CN104812406A - Chlamydia antigen compositions and uses thereof - Google Patents

Abstract

The present invention provides, in part, fusion proteins derived from Chlamydia. The present invention also partly provides a method for using the fusion protein to treat or prevent Chlamydia infection.

Description

Chlamydia antigen composition and use thereof

technical field

The present invention relates to bacterial infections. More specifically, the present invention provides, in part, a fusion protein for resisting Chlamydia infection.

Background technique

Chlamydia trachomatis is an intracellular pathogen that causes more than 92 million sexually transmitted infections and 85 million ocular infections per year worldwide (Starnbach, M.N., and N.R. Roan. 2008. Conquering sexually transmitted diseases. Nat Rev Immunol 8:313-317.). Sexually transmitted Chlamydia trachomatis is a major cause of medium and long-term disease sequelae such as infertility and ectopic pregnancy in women (Brunham, R.C, D.J. Zhang, X. Yang, and G.M. McClarty. 2000. The potential for vaccine development against chlamydial infection and disease . J Infect Dis 181 Suppl 3:S538-543; Igietseme, J.U., C.M. Black, and H.D. Caldwell. 2002. Chlamydia vaccines: strategies and status. BioDrugs 16:19-35). Chlamydia trachomatis infection in women is often overlooked until severe reproductive impairment (infertility, pelvic inflammatory disease, ectopic pregnancy) has occurred. In addition, women infected with C. trachomatis have an increased risk of HIV infection after exposure.

"Seek and treat" programs for the prevention and control of sexually transmitted infections with Chlamydia trachomatis appear to be failing due to continued increases in prevalence and reinfection rates (Brunham, R.C, B. Pourbohloul, S. Mak, R. White , and M.L.Rekart.2005.The unexpected impact of a Chlamydiatrachomatis infection control program on susceptibility to reinfection.J InfectDis 192:1836-1844), may be due to early treatment interfering with the development of a protective immune response (Su,H., R.Morrison, R.Messer, W.Whitmire, S.Hughes, and H.D.Caldwell.1999.The effect of doxycycline treatment on the development of protective immunity in a murine model of chlamydial genital infection.J Infect Dis 180:2585)2- .

Previous attempts to vaccinate humans and murine models against C. trachomatis and C. muridarum infection using dead elementary bodies (EBs), which are the Non-replicating infectious particles released when cells rupture, providing limited protection (Grayston, J.T., and S.P.Wang. 1978. The potential vaccine against infection of the genital tract with Chlamydia trachomatis. Sex Transm Dis 5:73-77; Grayston ,J.T.,S.P.Wang,L.J.Yeh,and C.C.Kuo.1985.Importance of reinfection in the pathogenesis of trachoma.Rev Infect Dis 7:717-725;Lu,H.,Z.Xing,and R.C.Brunham.2002.GM- CSF transgene-based adjuvant allows the establishment of protective mucosal immunity following vaccination with inactivated Chlamydia trachomatis. J Immunol 169:6324-6331; Schachter, J., and H.D. Caldwell. 1980. Chlamydiae. Annuol 3 Rev: 9285bi-3 Rev. However, mice immunized with live Chlamydia trachomatis murine biotype EB have been shown to confer better protection (Lu, H., Z. Xing, and R.C. Brunham. 2002. GM-CSF transgene-based adjuvant allows the establishment of protective mucosalimmunity following vaccination with inactivated Chlamydia trachomatis. JImmunol 169:6324-6331; Su, H., R. Messer, W. Whitmire, E. Fischer, J.C. Portis, and H.D. Caldwell. pulsed ex vivo with nonviable Chlamydiae. J Exp Med 188:809-818).

Studies of the mechanisms underlying the efficient induction of immunity conferred by live C. murine biotypes compared to dead organisms suggest that dendritic cells (DCs) exposed to live or dead C. trachomatis develop different phenotypes. Specifically, DCs exposed to live C. murine biotypes matured and stimulated antigen-specific CD4 T cells, whereas DCs exposed to dead C. murine species were inhibited from acquiring a mature phenotype. Costimulation of DCs with dead EBs and CpG oligodeoxynucleotides has been shown to partially overcome the inhibition of DC maturation by dead EBs (Rey-Ladino, J., K.M. Koochesfahani, M.L. Zaharik, C. Shen, and R.C. Brunham. 2005. Alive and inactivated Chlamydia trachomatis mouse pneumonitis strain induces the maturation of dendritic cells that are phenotypically and immunologically distinct. Infect Immun 73:1568-1577). Investigation of the transcriptional responses of bone marrow-derived DCs following exposure to live and dead Chlamydia murinae biotypes using GeneChip microarrays reveals a marked shift in the CXC chemokine profile in DCs exposed to live or dead organisms Difference (Zaharik, M.L., T.Nayar, R.White, C.Ma, B.A. Vallance, N.Straka, X.Jiang, J.Rey-Ladino, C.Shen, and R.C.Brunham.2007. Genetic profiling of dendritic cells exposed to live-or ultraviolet-irradiated Chlamydia muridarum reveals marked differences in CXC chemokine profiles. Immunology 120:160-172). Taken together, the data show that DCs exposed to live EBs are phenotypically and functionally distinct from DCs generated by exposure to dead EBs.

Immunity to C. trachomatis infection in mice is thought to be primarily cell-mediated and thus dependent on Chlamydia-derived peptides presented to CD4 T cells by MHV molecules on antigen-presenting cells (Brunham, R.C, and J. Rey-Ladino. 2005 . Immunology of Chlamydia: implications for a Chlamydia trachomatis vaccine. Nat Rev Immunol 5:149-161; Steinman, R.M., and M. Pope. 2002. Exploiting dendritic cells to improve vaccine efficacy. J Clin Invest 109,15:1251; H., and H.D. Caldwell. 1995. CD4+T cells play a significant role in adoptive immunity to Chlamydiatrachomatis infection of the mouse genital tract. Infect Immun 63:3302-3308; Morrison, S.G., H.Su, H.D. Caldwell. Morrison.2000.Immunity tomurine chlamydial genital tract reinfection involves B cells and CD4(+)T cells but not CD8(+)T cells.Infect Immun 68:6979-6987; Morrison,R.P.,and H.D.Caldwell to lamurine.2002.Immunity genital infection. Infect Immun 70:2741-2751; Igietseme, J.U., K.H. Ramsey, D.M. Magee, D.M. Williams, T.J. Kincy, and R.G. Rank. 1993. Resolution of murine chlamydial genital infection by the adoptive Thoptive transfer -specific mphovarcy of lya biovar . Reg Immunol 5:317-324).

An immunoproteomic approach to the identification of T-cell antigens from Chlamydia trachomatis biotypes in mice (Hunt, D.F., R.A. Henderson, J. Shabanowitz, K. Sakaguchi, H. Michel, N. Sevilir, A.L. Cox, E. Appella, and V.H. Engelhard.1992.Characterization of peptidesbound to the class I MHC molecule HLA-A2.1by mass spectrometry.Science255:1261-1263; de Jong,A.1998.Contribution of mass spectrometry to contemporary immunology.Mass Spectrom3-35:1 Rev 1 Olsen, J.V., L.M.de Godoy, G.Li, B. Macek, P. Mortensen, R. Pesch, A. Makarov, O. Lange, S. Horning, and M. Mann. 2005. Parts per million mass accuracy on an Orbitrapmass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010-2021) Isolation Based on Pathogen-Derived Peptides Binding to MHC Class II Molecules Presented on the DC Surface After Pulsed with Live EBs and sequencing, which led to the identification of many C. trachomatis murine biotypes derived from 8 novel epitopes (Karunakaran, K.P., J.Rey-Ladino, N.Stoynov, K.Berg, C.Shen, X.Jiang, B.R. Gabel, H. Yu, L.J. Foster, and R.C. Brunham. 2008. Immunopreteomic discovery of novel T cell antigens from the obligate intracellular pathogen Chlamydia. J Immunol 180:2459-2465). These peptides were recognized in vitro by antigen-specific CD4 T cells, and recombinant proteins containing MHC-binding peptides were able to partially induce protection in vivo by immunization against C. trachomatis infection in mice (Yu, H, X. Jiang, C. Shen , K.P.Karunakaran, and R.C.Brunham.2009.Novel Chlamydia muridarum T cell antigens induce protective immunity against lung and genital tract infection in murine models.J Immunol 182:1602-1608).

For example in US 6030799, US 6696421, US 6676949, US 6464979, US6653461, US 6642023, US 6887843 and US 7459524; or; in the sequences disclosed in US Patent Publications 2005/0232941, 2009/0022755 and 2008/0102112 and peptides). Specific Chlamydia antigens are disclosed, for example, in PCT Publication Nos. WO 2010/085896 and WO 2013/044398.

Contents of the invention

The present disclosure provides, in part, fusion proteins derived from Chlamydia. The present invention also partly provides a method for using the fusion protein to treat or prevent Chlamydia infection.

In one aspect, there is provided an immunogenic composition comprising a fusion protein comprising at least two Chlamydia proteins selected from the group consisting of polymorphic membrane protein G (PmpG), polymorphic membrane protein G (PmpG), and a physiologically acceptable carrier. Membrane protein F (PmpF), polymorphic membrane protein E (PmpE), polymorphic membrane protein H (PmpH), ribosomal protein L6 (RplF), anti-anti-sigma factor (Anti-anti-sigma factor, Aasf), Translocated phosphoactin (Tarp), hypothetical protein corresponding to locus tag CT143/TC0420, metalloprotease, insulinase family (CT806/TC0190), hypothetical protein corresponding to locus tag CT538/TC0825, corresponding to locus A hypothetical protein of the tag CT017/TC0285, a hypothetical protein corresponding to the locus tag CT619, or MOMP, or an immunogenic fragment thereof.

In alternative embodiments, the fusion protein comprises the combination: PmpG and MOMP, PmpG and PmpF, PmpG and PmpH, or PmpE and PmpF.

In alternative embodiments, the composition further comprises an adjuvant, such as DDA/TDB, DDA/MMG or DDA/MPL.

