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WO2024118544A2 - Vaccins contenant de nouveaux supports de nanoparticules - Google Patents

Vaccins contenant de nouveaux supports de nanoparticules Download PDF

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Publication number
WO2024118544A2
WO2024118544A2 PCT/US2023/081246 US2023081246W WO2024118544A2 WO 2024118544 A2 WO2024118544 A2 WO 2024118544A2 US 2023081246 W US2023081246 W US 2023081246W WO 2024118544 A2 WO2024118544 A2 WO 2024118544A2
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seq
sequence
nanoparticle
vaccine construct
nanoparticle vaccine
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PCT/US2023/081246
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WO2024118544A3 (fr
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Linling HE
Jiang Zhu
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The Scripps Research Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A61K2039/645—Dendrimers; Multiple antigen peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Influenza viruses belong to the Orthomyxoviridae family and can be classified into four types: A, B, C, and D. All influenza viruses are enveloped negative- sense single-stranded RNA viruses with a segmented genome, with influenza A and B viruses (IAV and IBV) containing 8 gene segments, which encode for at least 17 proteins. The most abundant surface glycoprotein, hemagglutinin (HA), allows the virus to bind to the host cell receptors and mediates the cell entry. Neuraminidase (NA) aids in the release of viral particles through cleavage of residues on the host cell’s surface.
  • HA hemagglutinin
  • NA Neuraminidase
  • Matrix-1 protein (M1) aids in the budding of the virus from the plasma membrane of infected cells
  • matrix-2 protein (M2) facilitates the maintenance of pH during viral entry and viral replication in host cells.
  • IAVs can be further classified by subtype, based on the antigenic properties of the two surface glycoproteins, HA (HA1-18) and NA (NA1-11). IAVs can infect many hosts, whereas IBVs are restricted to humans and have diverged into two lineages (Victoria and Yamagata) through the intra-host evolution. Influenza viruses of avian origin recognize ⁇ -2,3 sialic acid receptors, whereas human influenza viruses bind preferably to ⁇ -2,6 sialic acid receptors in the upper respiratory tract.
  • Influenza virus utilizes two mechanisms to evade the immune system.
  • Antigenic drift consists of small changes introduced into HA and NA under the immune pressure and is the cause of annual epidemics of human influenza.
  • Antigen shift occurs when a complete change in HA and/or NA genes occurs in IAVs due to their large animal reservoirs.
  • Antigen shift results in novel IAV strains with increased transmission in humans and is a major cause of pandemics.
  • seasonal influenza vaccines have been used as an efficient and cost-effect tool to minimize influenza epidemics and improve public health.
  • Current vaccines use inactivated or live-attenuated strains. The most common types of inactivated virus vaccines are referred to as split vaccines where detergents or chemicals are used to disrupt viral particles.
  • Live-attenuated vaccines use cold-adapted live viruses that do not replicate at human body temperature and are generally administered intranasally to induce strong local immunity.
  • Subunit vaccines utilize viral HA or NA proteins that are partially purified after chemical or detergent splitting.
  • Virus strains selected for quadrivalent vaccines which contain an H1N1, H3N2, and two Flu B strains are produced in chicken eggs.
  • current influenza vaccines must be updated yearly to include predicted strains. Strain mismatch often results in low efficacy, highlighting the need for better vaccines.
  • a strong unmet need exists in the medical field for more reliable and effective influenza vaccines. The instant invention is directed to this and other unmet needs in the art.
  • the invention provides N-terminally extended I3-01 nanoparticle scaffold sequence. These scaffold sequences contain an extended N- terminal helix relative to the N-terminal helix in the original or wildtype I3-01 scaffold sequence.
  • the novel I3-01 derived NP scaffold sequences of the invention contain a heterologous helical motif of about 6 to about 12 amino acid residues that is fused to the N-terminus of the I3-01 scaffold sequence set forth in SEQ ID NO:27 (I3-01v9).
  • the inserted heterologous helical motif contains AKLAEELQK (SEQ ID NO:25), a conservatively modified variant or a substantially identical sequence thereof.
  • N-terminally extended I3-01 nanoparticle scaffold sequences of the invention contain SEQ ID NO:4, a conservatively modified variant or a substantially identical sequence thereof.
  • the invention provides self-assembling nanoparticles that are formed with the novel N-terminally extended I3-01 nanoparticle scaffold sequences of the invention.
  • the invention provides nanoparticle vaccine constructs that contain an immunogenic protein or a polypeptide immunogen that is fused to an N- terminally extended I3-01 nanoparticle scaffold sequence.
  • the N-terminally extended I3-01 nanoparticle scaffold sequence in these NP vaccine constructs contains an extended N-terminal helix relative to the N-terminal helix in the original or wildtype I3- 01 scaffold sequence.
  • the N-terminally extended I3-01 nanoparticle scaffold sequence contains a heterologous helical motif of about 6 to about 12 amino acid residues that is fused to the N-terminus of the I3-01 scaffold sequence set forth in SEQ ID NO:27 (I3-01v9).
  • the extending heterologous helical motif contains AKLAEELQK (SEQ ID NO:25), a conservatively modified variant or a substantially identical sequence thereof.
  • the N-terminally extended I3-01 nanoparticle scaffold sequence contains SEQ ID NO:4, a conservatively modified variant or a substantially identical sequence thereof.
  • the displayed immunogenic protein in the vaccine constructs is fused via a linker at its C-terminus to the N-terminus of the N-terminally extended I3-01 nanoparticle scaffold sequence.
  • the employed linker contains GGGGS (SEQ ID NO:3).
  • the displayed polypeptide immunogen is an influenza fusion polypeptide containing 2 or more tandem repeats of influenza M2 protein ectodomain (M2e).
  • the displayed polypeptide immunogen is an HCV immunogenic protein.
  • the displayed tandem influenza M2e repeats are separated by a peptide spacer, e.g., GGGG (SEQ ID NO:9).
  • At least one of the displayed M2e tandem repeats contains missense mutations at the conserved Cys17 and Cys19 residues.
  • the missense mutations contain substitutions of each of the two Cys residues with an amino acid residue with uncharged polar side chains.
  • each of the Cys residues is independently replaced with an amino acid residue selected from the group consisting of serine, glycine, asparagine, glutamine, threonine and tyrosine.
  • the two Cys residues in the same M2e sequence are both replaced with Ser.
  • the displayed immunogenic protein contains 3 tandem M2e sequences.
  • the 3 tandem M2e sequences are independently a human M2e sequence, an avian/swine consensus M2e sequence, or a human/swine consensus M2e sequence, except for missense mutations at residues Cys17 and Cys19 in at least 2 of the 3 tandem M2e sequences.
  • the displayed influenza fusion polypeptide contains, in any order, a human M2e (SEQ ID NO:2), an avian/swine consensus M2e sequence with Cys17 and Cys19 each replaced with a Ser residue (SEQ ID NO:30), and a human/swine consensus M2e sequence with Cys17 and Cys19 each replaced with a Ser residue (SEQ ID NO:31).
