Leptospirosis vaccine

Abstract

Leptospirosis is a disease endangering life. The present disclosure provides compositions comprising an effective amount of a plurality of leptospirosis proteins or immunogenic fragments thereof. The disclosure also provides methods of generating an immune response against leptospirosis using a composition comprising the plurality of leptospirosis proteins or immunogenic fragments thereof and an adjuvant.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 302,817, filed on January 25, 2022 (which is incorporated herein by reference in its entirety).

[0003] STATEMENT OF RIGHTS TO INVENTS FROM FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with government support under Grants AI088752 and AI121207 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Leptospirosis is a life-threatening disease that can occur in a variety of epidemiological settings. Current leptospirosis control measures have been ineffective in addressing the enormous human and animal health burden caused by leptospirosis worldwide. The major challenge is to develop a vaccine to protect against all 38 genetically diverse pathogenic species and more than 300 serovars, which are potential agents of leptospirosis. All attempts to identify candidate vaccines that can be used as a broadly applicable vaccine against leptospirosis have failed.

[0006] Therefore, there is a need in the art for an effective leptospirosis vaccine that protects against multiple genetically diverse pathogenic species and serovars. The present invention addresses this need. Summary of the invention

[0007] In one aspect, the invention provides a composition comprising a plurality of Leptospira peptides, each Leptospira peptide having 80% or greater sequence identity to one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0008] In another aspect, the present invention provides a method of conditioning an immune response against Leptospirosis in a subject, the method comprising administering to the subject an effective amount of a composition comprising a plurality of Leptospira peptides, each Leptospira peptide having 80% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0009] In various embodiments, each Leptospira peptide has 90% or greater sequence identity to one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0010] In various embodiments, each Leptospira peptide has 95% or greater sequence identity to one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0011] In various embodiments, the composition further comprises an adjuvant.

[0012] In various embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0013] In various embodiments, the composition includes a plurality of Leptospira peptides, including a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 1, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 2, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 3, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 4.

[0014] In various embodiments, the composition includes a plurality of Leptospira peptides, including a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:7, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:8.

[0015] In various embodiments, the composition includes a plurality of Leptospira peptides, including a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:3, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:4, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6.

[0016] In various embodiments, the composition is administered to the subject in three separate doses.

[0017] In various embodiments, the subject is a mammal.

[0018] In various embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A-1B Depicted L1-130 fcpA - Spread of mutants in animal tissues. Figure 1A : Vaccination 10 7 Figure 2. Infection kinetics of L1-130 WT, L1-130 fcpA-vaccine, and L1-130 heat-killed vaccine in hamster blood, kidney, liver, and brain after 10 days of infection. All animals infected with the WT strain died 5 to 6 days after infection. Figure 1B :L1-130 WT (10 7 Leptospira) and L1-130 fcpA - Attenuated vaccine (dose range 10 7 Up to 10 1 The results are expressed as log genome equivalents per gram or milliliter of tissue and the mean and standard deviation. In both models, all doses were administered subcutaneously.

[0020] Figure 2A-2G Depicted L1-130 fcpA - Efficacy of attenuated vaccine model. Animals were given 10 7 Leptospira (hamster) dose or 10 7 Up to 10 1 Vaccination was performed with a range of Leptospira (mouse) doses. Animals were bled one day before immunization (day -1) and on the 20th day after immunization ( Figure 2A ). Hamsters were challenged with either the homologous strain or a different heterologous strain by the conjunctival route. Mice were challenged with a heterologous serovar of the Manilae type of L. interrogans by the intraperitoneal route. By combining all vaccine experiments performed, the efficacy of hamsters ( Figure 2B and Figure 2D ) and mice ( Figure 2C and Figure 2E ) vaccine against mortality and colonization and are presented as percentages and 95% CIs based on the frequency of results compared with animals immunized with PBS. - Results after vaccination with attenuated vaccine (red) and heat-inactivated vaccine (blue). Figure 2F ) and mice ( Figure 2G ) and in animals immunized with PBS (blue) and with fcpA - Comparisons were made between animals immunized with attenuated mutants (red). Results are expressed as log genome equivalents per gram of kidney tissue and as mean and standard deviation. Asterisks indicate statistical significance calculated by t-test: *p<0.01, ***p<0.0001. See also Tables 3 and 4.

[0021] Figures 3A-3G Depicted L1-130 fcpA - Immunogenicity and immune correlates of attenuated vaccine models. 7 Leptospira (hamster) or doses ranging from 10 7 Up to 10 1 Individual sera from hamsters and mice were obtained using attenuated Leptospira vaccine (mouse). Microscopic agglutination test (MAT) was performed using all strains used for challenge in hamsters and mice as antigens. Figure 3A and Figure 3C ) and Western blot ( Figure 3B and Figure 3D In addition, ELISA was also used to determine ( Figure 3E )—whole cell extracts of Manilae serovars treated with (red) and without (blue) proteinase K as antigens—were tested on mouse sera. 7 fcpA - A pool of hamster immune sera with attenuated vaccine Leptospira doses was used for passive transfer experiments. 2 mL or 0.5 mL of sera were passively transferred to naive hamsters ( Figure 3F ) or mice ( Figure 3G ), and then via the conjunctival (CJ) or intraperitoneal (IP) route, with 10 8 Manilae type heterologous serovar Leptospira challenge. Results showed that passive transfer of fcpA - Survival curves of animals treated with antiserum (red) and control hamster serum (blue) are represented.

[0022] Figures 4A-4D Depicts targeting L1-130 fcpA - Proteomic array analysis of immune sera from attenuated vaccines. Using statistical modeling, t statistics were calculated for each individual antigen used in the proteomic array based on three groups (660 for hamsters and 330 for mice): 10 7Leptospira vaccine doses in vaccinated and unvaccinated hamsters ( Figure 4A ) or mice ( Figure 4B ) between the two groups; each antigen has 10 7 Up to 10 1 Dose-response relationship in mice with a range of attenuated Leptospira vaccines ( Figure 4C ). The results are ranked according to the individual t-statistics for each antigen, and the dashed lines represent the antigen selection points based on the Bhp test. Venn diagram ( Figure 4D ) shows the relationship of all 154 antigens identified in these three groups. The subset of antigens selected for further characterization is highlighted in color. See Figure 7.

[0023] Figures 5A-5D In silico analysis of 154 protein targets is depicted. Using PSORB information and the Genoscope database, the 154 protein targets identified in this study were sorted by their putative localization in the cell ( Figure 5A ) and its clusters of orthologous groups (COG) classification ( Figure 5C The group classification of proteins is as follows: proteins identified in all three groups (7, red); 7 Proteins identified in hamsters and mice immunized with attenuated vaccine Leptospira (31, yellow); 7 Proteins identified between groups of mice immunized with different doses of Leptospira (2, green); 7 Proteins identified between groups of hamsters immunized with different doses of Leptospira (1, blue); proteins identified only in groups of mice immunized with different doses (3, purple); 7 Proteins identified in the group of mice immunized with 10 Leptospira doses (16, orange); and only in the group of mice immunized with 10 7 Proteins identified in the hamster groups immunized with each dose of Leptospira (94 proteins, brown). Based on PSORB localization ( Figure 5B ) and COG( Figure 5D ), and the enrichment analysis of reactive antigens compared with the entire proteome of Leptospira was performed. Statistical results are indicated by *(p<0.05), **(p<0.001) and ***(p<0.0001). See Table 5.

[0024] Figure 6 The mouse dose-response relationship is depicted, showing different doses of attenuated L1-130 fcpA in mice -Correlation between attenuated vaccines and the average signal response intensity against all different proteins.

[0025] FIG. 7 depicts the results of the experiment using attenuated L1-130 fcpA - Heat map of 41 serum-reactive proteins recognized by hamsters and mice immunized with attenuated vaccines. Proteins were selected based on the groups shown in Figure 4 and Table 5: present in all three analyzed groups (red), present in 7 In hamsters and mice immunized with Leptospira (yellow), 7 Leptospira in hamsters and mice immunized with a range of doses (blue), as well as in mice immunized with 10 7 The proteins are identified by their L. interrogans serovar Copenhageni ORF number, and the heat map shows that vaccination with fcpA - Signal intensity (based on log fold change) of the antibody response in mutants, all animals used for this analysis (37 hamsters and 34 mice). The right panel shows the amino acid sequence identity of the corresponding ORF among representatives of all pathogenic Leptospira species.

[0026] FIG8 depicts the attenuated L1-130 fcpA - Complementation heatmap of 41 serum reactive proteins recognized by hamsters and mice immunized with attenuated vaccines. Proteins were selected based on the groups shown in Figure 4 and Table 5: present in all three analyzed groups (red), present in 7 In hamsters and mice immunized with Leptospira (yellow), 7 Leptospira in hamsters and mice immunized with a range of doses (blue), as well as in mice immunized with 10 7 The leptospira immunized mice and mice immunized with a dose range (green). These proteins are identified by their interrogative leptospira Copenhageni type serovar ORF numbering, and the heat map shows the signal intensity (based on the logarithm of the multiple change) of the antibody response in all control animals (14 hamsters vaccinated with heat-inactivated vaccines, 37 PBS control hamsters and 4 PBS control mice) for this analysis. The heat map also shows the results of 30 leptospirosis patients. The right figure shows the amino acid sequence identity of the corresponding ORF among the representatives of all intermediate and saprophytic leptospira species.

[0027] Fig. 9Depicted are 8 targets selected from a list of outer membrane proteins (OMPs) identified by attenuated vaccines: LIC20250 (SEQ ID NO: 1), LIC12631 (SEQ ID NO: 2), LIC10050 (SEQ ID NO: 3), LIC10054 (SEQ ID NO: 4), LIC11436 (SEQ ID NO: 5), LIC10486 (SEQ ID NO: 6), LIC11959 (SEQ ID NO: 7), and LIC12544 (SEQ ID NO: 8). The combination "OMP1" includes LIC20250, LIC12631, LIC10050, and LIC10054; the combination "OMP2" includes LIC11436, LIC10486, LIC11959, and LIC12544; and the combination "OMP3" includes LIC10050, LIC10054, LIC11436, and LIC10486.

[0028] Figures 10A-10B Depicted are survival and renal colonization following vaccination with "OMP1" and "OMP2" in a hamster model. Fig. 10A , survival curve; Fig. 10B , Kidney colonization determined by qPCR after challenge with Copenhageni type serovars.