In an alternative aspect, there is provided a method for inducing an immune response in an animal against Chlamydia or a component thereof by administering to the animal an effective amount of a composition described herein to induce in the animal immune response. The immune response may be a cellular immune response.

In an alternative aspect, there is provided a method of treating or preventing a Chlamydia infection in an animal by administering to the animal an effective amount of a composition described herein, thereby treating or preventing a Chlamydia infection in the animal .

In alternative embodiments, the Chlamydia spp. may be Chlamydia trachomatis or Chlamydia trachomatis.

In alternative embodiments, the animal may be a human.

In an alternative aspect there is provided use of a composition described herein for inducing an immune response against Chlamydia or a component thereof in an animal. The immune response may be a cellular immune response.

In an alternative aspect, there is provided use of a composition described herein for treating or preventing infection by Chlamydia or components thereof in an animal. The Chlamydia genus may be Chlamydia trachomatis or Chlamydia murine biotype. The animal can be a human.

This summary does not necessarily describe all features of the invention.

Description of drawings

These and other features of the present disclosure will become more apparent from the following description taken with reference to the accompanying drawings, in which:

Figures 1A-II show the amino acid sequence and corresponding nucleic acid sequence (SEQ ID No: 1-35) of mouse biotype Chlamydia trachomatis and Chlamydia trachomatis protein.

Figure 2A～F shows the amino acid and nucleic acid sequence of PmpE-PmpF&PmpG-PmpH fusion protein (the sequence in italics represents the second protein; The sequence that adds underline represents the α-helix linker that connects two protein structural domains; SEQ ID NO: 36 ~41).

Figures 3A-B are graphs showing protection against Chlamydia genital tract infection by fusion proteins at day 6 (A) and day 12 (B).

Figures 4A-C are graphs showing PmpE, F, G, H plus MOMP in C57 (A), Balb/c (B) or C3H (C) mice after immunization with individual (mixed) or fusion forms , A graph of the mouse C. trachomatis-specific cytokine response.

Figures 5A-C are graphs showing that after immunization with individual (mixed) or fusion forms of PmpE, F, G, H plus MOMP, in C57 (A), Balb/c (B) or C3H (C) small A graph of the antigen-specific IFN-γ responses of C. murine biotypes trachomatis in mice.

Figures 6A-C are graphs showing that after immunization with individual (mixed) or fusion forms of PmpE, F, G, H plus MOMP, the vaccines in C57 (A), Balb/c (B) or C3H (C) Graph of protection against genital tract infection with C. murine biotypes elicited in mice.

Figures 7A-C are graphs showing that after immunization with individual (mixed) or fusion forms of PmpE, F, G, H plus MOMP, the vaccines in C57 (A), Balb/c (B) or C3H (C) Graph of protection against genital tract infection with C. murine biotypes elicited in mice.

Detailed ways

The present disclosure provides, in part, fusion proteins derived from Chlamydia proteins. The present disclosure also provides, in part, a method for treating or preventing Chlamydia infection using the fusion protein.

In some embodiments, these fusion proteins are useful as vaccines for the prevention or treatment of Chlamydia infection.

Chlamydia spp.

"Chlamydia" means the genus of bacteria that are obligate intracellular parasites. The genus Chlamydia includes C. trachomatis (human pathogen) and C. trachomatis muris (causative to mice and hamsters). Since C. trachomatis and C. trachomatis are highly orthologous pathogenic microorganisms that co-evolved with their host species, C. trachomatis has been used as a robust animal for the study of cellular immunity and vaccine development Model.

In some embodiments, Chlamydia trachomatis includes, without limitation, Chlamydia trachomatis serotypes D/UW-3/CX, as well as serotypes A, B, Ba, C (involved in trachoma), serotypes D, E, F, G, H, I, J, K (involved in genitourinary tract infections) and LI, L2, L3 (lymphogranuloma venereum serotypes).

In some embodiments, the C. muridiformis trachomatis comprises the C. muridiformis trachomatis mouse pneumonia (MoPn) strain Nigg.

The genome sequences of various Chlamydia species have been determined. For example, the genome sequence of Chlamydia trachomatis strain D/UW-3/CX is described in Stephens, R.S. et al., 1998 (Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282(5389):754-759) , and provided under GenBank accession number NC_000117.1, GI:15604717; referred to herein as "Chlamydia trachomatis genome sequence".

For example, in Read, T., et al., 2000 (Genome sequences of Chlamydiatrachomatis muridarum MoPn and Chlamydia pneumoniae AR39Nucleic AcidsRes.28 (6): 1397-1406) described the genome sequence of mouse biotype Chlamydia trachomatis, and in GenBank Accession No. NC_002620.2, GI: 29337300; referred to herein as "Mouse biotype Chlamydia trachomatis genome sequence".

Chlamydia fusion polypeptide and nucleic acid molecule

Compounds for use in the compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising sequences of Chlamydia polypeptides described in two or more clauses, such as those listed in Table 1 or Table 2, or Figures 1A-II proteins or polypeptides, or immunogenic fragments thereof, and nucleic acid molecules encoding such fusion proteins.

Table 1: Homology of source proteins derived from mouse biotype C. trachomatis relative to C. trachomatis, other bacteria and humans

In some embodiments, the compounds for use in the compositions and methods according to the present disclosure include, without limitation, Chlamydia trachomatis muris or derived amino acid sequences, e.g., containing polypeptides described herein (e.g., Table 1 or 2, or those listed in Figures 1A-II, or immunogenic fragments thereof) two or more fusion proteins with substantially the same amino acid sequence.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, Chlamydia trachomatis muris or a Chlamydia trachomatis-derived nucleic acid molecule, such as a nucleic acid sequence encoding a fusion protein comprising Amino acid sequences that are substantially identical to the sequences of two or more of the polypeptides described herein (eg, those listed in Table 1 or Table 2, or Figures 1A-II, or immunogenic fragments thereof).

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, Chlamydia trachomatis muris or a Chlamydia trachomatis-derived nucleic acid molecule, e.g., with a polypeptide described herein (e.g., Table 1 or Table 2 , or those listed in Figure 1A-II, or immunogenic fragments thereof), the nucleic acid sequences of two or more of which are substantially identical.

In alternative embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising two or more Chlamydia polypeptides, such as polymorphic membrane protein G (PmpG ), polymorphic membrane protein F (PmpF), polymorphic membrane protein E (PmpE), polymorphic membrane protein H (PmpH), ribosomal protein L6 (RplF), anti-anti-sigma factor (Aasf), translocated phospho Actin (Tarp), hypothetical protein corresponding to locus tag CT143/TC0420, metalloprotease, insulinase family (CT806/TC0190), hypothetical protein corresponding to locus tag CT538/TC0825, corresponding to locus tag CT017/ A hypothetical protein of TC0285, a hypothetical protein corresponding to the locus tag CT619, or MOMP, or an immunogenic fragment thereof.

In alternative embodiments, compounds for use in the compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising two or more Chlamydia-derived amino acid sequences, e.g. or a fusion protein of more amino acid sequences having substantially the same sequence of the following polypeptides: polymorphic membrane protein G (PmpG), polymorphic membrane protein F (PmpF), polymorphic membrane protein E (PmpE), polymorphic membrane protein H (PmpH), ribosomal protein L6 (RplF), anti-anti-sigma factor (Aasf), translocated phosphoactin (Tarp), hypothetical protein corresponding to locus tag CT143/TC0420, metalloprotease, insulinase Family (CT806/TC0190), hypothetical protein corresponding to locus tag CT538/TC0825, hypothetical protein corresponding to locus tag CT017/TC0285, hypothetical protein corresponding to locus tag CT619, or MOMP, or an immunogenic fragment thereof .

In alternative embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins encoded by two or more Chlamydia-derived nucleic acid sequences, for example encoding a sequence containing and The nucleic acid sequence of the fusion protein of two or more of the following polypeptides having substantially the same amino acid sequence: polymorphic membrane protein G (PmpG), polymorphic membrane protein F (PmpF), polymorphic membrane protein E (PmpE) , polymorphic membrane protein H (PmpH), ribosomal protein L6 (RplF), anti-anti-sigma factor (Aasf), translocated phosphoactin (Tarp), hypothetical protein corresponding to the locus tag CT143/TC0420, metalloprotease, insulinase family (CT806/TC0190), hypothetical protein corresponding to locus tag CT538/TC0825, hypothetical protein corresponding to locus tag CT017/TC0285, hypothetical protein corresponding to locus tag CT619, or MOMP, or its immunogenic fragments.

In alternative embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins encoded by two or more Chlamydia-derived nucleic acid Nucleic acid sequences in which the nucleic acid sequences of one or more of the following polypeptides are substantially identical: polymorphic membrane protein G (PmpG), polymorphic membrane protein F (PmpF), polymorphic membrane protein E (PmpE), polymorphic membrane protein H (PmpH), ribosomal protein L6 (RplF), anti-anti-sigma factor (Aasf), translocated phosphoactin (Tarp), hypothetical protein corresponding to the locus tag CT143/TC0420, metalloproteases, insulinase family (CT806/TC0190), a hypothetical protein corresponding to the locus tag CT538/TC0825, a hypothetical protein corresponding to the locus tag CT017/TC0285, a hypothetical protein corresponding to the locus tag CT619, or MOMP, or an immunogenic fragment thereof.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising PmpG, PmpF, PmpE, PmpH, RplF, Aasf, Tarp, TC0420, TC0190, TC0825 , TC0285, CT619, MOMP, or an immunogenic fragment thereof, or two or more of nucleic acid molecules encoding such fusion proteins.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising PmpG, PmpF, PmpE, PmpH, RplF, Aasf, Tarp, TC0420, TC0190, TC0825 , TC0285, CT619, MOMP, or an immunogenic fragment thereof, or three or more of nucleic acid molecules encoding such fusion proteins.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising PmpG, PmpF, PmpE, PmpH, RplF, Aasf, Tarp, TC0420, TC0190, TC0825 , TC0285, CT619, MOMP, or an immunogenic fragment thereof, or four or more of nucleic acid molecules encoding such fusion proteins.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising two or more of the following Chlamydia proteins/antigens: PmpG, PmpE, PmpF , PmpH, and optionally MOMP, or an immunogenic fragment thereof, or a nucleic acid molecule encoding such a fusion protein.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising two or more of the following Chlamydia proteins/antigens: PmpG, PmpE, PmpF and TC0420, and optionally MOMP, or an immunogenic fragment thereof, or a nucleic acid molecule encoding such a fusion protein.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising the following Chlamydia proteins/antigens: PmpG and MOMP, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins. In alternative embodiments, the fusion protein contains only the following Chlamydia proteins/antigens: PmpG and MOMP, or immunogenic fragments thereof, or nucleic acid molecules encoding such fusion proteins.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising the following Chlamydia proteins/antigens: PmpG and MOMP, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins. In alternative embodiments, the compounds for use in the compositions and methods according to the present disclosure comprise fusion proteins comprising only the following Chlamydia proteins/antigens: PmpG and PmpF, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising the following Chlamydia proteins/antigens: PmpG and PmpH, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins. In alternative embodiments, the compounds for use in the compositions and methods according to the present disclosure comprise fusion proteins comprising only the following Chlamydia proteins/antigens: PmpG and PmpH, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising the following Chlamydia proteins/antigens: PmpE and PmpF, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins. In alternative embodiments, the compounds for use in the compositions and methods according to the present disclosure comprise fusion proteins comprising only the following Chlamydia proteins/antigens: PmpE and PmpF, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins.