  • the displayed influenza M2e tandem repeat fusion polypeptide contains, in any order, a human M2e with Cys17 and Cys19 each replaced with a Ser residue (SEQ ID NO:29), an avian/swine consensus M2e sequence with Cys17 and Cys19 each replaced with a Ser residue (SEQ ID NO:30), and a human/swine consensus M2e sequence with Cys17 and Cys19 each replaced with a Ser residue (SEQ ID NO:31).
  • the displayed influenza M2e tandem fusion polypeptide contains SEQ ID NO:23, SEQ ID NO:24, a conservatively modified variant or a substantially identical sequence thereof.
  • influenza vaccine constructs contain a subunit or scaffold sequence shown in SEQ ID NO:11, SEQ ID NO:12, a conservatively modified variant or a substantially identical sequence thereof.
  • novel I3-01 scaffold based vaccine constructs of the invention can additionally include a locking domain and a T-cell epitope at the C-terminus.
  • they can contain at the C-terminus the locking domain shown in SEQ ID NO:5 and the T-cell epitope shown in SEQ ID NO:6.
  • Some of these vaccine constructs contain a subunit or scaffold sequence shown in SEQ ID NO:14, SEQ ID NO:15, a conservatively modified variant or a substantially identical sequence thereof.
  • Some vaccine constructs of the invention can additionally include an N-terminal leader sequence. Some of these vaccine constructs contain a subunit or scaffold sequence shown in SEQ ID NO:17, SEQ ID NO:18, a conservatively modified variant or a substantially identical sequence thereof. Some vaccine constructs of the invention can additionally include an N-terminal leader sequence, and a locking domain and a T-cell epitope at the C-terminus. Some of these vaccine constructs contain a subunit or scaffold sequence shown in SEQ ID NO:20, SEQ ID NO:21, a conservatively modified variant or a substantially identical sequence thereof. [0011] In some vaccine constructs of the invention, the displayed immunogenic protein is an HCV immunogen, e.g., an E2 core or an E1E2 dimer protein.
  • HCV immunogen e.g., an E2 core or an E1E2 dimer protein.
  • the displayed protein contains a HCV E2 core as shown in any one of SEQ ID NOs:32-35.
  • the displayed HCV immunogenic protein contains a tandem copy of 2 E2 core sequences.
  • the 2 E2 core sequences are from different HCV isolates.
  • the 2 tandem E2 core sequences can respectively contain SEQ ID NOs:32 and 33, or SEQ ID NOs:34 and 35.
  • Some of the HCV vaccine constructs of the invention additionally contain a locking domain and a T-cell epitope at the C-terminus.
  • the vaccine constructs can include the locking domain shown in SEQ ID NO:5, and/or the T-cell epitope shown in SEQ ID NO:6.
  • HCV vaccines contain a subunit or scaffold sequence that has from the N-terminus to the C-terminus different structural motifs respectively shown in (a) SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:5 and SEQ ID NO:6, or (b) SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:5 and SEQ ID NO:6.
  • Some other constructs contain a subunit or scaffold sequence that is a conservatively modified variant or substantially identical sequence of one of these exemplified scaffold sequences.
  • the invention provides polynucleotide sequences that encode the subunit or scaffold sequence of one of the nanoparticle vaccine constructs described herein.
  • FIG. 1 M2e-based vaccine construct design. Left: hM2e structure and sequence (SEQ ID NO:2). Middle: model of the hM2e-5GS-1TD0 trimer; Right: models of hM2e-5GS-FR, hM2e-5GS-E2p-LD4-PADRE, and hM2e-5GS-I3-01v9a- LD7-PADRE 1c-SApNPs.
  • C SEC profiles of hM2e trimer and 1c-SApNPs.
  • D Micrographs of Fab148-purified hM2e 1c-SApNPs by negative-stain EM..
  • FIG. 1 Mouse immunization/challenge schedule.
  • B Survival and weight loss after A/Puerto Rico/8/1934 (PR8) H1N1 virus challenge.
  • C Survival and weight loss after A/Hong Kong/1/1968 (HK68) H3N2 virus challenge.
  • D ELISA of hM2e-specific antibody responses in mouse sera against an hM2e probe.
  • Figure 5. Negative-stain EM images of tandem M2e 1c-SApNPs. A M2ex3-5GS-FR sample that was held at 70°C for 10 min showed no visible change in structure and bound to anti-M2e antibodies with nearly identical affinity.
  • M2e The ectodomain of M2 protein (M2e) is a highly conserved target for universal IAV vaccines. Although M2e is small ( ⁇ 23 aa) and non-immunogenic, it can be attached to large carriers to elicit cross-protection and reduce virus replication. Internal proteins such as nucleoprotein and M1 have been explored for T cell targeting. The use of adjuvants, such as MF59 and AS03, has been shown to significantly improve the efficacy of influenza vaccines. Therefore, adjuvant effect must be carefully examined in the development of universal flu vaccines to target the subdominant HA stem and M2e.
  • M2e-based vaccines confer protection via mechanism such as antibody- dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), which eliminate virus-infected cells.
  • ADCC antibody- dependent cellular cytotoxicity
  • ADCP phagocytosis
  • M2e has been attached to various carriers for vaccine development.
  • One of the early M2e vaccines used hepatitis B core protein (HBc) as a carrier.
  • Tobacco mosaic virus (TMV) coat protein, keyhole limpet hemocyanin (KLH), rotavirus NSP4, GCN4, bacterial flagellin, and liposome have since been tested in M2e vaccine development.
  • TMV hepatitis B core protein
  • KLH keyhole limpet hemocyanin
  • NSP4 rotavirus NSP4
  • GCN4 bacterial flagellin, and liposome
  • the adjuvanted M2e-HBc fusion protein induced anti-M2e antibodies in 90% of the cases and was well tolerated in the Phase-I trial (clinicaltrials.gov: NCT00819013). However, the vaccine induced anti-M2e antibody response declined rapidly.
  • the M2e-flagellin fusion vaccine was highly immunogenic but caused undesirable side effects, such as fever, diarrhea, fatigue, headache, and muscle pain, at higher doses in the Phase-I trial (clinicaltrials.gov NCT00921206).
  • the vaccine that combines M2e with multiple cytotoxic T lymphocyte (CTL) epitopes could stimulate strong cellular immunity in humans (clinicaltrials.gov: NCT01181336), but the T cell response was narrow and slow, making this vaccine unsuitable in the event of an emerging pandemic. Therefore, the carrier, the adjuvant, and the right balance between antibody and T-cell responses, are the major challenges facing M2e vaccine development.
  • the present inventors designed multilayered single-component self- assembling protein nanoparticles (1c-SApNP) based on E2p and I3-01, two bacterial proteins that self-assemble into 60-mers of 22-25 nm, as carriers of foreign antigens for vaccine development.
