 91), and Pol ³⁰⁰ oridel CpG (SEQ ID NO: 92). Each vector was assessed for transgene stability by PCR after serial passaging of the vector containing the supernatant, with genetic stability defined by a major band demonstrating the correct size of the full-length transgene (TG). The results are presented in Table 14. Surprisingly, major differences in the stability of Pol ³⁰⁰ transgenes in VV2 vectors were evident between the different nucleotide sequences despite encoding an identical polypeptide antigen, with the Pol ³⁰⁰ dint, Pol ³⁰⁰ ori, and Pol ³⁰⁰ oridel CpG polynucleotide sequences demonstrating the greatest stability, for example, after at least five passages.

Table 14. Comparative table for assessing the genetic stability of transgenes ___________________VV2-capsid-P2A-sAg and Pol ³⁰⁰ ___________________

Genotype Nucleic acid SEQ ID NO: Vector Stable TG insertion up to GT-D 37 W2-iCore-P2A-sAg P5 GT-B 29 W2-Pol ³⁰⁰ Pl GT-B 94 W2 -Ро13 OOIDTCpGdel P3 GT-B 91 W2-Pol ³⁰⁰ huCo lowGC P4 GT-B 89 W2-Pol ³⁰⁰ ori P5 GT-B 92 W2-Pol ³⁰⁰ oridel CpG P5 GT-B 90 W2-Pol ³⁰⁰ dint P5

*VV2 denotes replication-incompetent PICV vectors

P# stands for the number of passages (for example, P1 equals 1 passage)

Next, to assess potential differences in immunogenicity between vectors carrying Pol ³⁰⁰ dint and Pol ³⁰⁰ ori transgenes, C57BL/6 mice were immunized twice at day 0 and day 21 with replication-incompetent PICV (VV2) vectors encoding GT-B Pol ³⁰⁰ ori or GT-B Pol ³⁰⁰ dint. HBV-specific T cell responses from splenocytes were then measured by IFN-γ ELISPOT using Pol peptide pools. Surprisingly, V2-GT-B Pol ³⁰⁰ dint induced a much stronger T cell response than VV2-GT-B Pol ³⁰⁰ ori despite coding identical amino acid sequences (Fig. 25). Thus, VV2-GT-B Pol ³⁰⁰ dint is more immunogenic than VV2-GT-B Pol ³⁰⁰ ori in C57BL/6 mice.

- 101 047988

Example 14. Immunogenicity of replication-incompetent LCMV and PICV arenavirus vectors using homologous or heterologous prime/boost immunization cycles in C57BL/6 mice

The immunogenicity of replication-incompetent LCMV vectors (VV1) and PICV vectors (VV2) encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ was assessed using a homologous prime/boost immunization cycle (VV1 vector followed by VV1 vector) or a heterologous prime/boost immunization cycle (VV2 vector followed by VV1 vector) in C57BL/6 mice.

Methods

C57BL/6 mice were immunized twice with replication-incompetent LCMV and PICV vectors encoding GT-D iCore-P2A-sAg and GT-B Pol ³⁰⁰ as described in Table 15. HBV-specific T cell responses were measured on day 28 by IFN-γ ELISPOT using splenocytes. Anti-sAg antibody responses were quantified on day 28 by ELISA.

Table 15. Study groups in the immunogenicity study

Group N Immunization cycle Priming vector day 0 Reinforcing vector - day 21 Selection day Dose/vector 1 5 - Imitation Imitation 28 10 ⁶ FOB 2 5 Homologous priming/amplification VV1-GT-D iCore-P2A-sAg Wl-GT-D iCore- P2A-sAg 28 ΙΟ ⁶ FOB 3 5 Heterologous priming/amplification VV2-GT-D iCore-P2A-sAg Wl-GT-D iCore- P2A-sAg 28 ΙΟ ⁶ FOB 4 5 Homologous priming/amplification VV1-GT-B Pol ³⁰⁰ VV1-GT-B Pol ³⁰⁰ 28 ΙΟ ⁶ FOB 5 5 Heterologous priming/amplification VV2-GT-B Pol ³⁰⁰ dint VV1-GT-B Pol ³⁰⁰ 28 ΙΟ ⁶ FOB