In some embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising the following Chlamydia proteins/antigens: PmpG and TC0420, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins. In alternative embodiments, the compounds for use in the compositions and methods according to the present disclosure comprise fusion proteins comprising only the following Chlamydia proteins/antigens: PmpG and TC0420, or immunogenic fragments thereof, or Nucleic acid molecules encoding such fusion proteins.

In alternative embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, one or more of the fusion proteins depicted in Figures 2A-F.

It is understood that compositions according to the present disclosure may include fusion proteins and mixtures of individual (non-fusion) proteins or immunogenic fragments thereof, so long as at least one polypeptide in the mixture is a fusion protein.

In some embodiments, a composition according to the present disclosure includes, without limitation, a mixture of two or more fusion proteins, and optionally individual antigens, such as PmpG/PmpH and PmpE/PmpF and optionally MOMP; and/ Or a mixture of PmpG/TC0420 and PmpE/PmpF and optionally MOMP, or an immunogenic fragment thereof.

In alternative embodiments, compositions according to the present disclosure further include, without limitation, a mixture of fusion proteins, wherein MOMP or an immunogenic fragment thereof is part of the fusion protein.

In alternative embodiments, compounds for use in compositions and methods according to the present disclosure include, without limitation, fusion proteins comprising two or more Chlamydia trachomatis polypeptides, such as ribosomal protein L6 (RpIF , gi: 3328951), anti-anti-sigma factor (Aasf, gi: 15605151), polymorphic membrane protein G (PmpG, gi: 3329346), hypothetical protein (TC0420, gi: 15604862), polymorphic membrane protein F (PmpF , gi:3329345), or major outer membrane protein 1 (MOMP) (gi:3329133), or an immunogenic fragment or portion thereof. Examples of such fragments or portions of C. trachomatis polypeptides include, without limitation, amino acids 25-512 of PmpG (PmpG _25-512 ), amino acids 26-585 of PmpF (PmpF _26-585 ), or amino acids 22-393 of MOMP.

In alternative embodiments, the compounds for use in the compositions and methods according to the present disclosure further include, without limitation, fusion proteins comprising two or more murine biotype Chlamydia trachomatis polypeptides, such as ribose Body protein L6 (RpIF, gi: 15835415), anti-anti-sigma factor (Aasf, gi: 15835322), polymorphic membrane protein G (PmpG or PmpG-1, gi: 15834883), hypothetical protein TC0420 (gi: 15835038) , polymorphic membrane protein F (PmpF or PmpE/F, gi: 15834882), or major outer membrane protein 1 (MOMP, gi7190091), or an immunogenic fragment or protein thereof. Examples of such fragments or parts of murine C. trachomatis polypeptides include, without limitation, amino acids 25 to 500 of PmpG-1 (PmpG-1 _{25 to 500} ), amino acids 25 to 575 of PmpE/F-2 (PmpE/F -2 _25～575 ) or amino acid 23～387 of MOMP.

In alternative embodiments, the immunogenic fragment or portion of a Chlamydia polypeptide comprises a region of the polypeptide normally exposed on the surface of the polypeptide. In alternative embodiments, such a fragment or portion of a Chlamydia polypeptide comprises a passenger domain of a Pmp polypeptide, eg, a domain located between the signal sequence and the translocation unit.

In an alternative embodiment, the immunogenic fragment or part of the mouse biotype Chlamydia trachomatis polypeptide comprises the carrying domain of the mouse biotype Chlamydia trachomatis Pmp polypeptide or a part thereof, such as amino acids 18-667 of PmpE; amino acids of PmpE 18-575; amino acids 20-722 of PmpF; amino acids 20-575 of PmpF; amino acids 25-675 of PmpG; amino acids 25-555 of PmpG; amino acids 27-653 of PmpH; or amino acids 27-575 of PmpH. In alternative embodiments, the immunogenic fragment or portion of a C. trachomatis polypeptide comprises the load domain of a C. trachomatis Pmp polypeptide or a portion thereof. In alternative embodiments, the immunogenic fragment or portion of a Chlamydia polypeptide comprises the peptide sequences described in Table 1. In alternative embodiments, the cargo domain fragment may be about 550 amino acids, or about 600 amino acids, or less in length from the N-terminus of the Pmp polypeptide. In alternative embodiments, the immunogenic fragment may be about 25 to about 600 amino acids in length, such as about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, or any integer within these values.

In general, it is understood that the sequences of polypeptides or amino acids cited herein correspond to those indicated in the locus tags cited in the C. trachomatis genome sequence and/or the C. trachomatis genome sequence. It is also understood that the nucleic acid sequences corresponding to the loci tags can be obtained from the C. trachomatis genome sequence and/or the C. trachomatis genome sequence.

In some embodiments, fusion proteins for use in accordance with the present disclosure consist essentially of two Chlamydia polypeptides or immunogenic fragments thereof, as described herein.

In some embodiments, a fusion protein for use in accordance with the present disclosure consists essentially of a combination of three Chlamydia polypeptides or immunogenic fragments thereof, as described herein.

In some embodiments, a fusion protein for use in accordance with the present disclosure consists essentially of four Chlamydia polypeptides or immunogenic fragments thereof, as described herein.

In some embodiments, fusion proteins for use according to the present disclosure comprise at least two Chlamydia polypeptides or immunogenic fragments thereof, eg at least 2, 3, 4, 5 or more.

"Fusion protein" or "chimeric protein" means a recombinant protein or polypeptide in which at least two Chlamydia proteins or antigens (such as described herein or described in Table 1 or Table 2 or Figures 1A-II) are combined singly or recombinantly the presence of peptides. It is understood that the individual Chlamydia proteins or antigens that make up the fusion protein may be present in the fusion protein in any order or orientation. For example, in some embodiments, individual Chlamydia proteins or antigens may be present in the fusion protein in the opposite orientation (ie, N-terminally towards C-terminally) from the naturally occurring orientation. In some embodiments, fusion proteins may include full-length Chlamydia proteins or antigens. In alternative embodiments, the fusion protein may comprise a portion or fragment of a Chlamydia protein or antigen, such as a surface-exposed region of a Chlamydia protein or antigen (“carrier domain”), or a Chlamydia protein comprising an immunodominant epitope. or antigenic regions.

In some embodiments, fusion proteins may be provided in combination with heterologous peptides or polypeptides (eg, epitope tags).

In some embodiments, individual Chlamydia proteins or antigenic sequences may be linked directly to each other.

In some embodiments, the fusion protein may be provided in combination with a heterologous peptide or polypeptide (e.g., a linker or spacer) that, for example, enables proper folding and/or presentation and/or expression of the fusion protein . Linkers or spacers may be located between every single Chlamydia protein or antigen sequence, or may be located between only some of the individual Chlamydia protein or antigen sequences present in the fusion protein.

In alternative embodiments, the linker may be a heterologous linker, such as a sequence from another Chlamydia protein or antigen or from a non-adjacent position of the Chlamydia protein or antigen forming the fusion protein (eg, an alpha-helical sequence), or It may be a homologous linker, eg a sequence from one of the Chlamydia proteins or antigens forming the fusion protein and adjacent to the sequence used in the fusion protein (eg alpha helical sequence). For example, the cargo domain of a Pmp fusion partner can be linked to the polypeptide via an alpha-helical linker (shown underlined in Figure 2E-F). The linker polypeptide may be derived from the polypeptide sequence of one of the fusion protein partners. For example, linkers for PmpE-PmpF fusion proteins include sequences from PmpE, and linkers for PmpG-PmpH fusion proteins include sequences from PmpG (Figure 2E-F).

It is well known in the art that some modifications and changes can be made to the structure of a polypeptide without substantially altering the biological function of the polypeptide, for example, its ability to be cleaved into smaller peptides capable of binding to MHC proteins to obtain biological equivalent peptides. Accordingly, those skilled in the art will understand that numerical designations of amino acid positions within a sequence are relative to the specific sequence. Likewise, the same position may be assigned a different numerical designation depending on how the sequence is numbered and which sequence is selected. In addition, sequence variations such as insertions and deletions can alter the relative position, and subsequently the numerical designation, of specific amino acids at or near that position.

A "protein," "peptide," or "polypeptide" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogs, without regard to post-translational modifications (e.g., glycosylation or phosphorylation). An "amino acid sequence", "polypeptide", "peptide" or "protein" of the present invention may include peptides or proteins with abnormal linkages, cross-links and end caps, non-peptide bonds or alternatively modifying groups. Such modified peptides are also within the scope of the invention. The term "modifying group" is intended to include structures that are directly attached to the peptide structure (e.g., by covalent linkage), as well as those that are indirectly attached to the peptide structure (e.g., by a stable non-covalent linkage or by covalent linkage to another amino acid residues or mimetics, analogues or derivatives thereof, which may flank the core peptide structure). For example, a modifying group can be attached to the amino- or carboxyl-terminus of the peptide structure, or to a peptide or peptidomimetic region flanking the core domain.