  • the invention encompasses novel I3-01 derived NP platforms that have demonstrated activities for presenting immunogenic proteins such as influenza M2e proteins and HCV immunogens.
  • the invention also encompasses broadly protective vaccines that contain immunogenic proteins (e.g., HCV E2 core proteins and influenza M2e proteins) displayed on the novel NP scaffolds. Details for making and using the compositions and methods encompassed by the invention are described below. [0022]
  • the nanoparticle scaffolds and vaccine constructs described can have various applications in clinical setting.
  • the novel I3-01 derived NP scaffolds described herein can be used to present various other monomeric antigens in addition to HCV and influenza immunogenic proteins exemplified herein.
  • the vaccines thus constructed such as the HCV vaccines or tandem hM2e vaccines as exemplified herein, can be used as broadly protective vaccines.
  • influenza vaccine as an example, they can be added to seasonal vaccines (e.g., HCV or influenzas vaccines) as a “performance enhancer” to improve protection against endemic (human strains) and pandemic (swine and avian strains) influenza viruses.
  • novel I3-01 scaffold based vaccines e.g., M2e displaying vaccines
  • other vaccine modalities e.g., hemagglutinin (HA) stem-based vaccines
  • the vaccines of the invention also have a number of advantageous properties relative to related vaccines known in the art. Using influenza vaccines for illustration, the uniform distribution and the just-above-sea-level exposure of antigen anchoring sites on the surface make the novel I3-01 scaffolds described herein ideal nanoparticle platforms for presenting monomeric antigens.
  • 1c-SApNPs ideal carriers for multivalent display of a suitable antigen, e.g., influenza M2e. Being a single segment or a tandem construct design, it can be optimally displayed on the nanoparticle surface and can generate high- quality antibody responses. Additionally, the genetic fusion combined with self- assembly will result in robust production of 1c-SApNPs in laboratory and industrial settings. As demonstrated herein, the vaccines can be produced in ExpiCHO cells with reasonable yield and extremely high purity after immunoaffinity (Fab148) purification.
  • Fab148 immunoaffinity
  • CHO is one of the principal mammalian cell lines used for industrial manufacture of protein therapeutics and vaccines and ExpiCHO is a transient version of this CHO cell line
  • the vaccines obtained from ExpiCHO cells e.g., influenza M2e vaccines
  • the multilayered structure will ensure the thermostability of the 1c-SApNPs (e.g., influenza M2e 1c-SApNPs) and allow various delivery routes and combined use with other related vaccines.
  • the high-temperature hold at 70°C for 10 min did not cause any structural change and ELISA showed nearly identical binding to M2e-specific antibodies such as Fab65 and Fab148.
  • the superior thermostability of the 1c-SApNPs of the invention e.g., influenza M2e 1c-SApNPs
  • the various compositions and methods of the invention can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art.
  • an Env-derived trimer can refer to both single or plural Env-derived trimer molecules, and can be considered equivalent to the phrase “at least one Env-derived trimer.”
  • the terms "antigen” or “immunogen” are used interchangeably to refer to a substance, typically a protein, which is capable of inducing an immune response in a subject.
  • the term also refers to proteins that are immunologically active in the sense that once administered to a subject (either directly or by administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein.
  • vaccine immunogen is used interchangeably with “protein antigen” or “immunogen polypeptide.”
  • immunogen polypeptide refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • Epitope refers to an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and/or T cells respond. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. [0030] Effective amount of a vaccine or other agent that is sufficient to generate a desired response, such as reduce or eliminate a sign or symptom of a condition or disease, such as seasonal flu.
  • this can be the amount necessary to inhibit viral replication or to measurably alter outward symptoms of the viral infection, such as increase of T cell counts in the case of an influenza infection. In general, this amount will be sufficient to measurably inhibit viral replication or infectivity.
  • a dosage When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in lymphocytes) that has been shown to achieve in vitro inhibition of viral replication.
  • an "effective amount" is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease, for example to treat influenza infection. In some embodiments, an effective amount is a therapeutically effective amount.
  • an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with the disease.
  • a fusion protein is a recombinant protein containing amino acid sequence from at least two unrelated proteins that have been joined together, via a peptide bond, to make a single protein.
  • the unrelated amino acid sequences can be joined directly to each other or they can be joined using a linker sequence.
  • proteins are unrelated, if their amino acid sequences are not normally found joined together via a peptide bond in their natural environment(s) (e.g., inside a cell).
  • Immunogen is a protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen can lead to protective immunity and/or proactive immunity against a pathogen of interest.
  • Immune response refers to a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • the response is specific for a particular antigen (an "antigen-specific response").
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • the response is a B cell response, and results in the production of specific antibodies.
  • Immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against virus expressing the immunogenic polypeptide, or induces a measurable B cell response (such as production of antibodies) against the immunogenic polypeptide.
  • amino acid numbering or amino acid numbering system refers to the numbering or linear positions of amino acid residues in an immunogenic protein or polypeptide (e.g., influenza M2e) from a prototype strain or species. With a normalized sequence alignment, it allows comparison of the sequences of different orthologs of the same immunogenic protein (e.g., M2e) from other strains or species, or engineered versions of the same protein described herein with that of the prototype sequence. Utilizing such a standard or normalized amino acid numbering, conserved amino acid residues in the immunogenic proteins from various viral strains or engineered proteins can be readily identified and designated.
  • immunogenic protein or polypeptide e.g., influenza M2e
  • amino acid numbering of the M2e protein can be based on the consensus sequence of human influenza M2e protein. With this numbering, the conserved Cys residues to be mutated are referred to as residues Cys17 and Cys19 for all influenza strains.
  • Sequence identity or similarity between two or more nucleic acid sequences, or two or more amino acid sequences is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are.
  • Two sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • subject refers to any animal classified as a mammal, e.g., human and non-human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.
  • the term “treating” or “alleviating” includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., an influenza infection), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder.
  • Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • Vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject.
  • the immune response is a protective immune response.
  • a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition.
  • a vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents.
  • vaccines or vaccine immunogens or vaccine compositions are expressed from fusion constructs and self-assemble into nanoparticles displaying an immunogen polypeptide or protein on the surface.
  • a vaccine refers to an immunogenic composition capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of a disease or infection (e.g., influenza virus infection).
  • a vaccine may include, for example, attenuated or killed (e.g., split) pathogen (e.g., a virus), virus-like particles (VLPs) and/or antigenic polypeptides or DNA derived from them, or any recombinant versions of such immunogenic materials.
  • virus-like particle refers to a non-replicating, viral shell, derived from any of several viruses.
  • VLPs are generally composed of one or more viral proteins, such as, but not limited to, those proteins referred to as capsid, coat, shell, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for producing particular VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art, such as by electron microscopy, biophysical characterization, and the like. See, for example, Baker et al. (1991) Biophys. J.60:1445-1456; and Hagensee et al. (1994) J. Virol.68:4503-4505.
  • VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density banding.
  • cryoelectron microscopy can be performed on vitrified aqueous samples of the VLP preparation in question, and images recorded under appropriate exposure conditions.
  • a self-assembling nanoparticle refers to a ball-shape protein shell with a diameter of tens of nanometers and well-defined surface geometry that is formed by identical copies of a non-viral protein capable of automatically assembling into a nanoparticle with a similar appearance to VLPs.
  • Known examples include ferritin (FR), which is conserved across species and forms a 24-mer, as well as B.
  • Thermotoga maritima encapsulin which all form 60-mers.
  • Self-assembling nanoparticles can form spontaneously upon recombinant expression of the protein in an appropriate expression system. Methods for nanoparticle production, detection, and characterization can be conducted using the same techniques developed for VLPs.
  • Novel NP scaffolds with improved activities [0044] The invention provides novel nanoparticle scaffold sequences that are suitable for presenting various viral immunogenic proteins for eliciting potent neutralizing antibody responses.
  • I3-01 is an engineered protein (SEQ ID NO:22) that can self-assemble into hyperstable nanoparticles.
  • SEQ ID NO:22 The original (“un- extended” or “wildtype”) I3-01 protein was described in Hsia et al., Nature 535, 136- 139, 2016.
  • hyperstable nanoparticle scaffolds derived from I3-01 were previously developed and employed for presenting viral proteins such as that from HIV-1 and HCV. See, e.g., WO21/021603, WO22/035739, US Patent No.10,906,944, and WO19/089817.
  • the inventors rationally designed and functionally tested the known I3-01 variant scaffolds.
  • the original I3-01 protein and variants known in the art i.e., un-extended I3-01 scaffold sequences
  • the novel scaffold sequences of the invention were obtained by extending the N-terminal helix of the existing I3-01 variant scaffolds, e.g., I3-01v9 (SEQ ID NO:27), via grafting a heterologous helical motif, followed by rational design with an ensemble- based protein design program.
  • novel scaffolds is I3-01v9a (SEQ ID NO:4), as exemplified herein.
  • the resulting novel variant I3-01 scaffolds e.g., SEQ ID NO:4 are able to provide the optimal surface display of monomeric protein antigens.
  • the novel I3-01 derived NP scaffolds of the invention contains an I3-01 variant sequence (e.g., SEQ ID NO:27) except for the addition of a helix motif of about 6 to about 12 amino acid residues at the N-terminus.
  • the inserted helical motif leads to extension of the original N-terminal helix KMEELFKKHK (SEQ ID NO:26) in the I3-01 protein.
  • the inserted helical motif contains AKLAEELQK (SEQ ID NO:25), a conservatively modified variant or a substantially identical sequence thereof.
  • I3-01v9 (SEQ ID NO:27) (N-terminal helix underlined): [0048] KMEELFKKHKIVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPD ADTVIKELSFLKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEK GVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVP TGGVNLDNVCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE [0049] I3-01v9a (SEQ ID NO:4) (extension of N-terminal helix underlined): [0050] AKLAEELQKKMEELFKKHKIVAVLRANSVEEAKMKALAVFVGGV HLIEITFTVPDADTVIKELSFLKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHL DEEISQFCKEKGVFYMPGVMTP
  • the helical motif inserted at the N-terminus is identical to SEQ ID NO:25, while the rest of the scaffold sequence is a conservative modified variant or a substantially identical sequence of SEQ ID NO:27.
  • the entire extended N-terminal helix of the novel I3-01 variant scaffold is identical to the N-terminal helix in I3-01v9a, i.e., AKLAEELQKKMEELFKKHK (SEQ ID NO:28), while the remaining sequence is a conservative modified variant or a substantially identical sequence of the corresponding sequence of SEQ ID NO:27 (i.e., SEQ ID NO:27 minus the N-terminal helix).
  • Immunogenic polypeptides or proteins for generating vaccine compositions can be used to construct vaccines that present many different immunogenic proteins, including monomeric polypeptides and multimeric proteins. These include any proteins or polypeptides from pathogens against which an elicited immune response may be desired.
  • the vaccine compositions of the invention can utilize immunogenic polypeptides that are derived from any viruses, bacteria or other pathogenic organisms. Suitable immunogenic polypeptides for the invention can also be derived from non- pathogenic species, including human proteins, against which an elicited immune response may have a therapeutic effect, alleviate disease symptoms, or improve general health.
  • the immunogenic polypeptide can be any structural or functional polypeptide or peptide that contains at least about 10 amino acid residues. In some embodiments, the immunogenic polypeptides contains between about 10 to about 10,000 amino acid residues in length. In some embodiments, the immunogenic polypeptides contains between about 25 to about 2,000 amino acid residues in length. In some embodiments, the immunogenic polypeptides contains about 50 to about 500 amino acid residues in length.
  • the immunogenic polypeptides or proteins suitable for the invention can have a molecular weight of from about 1 kDa to about 1,000 kDa, and preferably from about 2.5 kDa to about 250 kDa.
  • the employed immunogenic polypeptide has a molecular weight of about 5 kDa to about 25 kDa or 50 kDa.
  • the immunogenic polypeptide or protein used in the vaccine compositions of the invention can be derived from a viral surface or core protein (target polypeptide). There are many known viral proteins that are important for viral infection of host cells.
  • glycoproteins or surface antigens, e.g., GP120 and GP41
  • capsid proteins or structural proteins, e.g., P24 protein
  • HIV surface antigens or core proteins of hepatitis A, B, C, D or E virus (e.g., small hepatitis B virus surface antigen (S-HBsAg) and the core proteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoproteins gp350/220 of Epstein- Barr virus (EBV), glycoprotein (G-protein) or the fusion protein (F-protein) of respiratory syncytial virus (RSV); surface and core proteins of herpes simplex virus HSV-1 and HSV-2 (e.g., glycoprotein D from HSV-2), surface proteins (e.g., gB, gC, gD, gH and gL) of poliovirus, envelope glycoproteins hemagglutin
  • the immunogens or immunogenic proteins displayed on the novel I3-01 NP scaffolds are monomeric proteins.
  • such proteins include, e.g., influenza M2 ectodomain (M2e) proteins exemplified herein.
  • influenza vaccines of the invention encompass NP vaccines that contain a novel I3-01 scaffold (e.g., SEQ ID NO:4) that displays a tandem repeat (e.g., 2, 3, 4 or more copies) of the M2e protein.
  • a tandem repeat e.g., 2, 3, 4 or more copies of the M2e protein.
  • one or more of the tandem M2e copies contain substitutions at the conserved Cys17 and Cys19 residues to prevent formation of random disulfide bonds.
  • HCV vaccines containing the novel I3-01 NP scaffolds (e.g., SEQ ID NO:4) that display an HCV immunogenic protein.
