Leptospiral virulence modulating protein epitopes and uses thereof

Abstract

Provided are Leptospiral virulence modifying (VM) protein epitopes, and their use as vaccines and for generating therapeutic antibodies.

Description

LEPTOSPIRAL VIRULENCE MODULATING PROTEIN EPITOPES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 571,551, filed March 29, 2024, the contents of which are incorporated by reference herein in their entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. All 15658 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Leptospirosis is a globally neglected zoonotic disease— so neglected that WHO has declined repeatedly to put it onto the Neglected Disease list despite petitions from the field. The disease is conservatively estimated to affect more than 1 million people annually with a 20% case fatality rate. Leptospirosis is caused by the genus Leptospira, recognized as important emerging pathogens. Strains (serovars) of Leptospira interorgans are virtually the only Leptospira that causes severe human disease. Mechanism of pathogenesis of leptospirosis remains elusive ever since the initial descriptions of the etiology. There is no safe and effective vaccine approved for humans to date, indicating the urgent need to identify an effective strategy for controlling and treating leptospirosis. Recently a novel paralogous PF07598 gene family comprised of multiple highly conserved members — so-called virulence modifying (VM) proteins — restricted to group I pathogenic Leptospira was discovered. PF07598 genes encode secreted exotoxins, with a single polypeptide encoding an AB-toxins. VM proteins are comprised of bona fide N-terminal ricin B domains and C-terminal DNase / toxin domains. In a mouse model recapitulating human disease, as few as two VM proteins protected animals from lethal challenge infection.

[0004] There remains a need in the art for novel compositions that have vaccine potential against pathogenic Leptospira. The current invention satisfies this unmet need.SUMMARY OF THE INVENTION

[0001] The invention relates to Leptospiral virulence modifying (VM) protein epitopes, and variants and fragments thereof, vaccine compositions comprising the same, and the use thereof for the generation of antibodies for the treatment of Leptospira spp.

[0002] In one embodiment, the invention relates to compositions comprising at least one Leptospiral virulence modifying (VM) protein epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID N0:9, SEQ ID NOTO, SEQ ID NO : 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO 32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof.

[0003] In one embodiment, the composition is a fusion protein comprising a Leptospiral VM protein epitope fused to a targeting domain specific for binding to a target molecule. In one embodiment, the target molecule is selected from the group consisting of a bacterial antigen, viral antigen, parasitic antigen, cancer antigen, tumor-associated antigen, and tumor-specific antigen.

[0004] In one embodiment, the composition comprises a combination of two or more Leptospiral VM protein epitopes. In one embodiment, the composition comprises at least one lipid nanoparticle (LNP) comprising at least one Leptospiral VM protein epitope. In one embodiment, the composition comprises a combination of at least two LNP comprising at least two VM protein epitopes.

[0005] In one embodiment, the invention relates to compositions comprising at least one nucleic acid molecule encoding at least one Leptospiral virulence modifying (VM) protein epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ IDN0:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO 36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof. In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a Leptospiral VM protein epitope fused to a targeting domain specific for binding to a target molecule. In one embodiment, the target molecule is selected from the group consisting of a bacterial antigen, viral antigen, parasitic antigen, cancer antigen, tumor-associated antigen, and tumor-specific antigen. In one embodiment, the composition comprises at least one lipid nanoparticle (LNP) comprising at least one nucleic acid molecule encoding at least one VM protein epitope. In one embodiment, the nucleic acid molecule comprises an mRNA molecule encoding the at least one VM protein epitope. In one embodiment, the composition comprises a vaccine.

[0006] In one embodiment, the invention relates to anti-Leptospiral VM protein antibodies specific for binding to an epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO 21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO 28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof. In one embodiment, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and an scFv antibody fragment.

[0007] In one embodiment, the invention relates to compositions comprising an antibody specific for binding to an epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20,SEQ ID N0:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and an scFv antibody fragment. In one embodiment, the composition comprises polyclonal anti-serum.

[0008] In one embodiment, the invention relates to a nucleic acid molecule encoding an antibody specific for binding to an epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NON, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO 28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and an scFv antibody fragment.

[0009] In one embodiment, the invention relates to methods of inducing an immune response in a subject, the method comprising administering the compositions of the present invention. In one embodiment, the subject is currently infected with Leptospira sp and the composition induces an immune response against Leptospira sp.

[0010] In one embodiment, the invention relates to methods of treating or preventing a disease or disorder in a subject, comprising administering the antibody specific for binding to an epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NON, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NON, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NON2, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO 29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID NO:32, SEQ ID NO:33, SEQID NO:34, SEQ ID NO 35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and an scFv antibody fragment, a composition comprising the antibody, wherein the composition comprises polyclonal anti-serum, or a nucleic acid molecule encoding the antibody to the subject. In one embodiment, the disease or disorder is at least one selected from the group consisting of cancer, a bacterial infection, a viral infection, and a parasitic infection.

[0011] In one embodiment, the invention relates to methods for diagnosing infection of at least one pathogen in a human subject, comprising detecting IgG or IgM antibody produced after infection in the subject, comprising: a) determining the level of IgG and / or IgM antibody in the subject; wherein the determining comprises: i) mixing a biological sample from the subject with a first reagent; wherein the first reagent comprises at least one Leptospiral VM protein antigen coated on a solid phase support, and anti-human IgG antibody or anti-human IgM antibody conjugated to a label; wherein the antigen, IgG or IgM antibody from the sample, and the antihuman IgG antibody or anti-human IgM antibody conjugated to a label form a complex if the sample comprises IgG or IgM antibody that bind to the at least one antigen; ii) washing the complex after the mixing step;iii) incubating the complex with a second reagent, wherein the second reagent comprises a detection agent which produces a detectable signal when exposed to the label; iv) acquiring the detectable signal; and v) obtaining the level of the IgG or IgM antibody in the sample based upon the detectable signal; b) comparing the level of IgG or IgM antibody in the sample with a comparator control; c) diagnosing the subject as infected when the level of IgG or IgM antibody in the subject is elevated compared with the level of IgG or IgM antibody in the comparator control, wherein the at least one Leptospiral VM protein antigen comprises at least one Leptospiral VM protein antigen selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO 31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQID NO:36, SEQ ID NO:37, SEQ ID NO 38, SEQ ID NO:39 or SEQ ID NO 40, SEQ ID N0 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or a fragment or variant thereof.

[0012] In one embodiment, the at least one pathogen comprises at least one pathogen selected from the group consisting of L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri, and L. alexanderi. In one embodiment, the label comprises Horse Radish Peroxidase (HRP). In one embodiment, the detection agent comprises 3, 3’, 5, 5’ tetramethylbenzidine (TMB) substrate. In one embodiment, the acquiring comprises obtaining optical density (OD) measurements at 450nm. In one embodiment, the diagnosing occurs immediately following infection. In one embodiment, the diagnosing occurs at least 2 days following infection. In one embodiment, the biological sample comprises plasma or serum from the human subject. In one embodiment, the comparator control comprises the level of IgG and / or IgM in a plasma or serum sample from an uninfected human subject. In one embodiment, when the subject is diagnosed as infected, the subject is treated.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following detailed description of exemplary embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are exemplary. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0014] Figure 1 depicts the method of epitope mapping using overlapping synthetic peptides, 12-mer peptides with 4 amino acid overlap, synthesized with a biotinylated 4 amino acid N-terminal linker to enable attachment to streptavidin-coated 96 well ELISA plates.