Alternatively, the modifying group may be attached to the side chain of at least one amino acid residue of the peptide structure, or to a region of the peptide or peptidomimetic that flanks the core domain (e.g., through the epsilon amino group of a lysine residue, through the natural Carboxyl groups of aspartic acid residues or glutamic acid residues, hydroxyl groups through tyrosine residues, serine residues or threonine residues, or other suitable reactive groups on amino acid side chains). Modifying groups can be covalently attached to the peptide structure by means and methods known in the art for attachment to chemical structures including, for example, amide, alkylamino, carbamate or urea linkages.

In one aspect of the invention, the polypeptides of the invention also extend to biologically equivalent peptides or "variants", which differ from part of the sequence of the polypeptides of the invention by conservative amino acid substitutions, or by not affecting biological function ( e.g., non-conservative substitutions for immunogenicity). As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid for another amino acid at a specified position in a peptide, wherein a substitution can be made without substantial loss of the associated function. In making such changes, substitutions of similar amino acid residues can be made based on the relative similarity of the side chain substituents (e.g., their size, charge, hydrophobicity, hydrophilicity, etc.), and such substitutions can be analyzed by routine testing. The effect of substituents on the function of the peptide.

As used herein, the term "amino acid" means those L-amino acids, D-amino acids and modified amino acids normally found in naturally occurring proteins. Thus, amino acids of the present invention may include, for example: 2-aminoadipic acid; 3-aminoadipic acid; β-alanine; β-alanine; 2-aminobutyric acid; 4-aminobutyric acid; Glycolic acid; 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosin; 2, 2'-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; isohydroxylysine; 3-hydroxyproline; 4-Hydroxyproline; Isodesmosine; Alloisoleucine; N-Methylglycine; Sarcosine; N-Methylisoleucine; Valine; Pentine; Norleucine and Ornithine.

In some embodiments, conservative amino acid substitutions can be made, wherein an amino acid residue is substituted for another amino acid having a similar hydrophilicity value (e.g., within plus or minus 2.0, or plus or minus 1.5, or plus or minus 1.0, or plus or minus 1.0, or plus or minus within the range of values minus 0.5), where the following may be amino acids with a hydropathic index of about -1.6, such as Tyr (-1.3) or Pro (-1.6) assigned to amino acid residues (incorporated herein by reference Detailed in US Patent Application No. 4,554,101): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln ( +0.2); Gly(0); Pro(-0.5); Thr(-0.4); Ala(-0.5); His(-0.5); Cys(-1.0); Met(-1.3); Val(-1.5) ; Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

In alternative embodiments, conservative amino acid substitutions can be made, wherein an amino acid residue is substituted for another amino acid having a similar hydropathic index (e.g., within plus or minus 2.0, or plus or minus 1.5, or plus or minus 1.0, or plus or minus 0.5 values). In such embodiments, each amino acid residue can be assigned a hydrophilicity index based on its hydrophobicity and charge properties as follows: He (+4.5); Val (+4.2); Leu (+3.8); Phe (+ 2.8); Cys(+2.5); Met(+1.9); Ala(+1.8); Gly(-0.4); Thr(-0.7); Ser(-0.8); Trp(-0.9); Tyr(-1.3) ; Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9) and Arg (-4.5).

In alternative embodiments, conservative amino acid substitutions may be made using a family of publicly available similarity matrices (60, 70, 102, 103, 94, 104, 86). The PAM matrix is based on counts derived from evolutionary models, while the Blosum matrix uses counts derived from highly conserved blocks in the alignment. Conservative amino acid substitutions can be made using a similarity score greater than 0 in either the PAM or Blosum matrices.

In alternative embodiments, conservative amino acid substitutions can be made, wherein an amino acid residue is substituted for another amino acid residue of the same class, wherein amino acids are classified into non-polar, acidic, basic, and neutral classes as follows : Non-polar: Ala, Val, Leu, He, Phe, Trp, Pro, Met; Acidic: Asp, Glu; Basic: Lys, Arg, His; Neutral: Gly, Ser, Thr, Cys, Asn, Gln , Tyr.

Conservative amino acid changes may include substitution of L-amino acids with corresponding D-amino acids, conserved D-amino acids, or naturally occurring amino acids in non-genetically encoded forms, as well as conservative substitutions of L-amino acids. Naturally occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3-diaminopropionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, tert-butylalanine, tert-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, Norleucine, pentamylalanine, 2-naphthylalanine, pyridylalanine, 3-benzothienylalanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3 -Fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydroxy-isoquinoline-3-carboxylic acid, β-2-thienylalanine, methionine Sulfone, homoarginine, N-acetyllysine, 2-aminobutyric acid, 2-aminobutyric acid, 2,4,-diaminobutyric acid, p-aminophenylalanine, N-methylvaline acid, homocysteine, homoserine, sulfoalanine, ε-aminocaproic acid, δ-aminovaleric acid, or 2,3-diaminobutyric acid.

In alternative embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids are generally characterized as hydrophobic or hydrophilic primarily based on the nature of the amino acid side chain. Hydrophobic amino acids exhibit a hydrophobicity greater than zero, and hydrophilic amino acids exhibit a hydrophilicity less than zero, based on the standardized consensus hydrophobicity scale of Eisenberg et al. (Ann. Rev. Biochem. 53:595-623, 1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, He, Pro, Met, and Trp, and genetically encoded hydrophilic amino acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys. Non-genetically encoded hydrophobic amino acids include tert-butylalanine, while non-genetically encoded hydrophilic amino acids include citrulline and homocysteine.

Amino acids can be further subdivided into hydrophobic or hydrophilic amino acids based on the nature of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid having a side chain comprising at least one aromatic or heteroaromatic ring, which may include one or more substituents, such as -OH, -SH, -CN, -F, -CI, -Br , -I, -NO ₂ , -NO, -NH ₂ , -NHR, -NRR, -C(O)R, -C(O)OH, -C(O)OR, -C(O)NH ₂ , -C(O)NHR, -C(O)NRR, etc., wherein R is independently (-C ₆ ) alkyl, substituted Alkyl, (C C ₆ ) alkenyl, substituted (-C ₆ ) alkenyl, (Cj-C ₆ ) alkynyl, substituted Alkynyl, (C ₅ -C ₂₀ )aryl, substituted (C ₅ -C ₂₀ )aryl, (C ₆ -C ₂₆ )alkaryl, substituted (C ₆ -C ₂₆ )alkaryl, 5 ~20-membered heteroaryl, substituted 5-20-membered heteroaryl, 6-26-membered alkano-heteroaryl or substituted 6-26-membered alkano-heteroaryl. Genetically encoded aromatic amino acids include Phe, Tyr, and Trp, while non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, β-2-thienylalanine, 1,2,3,4-tetra Hydroxy-isoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine.

A non-polar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH, and which has a bond in which a pair of electrons shared by two atoms is equally occupied by the two atoms (ie, the side chain is non-polar). Genetically encoded non-polar amino acids include Gly, Leu, Val, He, Ala, and Met, while non-genetically encoded non-polar amino acids include cyclohexylalanine. Non-polar amino acids can be further subdivided to include aliphatic amino acids which are hydrophobic amino acids with aliphatic hydrocarbon side chains. Genetically encoded aliphatic amino acids include Ala, Leu, Val, and He, while non-genetically encoded aliphatic amino acids include norleucine.

Polar amino acids are hydrophilic amino acids with side chains that are uncharged at physiological pH, but which have a bond in which a pair of electrons shared by two atoms is more tightly occupied by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln, while non-genetically encoded polar amino acids include citrulline, N-acetyllysine, and methionine sulfoxide.

An acidic amino acid is a hydrophilic amino acid having a side chain with a pKa value of less than 7. Acidic amino acids generally have side chains that are negatively charged at physiological pH due to loss of hydrogen ions. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid having a side chain with a pKa value greater than 7. Basic amino acids generally have side chains that are positively charged at physiological pH due to association with hydrogen ions. Genetically encoded basic amino acids include Arg, Lys, and His, while non-genetically encoded basic amino acids include the acyclic amino acids ornithine, 2,3,-diaminopropionic acid, 2,4-diaminobutyric acid, and homo Leucine. Those skilled in the art will appreciate that the above classifications are not absolute and that amino acids may have more than one class classification. In addition, amino acids can be classified based on known behavior and or characteristic chemical, physical or biological properties based on specified assays or compared to previously identified amino acids. Amino acids can also include bifunctional moieties with amino acid-like side chains.

Conservative changes may also include the replacement of non-derivatized residues with chemically derivatized moieties by reaction of, for example, functional side chain groups of amino acids. Thus, such substitutions may include compounds whose free amino groups are derivatized to amine hydrochloride, p-toluenesulfonyl, benzyloxycarbonyl, t-butoxycarbonyl, chloroacetyl or formyl. Similarly, free carboxyl groups can be derivatized to form salts, methyl or ethyl esters, or other types of esters or hydrazides, and side chains to free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives, or to form The imidazole nitrogen of amino acid can be derivatized into N-im-benzylhistidine (N-im-benzylhistidine).

Peptides or peptide analogs can be synthesized by standard chemical techniques, for example by automated synthesis using solution or solid phase synthesis methods. Automated peptide synthesizers are commercially available and use techniques well known in the art. Also available are, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual. ^{3 rd} ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2000) or Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1987-2012), use recombinant DNA techniques to prepare peptides and peptide analogs.

Thus, as described herein, compounds for use according to the present disclosure comprise nucleic acid molecules encoding fusion proteins as described herein.