  • the HCV immunogenic protein to be displayed on the NP scaffold is derived from HCV glycoproteins E1 and E2, which form a heterodimer on the HCV envelope that mediates viral entry into host hepatocytes.
  • the displayed HCV protein contains E2 core.
  • E2 core as well understood in the art refers to a portion of E2 that forms a 3-dimensional structure that is recognized by broadly neutralizing antibody AR3C Fab (Law et al., Nat. Med. 2008;14:25, 2008).
  • the I3-01 variant scaffold of the invention can be used to display either a single copy of the E2 core protein or a tandem E2 core fusion protein.
  • One specific HCV E2 core protein that can be used in the HCV vaccine constructs of the invention is the redesigned E2mc3 protein as described in US Patent No.11,008,368.
  • E2mc3 derived from various HCV subtypes or isolates can be used, including E2mc3 sequences of HCV H77, J6, ED43 and UKN3A1.28c isolates (SEQ ID NOs:32-35, respectively) as exemplified herein.
  • the displayed HCV immunogenic protein is a tandem E2 core fusion protein containing SEQ ID NO:32 and SEQ ID NO:33, in any order.
  • the displayed HCV immunogenic protein is a tandem E2 core fusion protein containing SEQ ID NO:34 and SEQ ID NO:35, in any order.
  • the HCV immunogenic protein displayed by the NP scaffold contains an E1E2 heterodimer, e.g., a rationally redesigned HCV E1E2 dimer.
  • the novel I3-01 scaffold displayed HCV vaccines can additionally contain a locking domain and/or a T cell epitope.
  • the vaccine constructs can have a LD7 motif (SEQ ID NO:5) and a PADRE epitope (SEQ ID NO:6) at the C-terminus, as exemplified herein.
  • any E2 core protein sequences, tandem E2 core fusion molecules and E1E2 dimers that are known in the art or that can be readily engineered are suitable.
  • immunogenic proteins or polypeptides for display on the novel I3-01 NP scaffolds of the invention can be obtained or generated in accordance with the protocols exemplified herein or methods well known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003). V.
  • NP vaccines containing novel I3-01 NP scaffolds The invention provides nanoparticle vaccines bearing the novel I3-01 NP scaffolds disclosed herein. As noted above, some of the vaccine constructs display an HCV immunogenic protein such as a tandem E2 core protein. Some other vaccine constructs of the invention display a single copy of the influenza M2 protein ectodomain (M2e). A few examples of such influenza NP vaccines are exemplified herein. In still some other embodiments, a fusion polypeptide that contains tandem repeats of the influenza M2e protein is displayed on the novel I3-01 derived nanoparticle scaffolds.
  • HCV immunogenic protein such as a tandem E2 core protein.
  • M2e influenza M2 protein ectodomain
  • the displayed immunogenic protein displayed on the novel I3-01 scaffold sequence is a fusion polypeptide containing 2 or more tandem repeats of influenza M2e sequence.
  • at least one of the M2e tandem repeats contain missenses mutations at the conserved Cys17 and Cys19 residues to prevent random disulfide bond formation.
  • the engineered missense mutations are substitutions of each of the Cys residues with an amino acid residue that contains an uncharged polar side chains.
  • each of the two CYs residues in one or more of the tandem M2e repeats can be independently replaced with serine, glycine, asparagine, glutamine, threonine or tyrosine.
  • the tandem M2e fusion polypeptide sequence is fused to the N- terminus of the novel I3-01 scaffold sequence, e.g., via a linker motif such as GGGGS (SEQ ID NO:3) as exemplified herein.
  • the tandem M2e repeats in the displayed fusion polypeptide are separated by a short linker or spacer.
  • a GGGG (SEQ ID NO:9) spacer herein can be used to separate the different M2e sequences, as exemplified herein.
  • Some influenza NP vaccines of the invention contain 3 tandem M2e repeats.
  • the tandem M2e repeats displayed on the novel I3-01 scaffolds of the invention can be identical or different.
  • Ortholog M2e sequences from many species and modified versions thereof are known in the art. See, e.g., Mezhenskaya et al., J. Biomed. Sci.26, 76, 2019.
  • each of the M2e tandem repeats can be independently a human M2e sequence (SEQ ID NO:2), an avian/swine consensus M2e sequence (SEQ ID NO:7), or a human/swine consensus M2e sequence (SEQ ID NO:8).
  • SEQ ID NO:2e sequences from different sources are employed in the tandem M2e molecule, the different M2e motifs can be linked in any order.
  • the tandem M2e repeat sequence in the influenza vaccines of the invention can contain a human M2e sequence, an avian/swine consensus M2e sequence, and a human/swine consensus M2e sequence.
  • the 3 different Me2 sequences can be linked to the scaffold sequence in any of the 6 possible sequence orders.
  • at least 2 of the 3 tandem M2e repeats contain the substitutions at residues Cys17 and Cys19.
  • the human M2e sequence can retain unmutated residues at Cys17 and Cys19, while the avian/swine consensus M2e sequence and the human/swine consensus M2e sequence contain substituted residues (e.g., all with Ser) at Cys17 and Cys19.
  • the mutated Cys residues are all replaced with Ser residues.
  • the displayed M2e fusion polypeptide can contain, in any order, an unmutated human M2e (SEQ ID NO:2), a mutated avian/swine consensus M2e sequence SLLTEVETPTRNGWESKSSDSSD (SEQ ID NO:30), and a mutated human/swine consensus M2e sequence SLLTEVETPTRSEWESRSSGSSD (SEQ ID NO:31).
  • all 3 tandem M2e repeats contain the substitutions at residues Cys17 and Cys19.
  • the displayed M2e fusion polypeptide can contain, in any order, a mutated human M2e SLLTEVETPIRNEWGSRSNDSSD (SEQ ID NO:29), a mutated avian/swine consensus M2e sequence SLLTEVETPTRNGWESKSSDSSD (SEQ ID NO:30), and a mutated human/swine consensus M2e sequence SLLTEVETPTRSEWESRSSGSSD (SEQ ID NO:31).
  • the fusion M2e polypeptide can contain the sequence set forth in SEQ ID NO:23 or SEQ ID NO:24, a conservatively modified variant or a substantially identical sequence thereof.
  • the NP vaccines containing the novel I3-01 variant scaffolds of the invention may optionally contain a trimerization motif, e.g., SHP or foldon.
  • a trimerization motif e.g., SHP or foldon.
  • Some nanoparticle vaccine compositions can additionally contain other structural components that function to further enhance stability and antigenicity of the displayed immunogen.
  • a locking protein domain LD can be inserted into the nanoparticle construct, e.g., by covalently fused to the C-terminus of the nanoparticle subunit.
  • the locking domain can be any dimeric protein that is capable of forming an interface through specific interactions such as hydrophobic (van der Waals) contacts, hydrogen bonds, and/or salt bridges.