[0029] Figures 11A-11E Survival and renal colonization after vaccination with "OMP1" are depicted. Fig.11A , protection against mortality after challenge with Copenhageni type serovars (homologous); Fig. 11B , cross protection against mortality after challenge with Hardjo serovar (heterologous); Fig. 11C , renal colonization in hamsters challenged with Copenhageni type serovars; Fig.11D , renal colonization in hamsters challenged with Hardjo serovar; Fig.11E , Survival kinetics of Copenhageni type serovar (Fiocruz) in blood.

[0030] Figures 12A-12B The immunogenicity of multiple recombinant constructs of "OMP1" is depicted. Fig. 12A , the effect of enhancing the production of antibodies against Copenhageni type serovars in hamsters with "OMP1"; Fig. 12B , using "OMP1" to enhance the effect on the production of antibodies against the Hardjo serovar.

[0031] Fig.13Depicted is the cross-protection of "OMP1" multiple recombinant constructs against kidney colonization in hamsters by Leptospira interrogans (Copenhageni, Pomona, and Canicola serovars).

[0032] Figures 14A-14D The composition of the multiple recombinant constructs was shown to influence the ability to induce cross-protection against death and renal colonization. Fig.14A , survival of hamsters immunized with “OMP2” and “OMP3” after challenge with Copenhageni type serovars; Fig. 14B , survival of hamsters immunized with “OMP2” and “OMP3” after challenge with Hardjo serovar; Fig. 14C , renal colonization of hamsters immunized with “OMP2” and “OMP3” after challenge with Copenhageni type serovars; Fig.14D , kidney colonization of hamsters immunized with “OMP2” and “OMP3” after challenge with Hardjo serovar.

[0033] Fig.15 It is depicted that anti-"OMP3" sera induce high levels of cross-bactericidal activity (50-60%).

[0034] Fig.16 is a graph showing that "OMP3" induces cross-species sterilizing immunity in a hamster model.

[0035] Fig.17 is a graph showing that sera from animals vaccinated with "OMP3" induce higher levels of cross-bactericidal activity than sera from animals vaccinated with "OMP1" or "OMP2".

[0036] Fig.18 is a graph showing that sera of animals vaccinated with "OMP3" induce a high level of cross-bactericidal activity against Leptospira spp. strains P1 and P2, for which animal experiments were not possible.

[0037] Fig.19 is a graph showing that the immune response to the "OMP3" construct induced a significant enhancement against all proteins and whole-cell Leptospira after each immunization. DETAILED DESCRIPTION

[0038] The present disclosure relates to compositions and methods for inducing an immune response against Leptospira to prevent and treat leptospirosis. On the one hand, the present disclosure provides a composition comprising a plurality of Leptospira peptides, each Leptospira peptide having 80% or higher sequence identity with one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In certain embodiments, the composition consists essentially of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, collectively referred to herein as "OMP3". In some embodiments, the composition consists essentially of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and an adjuvant.

[0039] definition

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with its defined manner, where definitions are provided.

[0041] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] It is also to be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein, as such methods and conditions may vary.

[0043] In addition, unless otherwise indicated, the experiments described herein use conventional molecular and cell biology and immunological techniques within the technical scope of the art. Such techniques are well known to those skilled in the art and are fully described in the literature. See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (4th edition) MR Green and J. Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0044] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. In the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Unless otherwise indicated, the use of "or" means "and / or". The use of the term "including" and other forms such as "includes" and "included" is not restrictive.

[0045] In general, the nomenclature used for cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. Unless otherwise indicated, the methods and techniques provided herein are generally carried out according to conventional methods described in various general and more specific references as quoted and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as usually completed in this area or as described herein. The nomenclature used for analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein, and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery and the treatment of patients.

[0046] The compounds of the present disclosure may include one or more asymmetric centers and may thus give rise to enantiomers, diastereomers and other stereoisomeric forms. The present disclosure is intended to encompass all such possible forms, as well as their racemic and resolved forms and mixtures thereof.

[0047] In order to facilitate understanding of the present disclosure, selected terms are defined below.

[0048] As used herein, the articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0049] As used herein, when referring to a measurable value such as an amount, a time duration, etc., the term "about" is intended to encompass variations within ±20% or 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value, as long as such variations are suitable for performing the disclosed methods. Unless the context indicates otherwise, all numerical values ​​provided herein are modified by the term "about".

[0050] The term "antibody" or "Ab" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to a specific epitope on an antigen. An antibody can be an intact immunoglobulin derived from a natural source or a recombinant source, and can be an immunoreactive portion of an intact immunoglobulin. Antibodies useful in the present invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1998, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426). Antibodies may be derived from natural or recombinant sources. Antibodies are generally tetramers of immunoglobulin molecules.

[0051] As used herein, the term "antigen" or "Ag" is defined as a molecule that stimulates an immune response. This immune response may involve the production of antibodies, or the activation of specific immunocompetent cells, or both. It will be understood by those skilled in the art that any macromolecule, including almost all proteins or peptides, can serve as an antigen. In addition, antigens can also be derived from recombinant or genomic DNA. It will be understood by the technician that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response thus encodes an "antigen", as the term is used herein. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. It is obvious that the present invention includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to induce the desired immune response. In addition, it will be understood by the skilled technician that antigens do not need to be encoded by a "gene" at all. It is obvious that antigens can be generated, synthesized, or derived from biological samples. Such biological samples may include but are not limited to tissue samples, tumor samples, cells or biological fluids.

[0052] As used herein, "heterologous antigen" refers to an antigen that is not endogenous to the organism that includes or expresses the antigen. As used herein, the term "heterologous protein" refers to a protein that elicits a beneficial immune response in a subject (ie, a mammal), regardless of its origin.

[0053] The terms "specific binding", "selective binding" or "binding specificity" refer to the ability of a humanized antibody or binding compound of the invention to bind to a target epitope with a higher affinity than when binding to a non-target epitope. In certain embodiments, specific binding refers to binding to a target with an affinity that is at least 10, 50, 100, 250, 500, or 1000 times greater than the affinity for a non-target epitope.

[0054] As used herein, "combination therapy" means that a first agent is administered in conjunction with another agent. "In combination with" or "in conjunction with" means that in addition to one treatment modality, another treatment modality is administered. Thus, "in combination with" means that before, during, or after one treatment modality is delivered to an individual, another treatment modality is administered. Such combinations are considered part of a single treatment regimen or regime.

[0055] "Humoral immunity" or "humoral immune response" both refer to B cell-mediated immunity and are mediated by highly specific antibodies produced and secreted by B lymphocytes (B cells).

[0056] "Incorporated into" or "encapsulated within" refers to an antigenic protein or peptide within a delivery vehicle, such as microparticles, bacterial ghosts, attenuated bacteria, virus-like particles, attenuated viruses, ISCOMs, liposomes, preferably virosomes.

[0057] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must include at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein that includes two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (also commonly referred to as, for example, peptides, oligopeptides and oligomers in the art); and longer chains (commonly referred to as proteins in the art, which have various types). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0058] The term "isolated" as used herein in connection with a polypeptide, as in "isolated polypeptide," refers to a polypeptide separated from other cellular proteins, and is meant to encompass both purified and recombinant polypeptides. Thus, the term "isolated polypeptide(s)" refers to a polypeptide / polypeptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or free of chemical precursors or other chemicals when chemically synthesized.

[0059] As used herein, "identity" refers to the subunit sequence identity between two polymer molecules, in particular between two amino acid molecules, such as between two polypeptide molecules. When two amino acid sequences have the same residue at the same position, for example, if the position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. When aligning, the identity or degree of having the same residue at the same position of two amino acid sequences is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., five positions in a polymer of ten amino acids in length) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or identical, the two amino acid sequences are 90% identical.

[0060] As used herein, a "subject" or "patient" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human. In some embodiments, the subject is a domestic pet or livestock. In some embodiments, the subject is a mouse. In some embodiments, the subject is a hamster.

[0061] The term "biological sample" refers to a sample obtained from an organism or from a component (e.g., a cell) of an organism. The sample can be any biological tissue or fluid. Typically, the sample will be a "clinical sample", i.e., a sample derived from a patient. These samples include, but are not limited to, bone marrow, heart tissue, sputum, blood, lymph, blood cells (e.g., leukocytes), tissue or fine needle aspiration biopsy samples, urine, peritoneal fluid and pleural fluid, or cells thereof. Biological samples may also include tissue sections, such as frozen sections obtained for histological purposes.

[0062] "Vaccination" refers to the process of administering at least one antigen to a subject to elicit an immune response in the subject, which helps prevent or treat a disease or disorder associated with COVID-19. The term "immunization" is used interchangeably with vaccination herein.

[0063] "Leptospira bacterium" or "Leptospira bacteria" refers to one or more spirochetal bacteria. Leptospira bacteria are very slender, tightly coiled, obligately aerobic spirochetes characterized by a unique tortuosity motility. Leptospira bacteria are gram-negative spirochetes with internal flagella. The genus is divided into 64 species: pathogenic Leptospira represented by P1 and P2 and free-living Leptospira represented by S1 and S2. Serotypes P1 and P2 are the agents of leptospirosis, a zoonosis.

[0064] "Pathogen" refers to an infectious agent that is capable of causing infection, producing toxins and / or causing disease in a host, such as a bacterium of the genus Leptospira.

[0065] "Flagellar-coiling protein A" or "FcpA" refers to an abundant protein from Leptospira that is exposed on the surface of the periplasmic flagellum. The gene encoding FcpA is highly conserved in Leptospira and is not present in other bacteria. FcpA(-) mutants obtained from clinical isolates or by allelic exchange are unable to generate translational motility and do not cause disease in the standard hamster leptospirosis model.

[0066] As used herein, "homologous" when applied to nucleic acids or proteins refers to sequences having about 50% sequence identity. More preferably, homologous sequences have about 75% sequence identity, and even more preferably, have at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0067] As used herein, the term "immunogenicity" refers to the innate ability of an antigen or organism to elicit an immune response in an animal when the antigen or organism is administered to the animal. Thus, "enhancing immunogenicity" refers to increasing the ability of an antigen or organism to elicit an immune response in an animal when the antigen or organism is administered to the animal. The increased ability of an antigen or organism to elicit an immune response can be measured by a greater number of antibodies that bind to the antigen or organism, a greater diversity of antibodies against the antigen or organism, a greater number of T cells specific for the antigen or organism, a greater cytotoxic or helper T cell response to the antigen or organism, greater expression of cytokines in response to the antigen, and the like.