[0015] Figure 2 depicts epitope mapping of monoclonal antibodies 5F8, 5G10 and 6A5 on LA 0591.

[0016] Figure 3 A depicts epitope mapping of polyclonal antibodies Anti-ALA0591 andAnti-LA1402 on LA_0591.

[0017] Figure 3B depicts epitope mapping of polyclonal antibodies Anti-ALA0591 and Anti-LA1402 on LA_0591.

[0018] Figure 4 depicts the LA0591 sequence and representation of respective epitopes (polyAbs and mAbs) on a predicted 3-D structure of the protein.

[0019] Figure 5 depicts the results from Sequential B-Cell Epitope Predictor onLA 0591.

[0020] Figure 6 depicts the results from Sequential B-Cell Epitope Predictor on LA_1402.

[0021] Figure 7 depicts the purification of soluble VM proteins.

[0022] Figure 8A and Figure 8B depict the high level of VM protein peptide sequence epitope conservation recognized by the mAbs 5F8 and 6A5 of the PF07598 gene family in two different L. interrogans serovars and two different Leptospira species (L. interrogans Lai and Copenhageni and L. borgpetersenii serovar Hardjo Bovis). These are just examples and epitomize the entire genus of pathogenic Leptospira for which the high degree of epitope conservation is true.

[0023] Figure 9 depicts data demonstrating that Anti-LA0591 mAbs 5F8 and 6A5 crossreact with multiple recombinant full length Leptospira interrogans VM proteins further demonstrating their conservation and targets of protective immunity.

[0024] Figure 10 depicts data demonstrating that upregulation of VM proteins in humaninfecting lethal serovars imply potential role in severe human leptospirosis and therapeutic target and offers a potential explanation of why Leptospira interrogans serovar Copenhageni uniquely causes severe disease in humans.

[0025] Figure 11A through Figure 1 ID depicts data demonstrating that detection of VM proteins in serum of Leptospira interrogans serovar Copenhageni-infected hamsters using a capture ELISA with mAb 6A5 capture and mAb 5F8 detecting antibody, demonstrating that VM proteins are a diagnostic target for VM protein antigen detection and quantification, which would indicate the need to adjunct therapy using therapeutic neutralizing mAbs or to-be-discovered small molecules that inactivate VM toxin function. Figures 11 A and 1 IB are standard curves with Log transformation. Figure 11C demonstrates VM protein detection in 15 hamsters, three groups of N=5 infected with different amounts of Leptospira. Figure 1 ID presents a representative depiction of the capture ELISA protocol.

[0026] Figure 12 depicts data demonstrating that mAbs 5F8 + 6A5 neutralize VM protein cytotoxic activity on Hela cells. Rabbit polyclonal anti-LA0591+LA1402 antisera neutralize VM protein cytotoxic activity on Hela cells.

[0027] Figure 13, comprising Figure 13A and Figure 13B, depicts representative imagesof the schematic organization and immunoreactivity of VM proteins. Figure 1 A depicts a representative illustration of the domain architecture of LA3490 (full-length) (SEQ ID NO: 1), RBLs (N-terminal ricin binding lectin domains RBL1 and RBL2, amino acids 41-335) (SEQ ID NO:2), and LA0591 (C-terminal domain lacking RBL1 and RBL2) (SEQ ID NO: 3). Figure IB depicts representative data presenting the immunoreactivity of LA3490 (SEQ ID NO: 1), LA0591 (SEQ ID NO:3), and RBLs (SEQ ID NO:2). RBLs was detected with polyclonal antibodies generated against full-length VM protein; LA3490 and LA0591 were detected with monoclonal 5G10 antibody generated against LA0591. The arrow represents the reactivity and molecular weight of VM proteins. M shows the molecular weight.

[0028] Figure 14, comprising Figure 14A through Figure 14C, depicts representative distribution of mean OD values by qPCR (Figure 14A) and MAT results (Figure 14B), and final disease confirmation (Figure 14C) among suspected cases of leptospirosis and healthy controls.

[0029] Figure 15 depicts representative distribution of VM protein antibody OD values among patients grouped by MAT results with paired serum samples.

[0030] Figure 16 depicts representative distribution of VM protein antibody OD values by duration of illness.

[0031] Figure 17 depicts representative Receiver Operating Characteristic (ROC) curves presenting the diagnostic accuracy of VM protein antibodies to detect Leptospira infection.

[0032] Figure 18 depicts a representative schematic of the study design.

[0033] Figure 19, comprising Figure 19A through Figure 19E, depicts representative assessment of physical and biochemical parameters of the non-human primate (NHP) subjects Xena, Skeeter, and Melody used in the experimental procedure over 21 days.

[0034] Figure 20, comprising Figure 20A through Figure 20E, depicts representative OD readings from NTA-ELISA experiments detecting anti-LA0591, anti-LA1400, anti-LA1402, anti-LA3490, and anti -RBLs antibodies in the infected NHP subjects.

[0035] Figure 21, comprising Figure 21 A through Figure 21E, depicts representative OD readings from NTA-ELISA experiments detecting anti-LA0591, anti-LA1400, anti-LA1402, anti-LA3490, and anti -RBLs antibodies in the infected NHP subjects.

[0036] Figure 22, comprising Figure 22A through Figure 22D, depicts representative H&E stained tissue sections of lung, liver, kidney, and spleen tissue following dissection of thoracic and abdominal cavities of NHP subjects.

[0037] Figure 23, comprising Figure 23A through Figure 23C, depicts representative cytokine measurements performed using luminex multiplex bead array kits in the NHP subjects.

[0038] Figure 24, comprising Figure 24A through Figure 24H, depicts representative clinical analyses of the NHP subjects; Xena: Figures 24A-24B, Skeeter: Figures 24C-24D, Melody: Figures 24E-24F, reference: Figures 24G-24H.

[0039] Figure 25 depicts representative images of the inoculation site, at 4 days postinfection in two of the NHP subjects.

[0040] Figure 26, comprising Figure 26A and Figure 26B, depicts representative H&E stained tissue sections of lung, liver, and kidney, at 4 and 7 days post-infection.

[0041] Figure 27, comprising Figure 27A through Figure 27C, depicts representative cytokine measurements performed using luminex multiplex bead array kits in the NHP subjects.

[0042] Figure 28 depicts a representative prototype leptospiral lateral flow rapid diagnostic test.