  • One example of locking domains that can be used in the vaccines of the invention is LD7 (SEQ ID NO:5) as exemplified herein.
  • General guidance on selecting locking domains and various other examples are described in the art, e.g., WO19/241483, US Patent NO.10,906,944, and US Patent NO.11,305,004.
  • scaffolded influenza vaccines of the invention can also contain a T-cell epitope to promote robust T-cell responses and to steer B cell development towards bNAbs.
  • the T-cell epitope can be located at any position in relation to the other structural components as long as it does not impact presentation of the engineered HA protein on the nanoparticle surface.
  • Any T-cell epitope sequences or peptides known in the art may be employed in the practice of the present invention. They include any polypeptide sequence that contain MHC class-II epitopes and can effectively activate CD4+ and CD8+ T cells upon immunization, e.g., T-helper epitope that activates CD4+ T helper cells. See, e.g., Alexander et al., Immunity 1, 751- 761,1994; Ahlers et al., J. Clin.
  • the T cell epitope inserted into the nanoparticle vaccine construct is a universal pan DR epitope peptide (PADRE), AKFVAAWTLKAAA (SEQ ID NO:6), as exemplified herein for influenza and HCV vaccines. More detailed information of T-cell epitopes suitable for the invention are described in, e.g., Hung et al., Mole.
  • Other examples of suitable T-cell epitope are also described in the art, e.g., the D and TpD epitope (Fraser et al., Vaccine 32, 2896-2903, 2014).
  • the novel I3-01 scaffold based nanoparticle vaccines of the invention can be constructed in accordance with standard recombinant techniques and other methods that have been described in the art, e.g., He et al., Nat. Comm.7, 12041, 2016; Kong et al., Nat.
  • the novel I3-01 scaffold based nanparticle vaccines can be constructed by fusing an immunogenic protein of interest (e.g., tandem HCV E2 core or tandem influenza M2e polypeptide) to the I3-01 scaffold subunit.
  • an immunogenic protein of interest e.g., tandem HCV E2 core or tandem influenza M2e polypeptide
  • C-terminus of the immunogenic protein sequence is fused to the N-terminus of the nanoparticle subunit sequence.
  • a short peptide linker or spacer (e.g., SEQ ID NOs:3 and 9) can be inserted between the immunogenic protein sequence and the nanoparticle subunit sequence or between the tandem copies of the immunogenic protein.
  • the nanoparticle vaccines of the invention can be substantially purified by any of the routinely practiced procedures. See, e.g., Guide to Protein Purification, Ed. Manualr, Meth. Enzymol.185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Substantial purification denotes purification from other proteins or cellular components.
  • a substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure.
  • antigenicity and other properties of the vaccines can also be readily examined with standard methods, e.g., antigenic profiling using known bNAbs and non-NAbs, differential scanning calorimetry (DSC), electron microscopy, binding analysis via ELISA, Biolayer Interferometry (BLI), Surface Plasmon Resonance (SPR), and co- crystallography analysis.
  • novel I3-01 scaffolds and vaccines based thereon of the invention are typically produced by first generating expression constructs (i.e., expression vectors) that contain operably linked coding sequences of the various structural components described herein.
  • expression constructs i.e., expression vectors
  • vaccine compositions of the invention are polynucleotide based (e.g., mRNA based vaccines).
  • the invention provides polynucleotides (e.g., DNA or RNA) that encode the novel I3-01 scaffolds or the subunit sequence of nanoparticle vaccines based on the scaffolds, expression vectors that harbor such polynucleotides, and host cells for producing the novel NP scaffolds and the vaccines (e.g., ExpiCHO cells as exemplified herein).
  • the fusion polypeptides encoded by the polynucleotides or expressed from the vectors are also encompassed by the invention.
  • the polynucleotides and related vectors can be readily generated with standard molecular biology techniques or the protocols exemplified herein.
  • Vectors useful for the invention may be autonomously replicating, that is, the vector exists extrachromosomally and its replication is not necessarily directly linked to the replication of the host cell's genome.
  • the replication of the vector may be linked to the replication of the host's chromosomal DNA, for example, the vector may be integrated into the chromosome of the host cell as achieved by retroviral vectors and in stably transfected cell lines.
  • Both viral-based and nonviral expression vectors can be used to produce the immunogens in a mammalian host cell.
  • Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet.15:345, 1997).
  • Useful viral vectors include vectors based on lentiviruses or other retroviruses, adenoviruses, adenoassociated viruses, cytomegalovirus, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev.
  • the host cell can be any cell into which recombinant vectors carrying a fusion of the invention may be introduced and wherein the vectors are permitted to drive the expression of the fusion polypeptide is useful for the invention. It may be prokaryotic, such as any of a number of bacterial strains, or may be eukaryotic, such as yeast or other fungal cells, insect or amphibian cells, or mammalian cells including, for example, rodent, simian or human cells.
  • Cells expressing the fusion polypeptides of the invention may be primary cultured cells or may be an established cell line.
  • a number of other host cell lines capable well known in the art may also be used in the practice of the invention. These include, e.g., various Cos cell lines, HeLa cells, HEK293, AtT20, BV2, and N18 cells, myeloma cell lines, transformed B-cells and hybridomas.
  • fusion polypeptide-expressing vectors may be introduced to the selected host cells by any of a number of suitable methods known to those skilled in the art. For the introduction of fusion polypeptide-encoding vectors to mammalian cells, the method used will depend upon the form of the vector.
  • DNA encoding the fusion polypeptide sequences may be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation. These methods are detailed, for example, in Brent et al., supra. Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. For example, LipofectAMINETM (Life Technologies) or LipoTaxiTM (Stratagene) kits are available.
  • fusion polypeptide-encoding sequences controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and selectable markers.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant vector confers resistance to the selection and allows cells to stably integrate the vector into their chromosomes.
  • selectable markers include neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., J. Mol. Biol., 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30: 147, 1984).
  • the transfected cells can contain integrated copies of the fusion polypeptide encoding sequence.
  • compositions and therapeutic applications provide pharmaceutical or immunogenic compositions and related therapeutic methods of using the vaccines based on the novel I3-01 scaffolds, e.g., influenza vaccines or HCV vaccines.
  • the vaccine compositions can be used for preventing and treating a disease or an infection (e.g., HCV infection or flu).
  • a nanoparticle displaying an immunogenic protein e.g., tandem M2e or HCV E2 core protein
  • the pharmaceutical composition can be either a therapeutic formulation or a prophylactic formulation.
  • the composition additionally includes one or more pharmaceutically acceptable vehicles and, optionally, other therapeutic ingredients (for example, antibiotics or antiviral drugs).
  • compositions of the invention are vaccines.
  • appropriate adjuvants can be additionally included.
  • suitable adjuvants include, e.g., aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-12.
  • the novel I3-01 scaffold based vaccines of the invention can be formulated as a controlled-release or time-release formulation. This can be achieved in a composition that contains a slow release polymer or via a microencapsulated delivery system or bioadhesive gel.