[0068] A "fragment" of a polypeptide refers to at least about 5 to about 25 consecutive amino acids of a polynucleotide. It should be understood that a portion of a polypeptide may include every amino acid residue of the polypeptide. An "immunogenic fragment" is a fragment that is capable of inducing an immune response in a subject.

[0069] An "adjuvant" is a substance that enhances the immunogenicity of an antigen. An adjuvant can be a single substance or a mixture of substances and works by acting directly on the immune system or by providing a slow release of the antigen. Examples of adjuvants are aluminum salts, polyanions, bacterial glycopeptides, and sustained-release agents such as Freund's incomplete adjuvant or AddaVax TM , which is an oil-in-water nanoemulsion adjuvant.

[0070] "Delivery vehicle" refers to a composition that helps target antigens to specific cells and promotes the immune system to effectively recognize antigens. The best-known delivery vehicles are liposomes, virosomes, microparticles (including microspheres and nanospheres), polymers, bacterial ghosts, bacterial polysaccharides, attenuated bacteria, virus-like particles, attenuated viruses and ISCOMS. "Attenuation" refers to bacteria with reduced virulence relative to wild-type bacteria. Specifically, the bacteria have an attenuated virulence with a virulence reduction of about 10, 20, 30, 40, 50, 60, 70, 80% or more compared to wild-type bacteria.

[0071] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound useful in the present invention and other chemical components such as carriers, stabilizers, diluents, adjuvants, dispersants, suspending agents, thickeners and / or excipients. Pharmaceutical compositions facilitate the administration of compounds to organisms. There are a variety of techniques for administering compounds in the art, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration.

[0072] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or transporting the compound (s) of the invention into the body of a subject or subject so that it can perform its intended function. Generally speaking, such compounds are carried or transported from one organ or part of the body to another organ or part of the body. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and lauryl glycol. Acid ethyl ester; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; flavoring agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic compatible substances for pharmaceutical preparations, or any combination thereof. As used herein, "pharmaceutically acceptable carriers" also include any and all coatings, antibacterial and antifungal agents, and absorption delaying agents that are compatible with the activity of the compound and are physiologically acceptable to the subject. Supplementary active compounds can also be incorporated into the composition.

[0073] An "effective amount" refers to an amount required to reduce or ameliorate at least one symptom of a disorder, condition, or disease relative to an untreated patient. An effective amount for therapeutic treatment of a condition or disease or stimulation of an immune response varies depending on the specific disorder, condition, or disease, the extent of the disorder, condition, or disease, the administration of the cells, and the age, weight, and general health of the subject.

[0074] The term "vaccine" as used herein refers to a composition, bacterium, protein or nucleic acid of the invention, which is used to protect an animal from Leptospira bacterial disease and / or to treat an animal already infected with Leptospira bacteria, compared to an otherwise identical animal not administered the vaccine or compared to the animal prior to administration of the vaccine.

[0075] "Virulent" refers to the degree of pathogenicity of a given pathogen or the ability of an organism to cause disease in another organism. Virulence refers to the ability to invade a host organism, cause disease, evade immune responses, and produce toxins.

[0076] "Bacterial virulence" refers to the degree of pathogenicity of bacteria (e.g., Leptospira bacteria). Bacterial virulence includes causing infection or disease in a host, producing an agent that causes or enhances disease in a host, producing an agent that causes or enhances the spread of disease to another host, and causing infection or disease in another host.

[0077] "Virulent" or "pathogenic" refers to the ability of a bacteria to cause serious illness.

[0078] "Non-pathogenic" means incapable of causing disease.

[0079] "Wild type" refers to the non-mutated form of a gene, allele, genotype, polypeptide or phenotype, or a fragment of any of these. It can occur in nature or be recombinantly produced.

[0080] "Infection" refers to bacterial colonization of a host. Infection of a host occurs when one or more bacteria enter through a break in a barrier epithelial surface, such as an unhealed break in the skin, eye, or mucous membrane.

[0081] "Infectious disease" refers to a disease or condition in a subject caused by a pathogen that can be transmitted or transferred to an uninfected subject. Non-limiting examples of infectious diseases include bacterial infections, viral infections, fungal infections, and the like.

[0082] In the present disclosure, "comprises", "comprising", "containing", and "having", etc. may have the meanings assigned to them by U.S. Patent Law, and may mean "includes", "including", etc.; "consisting essentially of" or "consists essentially of" also have the meanings assigned to them by U.S. Patent Law, and the term is open-ended, allowing for more features than those recited, as long as the basic or novel characteristics of the recited features are not changed by the presence of more features than those recited, but the prior art embodiments are excluded. In some embodiments, the term "consisting essentially of" refers to a composition whose only active ingredient is the indicated active ingredient(s) (e.g., the indicated peptide), however, other compounds may be included for stabilizing the formulation, preserving the formulation, etc., but not directly involved in the therapeutic effect of the indicated active ingredient. The use of the transitional phrase "consisting essentially of" means that the scope of the claim should be interpreted to include the specified materials or steps recited in the claim, as well as those materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not meant to be interpreted as equivalent to "comprising".

[0083] As used herein, the terms "control" or "reference" are used interchangeably and refer to a value used as a standard for comparison. A control subject is, for example, a subject immunized with an adjuvant alone, or a subject immunized with a peptide or protein that is not expected to elicit an immune response.

[0084] As used herein, the term "eliciting an immune response" or "immunization" refers to the process of generating a B cell and / or T cell response against a foreign protein.

[0085] As used herein, the terms "treat," "treating," "ameliorating," "treatment," and the like refer to the reduction or improvement of an infectious disease or condition and / or one or more symptoms associated therewith. It should be understood that, although not excluded, treatment of an infectious disease or condition and / or one or more symptoms associated therewith does not require complete improvement or elimination of the disorder, condition, disease, or symptom associated therewith. It is meant that the clinical signs and / or symptoms associated with the disease are alleviated as a result of the actions performed. The signs or symptoms to be monitored will be known to the skilled clinician.

[0086] "Prevention" refers to vaccination against a disorder using a pharmaceutical composition.

[0087] "Immune response" means the actions taken by the host to defend itself against pathogens or abnormalities. Immune responses include innate (natural) immune responses and adaptive (acquired) immune responses. Innate responses are antigen-nonspecific. Adaptive immune responses are antigen-specific. The immune response in an organism provides protection to the organism against infection with a Leptospira bacterium when compared to an otherwise identical subject or a person prior to such administration to which the composition or cell has not been administered.

[0088] Range: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0089] Recitation herein of a variant or aspect of an embodiment includes that embodiment as any single embodiment or in combination with any other embodiment or portions thereof.

[0090] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0091] Composition

[0092] Without being limited by theory, the present invention is based in part on the discovery that, in general, vaccination with certain Leptospira peptides can provide immunity to Leptospirosis regardless of serovar. The present disclosure provides a universal vaccine against Leptospirosis using multiple recombinant protein constructs. In some embodiments, the multiple recombinant protein constructs include an effective amount of 2, 3, 4, 5, 6, 7 or 8 polypeptides selected from the following, or fragments thereof:

[0093] SEQ ID NO: 1 (LIC20250; uncharacterized OmpA-like protein);

[0094] SEQ ID NO: 2 (LIC12631; hemolysin);

[0095] SEQ ID NO: 3 (LIC10050; uncharacterized OmpA-like protein);

[0096] SEQ ID NO:4 (LIC10054; uncharacterized lipoprotein);

[0097] SEQ ID NO: 5 (LIC11436; uncharacterized FecR domain-containing protein);

[0098] SEQ ID NO: 6 (LIC10486; uncharacterized protein);

[0099] SEQ ID NO:7 (LIC11959; uncharacterized protein); and

[0100] SEQ ID NO: 8 (LIC12544; uncharacterized DNA binding protein).

[0101] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1.

[0102] MAKKENYYITIKGRKYDRKLIQLAEEFTSGKRDGKISINDAKRLLKIVKDNNAYTDIEKHTIEYIRENYKFTEKSDEWFRSEIRKWAAKKVQEAKKKSDVESILVDDSEAPEINFPSSWGEDKTEVVEITQTSKIDWRENSNFSSATSHSKKNKKIIPTLIFLSGFLILVGLVYFFRTLFYKEDLEQVVKTNS EIVSNSKEKQSDVSIEKAESTKEVRKKNVRSKKEESEIPKNALTILKPQTGKKLESKSLFSSLTNQNSTEEFSSNPQFREIESNVIRFEKNSIQIHKESRPSLNRLARWMKQDSSIRVKVIGHTSLEGSEDANQKVSLLRAQTVRNYIAGNGISKDRFEIIPKGASVPIGDNSKEEGKEMNRRVELRIYN(SEQ ID NO:1)

[0103] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:2.

[0104] (SEQ ID NO:2).

[0105] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3.

[0106] (SEQ ID NO:3).

[0107] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:4.

[0108] MVFSRSTDLFKGINRTEETMKQIAILTALIIFTSCASVESKRSVSASGDPSEIFFEKEIIPMDSNSNFVSQKPARRSFEEELSVEKYAKAQPPEKTNSSGDFDEVGMSSWYGAKFHGKPTASGEKFDKTKLTAAHPTLPLGSIIRVQNLENQKEVIVRVNDR GPFVKDRIIDLSEKAADTLDFKDVGIAKVGIKVVKRGGAANEESEDLENSDDEEALLEDGKPEKLNPQKSDYQNKPIAGGKYIKGAPKGYTVQVGVFREQSRAESYKSNLGQEYGEKTFLFTRDGLFVIQLGDFASRTEAESLKSKLKNDGIDCFIPKK(SEQ ID NO:4).

[0109] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5.

[0110] (SEQ ID NO:5).

[0111] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:6.