[0043] Figure 29, comprising Figure 29A through Figure 29D, depicts representative SDS-PAGE periodate silver stain and Western immunoblot analysis comparing the new method (Figures 29A and 29B) with a published method (Figures 29C and 29D) of purifying L. interrogans serovar Copenhageni and serovar Lai lipopolysaccharides (LPS). Figure 29A depicts a periodate silver stain of an SDS-PAGE gel. Figure 29B depicts a Western immunoblot. Figure 29A-29B: lane 1: E. coli LPS (Sigma), lanes 2: serovar Copenhageni LPS, and lanes 3: serovar Lai LPS. The new method yields high MW LPS that ladders with repeat units of at least 20kDa to a MW > 250kDa (Figure 29A). These repeats are immunoreactive with polyclonal rabbit antisera raised against L. interrogans serovar Copenhageni and Lai (Lai not shown but similar to the antisera against Copenhageni indicating they strongly cross react). Figures 29C and 29D (modified from Nally et al., Infect. Immun., 2005., 6: 3251-6. Figure 29C, lanes 3 and 4, LPS extraction from two different Copenhageni strains analyzed by SDS-PAGE / periodate silver stain and Figure 29D lanes 3 and 4, LPS extraction from two different Copenhageni strains analyzed by Western blot using polyclonal rabbit antisera raised against L. interrogans serovat Copenhageni as in Figure 29B. Note: as seen in pervious publications, Copenhageni LPS is found in a band from 20-25 kDa but not in the high molecular weight laddering form typical of LPS, as demonstrated in Figures 29A and 29B.DETAILED DESCRIPTION

[0044] The invention relates to Leptospiral virulence modifying (VM) protein epitopes, and variants and fragments thereof, vaccine compositions comprising the same, and the use thereof for the generation of antibodies for the treatment of Leptospira spp. In some embodiments, Leptospiral VM protein epitopes are immunogenic and can thus be used as a vaccine or immunogenic composition to treat or prevent leptospirosis in a subject in need. In some embodiments, Leptospiral VM protein epitopes can be used to generate monoclonal antibodies or polyclonal anti-serum which can be used to treat or prevent leptospirosis in a subject in need.

[0045] In one embodiment the invention provides compositions and methods for inducing or enhancing an immune response. For example, in certain embodiments, the invention relates to inducing or enhancing cell-mediated and / or humoral immunity directed against a desired antigen.

[0046] In one embodiment, the composition of the invention serves as an antigen to induce immunity directed against a Leptospira sp. bacterium.

[0047] In certain embodiments, the compositions and methods are used to prevent, treat and diagnose infection by Leptospira. In certain embodiments, the compositions and methods are used to prevent or treat a disease or disorder associated with infection by Leptospira, including, but not limited to, leptospirosis, kidney damage, meningitis, liver failure, respiratory distress, and even death. In one embodiment, the composition of the invention is a vaccine that induces the cell-mediated and / or humoral immunity directed against at least one Leptospira sp protein.Definitions

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described.

[0049] As used herein, each of the following terms has the meaning associated with it in this section. 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.

[0050] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0051] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0052] The term “antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: 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).

[0053] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample.

[0054] As used herein, the term “autologous” is meant to refer to any material derivedfrom an individual to which it is later to be re-introduced into the same individual.

[0055] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response.

[0056] The term “agent” includes any substance, metabolite, molecule, element, compound, or a combination thereof. It includes, but is not limited to, e.g., protein, oligopeptide, small organic molecule, glycan, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent,” “substance,” and “compound” can be used interchangeably. Further, a “test agent” or “candidate agent” is generally a subject agent for use in an assay of the invention.

[0057] The term “binding” refers to a direct association between at least two molecules, due to, for example, covalent, electrostatic, hydrophobic, ionic and / or hydrogen-bond interactions.

[0058] “CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate). The structure and protein folding of the antibody may mean that other residues are considered part of the antigen binding region and would be understood to be so by a skilled person. See for example Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

[0059] A “chimeric antibody” refers to a type of engineered antibody which contains a naturally-occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.

[0060] “Contacting” refers to a process in which two or more molecules or two or more components of the same molecule or different molecules are brought into physical proximity such that they are able undergo an interaction. Molecules or components thereof may be contacted by combining two or more different components containing molecules, for example by mixing two or more solution components, preparing a solution comprising two or moremolecules such as target, candidate or competitive binding reference molecules, and / or combining two or more flowing components.

[0061] As used herein, by “combination therapy” is meant that a first agent is administered in conjunction with another agent. “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during, or after delivery of the other treatment modality to the individual. Such combinations are considered to be part of a single treatment regimen or regime.

[0062] As used herein, the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap temporally with each other.

[0063] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0064] The term “donor antibody” refers to an antibody (monoclonal, and / or recombinant) which contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner, so as to provide the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralizing activity characteristic of the donor antibody.

[0065] The term “acceptor antibody” refers to an antibody (monoclonal and / or recombinant) heterologous to the donor antibody, which contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and / or light chain framework regions and / or its heavy and / or light chain constant regions to the first immunoglobulin partner. In certain embodiments a human antibody is the acceptor antibody.

[0066] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0067] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0068] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0069] As used herein, the term “heavy chain antibody” or “heavy chain antibodies” comprises immunoglobulin molecules derived from camelid species, either by immunization with a peptide and subsequent isolation of sera, or by the cloning and expression of nucleic acid sequences encoding such antibodies. The term “heavy chain antibody” or “heavy chain antibodies” further encompasses immunoglobulin molecules isolated from an animal with heavy chain disease, or prepared by the cloning and expression of VH (variable heavy chain immunoglobulin) genes from an animal.

[0070] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0071] A “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad Sci USA, 86: 10029-10032; 1991, Hodgson et al., Bio / Technology, 9:421). A suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and / or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. The prior art describes several ways of producing such humanized antibodies (see for example EP-A-0239400 and EP-A-054951).

[0072] The term “immunoglobulin” or “Ig,” as used herein, is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0073] As used herein, the term “immune response” includes T-cell mediated and / or B- cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity, and B cell responses, e.g., antibody production. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Thl and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0074] As used herein, an “inhibitory-effective amount” is an amount that results in a detectable (e.g., measurable) amount of inhibition of an activity. In some instance, the activity is its ability to bind with another component.

[0075] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0076] A “mutation,” as used herein, refers to a change in nucleic acid or polypeptide sequence relative to a reference sequence (which is may be a naturally-occurring normal or “wild-type” sequence), and includes translocations, deletions, insertions, and substitutions / point mutations. A “mutant” as used herein, refers to either a nucleic acid or protein comprising a mutation.

[0077] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intradermal (i.d.) injection, or infusion techniques.

[0078] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0079] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibodyrecognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “X,” the presence of a molecule containing epitope X (or free, unlabeled A), in a reaction containing labeled “X” and the antibody, will reduce the amount of labeled X bound to the antibody.

[0080] By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0081] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of a disease state.

[0082] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or clinical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0083] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0084] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0085] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Description

[0086] The present invention relates to Leptospiral virulence modifying (VM) protein epitopes, and variants and fragments thereof.

[0087] In some embodiments, the present invention provides a composition comprising a Leptospiral VM protein epitope, variant thereof, or fragment thereof. In some embodiment, the composition comprises a fragment of a Leptospiral VM protein epitope.