The term "nucleic acid" or "nucleic acid molecule" includes both RNA (plus and minus strands) and DNA, including cDNA, genomic DNA, and synthetic (eg, chemically synthesized) DNA. A nucleic acid can be double-stranded or single-stranded. When single-stranded, the nucleic acid can be either the sense strand or the antisense strand. A nucleic acid molecule can be any chain of two or more covalently associated nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. "RNA" means a sequence of two or more covalently bonded naturally occurring or modified ribonucleotides. One example of a modified RNA encompassed by this term is phosphorothioate RNA. "DNA" means a sequence of two or more covalently bonded naturally occurring or modified deoxyribonucleotides. "cDNA"means complementary or replicated DNA produced from an RNA template by the action of an RNA-dependent DNA polymerase (reverse transcriptase). Thus, "cDNA clone" means a double-helical DNA sequence complementary to an RNA molecule of interest carried by a cloning vector. "Complementary" means that the two nucleic acids (eg, DNA or RNA) contain a sufficient number of nucleotides capable of forming Watson-Crick base pairs to create a double-stranded region between the two nucleic acids. Thus, adenine in one strand of DNA or RNA pairs with thymine of the reverse complementary DNA strand or uracil of the reverse complementary RNA strand. It is understood that not every nucleotide in a nucleic acid molecule must form a matching Watson-Crick base pair with a nucleotide in the reverse complementary strand to form a double helix. A nucleic acid molecule is "complementary" to another nucleic acid molecule if it hybridizes to the second nucleic acid molecule under highly stringent conditions.

A compound is "isolated" when the compound is separated from components with which it naturally accompanies. Typically, a compound is isolated when it is at least 50 wt % or 60 wt %, or more typically at least 70 wt %, 75 wt %, 80 wt %, 85 wt %, 90 wt %, 95 wt % or 99 wt % of the total material in the sample . Thus, for example, a polypeptide that is chemically synthesized or produced by recombinant techniques will generally be substantially free of its naturally associated components. In general, a nucleic acid molecule is substantially pure or "isolated" when it is not immediately adjacent (i.e., covalently linked) to a coding sequence that is present in the naturally occurring genome of the organism from which the DNA of the invention is derived. Usually contiguous. Thus, an "isolated" gene or nucleic acid molecule is intended to mean a gene or nucleic acid molecule that is not flanked by the nucleic acid molecules that normally (actually) flank the gene or nucleic acid molecule (e.g., in a genomic sequence) and/or Or have been completely or partially purified (eg in cDNA or RNA libraries) from other transcribed sequences. For example, an isolated nucleic acid of the invention may also be substantially isolated relative to the complex cellular environment in which it naturally occurs. Thus, the term includes, for example, recombinant nucleic acid inserted into a vector (such as an autonomously replicating plasmid or virus); or into the genomic DNA of a prokaryote or eukaryote, or present as a separate molecule independent of other sequences (such as cDNA or genomic DNA fragments generated by PCR or restriction endonuclease treatment). It also includes recombinant nucleic acids that are part of a hybrid gene encoding additional polypeptide sequences. Preferably, the isolated nucleic acid comprises at least about 50, 80, or 90 percent (on a molar basis) of all macromolecular species present. Accordingly, an isolated gene or nucleic acid molecule can include a gene or nucleic acid molecule produced chemically or by recombinant means. Recombinant DNA contained in a vector is included within the definition of "isolated" as used herein. Likewise, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also include in vivo and in vitro RNA transcripts of the DNA molecules of the invention.

Each gene and nucleic acid sequence of the present invention may be a recombinant sequence. The term "recombinant" means that something has been recombined such that when referring to a nucleic acid construct, the term refers to a molecule consisting of nucleic acid sequences joined together or produced using molecular biological techniques. The term "recombinant" when referring to a protein or polypeptide refers to a protein or polypeptide molecule expressed through the use of recombinant nucleic acid constructs produced by molecular biology techniques. A recombinant nucleic acid construct may include a nucleotide sequence that is linked or manipulated to become linked to a nucleic acid sequence that is not actually linked or that is actually linked to a different location. Thus, "recombinant" in reference to a nucleic acid construct means that the nucleic acid molecule has been manipulated using genetic engineering (ie, human intervention).

For example, recombinant nucleic acid constructs can be introduced into host cells by transformation. Such recombinant nucleic acid constructs may comprise sequences derived from the same host cell species or from a different host cell species, which sequences have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences can be incorporated into the host cell genome as an initial transformation of the host cell or as a result of subsequent recombination and/or repair events.

As used herein, a "heterologous" nucleic acid or protein is a molecule that has been manipulated by human intervention such that it is located at a location other than where it is naturally found. For example, a nucleic acid sequence from one species can be introduced into the genome of another species, or a nucleic acid sequence from one genome can be moved to another genomic or extrachromosomal site in the same species. A heterologous protein includes, for example, a protein expressed from a heterologous coding sequence, or a protein expressed from a recombinant gene in a cell that does not naturally express the protein. A heterologous fusion protein may include a protein in a non-natural location (ie, N to C), or may include a fragment or portion of a protein at a position within the protein other than where it is naturally found.

A "substantially identical" sequence has only one or more conservative substitutions with the reference sequence as described herein, or one or more non-conservative substitutions at sequence positions that do not disrupt the biological function of the amino acid or nucleic acid molecule , deletions or insertions of different amino acid or nucleotide sequences. Using, for example, AlignProgram or FASTA, such sequences may be 45% to 99%, or more usually at least 45%, 50, 55% or 60%, or at least 65% identical at the amino acid or nucleotide level to the sequence being compared. %, 75%, 80%, 85%, 90% or 95%, or almost any integer identity of 96%, 97%, 98% or 99%. For polypeptides, the sequences compared may be at least 2, 5, 10, or 15 amino acids, or at least 20, 25, or 30 amino acids in length. In alternative embodiments, the sequences compared may be at least 35, 40 or 50 amino acids in length, or more than 60, 80 or 100 amino acids in length. For nucleic acid molecules, the comparison sequences may be at least 5, 10, 15, 20 or 25 nucleotides in length, or at least 30, 40 or 50 nucleotides in length. In alternative embodiments, the compared sequences may be at least 60, 70, 80 or 90 nucleotides in length, or more than 100, 200 or 500 nucleotides in length. Publicly available sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 or BLAST software available from the National Library of Medicine, or as described herein can be used. of) easily measure sequence identity. Examples of useful software include the programs Pile-up and PrettyBox. Such software matches similar sequences by assigning degrees of homology to each substitution, deletion, substitution or other modification.

Alternatively or additionally, two nucleic acid sequences may be "substantially identical" if they hybridize under highly stringent conditions. In some embodiments, highly stringent conditions are, for example, conditions that allow hybridization comparable to that at a temperature of 65°C in a medium containing 0.5M NaHPO ₄ pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Part V). buffer, or at a temperature of 42°C in a solution containing 48% formamide, 4.8×SSC, 0.2M Tris-Cl, pH 7.6, l×Danhard’s solution, 10% dextran sulfate and 0.1% SDS Hybridization occurs using DNA probes of at least 500 nucleotides in length in buffer (these are typical conditions for highly stringent northern or Southern hybridization). Hybridization may be performed over a period of about 20-30 minutes, or about 2-6 hours, or about 10-15 hours, or over 24 hours or longer. Highly stringent hybridization also relies on the success of many techniques routinely used by molecular biologists (eg, highly stringent PCR, DNA sequencing, single-strand conformational polymorphism analysis, and in situ hybridization). In contrast to northern and Southern hybridizations, these are typically performed with relatively short probes (e.g., typically about 16 nucleotides or longer for PCR or sequencing, and about 40 nucleotides or longer for in situ hybridization) these technologies. The highly stringent conditions used in these techniques are well known to those skilled in the art of molecular biology (Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY., 1998).

For example, a substantially identical sequence may be a substantially identical sequence to a Chlamydia sequence described or referenced herein. For example, the substantially identical sequence can be a sequence substantially identical to any one of SEQ ID NO: 1-71, or any one of the sequences indicated by the locus tag quoted in the Chlamydia trachomatis genome sequence and/or the sequence indicated in the text The sequence represented by any one of the represented Chlamydia trachomatis genome sequence or its fragment or variant is substantially the same sequence. In some embodiments, for example, the substantially identical sequence may be a sequence with any one of the nucleic acid sequences described herein, or a sequence encoding any one of the amino acid sequences described herein, or with the sequence cited in the Chlamydia trachomatis genome sequence. Any one of the sequences indicated by the locus tag and/or the complementary or hybrid nucleotide sequence of the Chlamydia trachomatis murine biotype genome sequence or its fragments or variants indicated herein. In some embodiments, the substantially identical sequences may be derived from a Chlamydia genus, eg, Chlamydia trachomatis or Chlamydia trachomatis muris.

Pharmaceutical & Veterinary Compositions, Dosage and Administration

The compounds and compositions described herein are useful in the preparation of vaccines or other formulations and/or for inducing an immune response to Chlamydia antigens or epitopes. In some embodiments, the composition can be formulated as a mixture of fusion proteins consisting of two or more Chlamydia proteins or immunogenic fragments thereof, as described herein. In alternative embodiments, a single fusion protein may be used to formulate the composition. In alternative embodiments, the composition may comprise MOMP as part of a fusion protein or as a single protein admixed with a fusion protein as described herein.

Alone or in combination with other compounds ( For example nucleic acid molecules, small molecules, polypeptides, peptides or peptide analogs) provide compounds or compositions in combination. Treatment with compounds according to the invention may, if desired, be combined with more traditional and existing treatments for Chlamydia infection.

Routine pharmaceutical practice may be used to provide suitable formulations for administering a compound or composition to a subject infected with the Chlamydia pathogen. Any suitable route of administration may be used, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intrathecal, intraorbital, ocular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal , epidermal, transdermal, mucosal aerosol, nasal, rectal, vaginal, topical or oral administration. In some embodiments, a compound or composition described herein can be applied to the surface of an epithelium. Some epithelial surfaces may include mucous membranes, eg, oral cavity, gums, nose, trachea, bronchi, gastrointestinal tract, genitals, rectum, urethra, vagina, cervix, uterus, and the like. Some epithelial surfaces may include keratinized cells, eg, skin, tongue, gingiva, palate, and the like. In some embodiments, the Chlamydial infection can be in the lungs, genital tract, or eye, and a compound or composition described herein can be administered intranasally or by injection.