[0112] MEPSNQVNKLQEQANLMNLALESVLTEEQAVELIQGKIRDAYLLKIRIDIENRSGAVVALVTKYKNEVIEIFSLFSNSSLIRKIRSFQDSAAFALDMVEAAKSEPYDPGLSDSIGRIVYSKLTKAVLESSYANWDKNDASSLVNILENQIKTSLKVNIVRIQADVEFISSLKFRAKNVFT GIIPTVNRPVEEPSISQVPESQEKTPVQRQIDQFRKPFGRVILSKTVLAPVGGVDFDELTEGDRLYFQLPTGSMDEKAMAKTLGGIDEDGNVRNVVGEFIGIAAGKGEYHIFAKGPSGVLLQAFEERPVRLAKVKTKTSSSASTTKTETSGGGSLGIIIVAGVVLVVGLLVFLIMK(SEQ ID NO:6).

[0113] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7.

[0114] LLRFLNIQKILWINFLFLYISSLSVFAQEIHRAASTYRSSISLSEPRISDIKEALSSESPNFPNSLKLFFQELKGNYAIFYDWNGETVYYKYRINKFDKSKLKQVRKLSEGAAYEVNGLWEGLIVFQVSTVPLFKKASEISLEEKKEKSSIPVFDLVEFKELSLDEILY(SEQ ID NO:7)

[0115] In some embodiments, the composition for stimulating an immune response includes a polypeptide having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8.

[0116] LNQKRVGQILREAREEKKLSVKDVARETNISVKYILALETEDYSQFPGETFTMGFLKNYGGYLKLDTGQLINLYRGEKIEESQAPLEELTRPTASYYTKINVDKNKIFTIASVLIILISAYLIIDSFIGPSSDEDSVEESGKKLDIPENIDFLSRSIPESRSESFILTPDKGVSFSVSNQQCKLFID SVEKGGAVNTAVLAFNVYPELTVYKFRLSEGQEKILSYTIPEISSLRRNVRIISQAVTENSAKVLVTLSDEEQKQENNNSVDNTKTLGDVPIQVTLFFNKPSYAEFIIDGQMGFRGLVQTGETRSLEAKDRLELKVGDGSAVEMIQNGKPKITLGRPGKLVKKIFVKTQNPYDSTQSIIKELGE(SEQ ID NO:8).

[0117] In some embodiments, the composition that induces an immune response includes proteins of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and are collectively referred to as "OMP1". In some embodiments, the composition that induces an immune response includes proteins of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and are collectively referred to as "OMP2". In some embodiments, the composition that induces an immune response includes proteins of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and are collectively referred to as "OMP3".

[0118] In some embodiments, the composition includes at least four immunogenic fragments from any one of SEQ ID NO: 1 to SEQ ID NO: 8. The immunogenic domains in the protein can be identified with suitable software, including but not limited to AbDesigner. By using such software or equivalent software, the immunogenic characteristics of the protein can be identified based on the prediction model. The prediction of the immunogenic domain in the protein is based on multiple protein factors, including hydrophobicity, secondary structure, uniqueness, conservation between species and the presence or absence of post-translational modifications. The range of immunogenic peptides is generally about 10 to about 25 amino acids. In one embodiment, the composition for inducing an immune response includes one or more immunogenic fragments from any one of SEQ ID NO: 1 to SEQ ID NO: 8.

[0119] The present invention also includes a composition of a protein or an immunogenic fragment thereof, which further includes a pharmaceutically acceptable carrier. In another embodiment, the composition further includes an adjuvant, such as an oil-in-water emulsion, a saponin, cholesterol, a phospholipid, CpG, a polysaccharide, a variant thereof, and any combination thereof.

[0120] In one aspect, the present invention provides a composition comprising a plurality of Leptospira peptides, each Leptospira peptide having 80% or higher sequence identity with one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. As used herein, the term "Leptospira peptide" refers to a peptide found in Leptospira bacteria, such as the Leptospira peptides disclosed herein. In this regard, each Leptospira peptide has 80% or higher sequence identity with one of SEQ ID NO: 1-8.

[0121] In certain embodiments, the composition comprises a plurality of Leptospira peptides, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 1, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 2, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 3, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 4. In some embodiments, the composition consists essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In some embodiments, the composition consists essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and an adjuvant.

[0122] In certain embodiments, the composition comprises a plurality of Leptospira peptides, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:7, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:8. In some embodiments, the composition consists essentially of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the composition consists essentially of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and an adjuvant.

[0123] In certain embodiments, the composition comprises a plurality of Leptospira peptides, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:3, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:4, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6. In some embodiments, the composition consists essentially of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the composition consists essentially of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and an adjuvant.

[0124] Protein production

[0125] The protein of the present invention or its immunogenic fragment can be produced by any known method for producing linear amino acid sequences, such as recombinant DNA technology. The nucleic acid sequence encoding the protein or peptide of the present invention, or the polymer of the peptide is introduced into an expression vector. Suitable expression vectors are, for example, plasmids, cosmids, viruses and YAC (yeast artificial chromosomes) - which include control regions necessary for replication and expression. The expression vector can be stimulated to express in a host cell. Suitable host cells are, for example, bacteria, yeast cells and mammalian cells. Such techniques are well known in the art and are described, for example, by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. Other well-known techniques are degradation or synthesis by coupling one amino acid residue with the next amino acid residue in a liquid phase or preferably on a solid phase (resin) - for example, by so-called Merrifield synthesis. See, e.g., Barany and Merrifield in the Peptides, Analysis, Synthesis, Biology, Vol. 2, E. Gross and Meinhofer, Ed. (Acad. Press, NY, 1980), Kneib-Coronier and Mullen Int. J. Peptide Protein Res., 30, p. 705-739 (1987), and Fields and Noble Int. J. Peptide Protein Res., 35, p. 161-214 (1990).

[0126] Protein purification

[0127] In some embodiments, the protein of SEQ ID NO: 1 to SEQ ID NO: 8 or its immunogenic fragment is purified from the contaminant. The contaminant may be the cell protein remaining after cloning and expressing the protein of interest in the cell system, or the chemical substance remaining after the chemical synthesis of the protein or its fragment. Suitable methods for purifying proteins or peptides from a mixture of contaminants include, but are not limited to, dialysis, gel filtration chromatography, anion exchange chromatography, cation exchange chromatography, affinity chromatography or hydrophobic interaction chromatography such as C4 or C18 chromatography combined with high performance liquid chromatography (HPLC). The purity of the protein can be assessed by methods including, but not limited to, SDS polyacrylamide gel electrophoresis (PAGE), native PAGE, mass spectrometry, HPLC, FPLC and immunoblotting. In certain embodiments, the purity of the peptide (one or more) of the present invention is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.

[0128] In some embodiments, the present invention provides mRNA molecules encoding one or more of the proteins of SEQ ID NO: 1 to SEQ ID NO: 8, or mRNA encoding immunogenic fragments thereof. In some embodiments, the mRNA is chemically modified to increase stability.

[0129] Pharmaceutical compositions and preparations

[0130] The vaccines of the present invention may be formulated into pharmaceutical compositions. In some embodiments, the vaccines include one or more purified or isolated proteins from SEQ ID NO: 1 to SEQ ID NO: 8.

[0131] The pharmaceutical composition can be in a form suitable for administration to a subject (i.e., a mammal), and the pharmaceutical composition can also include one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The various components of the pharmaceutical composition can exist in the form of a physiologically acceptable salt, as is well known in the art, such as in combination with a physiologically acceptable cation or anion.

[0132] In one embodiment, the pharmaceutical composition used to practice the methods of the present invention may include an adjuvant. Non-limiting examples of suitable adjuvants are Freund's complete adjuvant, Freund's incomplete adjuvant, AddaVax TM , Quil A, Detox, ISCOM, Squalene, MPLA and CpG or other activators of TLR or inflammasome. The pharmaceutical composition or vaccine composition may include any one or more adjuvants described herein.

[0133] Pharmaceutical compositions useful in the methods of the invention can be appropriately developed for inhalation, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intravenous or other routes of administration. Other formulations contemplated include projected nanoparticles, liposome preparations, resealed erythrocytes containing active ingredients, and immunologically based formulations. The route of administration (one or more) will be apparent to the skilled person and will depend on many factors, including the type and severity of the disease being treated, the type and age of the animal or human patient being treated, etc.

[0134] Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals are well known, and such modifications can be designed and performed by an ordinary skilled veterinary pharmacologist with only ordinary experimentation, if any.

[0135] The compositions of the present invention may include from about 0.005% to 2.0% of a preservative, based on the total weight of the composition.Preservatives are used to prevent spoilage due to exposure to environmental contaminants.

[0136] Reagent test kit

[0137] In some embodiments, a kit is provided for treating, preventing or ameliorating a given disease, disorder or condition, or a symptom thereof as described herein, wherein the kit comprises: a) a composition as described herein; and optionally b) an additional agent or therapy as described herein. The kit may also include instructions or labels for using the kit to treat, prevent or ameliorate a disease, disorder or condition. In yet other embodiments, the invention extends to a kit assay for a given disease, disorder or condition, or a symptom thereof as described herein. Such a kit may, for example, comprise reagents from PCR or other nucleic acid hybridization techniques (microarrays) or reagents for immunologically based detection techniques (e.g., ELISpot, ELISA).

[0138] method

[0139] In another aspect, the present invention provides a method of modulating an immune response against leptospirosis in a subject, the method comprising administering to the subject an effective amount of a composition comprising a plurality of leptospiral peptides, each leptospiral peptide having 80% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. The immune response is enhanced by repeated administration of Leptospira proteins, such as by repeated administration of proteins of SEQ ID NO: 1 to SEQ ID NO: 8, and a transient antibody response can be maintained over a period of time. In one embodiment, a mixture of proteins comprising SEQ ID NO: 3, 4, 5, 6 ("OMP3") is used to immunize a subject and enhance the subject. The antibodies generated can confer protection against infection by homologous pathogens, i.e., the strain used for immunization (i.e., Leptospira Copenhageni serovar), or heterologous pathogens (e.g., different Leptospira bacteria) in the subject or in a new subject upon transfer. The antibodies produced can be of any class, such as IgG, IgM or IgA, or of any subclass, such as IgGl, IgG2a and other subclasses known in the art.

[0140] In certain embodiments, the composition comprises a plurality of Leptospira peptides, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 1, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 2, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 3, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 4. In some embodiments, the composition consists essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. In some embodiments, the composition consists essentially of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and an adjuvant.