[0088] In one embodiment, the composition comprises a fusion protein, comprising a first domain comprising a Leptospiral VM protein epitope, variant thereof, or fragment thereof. In one embodiment, the fusion protein comprises a second domain. In one embodiment, the second domain is a targeting domain, wherein the targeting domain directs the fusion protein to a specific cell or tissue of interest. For example, in one embodiment, the targeting domain comprises an antibody, antibody fragment, or peptide that specifically binds to an antigen (e g., tumor antigen) thereby directing the fusion protein to a cell or tissue expressing the antigen. In one embodiment, the second domain comprises a detectable protein or peptide (e.g., a fluorescent protein) that allows for the visualization of the fusion protein.

[0089] In one embodiment, the present invention provides an isolated nucleic acid molecule encoding a Leptospiral VM protein epitope, variant thereof, or fragment thereof. In some embodiments, the isolated nucleic acid molecule comprises DNA, cDNA, RNA, or mRNA encoding a Leptospiral VM protein epitope, variant thereof, or fragment thereof. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a Leptospiral VM protein epitope, variant thereof, or fragment thereof.

[0090] In one embodiment, the composition comprises an immunological composition comprising (a) at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof;or (b) a nucleic acid molecule encoding at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof. As demonstrated herein, in certain embodiments, a Leptospiral VM protein epitope, variant thereof, or fragment thereof induces a protective immune response that can treat or prevent Leptospiral infection or leptospirosis in a subject in need thereof. In one embodiment, the immunological composition comprises a vaccine. In one embodiment, the immunological composition comprises a bacterium (e.g., a bacterium from genus Leptospira) modified to express a Leptospiral VM protein epitope, variant thereof, or fragment thereof. In one embodiment, the bacterium is attenuated in that it has reduced pathogenicity, but is capable of inducing a protective immune response. The compositions are not only useful as a prophylactic therapeutic agent for immunoprotection, but are also useful as a therapeutic agent for treatment of an ongoing infection, disease, or disorder.

[0091] In one embodiment, the present invention relates to methods of inducing cell death or damage comprising administering to a cell a composition comprising (a) at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof. In one embodiment, the method comprises administering the composition to a tumor, thereby inducing tumor cell death or damage.

[0092] The present invention also provides methods of preventing, inhibiting, and treating infection caused by bacteria of genus Leptospira in a subject in need thereof. In one embodiment, the methods of the invention induce immunity against genus Leptospira in the subject, by generating an immune response in the subject directed to a Leptospiral VM protein epitope. In certain embodiments, the method induces broad immunity across genus Leptospira.

[0093] In one embodiment, the methods of the invention induce production of VM protein-specific antibodies in the subject. In one embodiment, the methods of the invention prevent Leptospira related pathology, such as leptospirosis (also known as Weil’s disease) in a subject in need thereof. In one embodiment, the methods of the invention comprise administering to the subject a composition comprising a) at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding at least one Leptospiral VM protein, variant thereof, or fragment thereofCompositions

[0094] The present invention provides compositions comprising or encoding at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof.

[0095] In one embodiment, the Leptospiral VM protein epitope is SEQ ID NOT, SEQ ID NOT, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO 27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof.

[0096] In one embodiment, the invention relates to a vaccine comprising a combination of at least two, three, four, five, or more than five VM protein epitopes. In one embodiment, the vaccine comprises a combination of at least two, three, four, five, or more than five of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOT, SEQ ID NO:8, SEQ ID NOT, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO 32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or fragments or variants thereof.

[0097] In one embodiment, the invention relates to a toxoid vaccine comprising a at least one, two, three, four, five, or more than five VM protein epitopes. In one embodiment, the toxoid vaccine comprises at least one, two, three, four, five, or more than five of SEQ ID NO:2, SEQ ID NOT, SEQ ID NOT, SEQ ID NOT, SEQ ID NOT, SEQ ID NOT, SEQ ID NO:8, SEQ ID NOT, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NOTO, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NOTO, SEQ ID NOT 1, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NOTO, or fragments or variants thereof.

[0098] In various embodiments, the invention provides a protein, or a fragment, ahomolog, a mutant, a variant, a derivative or a salt of a protein as elsewhere described herein, wherein the activity of the various domains of Leptospiral VM proteins (e.g., immunogenic activity or cytopathic activity or activity related to Leptospiral VM protein mechanism of action) is retained.

[0099] Proteins or peptides of the present invention can be prepared using well known techniques. For example, the proteins can be prepared synthetically, using either recombinant DNA technology or chemical synthesis. Proteins of the present invention may be synthesized individually or as longer proteins composed of two or more proteins. The proteins of the present invention can be isolated, i.e., substantially free of other naturally occurring host cell proteins and fragments thereof.

[0100] The proteins of the present invention may contain modifications, such as glycosylation, aglycosylation, side chain oxidation, or phosphorylation; so long as the modifications do not destroy the immunologic activity of the proteins. Other modifications include incorporation of D-amino acids or other amino acid mimetics that can be used, for example, to increase the serum half-life of the proteins.

[0101] The proteins of the invention can be modified whereby the amino acid is substituted for a different amino acid in which the properties of the amino acid side-chain are conserved (a process known as conservative amino acid substitution). Examples of properties of amino acid side chains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (D, N, E, Q); a base containing side-chain (R, K, H); and an aromatic containing side-chain (H, F, Y, W). Note that the parenthetic letters indicate the one- letter codes of amino acids. As used herein, X stands for any amino acid.

[0102] The present invention should also be construed to encompass “mutants,” “derivatives,” and “variants” of the proteins of the invention (or of the DNA encoding the same) which mutants, derivatives and variants are polypeptides which are altered in one or more amino acids (or, when referring to the nucleotide sequence encoding the same, are altered in one or more base pairs) such that the resulting protein (or DNA) is not identical to the sequences recited herein, but has the same biological property as the protein disclosed herein.

[0103] The invention should also be construed to include any form of a protein variant having substantial homology to an amino acid sequence disclosed herein. In one embodiment, a protein variant is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence disclosed herein.

[0104] The invention should also be construed to include any form of a fragment having a substantial length of an amino acid sequence disclosed herein. In one embodiment, a fragment is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of an amino acid sequence disclosed herein.

[0105] The invention should also be construed to include any form of a fragment of a protein variant, having both substantial homology to and a substantial length of an amino acid sequence disclosed herein. In one embodiment, a fragment of a protein variant is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to an amino acid sequence disclosed herein, and is at least about 50%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of an amino acid sequence disclosed herein.

[0106] The protein may alternatively be made by recombinant means or by cleavage from a longer protein. The protein may be confirmed by amino acid analysis or sequencing.

[0107] The variants of the proteins according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (e.g., a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the protein comprises an alternative splice variant of the proteins or domains described herein, (iv) fragments of the proteins or domains described herein and / or (v) one in which the protein is fused with another protein or peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include proteins or peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0108] As known in the art the “similarity” between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide to a sequence of a second peptide. Variants are defined to include peptide sequences different from the original sequence, e.g., different from the original sequence in less than 40% of residues per segment of interest, different from the original sequence in less than 25% of residues per segment of interest, different by less than 10% of residues per segment of interest, or different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides may be determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences may be determined by using the BLASTP algorithm (BLAST Manual, Altschul, S„ et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S„ et al., J. Mol. Biol. 215: 403-410 (1990)).