Results

Administration of a replication-incompetent LCMV vector (VV1) encoding GT-D iCore-P2AsAg or encoding GT-B Pol ³⁰⁰ using a homologous priming/boosting cycle (VV1/VV1) induced robust T cell responses in C57BL/6 mice (Figs. 26A–26C). Administration of a replication-incompetent PICV vector (VV2) followed by VV1 (heterologous VV2/VV1 priming/boosting cycle) resulted in higher levels of sAg-specific T cell responses (Fig. 26A) and similar capsid- and Pol-specific T cell responses (Figs. 26B–26C) compared with the VV1/VV1 cycle. Furthermore, although administration of the replication-incompetent LCMV vector using the homologous priming/boosting cycle (VV1/VV1) inconsistently induced anti-sAg antibodies at low levels, immunization using the heterologous priming/boosting cycle (VV2/VV1) unexpectedly resulted in robust and persistent induction of anti-sAg antibodies in all animals and approximately 1000-fold higher than the mean anti-sAg antibody titer (Fig. 27).

Example 15. Immunogenicity of replication-attenuated LCMV and PICV arenavirus vectors using homologous or heterologous prime/boost immunization cycles in C57BL/6 mice

In addition to the replication-incompetent arenavirus vectors LCMV (VV1) and PICV (VV2), replication-competent but attenuated LCMV (TT1) and PICV (TT2) vectors encoding HBV antigens can also be produced by genetic engineering techniques. Unlike the VV1 and VV2 vectors, the TT1 and TT2 vectors contain three genomic segments that allow genomic space to insert two HBV antigens (the GT-D capsid-P2A-sAg fusion protein and the GT-B Pol ³⁰⁰ protein) into the same vector. Since each antigen can be inserted into two different genomic segments, vectors covering different insert combinations in both arenavirus vectors were generated as follows: i) GT-D capsid-P2A-sAg was inserted into segment 1 and GT-B Pol ³⁰⁰ was inserted into segment 2 in the LCMV backbone (TT1-GT-D capsid-P2A-sAg/GT-B Pol ³⁰⁰ ), ii) GT-D capsid-P2A-sAg was inserted into segment 1 and GT-B Pol ³⁰⁰ was inserted into segment 2 in the PICV backbone (TT2-GT-D capsid-P2A-sAg/GT-B Pol ³⁰⁰ ), iii) GT-D capsidP2A-sAg was inserted into segment 2 and GT-B Pol ³⁰⁰ was inserted into segment 1 in the LCMV backbone (TT1GT-B Pol ³⁰⁰ /GT-D capsid-P2A-sAg) and iv) GT-D capsid-P2A-sAg was inserted into segment 2 and GT-B Pol ³⁰⁰ was inserted into segment 1 of the PICV backbone (TT2-GT-B Pol ³⁰⁰ /GT-D capsid-P2A-sAg). The immunogenicity of these 4 vectors was then assessed using homologous or heterologous prime/boost immunization cycles in C57BL/6 mice.

- 102 047988

Methods

C57BL/6 mice were immunized twice with replication-attenuated LCMV and PICV vectors encoding GT-D capsid-P2A-sAg and GT-B Pol ³⁰⁰ as described in Table 16. HBV-specific T cell responses were measured on day 28 by IFN-γ ELISPOT using splenocytes.