The preparation can be in the form of liquid solution or suspension; tablet or capsule; powder, nasal drops or aerosol. Methods for making formulations are well known in the art (Berge et al. 1977. J. Pharm Sci. 66:1-19); Remington - The Science and Practice of Pharmacy, ^21st edition. Gennaro et aleditors. Lippincott Williams & Wilkins Philadelphia.). Such excipients may include, for example, salts, buffers, antioxidants, complexing agents, tonicity agents, cryoprotectants, lyoprotectants, suspending agents, emulsifying agents, antibacterial agents, preservatives Agents, chelating agents, binders, surfactants, wetting agents, anti-adhesive agents, dispersants, coating agents, glidants, deflocculants, anti-nucleating agents, surfactants, stabilizers, no Water vehicle (e.g., fixed oil), polymer or sealant for sustained or controlled release, ointment base, fatty acid, ointment base, emollients, emulsifiers, thickeners, preservatives, solubilizers, humectants, water , Alcohols, etc.

For example, formulations for parenteral administration may contain excipients, sterile water or saline, polyalkylene glycols (eg, polyethylene glycol), oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible biodegradable lactide polymers, lactide/glycolide copolymers, or polyethylene glycol-polypropylene glycol copolymers can be used to control the release of the compound or composition. Other potentially useful parenteral delivery systems for modulating compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable perfusion systems, and liposomes. Formulations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyethylene glycol-9-lauryl ether, glycocholate and deoxycholate, or may be administered as nasal drops. form or an oily solution administered as a gel.

For therapeutic or prophylactic compositions, the compound or composition is administered to the animal in an amount effective to stop or slow Chlamydia infection.

An "effective amount" of a compound according to the present invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result (eg, reduction of Chlamydia infection or induction of an immune response to a Chlamydia antigen or epitope), at dosages and for periods of time required. A therapeutically effective amount of a compound can vary depending on factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective to obtain a desired prophylactic result (eg, preventing Chlamydia infection or inducing an immune response to Chlamydia antigens or epitopes) at the required dose and time period. Typically, a prophylactic dose is used in a subject prior to or prior to a disease, so a prophylactically effective amount may be less than a therapeutically effective amount. The suitable range of the therapeutically or prophylactically effective dose of the compound may be any integer from 0.1 nM to 0.1 M, 0.1 nM to 0.05 M, 0.05 nM to 15 μM or 0.01 nM to 10 μM.

In some embodiments, effective amounts can be calculated on a mass/mass basis (eg, micrograms or milligrams per kilogram of subject), or can be calculated on a mass/volume basis (eg, concentration, micrograms or milligrams per milliliter). Using mass/volume units, one or more peptides or polypeptides may be present from about 0.1 ug/ml to about 20 mg/ml or any amount therebetween, such as 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25 , 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 10000, 20000ug/ml, or Any amount therebetween; or from about 1 ug/ml to about 2000 ug/ml, or any amount therebetween, for example 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0, 60.0, 70.0, 80.0 , 90.0, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000, 1500, 2000ug/ml, or any amount therebetween; or about 10ug/ml to about 1000ug/ml, or any amount therebetween amount, for example 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000ug/ml, or Any amount therebetween; or from about 30 ug/ml to about 1000 ug/ml, or any amount therebetween, such as 30.0, 35.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160, 180, 200 , 250, 500, 750, 1000ug/ml.

Quantities and/or concentrations can be calculated on a mass/mass basis (eg, micrograms or milligrams per kilogram of subject), or can be calculated on a mass/volume basis (eg, concentrations, micrograms or milligrams per milliliter). When using mass/volume units, one or more peptides or polypeptides may be present in about 0.1 ug/ml to about 20 mg/ml or any amount therebetween, for example 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25 , 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 10000, 20000ug/ml, or Any amount therebetween; or from about 1 ug/ml to about 2000 ug/ml, or any amount therebetween, for example 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0, 60.0, 70.0, 80.0 , 90.0, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000, 1500, 2000ug/ml, or any amount therebetween; or about 10ug/ml to about 1000ug/ml, or any amount therebetween amount, for example 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000ug/ml, or Any amount therebetween; or from about 30 ug/ml to about 1000 ug/ml, or any amount therebetween, for example 30.0, 35.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160, 180, 200 , 250, 500, 750, 1000ug/ml.

Compositions according to various embodiments of the invention comprising therapeutic compositions may comprise an effective amount of one or more peptides or polypeptides for dosing. Doses may comprise from about 0.1 ug/kg to about 20 mg/kg (based on the mass of the subject), such as 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60 , 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 10000, 20000ug/kg, or any amount therebetween; or about 1 ug/kg kg to about 2000ug/kg, or any amount therebetween, such as 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100, 120, 140, 160, 180, 200, 250, 500, 750, 1000, 1500, 2000 ug/kg, or any amount therebetween; or from about 10 ug/kg to about 1000 ug/kg, or any amount therebetween, such as 10.0, 15.0, 20.0, 25.0,30.0,35.0,40.0,50.0,60.0,70.0,80.0,90.0,100,120,140,160,180,200,250,500,750,1000ug/kg, or any amount therebetween; kg to about 1000ug/kg, or any amount therebetween, e.g. kg.

If necessary, those skilled in the art can easily interchange the units to suit the desired form of application.

It is to be noted that dosage values may vary depending on the severity of the condition to be alleviated. For any particular subject, the particular dosing regimen can be adjusted over time according to the individual needs and the professional judgment of the person administering the composition or monitoring the administration of the composition. The dosage ranges stated herein are exemplary only and do not limit the range of dosages that can be selected by a medical practitioner. The amount of active compound in the composition may vary according to factors such as the disease state, age, sex and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.

When administered, the amount of the composition administered, the method of administration, and the schedule of administration may all contribute to the observed effect. As an example, the composition may be administered systemically, such as intravenously, but with toxicity or undesired effects, whereas subcutaneous or intranasal administration of the same composition may not produce the same undesired effects. In some embodiments, local stimulation of immune cells in lymph nodes close to the site of subcutaneous injection may be beneficial, whereas systemic immune stimulation may not be beneficial.

In general, use of the compound or composition should not cause substantial toxicity. The therapeutic index, i.e., the ratio of LD50 (the dose lethal to 50% of the population) to LD100 (the dose lethal to 100% of the population) can be determined using standard techniques, for example, by testing and determining the therapeutic index in cell culture or experimental animals. toxicity of the compound. However, in some cases, eg in severe conditions, it may be necessary to administer a large excess of the composition.

Compositions according to various embodiments of the invention may be presented in unit dosage form, or in bulk form suitable for formulation or dilution for use. Compositions according to various embodiments of the present invention may be administered to a subject in a single dose or in several doses administered over time. Dosage regimens may depend, for example, on the subject's condition, age, sex, body weight, route of administration, dosage form or general health. Dosage regimens can be calculated from measurements of absorption, distribution, metabolism, excretion, and toxicity in subjects, or, for use in human subjects, can be extrapolated from measurements in experimental animals such as rats and mice. Dosage and dosage are discussed, for example, in Goodman &Gilman's The Pharmacological Basis of Therapeutics 11 ^th edition. 2006. LL Brunton, editor. McGraw-Hill, New York and Remington-The Science and Practice of Pharmacy, 21 ^st edition. Gennaro et al editors. Lippincott Williams & Wilkins Philadelphia. Optimization of treatment regimens.

"Vaccine" refers to a composition containing substances that elicit a desired immune response. A desired immune response may include protection against infection by a Chlamydia pathogen. For example, the desired immune response may comprise anywhere between 10% and 100% in a vaccinated animal compared to an unvaccinated animal, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, protection against infection by pathogens of the genus Chlamydia.

An "immune response" can generally refer to responses of the adaptive immune system (eg, humoral responses) and cell-mediated responses. The humoral response is one aspect of immunity mediated by secreted antibodies produced in cells of the B lymphoid lineage (B cells). Secreted antibodies bind to antigens on the surface of invading microorganisms, such as viruses or bacteria, which tags and destroys them. Humoral immunity is commonly used to refer to antibody production and the process that accompanies it, as well as the effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell production, opsonin promotion of phagocytosis, pathogen elimination, etc. A cell-mediated response can refer to an immune response that does not involve antibodies, but instead involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and various antigen-responsive release of cytokines. Cell-mediated immunity can generally refer to activation of some Th cells, activation of Tc cells, and T cell-mediated responses.

Antigen-presenting cells (APCs), such as dendritic cells (DCs), take up polypeptides and present epitopes of such polypeptides to other immune cells, including CD4+ and CD8+ cells, within the context of DC MHC I and II complexes. An "MHC complex" or "MHC receptor" is a cell surface receptor encoded by the major histocompatibility complex of a subject, which has the function of presenting antigens to the immune system. MHC proteins can be found on several cell types, including antigen presenting cells (APCs), such as macrophages or dendritic cells (DCs), or other cells found in mammals. Epitopes associated with MHC class I may be about 8-11 amino acids in length, while epitopes associated with MHC class II may be longer, about 9-25 amino acids in length.

Thus, an "immune response" includes, but is not limited to, one or more of the following responses in a mammal: antibodies, B cells, T cells directed specifically to antigens in the composition or vaccine following administration of the composition or vaccine (including induction of helper T cells, suppressor T cells, neurotoxin T cells, and γδT cells). Thus, an immune response to a composition or vaccine generally involves the development of a cell and/or antibody-mediated response to the composition or vaccine of interest in the host mammal. Typically, the immune response will result in the prevention or slowing of Chlamydia infection. In some embodiments, an immune response is specifically a cell-mediated response. In some embodiments, the immune response refers specifically to a cell-mediated response against a pathogen of the genus Chlamydia. In some embodiments, the compounds and compositions described herein are useful for inducing a cell-mediated immune response against Chlamydia pathogens.

Vaccines according to the present disclosure may include the polypeptides and nucleic acid molecules described herein, or immunogenic fragments thereof, and may be administered using any administration form known in the art or described herein.

An "immunogenic fragment" of a polypeptide or nucleic acid molecule refers to an epitope or amino acid or nucleic acid sequence that elicits an immune response. The term "epitope" refers to an arrangement of amino acids in a protein or a modification thereon (eg, glycosylation). Amino acids may be arranged in a linear fashion, such as the primary sequence of a protein, or once a protein is partially or fully formed, in closely adjacent secondary or tertiary arrangements of amino acids. An epitope may be specifically bound by an antibody, antibody fragment, peptide, peptidomimetic, etc., or may be specifically bound by a ligand, or may be retained within an MHC I or MHC II complex. Epitopes may be present within larger fragments or sequences of the Chlamydia proteins described herein.