[0109] The protein of the invention may or may not be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction. A polypeptide or protein of the invention may be phosphorylated using conventional methods such as the method described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992).

[0110] The protein of the invention may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during polypeptide translation.

[0111] A protein of the invention may be conjugated with other molecules, such as polyethylene glycol (PEG). This may be accomplished by inserting cysteine mutations or unnatural amino acids that can be modified with a chemically reactive PEG derivative. In oneembodiment, the protein is conjugated to other proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins provided that the resulting fusion protein retains the functionality of the protein described herein.

[0112] Cyclic derivatives of the proteins of the invention are also part of the present invention. Cyclization may allow the protein to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bonds may be side chains of amino acids, nonamino acid components or a combination of the two. In an embodiment of the invention, cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

[0113] It may be desirable to produce a cyclic protein which is more flexible than the cyclic proteins containing peptide bond linkages as described above. A more flexible protein may be prepared by introducing cysteines at the right and left position of the polypeptide and forming a disulfide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The protein is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic protein can be determined by molecular dynamics simulations.

[0114] The invention also relates to a fusion protein. For example, in one embodiment, the fusion protein comprises a first domain comprising a Leptospiral VM protein, variant thereof, or fragment thereof. In one embodiment, the fusion protein comprises a second domain. In one embodiment, the second domain is a targeting domain, wherein the targeting domain directs the fusion protein to a specific cell or tissue of interest. For example, in one embodiment, the targeting domain comprises an antibody, antibody fragment, or peptide that specifically binds to an antigen (e.g., tumor antigen) thereby directing the fusion protein to a cell or tissue expressing the antigen. In one embodiment, the second domain comprises a detectable protein or peptide (e.g., a fluorescent protein) that allows for the visualization of the fusion protein.

[0115] In one embodiment, the fusion protein comprises a targeting domain capable ofdirecting the resulting protein to a desired cellular component or cell type or tissue. The chimeric or fusion proteins may also contain additional amino acid sequences or domains. The chimeric or fusion proteins are recombinant in the sense that the various components are from different sources, and as such are not found together in nature (i.e., are heterologous).

[0116] In one embodiment, the targeting domain can be a membrane spanning domain, a membrane binding domain, or a sequence directing the protein to associate, for example, with vesicles or with the cell surface. In one embodiment, the targeting domain can target a protein to a particular cell type or tissue. For example, the targeting domain can be a cell surface ligand or an antibody against a cell surface antigens of a target tissue. A targeting domain may target a protein of the invention to a cellular component.

[0117] In one embodiment, the targeting domain may comprises an antibody or antibody fragment thereof. An antibody may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: 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). In one embodiment, the targeting domain of a composition of the invention comprises an antibody fragment. In one embodiment, the targeting domain comprises an antibody fragment that comprises a scFv.

[0118] The VM-epitope containing fusion molecule of the invention can be generated to be reactive to any desirable antigen of interest, or fragment thereof, including, but not limited to a tumor antigen, a bacterial antigen, a viral antigen or a self-antigen. In the context of the present invention, “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder,” refers to antigens that are common to specific hyperproliferative disorders, such as cancer. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, nonHodgkin's lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0119] The antigens discussed herein are merely included by way of example. The list is not intended to be exclusive and further examples will be readily apparent to those of skill in the art.

[0120] A fusion protein of the invention may be synthesized by conventional techniques. For example, the proteins may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and R. B. Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254 for solid phase synthesis techniques; and M Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin 1984, and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, suprs, Vol 1, for classical solution synthesis). By way of example, a polypeptide of the invention may be synthesized using 9-fluorenyl methoxy carbonyl (Fmoc) solid phase chemistry with direct incorporation of phosphothreonine as the N-fluorenylmethoxy- carbonyl-O-benzyl-L-phosphothreonine derivative.

[0121] N-terminal or C-terminal fusion proteins comprising a VM protein epitope of the invention, conjugated with at least one other molecule, may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal end of the peptide or protein, and the sequence of a selected protein or selectable marker with a desired biological function. The resultant fusion proteins contain the Leptospiral VM protein, variant thereof, or fragment thereof, fused to the selected protein or marker protein as described herein. Examples of proteins which may be used to prepare fusion proteins include immunoglobulins and regions thereof, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0122] A protein of the invention may be developed using a biological expression system. The use of these systems allows the production of large libraries of random sequences and the screening of these libraries for sequences that bind to particular proteins. Libraries may be produced by cloning synthetic DNA that encodes random peptide sequences into appropriate expression vectors (see Christian et al 1992, J. Mol. Biol. 227:711; Devlin et al, 1990 Science 249:404; Cwirla et al 1990, Proc. Natl. Acad, Sci. USA, 87:6378). Libraries may also be constructed by concurrent synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

[0123] The protein of the invention may be converted into pharmaceutical salts byreacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.

[0124] The present invention further encompasses fusion proteins in which the VM epitopes of the invention or fragments thereof, are recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to heterologous proteins (i.e., an unrelated protein or portion thereof, e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, or at least 500 amino acids of the polypeptide) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences.

[0125] In one example, a fusion protein in which an epitope of the invention or a fragment thereof can be fused to sequences derived from various types of immunoglobulins. For example, a polypeptide of the invention can be fused to a constant region (e.g., hinge, CH2, and CH3 domains) of human IgG or IgM molecule, for example, as described herein, so as to make the fused protein or fragments thereof more soluble and stable in vivo. In another embodiment, such fusion proteins can be administered to a subject so as to inhibit interactions between a ligand and its receptors in vivo. Such inhibition of the interaction will block or suppress signal transduction which triggers certain cellular responses.

[0126] In one aspect, the fusion protein comprises an epitope of the invention which is fused to a heterologous signal sequence at its N-terminus. For example, the signal sequence naturally found in the protein of the invention can be replaced by a signal sequence which is derived from a heterologous origin. Various signal sequences are commercially available. For example, the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, Calif.) are available as eukaryotic heterologous signal sequences. As examples of prokaryotic heterologous signal sequences, the phoA secretory signal (Sambrook, et al., supra; and Current Protocols in Molecular Biology, 1992, Ausubel, et al., eds., John Wiley & Sons) and the protein A secretory signal (Pharmacia Biotech; Piscataway, N.J.) can be listed. Another example is the gp67 secretory sequence of the baculovirus envelope protein (Current Protocols in Molecular Biology, 1992, Ausubel, et al., eds., John Wiley & Sons).

[0127] In another embodiment, a protein of the invention can be fused to tag sequences, e.g., a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz, et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other examples of peptide tags are the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, et al., 1984, Cell 37:767) and the "flag" tag (Knappik, et al., 1994, Biotechniques 17(4):754-761). These tags are especially useful for purification of recombinantly produced proteins of the invention.

[0128] In one embodiment, an epitope of the invention can be fused to a detectable label, such as a fluorescent tag. Non-limiting examples of fluorescent tags include green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), eGFP, mCherry, hrGFP, hrGFPII, Alexa 488, Alexa 594, and the like. Fluorescent tags may also be photoconvertible, such as for example kindling red fluorescent protein (KFP-red), PS-CFP2, Dendra2, CoralHue Kaede and CoralHue Kikume. However, the invention should not be limited to a particular label. Rather, any detectable label can be used to tag the expressed protein.