Table 16. Study groups in the immunogenicity study

Group N Priming vector - day 0 Reinforcing vector day 21 Selection day Dose/vector (RCV/ΦΟΕ) 1 5 Imitation Imitation 28 - 2 5 TT1-GT-D capsid-P2AsAg/GT-B Pol ³⁰⁰ TT1-GT-D capsid-P2AsAg/GT-B Pol ³⁰⁰ 28 5xl0 ⁴ 3 5 TT2-GT-D capsid-P2AsAg/GT-B Pol ³⁰⁰ TT2-GT-D capsid-P2AsAg/GT-B Pol ³⁰⁰ 28 5xl0 ⁴ 4 5 TT2-GT-D capsid-P2AsAg/GT-B Pol ³⁰⁰ TT1-GT-D capsid-P2AsAg/GT-B Pol ³⁰⁰ 28 5xl0 ⁴ 5 5 TT1-GT-B Pol ³⁰⁰ /GT-D Kancng-P2A-sAg TT1-GT-B Pol ³⁰⁰ /GT-D Kancng-P2A-sAg 28 5xl0 ⁴ 6 5 TT2-GT-B Pol ³⁰⁰ /GT-D Kancmi-P2A-sAg TT2-GT-B Pol ³⁰⁰ /GT-D Kancmi-P2A-sAg 28 5xl0 ⁴ 7 5 TT2-GT-B Pol ³⁰⁰ /GT-D Kancmi-P2A-sAg TT1-GT-B Pol ³⁰⁰ /GT-D Kancmi-P2A-sAg 28 5xl0 ⁴

Results

Administration of all replication-competent vectors resulted in robust T cell responses specific for the three HBV antigens sAg, capsid, and Pol (Fig. 28A-28C). Thus, TT1 and TT2 vectors expressing HBV antigens are highly immunogenic in C57BL/6 mice.

Example 16. Immunogenicity of replication-incompetent LCMV and PICV arenavirus vectors using homologous or heterologous prime/boost immunization cycles in cynomolgus macaques

We assessed the immunogenicity of replication-incompetent LCMV vectors (VV1) and PICV vectors (VV2) encoding GT-D iCore-P2A-sAg and GT-B Po1300 using a homologous prime/boost immunization cycle (VV1 vector followed by VV1 vector) or a heterologous prime/boost immunization cycle (VV2 vector followed by VV1 vector) in cynomolgus macaques.

Methods

Cynomolgus macaques (n=5) were immunized with VV2 vectors ( ^5x106 FFU/vector) at week 0 and then immunized with VV1 vectors ( ^5x106 FFU/vector) at week 4, and HBV-specific T cell responses were measured in PBMCs by IFN-γ ELISPOT at week 6. Data were compared with ELISPOT from 10 cynomolgus macaques immunized with VV1 vectors alone ( ^5x106 FFU/vector) at week 0 and week 4 (homologous prime/boost cycle).

Results

Administration of replication-incompetent LCMV vectors (VV1) encoding GT-D iCore-P2AsAg and GT-B Po1300 using a homologous priming/boosting cycle (VV1/W1) induced HBV-specific T cell responses in 5 of 10 cynomolgus monkeys (Fig. 29). In contrast, administration of a replication-incompetent PICV vector (W2) followed by the addition of VV1 (heterologous priming/boosting cycle VV2/VV1) yielded statistically higher HBV-specific T cell responses in all 5 animals compared with the homologous priming/boosting cycle VV1/VV1 (Fig. 29).

Example 17. Immunogenicity of replication-incompetent LCMV and PICV arenavirus vectors using homologous or heterologous prime/boost immunization cycles at 1-week dosing intervals in cynomolgus monkeys

We assessed the immunogenicity of replication-incompetent LCMV vectors (VV1) and PICV vectors (VV2) encoding GT-D iCore-P2A-sAg and GT-B Po1300 using a homologous prime/boost immunization cycle (VV1 vector followed by VV1 vector) or a heterologous prime/boost immunization cycle (VV2 vector followed by VV1 vector) administered at 1-week dosing intervals in cynomolgus monkeys.

Methods

Cynomolgus macaques were immunized as described in Table 17. HBV-specific T cell responses were measured in PBMCs using IFN-γ ELISPOT at week 4.