  • the various ppharmaceutical compositions can be prepared in accordance with standard procedures well known in the art.
  • Therapeutic methods of the invention involve administering a suitable vaccine (e.g., influenza vaccine or HCV vaccine) of the invention to a subject having or at risk of developing a disease or an infection (e.g., flu or HCV infection).
  • a suitable vaccine e.g., influenza vaccine or HCV vaccine
  • the immunogenic composition of the invention is typically administered in an amount sufficient to induce an immune response against an influenza virus or a group of viruses.
  • the immunogenic composition is provided in advance of any symptom, for example in advance of infection.
  • the prophylactic administration of the immunogenic compositions serves to prevent or ameliorate any subsequent infection.
  • a subject to be treated is one who has, or is at risk for developing, an influenza viral infection, for example because of exposure or the possibility of exposure to a virus.
  • the subject can be monitored for viral infection, symptoms associated with viral infection, or both.
  • the immunogenic composition is provided at or after the onset of a symptom of disease or infection, for example after development of a symptom of flu, or after diagnosis of an viral infection.
  • the immunogenic composition can thus be provided prior to the anticipated exposure to a virus in order to attenuate the anticipated severity, duration or extent of an infection and/or associated disease symptoms, after exposure or suspected exposure to the virus, or after the actual initiation of an infection.
  • the appropriate amount of a vaccine can be determined based on the specific disease or condition to be treated or prevented, severity, age of the subject, and other personal attributes of the specific subject (e.g., the general state of the subject's health and the robustness of the subject's immune system).
  • compositions containing a suitable vaccine of the invention can be provided as components of a kit.
  • a kit includes additional components including packaging, instructions and various other reagents, such as buffers, substrates, antibodies or ligands, such as control antibodies or ligands, and detection reagents.
  • Example 1 Rational design of a novel I3-01v9a nanoparticle scaffold
  • I3-01v9a nanoparticle scaffold We rationally optimized the I3-01v9 nanoparticle scaffold to achieve the optimal surface display of monomeric protein antigens (Fig.1).
  • the N-termini of I3- 01v9 form a wide triangle, which is desirable for displaying monomeric antigens (Fig. 1A).
  • the first residue (the antigen anchoring site) is under the nanoparticle surface, and as such, a long linker must be used to connect the antigen to the I3-01v9 N-terminus, which will increase structural instability.
  • our goal is to extend the I3-01v9 N-terminal helix so that its first residue is at the same level as the nanoparticle surface.
  • the extended N-terminal helix was then truncated to 11 residues so that its first residue would be just above the nanoparticle surface (Fig.1C).
  • Example 2 Display of HCV antigens on I3-01v9a nanoparticle scaffold
  • This Example describes multivalent display of HCV E2 cores and tandem E2 cores on the I3-01v9a nanoparticle scaffold.
  • the newly designed I3-01v9a nanoparticle scaffold (SEQ ID NO:4) has been used to present monomeric HCV E2 cores of diverse genotypes (Fig.2A).
  • E2mc3 I3-01v9a-LD7- PADRE nanoparticles designed for H77 (genotype 1a), HCV1 (genotype 1b), and ED43 (genotype 4) showed well-formed nanoparticles (Fig.2B).
  • E2mc3 of H77 isolate (SEQ ID NO:32): [0079] QLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFDSSGCPERAS GHYPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLN NTGNWFGCTWMNSTGFTKVCGAPPGGPTDGGSGPWITPRCMVDYPYRLWHY PCTINYTIFKVRMYVGGVEHRLEAACN [0080] E2mc3 of J6 isolate (SEQ ID NO:33): [0081] QLVNTNGSWHINRTALNCNDSLHTGFIASLFYTHSFNSSGCPERASG HYPRQCGVVSAKTVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNS TGSWFGCTWMNSSGYTKTCGAPPGGPTDGGSGPWLTPRCLIDYPYRLWHYPC TVNYTIFKIRMYVGGVEHRLTAACN [0082] E2m
  • the Fab65-bound hM2e folds into a compact conformation containing a ⁇ -turn (T5-E8) and a 3 10 helix (I11-W15).
  • the Fab148-bound hM2e adopts a hook conformation with an N-terminal ⁇ -turn (S2-T5).
  • PDB ID: 1TD0 trimeric scaffold
  • Fig.3A 5GS spacer
  • 1TD0 has been used in our previous study as a C-terminal motif to stabilize EBOV GP trimers.
  • Structural modeling indicates that two hM2e peptides on the 1TD0 scaffold would span 9.1 nm measured at P10.
  • the hM2e peptide was then fused to 24-meric ferritin (FR) and two “multilayered” 1c-SApNPs, E2p-LD4-PADRE (or termed E2p-L4P), and I3-01v9a-LD7-PADRE (also termed I3-01v9a-L7P), resulting in vaccine particles of 20.9 nm, 29.1 nm, and 32.4 nm, respectively (Fig.3A).
  • the four hM2e immunogens, one trimer and three 1c-SApNPs, were transiently expressed in 25ml ExpiCHO cells and purified by immunoaffinity chromatography (IAC) using antibodies Fab65 and Fab148 (Fig.3B).
  • mice/group were immunized via intradermal injection into the footpads with 10 ⁇ g total (2.5 ⁇ g/footpad) of hM2e-5GS- 1TD0, hM2e-5GS-FR, hM2e-5GS-E2p-LD4-PADRE, or hM2e-5GS-I3-01v9a-LD7- PADRE mixed with aluminum phosphate.
  • Mice were immunized twice, 3 weeks apart, and blood collected 2 weeks after each injection.
  • a group of na ⁇ ve mice was included as a negative control and a second group of mice was immunized with beta-propiolactone (BPL)-inactivated PR8 H1N1 virus (a.k.a.
  • mice were challenged intranasally (i.n.) with vaccine-matched A/Puerto Rico/8/1934 (PR8) H1N1 virus at 10 ⁇ LD 50 , the median lethal dose (determined in a previous study). Mice were weighed daily and monitored for visible symptoms of infection (including ruffled fur, hunched posture, and/or reduced activity) for 14 days post-infection (dpi). Mice that were visibly in distress or lost 75% of their original body weight were euthanized.
  • Fig.4A After the first challenge (Fig.4B), all na ⁇ ve mice and 8 of 10 mice in the 1TD0 trimer group died by 8 dpi. In contrast, survival rate was 100% for all three 1c- SApNP groups, as well as the group which received the inactivated PR8 H1N1 vaccine.
  • mice in the group which received the inactivated PR8 H1N1 vaccine died, whereas all hM2e-immunized mice survived the H3N2 challenge.
  • mice that received the inactivated H1N1 virus vaccine lost notably more weight upon the H3N2 challenge and started to regain their weight one day later (at dpi 6) and with a slower pace.