[0129] In some embodiments, the invention provides compositions comprising a lipid nanoparticle (LNP) or liposome conjugated to or encapsulating at least one VM protein epitope or peptide of the invention. In one embodiment, the composition comprises a combination of two or more LNPs encapsulating a combination of two or more VM protein epitopes. In some instances, the LNPs enhances cellular uptake of the VM protein epitopes.

[0130] In some embodiments, the composition comprises a scaffold, such as a tissue engineering scaffold, comprising a nucleic acid molecule encoding at least one VM protein epitope. For example, in one embodiment, the scaffold comprises LNP encapsulating the nucleic acid molecule encoding at least one VM protein epitope. In one embodiment, the scaffold comprises a cell or cell population comprising a nucleic acid molecule encoding at least one VM protein epitope. In some embodiments, the scaffold comprises a hydrogel, electrospun scaffold or the like comprising a biopolymer, synthetic polymer or combination thereof.

[0131] The present invention also provides isolated nucleic acid molecules that encode the VM protein epitope described herein. Therefore, in one embodiment, the composition of theinvention comprises an isolated nucleic acid molecule encoding a Leptospiral VM protein epitope, variant thereof, or fragment thereof.

[0132] The one or more isolated nucleic acid molecule may comprise any type of nucleic acid, including, but not limited to DNA, cDNA, and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a protein or functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule encoding a protein or a functional fragment thereof.

[0133] The nucleic acid sequences include both the DNA sequence that is transcribed into RNA and the RNA sequence that is translated into a protein. According to other embodiments, the nucleic acid sequences of the invention are inferred from the amino acid sequence of the proteins of the invention. As is known in the art several alternative nucleic acid sequences are possible due to redundant codons, while retaining the biological activity of the translated proteins.

[0134] Further, the invention encompasses an isolated nucleic acid molecule encoding a protein having substantial homology to the VM protein epitopes disclosed herein. In some embodiments, the present invention encompasses an isolated nucleic acid molecule encoding a protein comprising an amino acid sequence having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology with an amino acid sequence an amino acid sequence of the proteins disclosed herein. In some embodiments, the nucleic acid sequence encoding a protein of the invention is “substantially homologous,” that is about 50% homologous, about 70% homologous, about 80% homologous, about 90% homologous, about 91% homologous, about 92% homologous, about 93% homologous, about 94% homologous, about 95% homologous, about 96% homologous, about 97% homologous, about 98% homologous, or about 99% homologous to a nucleic acid sequence described herein.

[0135] It is to be understood explicitly that the scope of the present invention encompasses homologs, analogs, variants, fragments, derivatives and salts, including shorter and longer proteins and nucleic acid molecules, as well as protein and nucleic acid molecule analogs with one or more amino acid or nucleic acid substitution, as well as amino acid or nucleic acid derivatives, non-natural amino or nucleic acids and synthetic amino or nucleic acids as areknown in the art, with the stipulation that these modifications must preserve the activity of the original molecule. Specifically, any active fragments of the active proteins and nucleic acid molecules, as well as extensions, conjugates and mixtures are included and are disclosed herein according to the principles of the present invention.

[0136] The invention should be construed to include any and all isolated nucleic acid sequences which are homologous to the nucleic acid sequences described and referenced herein, provided these homologous nucleic acid sequences encode proteins having the biological activity of the proteins disclosed herein.

[0137] The skilled artisan would understand that the nucleic acid sequences of the invention encompass an RNA or a DNA sequence encoding a protein of the invention, and any modified forms thereof, including chemical modifications of the DNA or RNA which render the sequence more stable when it is cell free or when it is associated with a cell. Chemical modifications of nucleotides may also be used to enhance the efficiency with which a nucleic acid sequence is taken up by a cell or the efficiency with which it is expressed in a cell. Any and all combinations of modifications of the nucleic acid sequences are contemplated in the present invention.

[0138] Further, any number of procedures may be used for the generation of mutant, derivative or variant forms of a protein of the invention using recombinant DNA methodology well known in the art such as, for example, that described in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Procedures for the introduction of amino acid changes in a polypeptide or polypeptide by altering the DNA sequence encoding the polypeptide are well known in the art and are also described in these, and other, treatises.

[0139] The nucleic acid molecules of the present invention can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and / or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the invention. For example, in order to enhance the stability, the 3’- residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2’-deoxythymidine is tolerated and does not affect function of the molecule.

[0140] In one embodiment of the present invention the nucleic acid molecule may contain at least one modified nucleotide analogue. For example, the ends may be stabilized by incorporating modified nucleotide analogues.

[0141] Non-limiting examples of nucleotide analogues include sugar- and / or backbone- modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2’ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is C1-C6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0142] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. The above modifications may be combined.

[0143] In some instances, the nucleic acid molecule comprises at least one of the following chemical modifications: 2’-H, 2’-O-methyl, or 2’-OH modification of one or more nucleotides. In some embodiments, a nucleic acid molecule of the invention can have enhanced resistance to nucleases. For increased nuclease resistance, a nucleic acid molecule, can include, for example, 2’-modified ribose units and / or phosphorothioate linkages. For example, the 2’ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. For increased nuclease resistance the nucleic acid molecules of the invention can include 2’-O-methyl, 2’-fluorine, 2’ -O-m ethoxy ethyl, 2’-O-aminopropyl, 2’-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2’-4’-ethylene-bridged nucleic acids, and certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to atarget.

[0144] In one embodiment, the nucleic acid molecule includes a 2’-modified nucleotide, e.g., a 2’-deoxy, 2 ’-deoxy-2’ -fluoro, 2’-O-methyl, 2’-O-methoxyethyl (2’-O-MOE), 2’-O- aminopropyl (2’-O-AP), 2’-O-dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), or 2’-O-N- methylacetamido (2’-0-NMA). In one embodiment, the nucleic acid molecule includes at least one 2’-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2’-O-methyl modification.

[0145] Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA as well as RNA and DNA that have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, for example as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs, often termed modified RNAs, are typically the result of a post-transcriptional modification and are within the term unmodified RNA as used herein. Modified RNA, as used herein, refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, for example different from that which occurs in the human body. While they are referred to as “modified RNAs” they will of course, because of the modification, include molecules that are not, strictly speaking, RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non- ribophosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone.

[0146] Modifications of the nucleic acid of the invention may be present at one or more of, a phosphate group, a sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0147] The present invention also includes a vector in which the isolated nucleic acid of the present invention is inserted. The art is replete with suitable vectors that are useful in the present invention.

[0148] In brief summary, the expression of natural or synthetic nucleic acids encoding aprotein is typically achieved by operably linking a nucleic acid encoding the protein or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0149] The vectors of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therapy vector.

[0150] The isolated nucleic acid of the invention can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0151] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0152] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0153] For example, vectors derived from retroviruses such as the lentivirus are suitabletools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

[0154] In some embodiments, the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and / or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissuespecific, are known in the art and may be utilized.

[0155] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elementscan be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0156] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor -la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0157] Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.