- 103 -

Claims (46)

Table 17. Study groups in the immunogenicity study Group N Primer vaccine Booster vaccine Dose/vector Immunization schedule (week) Assay ELISPOT 1 5 VV1 W1 10 ⁸ FUE 0(Wl) 1 (W1) 2 (Wl) 3 (Wl) 4th week 2 5 VV2 W1 10 ⁸ FUE 0(W2) 1 (Wl) 2(W2) 3 (Wl) 4th week Results Administration of a replication-incompetent PICV vector (VV2) followed by addition of VV1 (VV2/VV1 heterologous priming/boosting cycle) resulted in higher HBV-specific T cell responses compared to vaccination with VV1 vector alone (Fig. 30). It is understood that the examples and embodiments described herein are intended for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and are intended to be included within the spirit and scope of this application and the scope of the appended claims. All publications, patent applications cited herein are incorporated by reference in their entirety and for all purposes into this specification. CLAUSE OF INVENTION 1. An immunogenic composition comprising a first viral expression vector and a second viral expression vector, wherein said first viral expression vector comprises a polynucleotide encoding a truncated mutant hepatitis B virus (HBV) polymerase polypeptide, wherein said polypeptide is no more than 600 amino acids in length, does not contain the entire terminal protein (TP) domain and does not contain all or part of the spacer domain, wherein said polypeptide comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 13-14; and said second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising, in sequence from N-terminus to C-terminus, a HBV capsid polypeptide and a HBV small surface antigen (sAg) polypeptide, wherein said fusion protein has a length of no more than 450 amino acids and wherein said capsid polypeptide belongs to genotype D of HBV, and the sAg polypeptide belongs to genotype D of HBV, wherein said fusion protein comprises or consists of an amino acid sequence that is at least 97% identical to the full-length sequence according to any SEQ ID NO: 40-41. 2. An immunogenic composition according to claim 1, characterized in that said fusion protein comprises or consists of an amino acid sequence that is at least 98 or 99% identical to the full-length sequence according to any SEQ ID NO: 40-41. 3. An immunogenic composition according to claim 1 or 2, characterized in that said fusion protein comprises or consists of an amino acid sequence according to any SEQ ID NO: 40-41. 4. An immunogenic composition according to any one of claims 1 to 3, comprising a first viral expression vector and a second viral expression vector, wherein said first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 13; and said second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence that is at least 97, 98 or 99% identical to the full-length sequence of SEQ ID NO: 41. 5. An immunogenic composition according to any one of claims 1 to 4, comprising a first viral expression vector and a second viral expression vector, wherein: a) said first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence according to any one of SEQ ID NOs: 29, 89, 90, 91, 92, 93 and 94 or a nucleic acid sequence that is at least 99% identical to the full-length sequence of any one of SEQ ID NOs: 29, 89, 90, 91, 92, 93 and 94; b) said second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence according to any of SEQ ID NOs: 35-37 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of any of SEQ ID NOs: 35-37. 6. An immunogenic composition according to any one of claims 1 to 5, comprising a first viral expression vector and a second viral expression vector, wherein: a) said first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence - 104 047988 of the acid of SEQ ID NO: 29, 89, 90 or 92, or a nucleic acid sequence that is at least 99% identical to the full-length SEQ ID NO: 29, 89, 90 or 92; and b) said second viral expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of SEQ ID NO: 37. 7. An immunogenic composition according to any one of claims 1 to 6, characterized in that said first viral expression vector and said second viral expression vector independently originate from a taxonomic family selected from adenoviruses, arenaviruses, herpes viruses, poxviruses, flaviviruses, rhabdoviruses and togaviruses. 8. An immunogenic composition according to any one of claims 1 to 7, characterized in that said first viral expression vector and said second viral expression vector originate from the same taxonomic family. 9. An immunogenic composition according to any one of claims 1 to 8, characterized in that said first viral expression vector and said second viral expression vector are derived from arenaviruses (Arenaviridae). 10. An immunogenic composition according to any one of claims 1 to 9, characterized in that said first viral expression vector and said second viral expression vector are independently derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV), Pichinde mammarenavirus (PICV), Guanarito virus (GTOV), Junin virus (JUNV), Lassa virus (LASV), Luyo virus (LUJV), Machupo virus (MACV), Sabia virus (SABV) and Whitewater Arroyo virus (WWAV). 