[0141] In certain embodiments, the composition comprises a plurality of Leptospira peptides, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:7, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:8. In some embodiments, the composition consists essentially of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In some embodiments, the composition consists essentially of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and an adjuvant.

[0142] In certain embodiments, the composition comprises a plurality of Leptospira peptides, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:3, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:4, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6. In some embodiments, the composition consists essentially of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the composition consists essentially of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and an adjuvant.

[0143] Antigens that stimulate an immune response, but do not produce pathogenic disease in a subject are exemplary vaccine candidates.An immunogenic Leptospira protein or immunogenic fragment thereof that stimulates an immune response is included in the methods of the present invention.

[0144] The methods further include administering adjuvants together with the compositions of the invention, such as oil-in-water emulsions, saponins, cholesterol, phospholipids, CpG, polysaccharides, variants thereof, and combinations thereof - either separately or in tandem with the compositions of the invention.

[0145] In various embodiments, the composition is administered to a subject in three doses. In various embodiments, the subject is a mammal. In various embodiments, the subject is a human.

[0146] Pharmaceutical formulations useful in the methods of the present invention can be appropriately developed for inhalation, oral, parenteral, pulmonary, intranasal, intravenous, or other routes of administration. Other contemplated formulations include modified nanoparticles, liposome preparations, and immunologically based formulations. The route of administration (one or more) will be apparent to the skilled person and will depend on many factors, including the type and severity of the disease being treated, the type and age of the animal or human patient being treated, and the like.

[0147] The pharmaceutical preparations described herein can be prepared by any method known in the art of pharmacology or developed thereafter. In general, such preparation methods include the step of associating the cell with a carrier or one or more other auxiliary ingredients, and then, if necessary or desired, shaping or packaging the product into a desired single or multiple dosage unit.

[0148] In one embodiment, the cells of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical preparation of the cells of the invention comprises a therapeutically effective amount of the cells of the invention and a pharmaceutically acceptable carrier. Useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0149] Administration / Dosage

[0150] In a clinical setting, the delivery system of the compositions described herein can be introduced into a subject by any of a variety of methods, each of which is familiar in the art. For example, the pharmaceutical formulation of the composition can be administered by inhalation or systemically, such as by intravenous injection.

[0151] The administration schedule may affect what constitutes an effective amount. The therapeutic formulation may be administered to a subject before or after the onset of symptoms associated with the disease or condition. In addition, several divided doses and staggered doses may be administered daily or sequentially, or the dose may be a continuous infusion or a bolus injection. In addition, the dose of the therapeutic formulation may be increased or decreased proportionally as indicated by the urgency of the therapeutic or preventive situation.

[0152] The administration of the composition of the present invention to a subject (preferably a mammal, more preferably a human) can be carried out using known procedures, at a dosage and time effective to treat the disease or condition of the subject. The effective amount of the composition required to achieve a therapeutic effect may vary according to factors such as the degree of implantation; the time of administration; the duration of administration; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder; the age, sex, weight, condition, general health and previous medical history of the subject being treated; and similar factors known in the medical field. The dosage regimen can be adjusted to provide the best therapeutic response. For example, it can be administered several times a day, or the dosage can be reduced proportionally as indicated by the urgency of the treatment situation. One of ordinary skill in the art will be able to study the relevant factors and determine the effective amount of the composition without undue experimentation.

[0153] Actual dosage levels of the pharmaceutical formulations of the invention may be varied so as to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the subject.

[0154] Route of administration

[0155] Routes of administration of the compositions of the present invention include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, buccal, urethral, ​​vaginal (e.g., vaginal and perivalbina), nasal, and rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0156] Suitable composition formulations and dosages include, for example, dispersions, suspensions, solutions, beads, pellets, magmas, creams, pastes, ointments, lotions, discs, suppositories, liquid sprays for nasal or oral administration, aerosolized formulations for inhalation, compositions and formulations for intravesical administration, and the like.

[0157] It should be understood that the formulations and compositions useful in the present invention are not limited to the specific formulations listed in the examples. The following examples are presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to prepare and use the cells, differentiation methods, engineered tissues, and therapeutic methods of the present invention, and are not intended to limit the scope of what the inventors consider to be their invention.

[0158] Example

[0159] The present invention is further described in detail with reference to the following experimental examples. Unless otherwise stated, these examples are for illustrative purposes only and are not restrictive. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all variations that become apparent due to the teachings provided herein.

[0160] Without further description, it is believed that those of ordinary skill in the art can utilize the foregoing description and the following illustrative examples to prepare and utilize the compounds of the present invention and to implement the claimed methods. Therefore, the following working examples specifically point out embodiments of the present invention and should not be construed as limiting in any way.

[0161] Example 1: Multiple recombinant constructs as broadly applicable leptospirosis vaccines

[0162] Here, we developed multiple recombinant protein constructs based on biologically relevant targets identified from attenuated vaccine models that, when combined and used as vaccines, elicited cross-immune protection against mortality and kidney colonization in a hamster infection model.

[0163] Previous results with single recombinant vaccines using selected outer membrane / extracellular (OMP) proteins identified in an attenuated vaccine model did not elicit significant protection against mortality or colonization in the hamster model, demonstrating that single protein approaches against leptospirosis may not be effective. To this end, this paper demonstrates a multiple recombinant protein approach as a leptospirosis vaccine. Focusing on a list of 154 proteins obtained from the attenuated vaccine model described herein, eight OMP proteins were selected as potential candidates for a multiple recombinant protein vaccine for leptospirosis ( Fig. 9 and SEQ ID NOs: 1-8). In addition to the fact that these candidates are surface exposed / extracellular proteins, an important feature for vaccine candidates, the selection process also included results regarding: attenuation in a hamster model after protein disruption (5 / 8); upregulation of transcription levels under host conditions based on growth in rats using a dialysis membrane chamber (DMC) method (3 / 8); and presence and conservation in all pathogenic species of the genus Leptospira (6 / 8). Four (4) non-outer membrane proteins (non-OMPs) were selected for use as controls. All selected proteins were expressed based on the genomic sequence of Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130.

[0164] The materials and methods used in performing the experiments disclosed herein are now described.

[0165] Vaccine and challenge strains

[0166] Leptospira were cultured in liquid EMJH medium supplemented with 1% rabbit serum. Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 fcpA -Mutants and all 7 different strains used for challenge experiments (Table 1) were incubated at 29°C for up to 7 days until they reached logarithmic phase (4 to 5 days in culture). For all immunization or infection experiments, the correct number of Leptospira was determined by Petroff-Hausser counting chamber (Fisher Scientific).

[0167] The heat-killed vaccine was prepared by heat-inactivating a preparation of Leptospira interrogans strain Fiocruz L1-130 at 56°C for 20 min.

[0168] Table 1. FcpA in L1-130 vaccinated in this study - Leptospira strains used for challenge after mutation

[0169]

[0170] ￡ Results are the average of two independent experiments

[0171] * This strain has been described to cause only kidney colonization in hamsters following intraperitoneal (IP) infection, thus having no IP route LD 50 However, in two experiments, infection via the conjunctiva (CJ) for 10 8 Leptospira, the fatality rate is 25%

[0172] Animal experiments

[0173] Communication Research

[0174] To perform Fiocruz L1-130 fcpA in hamsters - Transmission experiments of mutants and L1-130 heat-inactivated vaccine were performed using 10 7 Groups of 15 3-week-old male Golden Syrian hamsters (Envigo) were inoculated subcutaneously with a dose of Leptospira. Three groups of animals were euthanized on days 1, 4, 7, 14, and 21 after infection. As a control, groups of nine animals were infected with Fiocruz L1-130 WT using the same route and dose, and the animals were euthanized on days 1 and 4 after infection. The last group was euthanized at the onset of the disease. After the animals were euthanized, blood, kidneys, livers, and brains were carefully removed, collected in cryovials, and immediately placed in liquid nitrogen and then stored at -80°C until DNA was extracted. If necessary, kidneys and blood were inoculated in EMJH for Leptospira culture.

[0175] For mouse experiments, groups of three 4-week-old female C57BL / 6 mice (Jackson laboratory) were subcutaneously inoculated with different doses of vaccine (10 7 , 10 5 , 10 3 , 10 2 and 10 1 ), and the three animals in the control group were inoculated with 10 7 Blood was collected by retro-orbital bleeding at 1, 4, 8, 13, 15, 18, and 21 days after infection.

[0176] Immunity and attack

[0177] Some vaccination experiments were performed using 3-week-old male Syrian golden hamsters (Envigo) or 4-week-old female C57BL / 6 mice ( Figure 2A ), and the mice were divided into groups of 6 to 9 or 4 to 8 animals. Animals were vaccinated subcutaneously with Fiocruz L1-130 fcpA - Mutants. A single dose of 10 7 Leptospira, and mice were inoculated with a series of doses (10 7 , 10 5 , 10 3 , 10 2 and 10 1 In hamsters, a single subcutaneous dose of 10 7 Leptospira was vaccinated with heat-killed vaccine as a control group. In addition, each group of animals was injected with phosphate buffered saline (PBS) and served as an unvaccinated control. Blood samples were collected one day before immunization and 20 days after immunization by retro-orbital bleeding.

[0178] Animals were challenged on day 21 after immunization. Hamsters were challenged by conjunctival inoculation, which simulated the natural route of infection, using a range of lethal doses (10 8 Leptospira (Table 1). Hamsters vaccinated with heat-killed vaccines were challenged with either Fiocruz L1-130 (homologous) or Manilae type L495 (heterologous) strains only. Whole-cell inactivated vaccines (bacterins) against leptospirosis are known to lack cross-reactive protection, so only one heterologous serovar was used for comparison. Mice were vaccinated intraperitoneally with Leptospira interrogans serovar Manilae type L495 (10 8 After the animals were euthanized, kidneys were harvested and stored as described above.

[0179] Passive transfer experiment

[0180] The Fiocruz L1-130 fcpA gene was generated by immunizing groups of 10 3-week-old male Syrian golden hamsters using the same protocol as above. - Immune sera of mutants. Groups of 10 animals were injected with PBS to obtain control sera. On day 21 after immunization, the animals were inhaled with CO 2 The animals were euthanized. Blood was obtained by cardiac puncture and serum was separated and subsequently pooled as immune serum (hamster α-fcpA-) and control serum.