[0158] In order to assess the expression of a protein, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0159] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0160] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0161] Physical methods for introducing a peptide or protein into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0162] Biological methods for introducing a peptide or protein of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0163] Chemical means for introducing a peptide or protein into a host cell includecolloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0164] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0165] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0166] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular polypeptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0167] In one embodiment, the present invention provides a delivery vehicle comprising a protein, or a nucleic acid molecule encoding protein. Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymerosomes, liposomes, and micelles. For example, in some embodiments, the delivery vehicle is loaded with protein, or a nucleic acid molecule encoding a protein. In some embodiments, the delivery vehicle provides for controlled release, delayed release, or continual release of its loaded cargo. In some embodiments, the delivery vehicle comprises a targeting moiety that targets the delivery vehicle to a treatment site.

[0168] In one embodiment, the present invention provides an implantable scaffold or device comprising the protein or nucleic acid molecule encoding the protein. For example, in some embodiments, the present invention provides a tissue engineering scaffold, including but not limited to, a hydrogel, electrospun scaffold, polymeric matrix, or the like, comprising the protein or nucleic acid molecule encoding the protein in or on the scaffold.

[0169] In some embodiments, the invention provides compositions comprising a lipid nanoparticle (LNP) or liposome conjugated to or encapsulating at least one nucleic acid molecule encoding at least one VM protein or peptide of the invention. In one embodiment, thecomposition comprises a combination of two or more LNPs encapsulating a combination of two or more nucleic acid molecules encoding at least two or more VM proteins or peptides of the invention. In one embodiment, the nucleic acid molecule comprises an mRNA molecule encoding at least one VM protein or peptide of the invention. Therefore, in some embodiments, the invention provides at least one LNP or liposome conjugated to or encapsulating at least one mRNA molecule encoding at least one VM protein. In one embodiment, the invention provides a combination of LNP or liposomes conjugated to or encapsulating mRNA molecules encoding LIC 12340 and LIC 12985. In one embodiment, the invention provides a combination of LNP or liposomes conjugated to or encapsulating mRNA molecules encoding LIC_12340, LIC 12985, LA_3490, LA_0620, and LA_1402.

[0170] In certain aspects, the present invention encompasses compositions, including polypeptides, nucleotides, vectors, bacteria, and vaccines, that when administered to a subject, elicit or enhance an immune response. In certain instances, the composition elicits an immune response directed against a bacteria of genus Leptospira including an immune response directed against a Leptospiral VM protein. Further, when the compositions are administered to a subject, they elicit an immune response that serves to protect the inoculated subject against conditions associated with Leptospira infection.

[0171] In one embodiment, the present invention provides compositions that are useful as immunomodulatory agents, for example, for stimulating immune responses and in preventing Leptospira related pathology. In various embodiments, the immunomodulatory agents comprise (a) a Leptospiral VM protein, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, variant thereof, or fragment thereof. In one embodiment, the immune response is not detrimental to the host and therefore the compositions of the invention are useful as a vaccine. In one embodiment, the immunomodulatory agents are administered in combination with an adjuvant. In one embodiment, the adjuvant is glucopyranosyl lipid A (GLA), formulated in a stable oil-in-water nano-emulsion (SE), referred to as a GLA-SE adjuvant. In another embodiment, the immunomodulatory agents are administered in the absence of an adjuvant.

[0172] In some embodiments, the compositions are used as immunostimulatory agents to induce or enhance the production of specific antibodies. In certain aspects, the immunostimulatory agents protect against Leptospiral induced pathology.

[0173] In one embodiment, the composition comprises a bacterium comprising (a) a Leptospiral VM protein, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, variant thereof, or fragment thereof. For example, in one embodiment, the composition comprises a bacterium of genus Leptospira comprising (a) a Leptospiral VM protein, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, variant thereof, or fragment thereof. In one embodiment, the composition comprises a bacterium that is not a bacterium of genus Leptospira, wherein the bacterium comprises (a) a Leptospiral VM protein, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, variant thereof, or fragment thereof.

[0174] A bacterium comprising a nucleotide sequence encoding a Leptospiral VM protein, variant thereof, or fragment thereof, can be generated using any method known in the art including, but not limited to allelic exchange and site-directed mutagenesis.

[0175] Any bacterium or bacterial strain which has at least one nucleotide sequence encoding a Leptospiral VM protein, variant thereof, or fragment thereof can be selected and used in accordance with the invention. In one embodiment, naturally occurring mutants or variants, or spontaneous mutants can be selected. In another embodiment, mutant bacteria can be generated by exposing the bacteria to mutagens, such as ultraviolet irradiation or chemical mutagens, or by multiple passages and / or passage in non-permissive hosts. Screening in a differential growth system can be used to select for those mutants having a mutation in a Leptospiral VM protein.

[0176] In another embodiment, mutations can be engineered into a bacterium, for example a Leptospira bacterium using “reverse genetics” approaches. In this way, natural or other mutations which confer an inactivated or attenuated phenotype can be engineered into strains. For example, deletions, insertions or substitutions of the coding region of the gene responsible for the Leptospiral VM protein can be engineered. Deletions, substitutions or insertions in the non-coding region of the gene responsible for the Leptospiral VM protein are also contemplated. To this end, mutations in the signals responsible for the transcription, replication, poly adenylation and / or packaging of the gene responsible for the Leptospiral VM protein can be engineered.

[0177] In one embodiment, the bacterium is engineered to be deficient, in which a Leptospiral VM protein is absent. For example, in certain embodiments, a toxin-deficient mutant bacterium or virus, where one or more Leptospiral VM protein is absent, is unable to causedisease but is able to induce an adaptive immune response against genus Leptospira.

[0178] Bacterium generated by the approaches described herein can be used in the vaccine and pharmaceutical formulations described herein. Reverse genetics techniques can also be used to engineer additional mutations to other genes important for vaccine production - i.e., the epitopes of useful vaccine strain variants can be engineered into the bacterium. Alternatively, completely foreign epitopes, including antigens derived from other pathogens can be engineered into the inactivated or attenuated strain.

[0179] The inactivated or attenuated bacterium of the present invention can itself be used as the active ingredient in vaccine or pharmaceutical formulations. In certain embodiments, the bacterium can be used as the vector or “backbone” of recombinantly produced vaccines. To this end, the “reverse genetics” technique can be used to engineer mutations or introduce foreign epitopes into the bacterium, which would serve as the “parental” strain. In this way, vaccines can be designed for immunization against strain variants, or in the alternative, against completely different infectious agents or disease antigens.

[0180] For example, in one embodiment, the immunological composition of the invention comprises a bacterium, engineered to express one or more epitopes or antigens of a given pathogen. For example, the bacterium can be engineered to express neutralizing epitopes of other preselected strains. Alternatively, epitopes of other pathogens can be built into the mutant bacterium.

[0181] In one embodiment, the bacterium is capable of inducing a robust immune response in the host - a feature which contributes to the generation of a strong immune response when used as a vaccine, and which has other biological consequences that make the bacterium useful as pharmaceutical agents for the prevention and / or treatment of an infection, disease, or disorder associated with an antigen. For example, in certain embodiments, the bacterium induces an anti-Leptospiral immune response.