11. An immunogenic composition according to any one of claims 1 to 10, characterized in that said first viral expression vector and said second viral expression vector are derived from an arenavirus vector selected from lymphocytic choriomeningitis mammarenavirus (LCMV) or Pichinde mammarenavirus (PICV). 12. An immunogenic composition comprising a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: a) a first LCMV arenavirus expression vector comprises a polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, wherein said polypeptide is no more than 600 amino acids in length and does not comprise all of the terminal protein (TP) domain and does not comprise all or a portion of the spacer domain, wherein said polynucleotide comprises or consists of a nucleic acid sequence that is at least 90% identical to the full-length sequence of SEQ ID NO: 29; and b) the second LCMV arenavirus expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising, in sequence from N-terminus to C-terminus, a genotype D HBV capsid polypeptide and a genotype D HBV small surface antigen (sAg) polypeptide, wherein said fusion protein has a length of no more than 450 amino acids and wherein said polynucleotide comprises or consists of a nucleic acid sequence that is at least 90% identical to the full-length sequence of SEQ ID NO: 37. 13. The immunogenic composition of claim 12, wherein said polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprises or consists of a nucleic acid sequence that is at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the full-length sequence of SEQ ID NO: 29, and said polynucleotide encoding a capsid-sAg fusion protein comprises or consists of a nucleic acid sequence that is at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the full-length sequence of SEQ ID NO: 37. 14. An immunogenic composition according to any one of claims 12, 13, characterized in that said polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide contains or consists of the nucleic acid sequence of SEQ ID NO: 29, and said polynucleotide encoding a capsid-sAg fusion protein contains or consists of the nucleic acid sequence of SEQ ID NO: 37. 15. An immunogenic composition comprising a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: a) a first Pichinde arenavirus expression vector comprises a polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, wherein said polypeptide is no more than 600 amino acids in length, lacks all of a terminal protein (TP) domain, and lacks all or a portion of a spacer domain, wherein said polynucleotide comprises or consists of a nucleic acid sequence that is at least 90% identical to the full-length sequence of SEQ ID NO: 90; and - 105 047988 b) the second Pichinde arenavirus expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising, in sequence from N-terminus to C-terminus, a genotype D HBV capsid polypeptide and a genotype D HBV small surface antigen (sAg) polypeptide, wherein said fusion protein is no more than 450 amino acids in length and wherein said polynucleotide comprises or consists of a nucleic acid sequence that is at least 90% identical to the full-length sequence of SEQ ID NO: 37. 16. The immunogenic composition of claim 15, wherein said polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprises or consists of a nucleic acid sequence that is at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the full-length sequence of SEQ ID NO: 90, and said polynucleotide encoding a capsid-sAg fusion protein comprises or consists of a nucleic acid sequence that is at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the full-length sequence of SEQ ID NO: 37. 17. An immunogenic composition according to any one of claims 15-16, characterized in that said polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprises or consists of the nucleic acid sequence of SEQ ID NO: 90, and said polynucleotide encoding a capsid-sAg fusion protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 37. 18. An immunogenic composition according to any one of claims 1 to 17, characterized in that said first viral expression vector and said second viral expression vector are replication-defective or replication-deficient. 19. An immunogenic composition according to any one of claims 1 to 17, characterized in that said first viral expression vector and said second viral expression vector are replication-attenuated. 20. An immunogenic composition according to any one of claims 1 to 7, characterized in that said first viral expression vector and said second viral expression vector originate from different taxonomic families. 21. An immunogenic composition according to any one of claims 1 to 20, characterized in that said first viral expression vector and said second viral expression vector are provided in a ratio in the range from 1:10 to 10:1. 22. An immunogenic composition according to any one of claims 1 to 21, comprising in the range of from about 10 ³ to about 10 ¹² focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (VP) per milliliter of each of said first viral expression vector and said second viral expression vector. 