  • Our challenge data thus highlight the effectiveness and broad protection of M2e 1c-SApNP vaccines.
  • mice that received the inactivated H1N1 virus vaccine also showed no signals, consistent with the low abundance of M2 in virions32.
  • the italicized sequence indicate the 23-residue hM2e (SEQ ID NO:2).
  • the two conserved Cys residues in the hM2e sequence that are mutated in some of the tandem M2e constructs discussed below are also underlined. It is noted that the first Met residue was removed from the hM2e sequence inserted into the NP constructs described herein. As a result, while respectively termed Cys17 and Cys19 herein (and also in the literature) based on the original full hM2e sequence, they are actually the 16 th and 18 th residues in the hM2e sequence present in the vaccine constructs. The two bold and underlined residues show the restriction site for PCR.
  • the double underlined sequence represents the I3-01v9a NP scaffold subunit sequence (SEQ ID NO:4).
  • the constructs can optionally also contain a locking domain and/or a T-cell epitope.
  • the employed locking domain can be LD7 (SEQ ID NO:5) (shown in double underlined and bold font)
  • the T-cell epitope can be the PADRE epitope (SEQ ID NO:6) (shown in double underlined and italicized font).
  • Linkers or spacers separating the different structural motifs of the nanoparticle constructs are shown in italicized and underlined residues in the construct sequences herein, e.g., GS, GGGG spacer (SEQ ID NO:9) and the 5GS linker (SEQ ID NO:3).
  • hM2e-5GS-I3-01v9a-LD7-PADRE construct without N-terminal leader and LD/PADRE motifs SEQ ID NO:10
  • M2e is highly conserved, there are small but important sequence differences between IAVs from different species, which have been shown to limit cross-protection. Therefore, an M2e-based universal influenza vaccine must protect against pandemic strains that often arise from avian or swine IAVs, in addition to seasonal endemic strains. Combined use of M2e sequences from multiple species has been previously reported. Here, we designed a tandem M2e ⁇ 3 construct that contains human, avian/swine, and human/swine M2e sequences, with a GGGG (SEQ ID NO:9) spacer between the consecutive M2e segments.
  • Cys17 and Cys19 in the second (avian/swine) and third (human/swine) repeats were mutated to Serine to avoid random disulfide bonds.
  • Cys17 and Cys19 in all three repeats can be mutated to serine as exemplified herein.
  • M2e ⁇ 3 antigen was fused to 1TD0 and three 1c-SApNPs with a 5GS spacer, resulting in four constructs named M2e ⁇ 3-5GS-1TD0, M2e ⁇ 3-5GS-FR, M2e ⁇ 3-5GS-E2p-LD4-PADRE (or M2e ⁇ 3-5GS- E2p-L4P), and M2e ⁇ 3-5GS-I3-01v9a-LD7-PADRE (or M2e ⁇ 3-5GS-I3-01v9a-L7P). These four tandem M2e immunogens were transiently expressed in ExpiCHO cells and purified by IAC using an Fab148 antibody column.
  • the Fab148-purified 1c-SApNP samples were analyzed using negative-stain EM at the Scripps EM Core. Consistent with the hM2e 1c-SApNPs, all tandem M2e 1c-SApNPs showing well-formed NPs (Fig.5A). [0099] The immunogenicity and protective efficacy of tandem M2e vaccines were assessed in a mouse study following a similar schedule to the study of hM2e immunogens (Fig.4A). Two adjuvants, aluminum hydroxide (AH) and an oil-in-water emulsion, AddaVax, were tested in this study.
  • AH aluminum hydroxide
  • AddaVax additives
  • tandem M2e immunogens exhibited broad protection with the tandem M2e-5GS-1TD0 trimer being notably better than its hM2e counterpart and I3-01v9a 1c- SApNP being the best performer among all immunogens when paired with AddaVax.
  • Example 7 Sequences of some tandem M2e ⁇ 3 immunogen constructs [00100] Amino acid sequences of two exemplified tandem M2e vaccine constructs (M2e ⁇ 3-5GS-I3-01v9a-LD7-PADRE; aka M2e ⁇ 3-5GS-I3-01v9a-L7P) are set forth below (SEQ ID NOs:11 and 12).
  • Each of the two constructs contains the rationally designed I3-01v9a variant scaffold (SEQ ID NO:4) and a tandem M2e molecule having 3 M2e sequences (SEQ ID NO:23 or SEQ ID NO:24).
  • the 3 M2e sequences are respectively a human M2e sequence SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:2; Cys17 and Cys19 underlined), an avian/swine consensus M2e sequence SLLTEVETPTRNGWECKCSDSSD (SEQ ID NO:7; Cys17 and Cys19 underlined), and a human/swine consensus M2e sequence SLLTEVETPTRSEWECRCSGSSD (SEQ ID NO:8; Cys17 and Cys19 underlined).
  • each of the constructs could additionally have a leader sequence at the N-terminus.
  • the leader sequence could contain MGILPSPGMPALLSLVSLLSVLLMGCVAE (SEQ ID NO:1) as exemplified herein.
  • the conserved Cys17 and Cys19 residues in the M2e sequences or the replacing Ser residues are also underlined.
  • the two bold and underlined residues show the restriction site for PCR.
  • Linkers or spacers connecting the M2e sequences and/or the different motifs of the constructs are italicized and underlined. These include the 5 aa GS linker GGGGS (SEQ ID NO:3) and the GGGG (SEQ ID NO:9) spacer separating the tandem M2e sequences.
  • the double underlined sequence represents the display I3-01v9a scaffold sequence. Sequence of the locking domain (LD7) is double underlined and bolded. Finally, the PATRE T-cell epitope is indicated with double underlined and italicized font.
  • Tandem M2e NP construct with Cys17/CYs19 in M2e repeats #2 and #3 mutated to serine, without N-terminal leader and C-terminal LD/PADRE motifs SEQ ID NO:11: [00107] SLLTEVETPIRNEWGCRCNDSSDGGGGSLLTEVETPTRNGWESKSSDSS DGGGGSLLTEVETPTRSEWESRSSGSSDASGGGGSAKLAEELQKKMEELFKKHKI VAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSFLKELGAIIG AGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVK AMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAG VLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE [00108] Tandem M2e NP construct with Cys17/CYs19 in M2

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Abstract

La présente invention concerne de nouvelles séquences de support de nanoparticules élaborées issues de la protéine 13-01. Par comparaison avec la protéine 13-01 connue ou ses variants, les nouvelles séquences de support 13-01 de l'invention contiennent une hélice N-terminale étendue. L'invention concerne également des constructions vaccinales contenant diverses protéines immunogènes affichées sur les nouvelles séquences de support de nanoparticules de la présente invention. Les constructions vaccinales de l'invention comprennent, par exemple, des nanoparticules présentant des répétitions en tandem des protéines M2e de la grippe ou des protéines centrales E2 du VHC.
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