Thus, an immunogenic fragment may include, without limitation, any portion of any sequence described herein that includes one or more epitopes (sites recognized by specific immune system cells, such as T cells), or sequences substantially identical thereto . For example, immunogenic fragments may include, without limitation, peptides having a length between 6 and 60 amino acids, or any value in excess of 60, such as 10 to 20 amino acids in length derived from any one or more of the sequences described herein , or a peptide of any value between 20 and 40 amino acids in length. Such fragments can be identified using standard methods known to those skilled in the art, for example using, for example, the Omiga version 1.0 program of the Oxford Molecular Group (see, e.g., U.S. Patent No. 4,708,871) (76, 77, 81, 92, 73) epitope Mapping technique or antigenicity or hydrophilicity map. Epitopes can have a size range—for example, a linear epitope can be as small as two amino acids, or can be larger, from about 3 amino acids to about 20 amino acids. In some embodiments, an epitope can be from about 5 amino acids to about 10 or about 15 amino acids in length. An epitope of a secondary or tertiary array of amino acids can contain as few as 2 amino acids, or can be larger, from about 3 amino acids to about 20 amino acids. In some embodiments, a secondary or tertiary epitope is about 5 amino acids to about 10 or about 15 amino acids proximal to some or other amino acids within the epitope. In some embodiments, fusion proteins described herein comprise multiple epitopes, in which case immunogenic fragments may comprise a substantial portion of the full protein present in the fusion protein, as described herein.

In some embodiments, the vaccine includes a suitable carrier (eg, an adjuvant), which is a preparation that acts in a non-specific manner to enhance the immune response to a specific antigen or group of antigens, such that any given vaccine Dosing can reduce the amount of antigen, or allow less frequent dosing to produce a desired immune response.

Exemplary adjuvants include, but are not limited to, aluminum hydroxide, alum, Alhydrogel ^™ (aluminum trihydrate) or other aluminum-containing salts, virosomes, nucleic acids containing CpG motifs (e.g., CpG oligodeoxynucleosides Acid (CpG-ODN), Squalene, Oil, MF59 (Novartis), LTK63 (Novartis), QS21, Various Saponins, Virus-Like Particles, Monomyristylglycerol (MMG), Monophosphoryl Lipid A (MPL) /trehalosedicorynomycolate, toll-like receptor agonists, copolymers (such as polyoxypropylene and polyoxyethylene), AblSCO, ISCOM (AbISCO-100), montanide ISA 51, Montanide ISA 720+CpG, etc. , or any combination thereof. In some embodiments, exemplary adjuvants include cationic lipid delivery agents such as dimethyl octadecyl ammonium bromide (DDA) and modified mycobacterial cord factors (mycobacterial cord factor) trehalose 6,6'-dibehenate (TDB) (DDA/TDB), DDA/MMG or DDA/MPL, or any combination thereof. Further adsorption to aluminum hydroxide (alum hydroxide ) with or without MPL, see, for example, U.S. Patent Application Nos. 6,093,406 and 6,793,923B2. In some embodiments, exemplary adjuvants include prokaryotic RNA. In some embodiments, exemplary adjuvants include Such as those described in US Patent Publication 2006/0286128. In some embodiments, exemplary adjuvants include DDA/TDB, DDA/MMG or DDA/MPL and prokaryotic RNA.

In some embodiments, vaccine compositions include, but are not limited to, fusion proteins described herein in combination with DDA/TDB, DDA/MMG, or DDA/MPL, and optionally prokaryotic RNA.

In alternative embodiments, vaccine compositions include, but are not limited to, fusion proteins described herein in combination with MOMP, in combination with DDA/TDB, DDA/MMG, or DDA/MPL, and optionally prokaryotic RNA.

In alternative embodiments, vaccine compositions include, but are not limited to, fusion proteins described herein in combination with MOMP, in combination with DDA/TDB, DDA/MMG, or DDA/MPL, and optionally prokaryotic RNA.

In an alternative embodiment, the vaccine composition comprises a) a recombinant fusion protein comprising the polypeptide sequences of the Chlamydia proteins PmpG, PmpE, PmpF and PmpH or immunogenic fragments thereof, b) the adjuvant DDA/MPL and c) prokaryotic RNA .

In an alternative embodiment, the vaccine composition comprises a) a recombinant fusion protein comprising the polypeptide sequences of the Chlamydia proteins PmpG, PmpE, PmpF and PmpH or immunogenic fragments thereof, b) the adjuvant DDA/TDB and c) prokaryotic RNA .

In an alternative embodiment, the vaccine composition comprises a) a recombinant fusion protein comprising the polypeptide sequences of the Chlamydia proteins PmpG, PmpE, PmpF and PmpH or immunogenic fragments thereof and b) the adjuvant DDA/MPL.

In an alternative embodiment, the vaccine composition comprises a) a recombinant fusion protein comprising the polypeptide sequences of the Chlamydia proteins PmpG, PmpE, PmpF and PmpH and b) the adjuvant DDA/TDB.

In an alternative embodiment, the vaccine composition comprises a formulation comprising a) a combination (mixture) of two separate fusion proteins such as PmpG/PmpH and PmpE/PmpF or immunogenic fragments thereof, respectively; b ) adjuvant DDA/MPL and c) prokaryotic RNA.

In an alternative embodiment, the vaccine composition comprises a formulation comprising a) a combination (mixture) of two separate fusion proteins of PmpG/PmpH and PmpE/PmpF or immunogenic fragments thereof, respectively; b) Adjuvant DDA/TDB and c) prokaryotic RNA.

In an alternative embodiment, the vaccine composition comprises a formulation comprising a) a combination (mixture) of two separate fusion proteins of PmpG/PmpH and PmpE/PmpF or immunogenic fragments thereof, respectively; and b ) adjuvant DDA/MPL.

In an alternative embodiment, the vaccine composition comprises a formulation comprising a) a combination (mixture) of two separate fusion proteins of PmpG/PmpH and PmpE/PmpF or immunogenic fragments thereof respectively and b) Adjuvant DDA/TDB.

In some embodiments, the compositions described herein can be used to vaccinate test subjects, such as animal models of Chlamydia infection, such as mice. Methods of experimentally inoculating experimental animals are known in the art. For example, detection of a Chlamydia vaccine may involve intranasally infecting a previously vaccinated mouse with an inoculum comprising an infectious strain of Chlamydia and assessing the development of pneumonia. For example Tammiruusu et al., 2007. Vaccine 25(2):283-290 or Rey-Ladino et al., Infection and Immunity 73:1568-1577, 2005 describe the detection of samples. It is within the ability of those skilled in the art to make any minor changes to the particular pathology model to accommodate such assays.

In another embodiment, testing a Chlamydia vaccine may involve serially inoculating female mice with the cloned and expressed candidate T cell antigens described above. A series of inoculations may comprise two, three or more consecutive inoculations. Candidate T cell antigens can be combined with an adjuvant. About three weeks after the last inoculation in the series, mice can be subcutaneously treated with 2.5 mg of Depo-Provera, and one week later, naive mice and immunized mice can be intravaginally infected with Chlamydia. mice. The number of shed organisms can be monitored at intervals of 2 to 7 days for 6 weeks after the course of infection. The amount of shed organisms can be determined by counting the formation of Chlamydial inclusions in HeLa cells, suitably using diluted vaginal wash samples. Immunity can be measured by the reduction in the amount of organisms shed by immunized mice compared to blank mice.

In some embodiments, the present disclosure also provides compositions for inducing an immune response in a subject. Compositions according to various embodiments of the present invention may be used as vaccines, or for the preparation of vaccines.

In another embodiment, the fusion protein described herein can be used to prepare medicines, such as vaccine compositions for preventing or treating Chlamydia infection. Treatment or treatment includes prophylaxis, unless prophylaxis is expressly excluded, as in alternative embodiments of the present disclosure. Treating or treating refers to reducing the severity of a chlamydial infection, in whole or in part, and/or delaying the onset of a chlamydial infection, and/or reducing the incidence of one or more symptoms or features of a chlamydial infection, including decreased survival, growth and/or transmission of Chlamydia spp. (eg, C. murine biotypes or C. trachomatis). In some embodiments, treatment includes inducing immunity in an animal subject. In an alternative embodiment, the treatment comprises inducing cellular immunity in the animal subject. For the purpose of reducing the risk of developing pathology associated with the disease, disorder and/or condition, treatment may be administered to subjects who do not exhibit symptoms of the disease, disorder and/or condition (asymptomatic subjects), and/or Subjects presenting only early symptoms of a disease, disorder and/or condition. In some embodiments, treatment includes delivery of an immunogenic composition (eg, vaccine) to a subject.

The compositions or medicaments are useful for preventing or treating a Chlamydia infection in a subject having or suspected of having such an infection. In some embodiments, the composition or medicament is useful for preventing or treating a genitourinary or ocular disorder. Urogenital disorders include, but are not limited to, urethritis, cervicitis, pharyngitis, proctitis, epididymitis, and prostatitis. Disorders of the eye include, but are not limited to, trachoma and conjunctivitis.

In some embodiments, the fusion proteins described herein, alone or in combination, are useful for diagnosing the presence of Chlamydia infection in a subject (eg, even an asymptomatic subject). Diagnosis can determine a T cell response and can be performed using any of the techniques described herein or known to the skilled artisan.

products

Also provided are articles of manufacture including packaging materials and compositions comprising one or more fusion proteins as provided herein. The composition includes a physiologically or pharmaceutically acceptable excipient, and may further include an adjuvant, a delivery agent, or both, and the packaging material may include an indication of the active ingredients of the composition (e.g., the presence of a fusion agent). protein, adjuvant or delivery agent). The label may further include the intended use of the composition, eg, a therapeutic or prophylactic composition for use in the manner described herein.