23. An immunogenic composition according to any one of claims 1 to 22, further comprising one or more of an adjuvant, a detergent, a micelle former and an oil. 24. An immunogenic composition according to any one of claims 1 to 23, formulated for administration by a route of administration selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous and mucosal routes of administration. 25. An immunogenic composition according to any one of claims 1 to 24, formulated in the form of an aqueous solution or suspension. 26. An immunogenic composition according to any one of claims 1 to 25, characterized in that said composition is lyophilized. 27. A kit for inducing an immune response against HBV, comprising one or more single doses of one or more immunogenic compositions according to any one of claims 1 to 26. 28. The kit according to claim 27, comprising a first viral expression vector and a second viral expression vector, wherein: a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 29 and 89-94 or a nucleic acid sequence that is at least 99% identical to the full-length sequence of any one of SEQ ID NOs: 29 and 89-94; and b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any of SEQ ID NOs: 35-37 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of any of SEQ ID NOs: 35-37. 29. A kit for inducing an immune response against HBV, comprising a first LCMV arenavirus expression vector and a second LCMV arenavirus expression vector, wherein: a) a first LCMV arenavirus expression vector comprises a polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, wherein said polypeptide is no more than 600 amino acids in length, does not comprise the entire terminal protein (TP) domain and does not comprise all or part of a spacer domain, wherein said polynucleotide comprises or consists of a nucleic acid sequence - 106 047988 new acid that is at least 90% identical to the full-length sequence of SEQ ID NO: 29; and b) the second LCMV arenavirus expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising, in sequence from N-terminus to C-terminus, a genotype D HBV capsid polypeptide and a genotype D HBV small surface antigen (sAg) polypeptide, wherein said fusion protein is no more than 450 amino acids in length, and wherein said polynucleotide comprises or consists of a nucleic acid sequence that is at least 90% identical to the full-length sequence of SEQ ID NO: 37. 30. The kit according to claim 29, characterized in that said polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H comprises or consists of a nucleic acid sequence that is at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the full-length sequence of SEQ ID NO: 29, and said polynucleotide encoding a capsid-sAg fusion protein comprises or consists of a nucleic acid sequence that is at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the full-length sequence of SEQ ID NO: 37. 31. A kit according to any one of claims 29, 30, characterized in that said polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide containing an inactivated reverse transcriptase domain and an inactivated RNase H contains or consists of the nucleic acid sequence of SEQ ID NO: 29, and said polynucleotide encoding the capsidsAg fusion protein contains or consists of the nucleic acid sequence of SEQ ID NO: 37. 32. The kit according to any one of claims 29-31, further comprising a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: a) the first Pichinde arenavirus expression vector comprises a polynucleotide encoding a truncated hepatitis B virus (HBV) polymerase polypeptide comprising an inactivated reverse transcriptase domain and an inactivated RNase H, wherein said polypeptide is no more than 600 amino acids in length, lacks all of the terminal protein (TP) domain, and lacks all or a portion of the spacer domain, wherein said polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 90 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the full-length sequence of SEQ ID NO: 90; and b) the second Pichinde arenavirus expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising, in sequence from N-terminus to C-terminus, a genotype D HBV capsid polypeptide and a genotype D HBV small surface antigen (sAg) polypeptide, wherein said fusion protein has a length of no more than 450 amino acids, and wherein said polynucleotide comprises or consists of the nucleic acid sequence of SEQ ID NO: 37 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of SEQ ID NO: 37. 33. A method for treating or preventing human hepatitis B virus (HBV) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of one or more immunogenic compositions according to any one of claims 1-26. 34. The method of claim 33, wherein the one or more immunogenic compositions comprise a mixture comprising a first viral expression vector and a second viral expression vector, wherein: a) the first viral expression vector comprises a polynucleotide encoding a mutant HBV polymerase polypeptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 13-14; and b) the second viral expression vector comprises a polynucleotide encoding a capsid-sAg fusion protein comprising or consisting of the amino acid sequence of any of SEQ ID NOs: 40-41 or an amino acid sequence that is at least 97, 98 or 99% identical to the full-length sequence of any of SEQ ID NOs: 40-41. 