[0181] Immune serum or control serum was passively transferred intraperitoneally to groups of 5 naive female mice and 7 naive male hamsters (6-7 weeks old) at doses of 0.5 and 2.0 mL, respectively. 24 hours later, 10 8 Manilae serovar L495 (heterologous strain) to challenge mice and hamsters.

[0182] Serology

[0183] Pre- and post-vaccination sera were obtained by centrifugation of clotted blood at 1000 g for 15 min at room temperature. Serum samples were frozen at -20°C until analyzed for the presence of Leptospira antibodies by MAT, ELISA, immunoblotting and proteomic array.

[0184] MAT was performed using standard practice and as previously described. Sera were diluted 1:100 and tested against all strains used in this project (Table 1). Positive samples were subsequently titrated.

[0185] For ELISA, cultures of Leptospira interrogans serovar Manilae type L495 and Fiocruz L1-130 (10 8Whole cell lysates were prepared by lysing the culture plates with 500 μL of PBS for 20 min. The pellets were washed twice with PBS and resuspended in 500 μL of PBS. The resuspended cultures were sonicated on ice for 6 cycles at 30 kHz and 300 W of power output. The lysates were quantified by Bradford assay and used as antigen at a concentration of 150 ng / well in 0.05 M carbonate buffer (pH 9.6). Flat-bottomed polystyrene microtiter plates (Corning) were coated with Leptospira antigens and incubated overnight at 4°C. The plates were washed three times with PBS-0.05% (vol / vol) Tween 20 (PBST) and incubated with blocking solution (5% blocking milk in 2% [wt / vol] bovine serum albumin) at 37°C for 2 hr. After washing four times with PBST, the wells were incubated for 1 hr at 37°C with mouse immune serum diluted 100-fold in 2% BSA. Secondary anti-mouse HRP conjugated antibody (Jackson ImmunoResearch) was used at a dilution of 50,000 (2% BSA) and incubated at 37°C for 1 hr. TMB SureBlue Reserve (SeraCare) was used for detection and the reaction was terminated by adding 100 μL of 2N H2SO4. The absorbance (450 nm) was recorded by a microplate reader (Biotek).

[0186] To evaluate the effect of proteolytic enzyme treatment on Leptospira antigens, a previously described protocol was used. Briefly, Leptospira antigens coated in assay wells were treated with 0.1 mg proteinase K (Invitrogen) at 37°C for 2 hr. Plates were washed three times with PBST to remove unbound proteins and then blocked and tested as described above.

[0187] qPCR

[0188] DNA was extracted from blood and tissue samples using a Maxwell 16 (Promega Corporation) instrument according to the manufacturer's instructions. Quantitative real-time PCR assays were performed on hamster and mouse tissues using an ABI 7500 instrument (Applied Biosystems) and Platinum Quantitative PCR Supermix-UDG (Invitrogen Corporation) with lipL32 primers and probe as described previously.

[0189] Western blotting

[0190] Immunoblotting was performed with whole-cell extracts of Leptospira strains as described previously. α-fcpA was detected with hamster or mouse immune sera at a dilution of 1:100.- The pools were subjected to Western blotting. For subsequent detection, HRP goat anti-mouse or anti-hamster serum (Jackson ImmunoResearch) was used at a dilution of 1:100,000. The blots were analyzed using a ChemiDoc imager (Bio-Rad).

[0191] Proteome Array

[0192] As described above, a complete ORF set (ORFeome) was amplified from the interrogans Copenhageni type serovar strain Fiocruz L1-130. ORFs greater than 150 bp were amplified from genomic DNA and then recombinantly cloned into a T7 expression vector. Genes greater than 3 kb were cloned into segments. A list of 660 most reactive antigens was selected from a previous study with human sera from patients with leptospirosis and used in hamster experiments. Mouse sera were tested in an array containing 330 proteins selected based on the latter. Proteins were expressed in an in vitro transcription / translation (IVTT) RTS100 E. coli HY system (5PRIME), and the synthesized crude proteins were printed on 3 pads of nitrocellulose-coated AVID slides (Grace Bio-Labs) using a Gene Machine OmniGrid 100 microarray printer (Genomic Solutions). In addition to the proteins expressed by IVTT, each array contained no DNA control spots, consisting of IVTT reactions of serial dilutions of purified IgG / spots without the addition of plasmids.

[0193] The array was probed for IgG reactivity. For serum samples, the array was probed at a 1 / 100 dilution in a protein array blocking buffer (GVS) supplemented with E. coli lysate (Genscript) and a final concentration of 10 mg / mL for blocking anti-E. coli antibodies. The array was incubated overnight with continuous agitation at 4°C. After overnight incubation, the array was washed three times with T-TBS, then incubated for 45 min at RT with a biotin-conjugated anti-human IgG secondary antibody (Jackson ImmunoResearch) diluted 1 / 400 in the array blocking buffer, and then incubated with Qdot 800 streptavidin conjugate (Thermo Fisher Scientific). The array was air-dried after a brief centrifugation. The IgG signal of Q800 was detected with the ArrayCam 400s microarray imaging system (Grace Bio-Labs). The array signal intensity was quantified using QuantArray software. The average pixel intensity was corrected for the local background of all spots. Protein expression was verified by microarray using monoclonal anti-polyhistidine (clone His-1, Sigma).

[0194] In addition, 30 sera (acute and convalescent) from human patients diagnosed with acute Leptospirosis in Salvador, Brazil were probed in an array containing the 660 proteins as described above. Patient samples were collected and selected as described previously.

[0195] Data analysis

[0196] Analysis of the logarithm of the proteome signal intensity before and after inoculation 10 Fold change (LFC). Before calculating LFC, the chip background was subtracted and negative values ​​were set to 1 (to avoid problems with taking logarithms). Three data sets were analyzed: hamster data, which used a single attenuated vaccine 10 7 Leptospira dose (hamster 10 7 ); mouse dose-response data, including all vaccine doses 10 1 , 10 2 , 10 3 , 10 5 and 10 7 (full dose in mice); and 10 7 A subset of mice (10 mice) were dosed with attenuated Leptospira vaccine 7 The decision of these analysis groups was based on the fact that high doses of the vaccine in mice (10 7 ) and hamster experiments (hamster 10 7 ) and was the only dose that gave 100% protection against death and colonization in mice. In addition, other doses (full dose in mice) provided different levels of protection when death and colonization were combined, and comparative analysis of all three groups will increase the likelihood of identifying potential protein targets that play a role in these results.

[0197] Exploratory analysis of the mouse data showed a dose-response relationship, with increasing vaccine doses associated with increasing mean signal intensity ( Figure 6 ) and reduction in mortality and colonization. A model was used that allowed quantification of a dose-response relationship when it existed, whereas if only a single dose was used, the comparison between vaccinated and unvaccinated animals was measured. Each antigen was modeled separately. For each antigen, the LFC in each animal A was fitted with a linear model, i.e., LFCA=experimental A+VA+Log dose A, where experimental was a factor at four levels for mice and two levels for hamsters. VA was an indicator variable for whether animal A received an attenuated vaccine or a control injection. These terms were included in all models. The log dose term was only included in the dose-response relationship analysis for mice and was the logarithm of the dose (0, 1, 2, 3, 5, and 7 for control animals). The indicator variable VA prevented the control animal's Log dose = 0 from being considered a true zero. The statistic of interest was the t statistic. For hamsters and mice 10 7Models were interpreted using VAt statistics, whereas for the full-dose dose-response model in mice, Log-dose t statistics were interpreted. The Benjamini–Hochberg (BHp) correction was used to control the false discovery rate to 0.05. This analysis was performed in RStudio software (RStudio).

[0198] Prism 8 (GraphPad software) was used to perform full statistical analysis of in vivo data. Fisher's exact test and analysis of variance (ANOVA) were used to evaluate the statistical differences between each group and multiple groups respectively. P value < 0.05 was considered significant. Binomial proportion confidence intervals were calculated to determine the efficacy of the vaccine in both hamsters and mice. Protein homology of Leptospira interrogans Copenhageni type serovar strain Fiocruz L1-130 was identified by BLAST search (http: / / www.ncbi.nlm.nih.gov / BLAST / ). Orthologous clusters (COG), pSortB positioning, transmembrane domain (TMhmm) and signal peptide (SignalP) information were obtained from the Genoscope platform (http: / / www.genoscope.cns.fr / agc / microscope / home / ). In the R environment, the p value of enrichment statistical analysis was calculated using Fisher's exact test.

[0199] Table 2. Strains used in vaccine experiments

[0200]

[0201] *LD for intraperitoneal inoculation in hamsters 50

[0202] Passive transfer experiment. Using a dose of Leptospira interrogans Fioruz L1-130 fcp1 - Syrian golden hamsters were immunized with live attenuated vaccines and the animals were bled 21 days later to obtain hyperimmune sera. 7 Sixteen hours before lethal challenge with Leptospira—a heterologous strain of Leptospira interrogans, Manilae—3 mL of hamster serum was transferred intraperitoneally (IP) to groups of three naive hamsters. A control group of three hamsters was passively transferred with 3 mL of serum from unvaccinated animals. Animals were monitored twice daily for clinical signs of leptospirosis and mortality up to 14 days post-infection. The animals were inoculated with CO by inhalation. 2 Moribund animals were sacrificed immediately.

[0203] Statistical Analysis. Data were plotted and analyzed using GraphPad Prism 5.0c (GraphPad Software, La Jolla, CA). Fisher's exact test and analysis of variance (ANOVA) were used to assess the statistical significance of differences between groups and multiple groups, respectively. p values ​​< 0.05 were considered significant.

[0204] The results of the experiments disclosed herein are now described.