Reagent test kit

In another embodiment, a kit for the preparation of a medicament is provided, the kit comprising a composition comprising one or more fusion proteins provided herein and instructions for its use. The instructions may include a series of steps for preparing a medicament for inducing a therapeutic or prophylactic immune response in a subject to which it is administered. The kit may further include instructions for use of the medicament in the treatment, prevention, or treatment of one or more symptoms of Chlamydia infection, and include, for example, dosage concentrations, dosage intervals, preferred methods of administration, and the like.

The invention is further described in the following examples.

Example

Example 1

Molecular cloning, expression and purification of recombinant fusion proteins

PmpE, pmpF, pmpG and pmpH DNA fragments were generated by PCR using genomic DNA isolated from C. trachomatis murine biotypes. After restriction enzyme digestion using standard molecular biology techniques, the DNA fragment generated by PCR was cloned stepwise into the pET32a expression vector (GE Healthcare). For all four pmp genes, only the region encoding the bearer domain of the Pmp protein was cloned into the expression vector. Fig. 1 shows the amino acid sequences of the PmpE, PmpF, PmpG and PmpH proteins of Chlamydia murine biotype. The portion of the bearer domain between amino acids 18-575, 20-575, 25-555 and 27-575 of the whole protein of PmpE, PmpF, PmpG and PmpH, respectively, was used. A C-terminal His-tag was introduced into all fusion proteins. The plasmid containing the pmp gene was transformed into E. coli strain BL21(DE3) (Strategene), wherein the lac promoter for expression of T7 RNA polymerase was induced by using isopropyl-b-D-thiogalactopyranoside, and Perform protein expression.

Soluble expressed fusion protein was purified from lysates of E. coli by using an N-terminal GST tag and by affinity chromatography using a Glutathione Sepharose 4 High Flow Purification System (GE Healthcare). The insoluble fusion protein was purified by using a histidine binding purification system (Qiagen) with a nickel column using a C-terminal His-tag and refolded by stepwise removal of urea. LPS was removed from these proteins by adding 0.1% Triton-114 to one of the elution buffers during purification. Figure 2 shows the amino acid sequences of recombinant fusion proteins between PmpE and PmpF (ie, PmpE-PmpF) and between PmpG and PmpH (ie, PmpG-PmpH).

Protection against Chlamydia genital tract infection in mice immunized with individual proteins/antigens, as well as combinations of proteins formulated with different adjuvants was evaluated. More specifically, eight C57BL/6 mice were immunized with Chlamydia proteins PmpG (G), PmpF (F), and MOMP (M) mixed or fused with DDA/TDB (D/T) adjuvant at intervals of 2 weeks. Groups of mice 3 times. A group of mice immunized with phosphate-buffered saline (PBS) was used as a negative control. Another group of mice infected once with 1,500 inclusion forming units (IFU) of live C. murine biotypes trachomatis (EB) was used as a positive control. The experimental groups are as follows: 1) PBS (negative control), 2) PmpG+PmpF mixed protein+DDA/TDB, 3) PmpG-PmpF fusion protein+DDA/TDB, 4) PmpG+MOMP mixed protein+DDA/TDB, 5 ) PmpG-MOMP fusion protein + DDA/TDB and 6) live EBs (positive control) at 1500 IFU (middle). All groups were challenged intravaginally with 1,500 IFU of live C. murine biotype EB 4 weeks after the final immunization or 8 weeks after infection. Uterovaginal washes were taken on days 6 and 12, and bacterial titers were measured on HeLa 229 cells.

The results showed that PmpG-PmpF and PmpG-MOMP fusion proteins formulated in the adjuvant DDA/TDB were protective against Chlamydia genital tract infection when assessed on days 6 and 12 (Fig. 3A-B).

Example 2

To evaluate protection against intragenital infection with Chlamydia trachomatis murine biotypes, C57, Balb/c or C3H mice were immunized with PmpE, F, G, H plus MOMP (single protein or fusion protein) in the following groups .

C57 mice: (1) PmpE+PmpF+PmpG+PmpH+MOMP (mixed); (2) PmpE-F fusion+PmpG-H fusion+MOMP (fusion); (3) PBS; (4) live EB .

Balb/c mice: (5) PmpE+PmpF+PmpG+PmpH+MOMP (mixed); (6) PmpE-F fusion+PmpG-H fusion+MOMP (fusion); (7) PBS; (8) live EB.

C3H mice: (9) PmpE+PmpF+PmpG+PmpH+MOMP (mixed); (10) PmpE-F fusion+PmpG-H fusion+MOMP (fusion); (11) PBS; (12) live EB .

Mice were immunized three times at 2-week intervals with the fusion protein formulated with DDA/MPL. PBS was used as a negative control, and mice infected once with 1,500 inclusion forming units (IFU) of live Chlamydia trachomatis murine (EB) intranasally were used as positive controls. Four weeks after the final immunization or 8 weeks after live Chlamydia infection, all groups were challenged intravaginally with 1,500 IFU of live Chlamydia trachomatis murine primary mitochondria. Uterovaginal washes were taken on day 12 and bacterial titers were measured on HeLa 229 cells to evaluate protection.

Two weeks after the final immunization, spleen cells of mice were harvested and stimulated with HK-EB (5×10 ⁵ IFU/ml). IFN-γ-producing or TNF-α-producing CD4 T cells were analyzed by multiparameter flow cytometry. Results are expressed as mean ± SEM for groups of four mice (Fig. 4).

After immunization with single (mixed) or fusion forms of PmpE, F, G, H plus MOMP, individual C. murine trachomatis in C57, Balb/c or C3H mice were determined by ELISPOT assay. Antigen-specific IFN-γ responses. Two weeks after the final immunization, mouse spleen cells were harvested and stimulated in vitro with 5×10 ⁵ IFU/ml of HK-EB or 1 μg/ml of the Chlamydia recombinant protein for 20 hours. Results are expressed as mean ± SEM for groups of four mice (Fig. 5A-C).

Vaccine-induced resistance to Chlamydia trachomatis murine biotypes in C57, Balb/c or C3H mice after immunization with single (mixed) or fusion forms of PmpE, F, G, H plus MOMP was evaluated Infection protection. Four weeks after the final immunization, mice were challenged intravaginally with 1,500 IFU of C. trachomatis muris. Cervicovaginal washes were taken on days 3, 6, 13, and 13 post-infection, and cell shedding was measured on HeLa 229 cells. PBS-immunized mice were used as negative controls, and mice intravaginally infected with 1,500 IFU of live C. murine biotypes were used as positive controls. ***P value <0.001 compared with PBS group (Fig. 6A-C).

Vaccine-induced in C57 (A), Balb/c (B) or C3H (C) mice after immunization with single (mixed) or fusion forms of PmpE, F, G, H plus MOMP were evaluated. Protection against reproductive tract infection with Chlamydia trachomatis in mice. Four weeks after the final immunization, mice were challenged intravaginally with 1,500 IFU of C. trachomatis muris. Cervicovaginal washes were taken on days 3, 6, 13, and 13 post-infection, and bacterial shedding was measured on HeLa 229 cells. PBS-immunized mice were used as negative controls, and mice intravaginally infected with 1,500 IFU of live C. murine biotypes were used as positive controls. Mean Chlamydia IFU±SD are shown (Fig. 7A-C).

Different mouse strains showed the same level of protection against genital tract infection with C. murine biotypes after immunization with individual or fusion forms of PmpE, F, G, H plus MOMP.

All citations are incorporated herein by reference.

The invention has been described with reference to one or more embodiments. However, it will be apparent to those skilled in the art that many changes and changes can be made without departing from the scope of the invention as defined in the claims.

Claims (17)

1. an immunogenic composition, it comprises fusion rotein and the upper acceptable carrier of physiology, described fusion rotein comprises at least two kinds and is selected from following I (chlamydia) protein: polymorphic memebrane protein G (PmpG), polymorphic memebrane protein F (PmpF), polymorphic memebrane protein E (PmpE), polymorphic memebrane protein H (PmpH), Ribosomal protein L6 (RplF), anti-sigma factor (Aasf), transposition phosphoric acid actin (Tarp), corresponding to the putative protein of locus label C T143/TC0420, metalloproteases, insulinase family (CT806/TC0190), corresponding to the putative protein of locus label C T538/TC0825, corresponding to the putative protein of locus label C T017/TC0285, corresponding to the putative protein of locus label C T619, MOMP, or its immunogenic fragments.

2. compositions according to claim 1, wherein, described fusion rotein comprises PmpG and MOMP.

3. compositions according to claim 1, wherein, described fusion rotein comprises PmpG and PmpF.

4. compositions according to claim 1, wherein, described fusion rotein comprises PmpG and PmpH.

5. compositions according to claim 1, wherein, described fusion rotein comprises PmpE and PmpF.

6. the compositions according to any one of claim 1 to 6, wherein, described compositions comprises adjuvant further.

7. compositions according to claim 6, wherein, described adjuvant is selected from DDA/TDB, DDA/MMG or DDA/MPL.

8. in animal, bring out a method for the immunne response of anti-chlamydiaceae or its component, described method comprises bestows described animal by the compositions described in any one of the claim 1 to 7 of effective dose, brings out immunne response whereby in described animal.

9. method according to claim 8, wherein, described immunne response is cellullar immunologic response.

10. treat or prevent a method for chlamydia infections in animal, described method comprises bestows described animal by the compositions described in any one of the claim 1 to 7 of effective dose, treats whereby or prevents the chlamydia infections in described animal.

Method described in 11. according to Claim 8 ~ 10 any one, wherein, described chlamydiaceae is chlamydia trachomatis or mice biotype chlamydia trachomatis.

Method described in 12. according to Claim 8 ~ 11 any one, wherein, described animal is the mankind.

13. compositionss according to any one of claim 1 ~ 7 are used for the purposes of the immunne response of bringing out anti-chlamydiaceae or its component in animal.

14. purposes according to claim 13, wherein, described immunne response is cellullar immunologic response.

15. compositionss according to any one of claim 1 ~ 7 are used for the treatment of or prevent the purposes of chlamydia infections in animal.

16. purposes according to any one of claim 13 ~ 15, wherein, described chlamydiaceae is chlamydia trachomatis or mice biotype chlamydia trachomatis.

17. purposes according to any one of claim 13 ~ 16, wherein, described animal is the mankind.