35. The method according to any one of claims 33, 34, wherein one or more immunogenic compositions comprise a mixture comprising a first viral expression vector and a second viral expression vector, wherein: a) the first viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence according to any one of SEQ ID NOs: 29 and 89-94, or a nucleic acid sequence that is at least 99% identical to the full-length sequence of any one of SEQ ID NOs: 29 and 89-94; and b) the second viral expression vector comprises a polynucleotide comprising or consisting of a nucleic acid sequence of any of SEQ ID NOs: 35-37 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of any of SEQ ID NOs: 35-37. 36. The method according to any one of claims 33, 34, wherein one or more immunogenic compositions comprise a mixture comprising a first LCMV arenavirus expression vector and a second expression vector - 107 047988 arenavirus LCMV, and: a) the first LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of SEQ ID NO: 29; and b) the second LCMV arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of SEQ ID NO: 37. 37. The method according to any one of claims 33, 34, wherein said one or more immunogenic compositions comprise or further comprise a mixture comprising a first Pichinde arenavirus expression vector and a second Pichinde arenavirus expression vector, wherein: a) the first Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 90 or a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the full-length sequence of SEQ ID NO: 90; and b) the second Pichinde arenavirus expression vector comprises a polynucleotide comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37 or a nucleic acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the full-length sequence of SEQ ID NO: 37. 38. The method according to any one of claims 33-37, wherein the composition is administered by a route selected from intravenous, intramuscular, intradermal, subcutaneous and mucosal. 39. The method according to item 38, characterized in that the said mucosal route is selected from transbuccal, intranasal, intrarectal, intravaginal. 40. The method according to any one of claims 33 to 38, comprising administering from about 10 ³ to about 10 ¹² viral focus-forming units (FFU) or plaque-forming units (PFU) or infectious units (IU) or viral particles (VP), from about 10 4 to about 10 ⁷ viral FFU or PFU or IU or IU, or IU, or from about 10 ³ to about 10 4, 10 ⁵ , 10 6, 10 ⁷ , 10 8, 10 ⁹ , 10 10 , 10 ¹¹ or 10 ¹² viral FFU or PFU or IU or IU, per administration. 41. The method according to any one of claims 33-40, comprising administering intravenously or intramuscularly from about 106 to about 108 viral FFU, or PFU, or IE, or VP per administration every two weeks (Q2W) or monthly (Q4W). 42. The method according to any one of claims 33-41, comprising a priming/boosting cycle comprising administering a priming composition at a first time point and administering one or more boosting compositions at one or more subsequent time points. 43. The method according to claim 42, wherein the priming/boosting cycle comprises: a) priming with a priming composition comprising one or more lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more Pichinde mammarenavirus viral expression vectors; b) priming with a priming composition comprising one or more viral expression vectors of Pichinde mammaryenavirus and boosting with a boosting composition comprising one or more viral expression vectors of lymphocytic choriomeningitis mammaryenavirus (LCMV); c) priming with a priming composition comprising one or more replication-deficient viral expression vectors of Pichinde mammaryenavirus and boosting with a boosting composition comprising one or more replication-deficient viral expression vectors of lymphocytic choriomeningitis mammaryenavirus (LCMV); or d) priming with a priming composition comprising one or more replication-deficient lymphocytic choriomeningitis mammarenavirus (LCMV) viral expression vectors and boosting with a boosting composition comprising one or more replication-deficient Pichinde mammarenavirus viral expression vectors. 44. The method according to any one of claims 42, 43, comprising priming with a priming composition comprising one or more viral expression vectors of Pichinde mammaryenavirus, and boosting with a enhancing composition comprising one or more viral expression vectors of lymphocytic choriomeningitis mammaryenavirus (LCMV). 45. The method of any one of claims 33-44, further comprising administering to the subject one or more additional therapeutic agents. 46. The method of claim 45, comprising administering an agonist of a receptor selected from the group consisting of fms-like tyrosine kinase 3 (FLT3), receptor stimulator of interferon genes (STING), DExD/Hbox helicase 58 (DDX58; also known as RIG-I), and nucleotide-binding oligomerization domain containing protein 2 (NOD2). - 108 -