[0205] Motility-deficient strains as attenuated vaccine candidates

[0206] A previously unidentified flagellar sheath protein (FcpA) that is essential for translational motility and, therefore, virulence was characterized. Although the phenotype was completely attenuated, L1-130 fcpA was observed - The mutant caused a transient systemic infection in hamsters after intraperitoneal inoculation for 10 8 In this study, hamsters were inoculated subcutaneously for 10 7 After 100 Leptospira, the presence of DNA of this mutant was detected by qPCR in all tested tissues except brain ( Figure 1A ). These results are similar to those observed previously, with the wild type achieving higher numbers of Leptospira in all tissues analyzed, leading to death or euthanasia of animals due to clinical signs of disease at 5–7 days post infection. - The signal for the mutant strain was not detected after 7 days, and all vaccinated animals survived with no detectable Leptospira in the kidney or blood as measured by qPCR and culture. Similarly, no detectable signal was observed for animals immunized with the L1-130 heat-killed strain ( Figure 1A fcpA was also tested in a mouse model using different infection doses. - Mutants ( Figure 1B Although the dose of wild-type strain was insufficient to produce disease and lethality in infected mice, all animals became colonized and the presence of Leptospira DNA was detectable in the blood by day 15 post-infection ( Figure 1B ). In addition, no dose of fcpA - The mutant resulted in colonization (data not shown) and had a significantly different extent of dissemination in the blood compared with the wild type ( Figure 1B ). Until 13 days and 8 days after infection, respectively, only 10 7 and 10 5 fcpA was observed in the blood of animals infected with -Altogether, these results suggest that although fcpA - The mutant was attenuated in both hamster and mouse models, but was found to be hematogenously transmitted by testing its DNA. The mutant appeared to be cleared by the immune system before causing observable disease or death in the animals. Furthermore, it was observed in the mouse model that the spread of the mutant was dose-dependent. However, it is important to note that although the mutant was attenuated at doses equal to or below 10 3 However, the theoretical detection limit of the qPCR assay used here is 100 Leptospira / mL blood, which may lead to false-negative results.

[0207] Cross-protection immunity model for leptospirosis

[0208] Considering previous findings, it is hypothesized that fcpA - Transient infection with mutants induces cross-protection responses. - Mutant immunity ( Figure 2A ) conferred complete protection against mortality from infection with both homologous and heterologous serovars in hamsters ( Figure 2B and Table 3). In contrast, immunization with heat-killed Leptospira conferred partial protection against homologous, but not heterologous, serovars ( Figure 2B Heat-killed vaccines can provide a high level of protection against homologous challenge, but vaccination regimens usually include at least a second dose of vaccine. Figure 2B and Table 3), probably due to the lack of vaccine booster. For the purpose of evaluating the efficacy of the attenuated vaccine after a single dose, it was decided to maintain the standard regimen of vaccination and therefore also use only one dose of heat-inactivated vaccine. It is worth mentioning that the Hardjo type 203 strain has been described to cause colonization only in the hamster model infected by the intraperitoneal route. However, in the LD50 experiments using the conjunctival route, a mortality rate of 25% was reproducibly observed (Table 2). In addition, in the unvaccinated group, a total mortality rate of 21.4% was observed after challenge with the Hardjo type 203 strain, but no deaths occurred in the vaccinated group, which explains the wide 95% CI range ( Figure 2B ).

[0209] After homologous infection, only fcpA - Protection against kidney colonization was observed in 80% of animals immunized with the mutant. Heterologous infection produced varying levels of protection, ranging from 0% to 35.7% ( Figure 2D and Table 3). Hamsters are highly susceptible to leptospirosis, so it is not surprising that the attenuated strain was found to be partially protective against colonization. -The efficacy of mutant vaccines in preventing colonization was tested using a mouse heterologous infection model with different doses of immunization. - The protection conferred by the mutant was dose-dependent. Against death, the vaccine conferred up to 10 3 fcpA - - 100% protection against mutant Leptospira doses ( Figure 2C and Table 4), but requires up to 10 7 Leptospira doses are required to obtain 100% protection against colonization ( Figure 2E and Table 4). In addition, quantitative analysis of kidney colonization also showed that although fcpA - The mutant failed to promote complete protection but did not protect hamsters after heterologous infection ( Figure 2F and Table 3) and at lower vaccine doses in the mouse model, which also revealed a dose-dependent phenotype ( Figure 2G and Table 4).

[0210] These findings suggest that a single dose of the live attenuated vaccine elicits cross-protective immunity against serovars belonging to Leptospira interrogans, Leptospira cruzi, and Leptospira borgpetersenii, species that cover most serovars of importance to human and animal health.

[0211]

[0212]

[0213] Antibodies to Leptospira proteins as cross-protective immune correlates

[0214] In hamsters ( Figure 3A ) and mice ( Figure 3C ) Of the two, fcpA - The attenuated vaccine induced weak agglutinating antibody responses to the homologous serovar (i.e., Copenhageni type) and undetectable microscopic agglutination test (MAT) titers to the heterologous serovar. Furthermore, in the mouse model, agglutinating antibodies were only detected after at least 10 5 Leptospira can be measured at doses of Figure 3C ). In contrast, a single dose of fcpA - The mutants were able to induce a robust immune response against Leptospira proteins, recognizing different species of Leptospira used in the hamster model ( Figure 3B ) and a dose of at least 10 3 Heterologous strains used in the mouse model of Leptospira ( Figure 3D ) proteins. In addition, the presence of detectable antibodies measured by ELISA was consistent with that in the mouse model (103 Leptospira), and the OD of all doses decreased when Manilae antigen was treated with proteinase K ( Figure 3E ). In addition, in hamsters ( Figure 3F ) and mice ( Figure 3G ), using the fcpA - Passive transfer experiments with hamster immune sera from the attenuated vaccine conferred 100% protection against heterologous lethal infection. Altogether, these results suggest that anti-Leptospiral protein antibodies, but not agglutinating antibodies, are the correlates of vaccine-mediated cross-protective immunity.

[0215] Highly conserved serum reactive proteins as potential targets for eliciting cross-protective responses

[0216] Reduced proteome arrays of 660 and 330 ORFs for hamster and mouse sera, respectively, were used to characterize antibody responses to attenuated vaccines. 7 Hamsters immunized with 10 doses of Leptospira 7 ) and mice (mouse 10 7 ) were analyzed and a total of 133 ( Figure 4A ) and 56 ( Figure 4B ) protein targets, while analysis of mice immunized with different doses of the attenuated vaccine (full dose in mice) identified a total of 13 protein targets ( Figure 4C The rationale for analyzing the results for mice alone is based on the following facts:10 7 A dose of attenuated Leptospira vaccine provided 100% cross protection against lethality and colonization ( Figure 2C and 2E When combined, these three different analyses yielded a total of 154 unique protein targets ( Figure 4D Among them, 55% (85) had no prediction of localization, while 23% (36), 14% (21) and 8% (12) were predicted to be plasma membrane-associated, outer membrane protein (OMP) and cytoplasmic ( Figure 5A ). Enrichment analysis showed that OMP and cytoplasmic membrane-related proteins were enriched 5.0 times (p = 4.51E-10) and 1.8 times (p = 2.92E-04), respectively ( Figure 5B In contrast, cytoplasmic proteins were underrepresented in the reactive antigen group—0.3-fold (p=2.91E-10) ( Figure 5B ).

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] Clusters of orthologous groups (COGs) of proteins were widely represented in these targets (Table 5), with at least one protein in each of the 18 functional categories. The most highly represented COGs were general function prediction only - 19 proteins (R), cell wall / membrane / envelope biogenesis - 17 proteins (M), intracellular transport, secretion and vesicle transport - 16 proteins (U), and cell motility - 14 proteins (N). However, the vast majority of proteins were not assigned a COG (59) ( Figure 5C ). Enrichment analysis identified proteins with predicted COG-U, COG-N, and COG-M functions as being highly enriched in reactive antigens, 4.9-fold (p = 2.27E-07), 3.1-fold (p = 8.35E-05), and 1.6-fold (p < 0.05), respectively ( Figure 5D In addition, proteins predicted to be involved in signal transduction mechanisms (COG-T) and amino acid transport and metabolism (COG-E) were significantly underrepresented in reactive antigens, 0.4-fold (p=0.016) and 0.3-fold (0.02), respectively ( Figure 5D ). In summary, the enrichment analysis validates the method disclosed herein for identifying candidate proteins relevant to cross-protective vaccine biology.

[0230] Based on the relationship between the three different groups analyzed by proteomic array, the 154 proteins identified (Figure 7) were narrowed down to 41 protein targets (Figure 8). Seven proteins were identified in all groups ( Figure 4D , Figure 8 and Table 5), while using 107 Thirty-one proteins were identified in both hamsters and mice vaccinated with attenuated Leptospira vaccine ( Figure 4D , Figure 8 and Table 5). In addition, three additional proteins were identified in the groups of mice immunized with different doses. 7 Two additional proteins were identified between the groups of mice immunized with different Leptospira doses ( Figure 4D , Figure 8 and Table 5), and in the use of 10 7 An additional protein ( Figure 4D , Figure 8 and Table 5). Hamster and mouse immune sera were highly reactive to most of the 41 proteins, in contrast to the low reactivity of control sera and animals vaccinated with the heat-inactivated vaccine (Figure 8), indicating the ability of the attenuated vaccine to induce immunity against Leptospira proteins. Reasonable vaccine candidates were identified among the 41 serum-reactive proteins (Figure 8), including 6 OMPs and known putative virulence factors such as LipL32, LipL41 and Lig proteins, providing supporting evidence for the use of proteomic arrays to identify such proteins. Not surprisingly, 40% of these targets were identified as hypothetical proteins with no described function. However, in all 13 pathogenic Leptospira species analyzed, the majority (70%) had high amino acid sequence identity (>80%) in their respective orthologs (Figure 8) and were therefore likely targets for eliciting cross-protective responses. Furthermore, sera from patients with confirmed acute leptospirosis reacted with 17 of the 41 Leptospira proteins recognized by sera from animals immunized with the attenuated vaccine ( FIG. 8 ).

[0231] In vivo testing of Leptospirosis protein constructs

[0232] Three different combinations of four different recombinant proteins were evaluated in hamsters - "OMP1", "OMP2" and "OMP3". Immunogenicity and protection studies were performed using groups of four 3-month-old Syrian hamsters as a proof-of-concept. Animals were vaccinated with 40 μg of each protein for a total of 160 μg of protein diluted in 100 μL PBS and mixed with 100 μL squalene-based adjuvant (AddaVax TM ,Invivogen) combination. AddaVax TM is a Compared with alum, the most commonly used adjuvant in experimental leptospirosis vaccines, AddaVax TM Promote a more balanced Th1 / Th2 response. Recently, AddaVax TMIt has been used in vaccine trials for leptospirosis research. Animals were given 2 booster doses 2 weeks apart and 10 8 Animals were challenged with doses of either the homologous Copenhageni serovar Fiocruz L1-130 or the heterologous Leptospira borgpetersenii serovar Hardjo. Negative control groups were vaccinated with LigA, four non-OMP proteins, or adjuvant alone.

[0233] As expected, all animals in the adjuvant-only and non-OMP groups that showed symptoms between days 8 and 9 post-challenge were euthanized ( Fig. 10A Animals vaccinated with "OMP1", "OMP2" and ligA survived homologous challenge with Copenhageni type serovars ( Fig. 10A However, only hamsters vaccinated with either "OMP1" or "OMP2" were protected against kidney colonization ( Fig. 10B ).

[0234] The "OMP1" multiple recombinant construct was used in the Copenhageni type serovar ( Fig.11A ) and Hardjo serovar ( Fig. 11B )-challenged hamsters. "OMP1" only protected hamsters challenged with the homologous strain (Copenhageni type) from renal colonization ( Fig. 11C ), but not against Hardjo-type attacks ( Fig.11D ). However, similar blood bacterial burden kinetics after challenge with Copenhageni and Hardychoceae suggest the potential of this approach ( Fig.11E ). The "OMP1" multiple recombinant construct is highly immunogenic: the "OMP1" construct produced a significant enhancement of the immune response to all proteins after each immunization. There were no significant differences between animals challenged with homologous or heterologous strains. Three doses ( Fig. 12A , 12B ).

[0235] Animals immunized with "OMP1" and challenged with Leptospira interrogans (serovars Copenhageni, Pomona, and Canis), Leptospira kluyveri (serovar Gastroenteroides), and Leptospira borgpetersenii (serovar Hardjo) survived the infection. In addition, animals challenged with Leptospira interrogans were also 100% protected from kidney colonization ( Fig.13 ).

[0236] Subsequently, "OMP3" was evaluated. "OMP3" includes two proteins from the "OMP1" group and two proteins from the "OMP2" group. The proteins were selected based on the level of amino acid identity between pathogenic species. Both constructs induced protection against homologous attack (Copenhagenii type) ( Fig.14A ). In addition, the "OMP3" construct, but not the "OMP2" construct, was also able to induce cross-protection against death after heterologous challenge with the Hardjo serovar ( Fig. 14B All hamsters vaccinated with the "OMP3" construct not only survived but also showed no evidence of kidney colonization after challenge with either Copenhageni or Hardjo. Fig. 14C , 14D The cross protection of “OMP1” and “OMP3” against various species, namely Leptospira interrogans (Copenhageni, Canis, and Pomona), Leptospira kluyveri (Hydrotyphoid), and Leptospira borgpetersenii (Hardjo) was further evaluated ( Fig.16 ). All seven groups of animals vaccinated with both "OMP1" and "OMP3" vaccines survived the infection ( Fig.16 Vaccination with "OMP1" alone induced cross protection only against Leptospira interrogans, whereas vaccination with "OMP3" induced cross protection against all species tested, and animals were 100% protected against kidney colonization ( Fig.16 ).

[0237] The sera of animals vaccinated with "OMP3" contain antibodies with high complement-dependent bactericidal activity (50-60%). There is also a significant killing activity against heterologous strains representing the main species of Leptospira. Fig.15 Sera from animals vaccinated with “OMP1” and “OMP2” also had antibodies with high complement-dependent bactericidal activity, although not as high as those from animals vaccinated with “OMP3” ( Fig.17 The anti-"OMP3" serum was further tested and showed high (40-50%) bactericidal activity against Leptospira strains P1 and P2, for which animal experiments were not possible ( Fig.18 ). In addition, each immunization with "OMP" induced a significant enhancement effect on all proteins and whole-cell Leptospira, indicating that the antibodies induced by the recombinant proteins recognized both the recombinant proteins and the native proteins ( Fig.19 ). These results support the use of the composition of the present invention as a universal vaccine for Leptospirosis.

[0238] In summary, the results disclosed herein are the first evidence of 100% cross protection against mortality and colonization due to leptospirosis using recombinant proteins, indicating that a universal vaccine for leptospirosis using recombinant proteins is feasible given the correct targets identified herein.

[0239] Vaccination is an effective method to block spillover infection from livestock to humans and is also the main method to control or prevent leptospirosis in livestock animals, reducing the impact of leptospirosis to increase animal production. However, there is no effective control for leptospirosis and no safe and effective vaccine is available for human use. Whole-cell vaccines are widely used for veterinary purposes, but have obvious limitations because immunity is serovar-specific and short-lasting. Currently available monovalent commercial vaccines are effective in preventing clinical disease, but not all vaccines can provide protection from urinary shedding, and they cannot protect across leptospiral serovars. Multivalent vaccines can effectively prevent clinical disease from different serovars, but cannot prevent renal colonization and renal pathology. In addition, these multivalent vaccines have not been able to achieve adequate coverage of the serovar spectrum that is important for global animal and human health. Therefore, research has focused on characterizing promising subunit vaccine reporter candidates, including surface-associated proteins and host-expressed proteins. Attempts to produce effective leptospirosis subunit vaccines are based on known potential virulence factors of highly expressed proteins, but have not been successful. Different strategies have been used for cross-protective immunity against renal clearance, but so far only a few proteins have been shown to be promising protective antigens. However, the protective results described are often inconsistent in different studies and are difficult to reproduce depending on the strategy and adjuvant used. Currently, no single candidate effectively provides cross-protection. Attenuated vaccine models have revealed a unique list of proteins that are biologically relevant for cross-protective immunity against infection and the pathogenesis of leptospirosis. Studies have shown that a combination of three recombinant proteins induces a more robust immune response than vaccines based on single components. The multiple recombinant protein constructs disclosed herein, particularly "OMP3", are the first constructs to show evidence of 100% cross-protection from mortality and colonization due to leptospirosis using recombinant proteins.

[0240] It is contemplated herein that the immunogenic compositions disclosed herein (e.g., including multiple recombinant "OMP1," "OMP2," or "OMP3" protein constructs) will be used in a general method for preventing leptospirosis that will protect against all pathogenic species of the genus Leptospira in different epidemiological settings throughout the world. The compositions of the present invention also eliminate the production and safety issues of currently available vaccines for leptospirosis, making them suitable for use as human vaccines. The compositions of the present invention additionally have the advantage of reduced costs for producing leptospirosis vaccines for domestic and livestock animals compared to current veterinary leptospirosis vaccines.

[0241] List of enumerated implementations

[0242] Embodiment 1. A composition comprising a plurality of Leptospira peptides, each Leptospira peptide having 80% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0243] Embodiment 2. The composition according to embodiment 1, wherein each Leptospira peptide has 90% or greater sequence identity with one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0244] Embodiment 3. The composition according to embodiment 1, wherein each Leptospira peptide has 95% or greater sequence identity with one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0245] Embodiment 4. The composition according to embodiment 1, further comprising an adjuvant.

[0246] Embodiment 5. The composition according to embodiment 1, further comprising a pharmaceutically acceptable carrier.

[0247] Embodiment 6. A composition according to embodiment 1, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 1, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 2, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 3, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 4.

[0248] Embodiment 7. A composition according to embodiment 1, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:7, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:8.

[0249] Embodiment 8. A composition according to embodiment 1, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:3, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:4, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6.

[0250] Embodiment 9. A method of modulating an immune response to leptospirosis in a subject, the method comprising administering to the subject an effective amount of a composition comprising a plurality of leptospiral peptides, each leptospiral peptide having 80% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0251] Embodiment 10. The method according to embodiment 9, wherein each Leptospira peptide has 90% or greater sequence identity with one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0252] Embodiment 11. The method according to embodiment 9, wherein each Leptospira peptide has 95% or greater sequence identity with one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0253] Embodiment 12. The method of embodiment 9, wherein the composition further comprises an adjuvant.

[0254] Embodiment 13. The method according to embodiment 9, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0255] Embodiment 14. A method according to embodiment 9, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 1, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 2, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 3, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 4.

[0256] Embodiment 15. A method according to embodiment 9, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:7, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:8.

[0257] Embodiment 16. A method according to embodiment 9, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:3, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:4, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6.

[0258] Embodiment 17. The method of embodiment 9, wherein the composition is administered to the subject in three separate doses.

[0259] Embodiment 18. The method of embodiment 9, wherein the subject is a mammal.

[0260] Embodiment 19. The method according to embodiment 9, wherein the subject is a human.

[0261] Other Implementations

[0262] The recitation of a list of elements in any definition of a variable herein includes defining that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment, or in combination with any other embodiment or portion thereof.

[0263] The disclosure of each and every patent, patent application and publication cited herein is hereby incorporated by reference in its entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by other skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims

1. A composition comprising a plurality of Leptospira peptides, each Leptospira peptide having 80% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

2. The composition of claim 1, wherein each Leptospira peptide has 90% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

3. The composition of claim 1, wherein each Leptospira peptide has 95% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:

8.

4. The composition according to claim 1, further comprising an adjuvant. The composition according to claim 1 , further comprising a pharmaceutically acceptable carrier.

6. The composition of claim 1, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 1, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 2, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 3, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:

4.

7. The composition of claim 1, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:7, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:

8.

8. The composition of claim 1, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:3, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:4, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:

6.

9. A method of modulating an immune response to leptospirosis in a subject, the method comprising administering to the subject an effective amount of a composition comprising a plurality of leptospiral peptides, each leptospiral peptide having 80% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:

8.

10. The method of claim 9, wherein each Leptospira peptide has 90% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

11. The method of claim 9, wherein each Leptospira peptide has 95% or greater sequence identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:

8.

12. The method of claim 9, wherein the composition further comprises an adjuvant.

13. The method of claim 9, wherein the composition further comprises a pharmaceutically acceptable carrier.

14. The method of claim 9, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 1, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 2, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO: 3, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:

4.

15. The method of claim 9, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:6, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:7, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:

8.

16. The method of claim 9, wherein the plurality of Leptospira peptides comprises a first Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:3, a second Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:4, a third Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:5, and a fourth Leptospira peptide having 80% or greater sequence identity to SEQ ID NO:

6.

17. The method of claim 9, wherein the composition is administered to the subject in three separate doses.

18. The method of claim 9, wherein the subject is a mammal.

19. The method of claim 9, wherein the subject is a human.

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