[0182] For an antigenic composition to be useful as a vaccine, the antigenic composition must induce an immune response to the antigen in a cell, tissue or subject (e.g., a human). In certain aspects the vaccine induces a protective immune response in the subject. As used herein, an “immunological composition” may comprise, by way of examples, an antigen (e.g., a protein), a nucleic acid molecule encoding an antigen (e.g., an antigen expression vector), or a cell expressing or presenting an antigen. In particular embodiments the antigenic compositioncomprises or encodes all or part of any protein antigen described herein, or an immunologically functional equivalent thereof. In other embodiments, the antigenic composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination. In certain embodiments, the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.

[0183] In the context of the present invention, the term “vaccine” (also referred to as an immunogenic composition) refers to a substance that induces immunity upon inoculation into an animal. In one embodiment, the vaccine induces anti-Leptospiral immunity. In various embodiments, the vaccine of the invention comprises

[0184] In one embodiment, the vaccine is administered in combination with an adjuvant. In another embodiment, the vaccine is administered in the absence of an adjuvant.

[0185] A vaccine of the present invention may vary in its composition of nucleic acid and / or cellular components. In a non-limiting example, a nucleic encoding an antigen might also be formulated with an adjuvant. Of course, it will be understood that various compositions described herein may further comprise additional components. For example, one or more vaccine components may be comprised in a lipid or liposome. In another non-limiting example, a vaccine may comprise one or more adjuvants. A vaccine of the present invention, and its various components, may be prepared and / or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

[0186] In one embodiment, the protein vaccine of the invention includes, but is not limited to at least one Leptospiral VM protein, variant thereof, or fragment thereof, optionally mixed with adjuvant substances. In some embodiments, the protein is introduced together with an antigen presenting cell (APC). The most common cells used for the latter type of vaccine are bone marrow and peripheral blood derived dendritic cells, as these cells express costimulatory molecules that help activation of T cells. WO00 / 06723 discloses a cellular vaccine composition which includes an APC presenting tumor associated antigen polypeptides. Presenting the protein can be effected by loading the APC with a polynucleotide (e g., DNA, RNA) encoding the protein or loading the APC with the protein itself.

[0187] For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. A foreign substance that enters the living body is presented to T cells and B cells by the action of APCs. T cells that respond to the antigen presented by APC in an antigen-specific manner differentiate into cytotoxic T cells (also referred to as cytotoxic T lymphocytes or CTLs) due to stimulation by the antigen. These antigen stimulated cells then proliferate. This process is referred to herein as “activation” of T cells. Therefore, CTL induction by a certain polypeptide or combination of polypeptides of the invention can be evaluated by presenting the polypeptide to a T cell by APC, and detecting the induction of CTL. Furthermore, APCs have the effect of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils and NK cells.

[0188] A method for evaluating the inducing action of CTL using dendritic cells (DCs) as APC is well known in the art. DC is a representative APC having the strongest CTL inducing action among APCs. In this method, the polypeptide or combination of polypeptides are initially contacted with DC and then this DC is contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contact with DC shows that the polypeptide or combination of polypeptides have an activity of inducing the cytotoxic T cells. Furthermore, the induced immune response can be also examined by measuring IFN-gamma produced and released by CTL in the presence of antigen-presenting cells that carry immobilized polypeptide or combination of polypeptides by visualizing using anti-IFN-gamma antibodies, such as an ELISPOT assay.

[0189] Apart from DC, peripheral blood mononuclear cells (PBMCs) may also be used as the APC. The induction of CTL is reported to be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0190] The polypeptide, or combination of polypeptides, confirmed to possess CTL inducing activity by these methods are polypeptides having DC activation effect and subsequent CTL inducing activity. Therefore, a polypeptide or combination of polypeptides that induce CTL against a Leptospiral VM protein are useful as vaccines against Leptospira associated pathology. Furthermore, CTL that have acquired cytotoxicity due to presentation of the polypeptide or combination of polypeptides by APC can be also used as vaccines against Leptospiral infection.

[0191] Generally, when using a polypeptide for cellular immunotherapy, efficiency of the CTL-induction can be increased by combining a plurality of polypeptides having differentstructures and contacting them with DC. Therefore, when stimulating DC with protein fragments, it is advantageous to use a mixture of multiple types of fragments.

[0192] The induction of immunity by a polypeptide or combination of polypeptides can be further confirmed by observing the induction of antibody production against the specific antigen. For example, when antibodies against a polypeptide or combination of polypeptides are induced in a laboratory animal immunized with the polypeptide or combination of polypeptides, and when Leptospiral associated pathology is suppressed by those antibodies, the polypeptide or combination of polypeptides are determined to induce anti-Leptospiral immunity.Methods

[0193] In various embodiments, the compositions of the invention can be used in biological assays, including methods of detecting a protein (e g. asialofetuin). Exemplary biological assays include, but are not limited to, an immunochromatography assay, an immunodot assay, a Luminex assay, an ELISA assay, an ELISPOT assay, a protein microarray assay, a Western blot assay, a mass spectrophotometry assay, a radioimmunoassay (RIA), a radioimmunodiffusion assay, a liquid chromatography -tandem mass spectrometry assay, an ouchterlony immunodiffusion assay, reverse phase protein microarray, a rocket immunoelectrophoresis assay, an immunohistostaining assay, an immunoprecipitation assay, a complement fixation assay, FACS, an enzyme-substrate binding assay, an enzymatic assay, an enzymatic assay employing a detectable molecule, such as a chromophore, fluorophore, or radioactive substrate, a substrate binding assay employing such a substrate, a substrate displacement assay employing such a substrate, and a protein chip assay (see also, 2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook, Gulf Professional Publishing; 1996, Diamandis and Christopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007). In some embodiments, the level of asialofetuin in the biological sample is measured with an assay that uses at least one Leptospiral VM protein, variant thereof, or fragment thereof; or a nucleic acid molecule encoding a Leptospiral VM protein, variant thereof, or fragment thereof of the invention, as described elsewhere herein.

[0194] In various embodiments, the present invention provides methods comprisingadministering a composition described herein, to a subject in need thereof. For example, in one embodiment, the method comprises administering to a subject a composition comprising a) a Leptospiral VM protein, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, variant thereof, or fragment thereof. In various embodiments, the compositions of the invention can be used as agents for inducing Leptospiral immunity or as cytotoxic agents for the treatment of a disease or disorder.

[0195] Compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject’s disease, although appropriate dosages may be determined by clinical trials. When “an effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, disease progression, and condition of the patient (subject). The optimal dosage and treatment regime for a particular subject can readily be determined by one skilled in the art of medicine by monitoring the subject for signs of disease and adjusting the treatment accordingly.

[0196] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.

[0197] Forms of administration that may be useful in the methods described herein include, but are not limited to, direct delivery to a desired organ, oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, intratumoral, subcutaneous, intradermal, and other parental routes of administration. Additionally, routes of administration may be combined, if desired. In one embodiment, the route of administration is intradermal injection or intratumoral injection. In one embodiment, one or more composition is administered to a treatment site during a surgical procedure, for example during surgical resection of all or part of a tumor.Methods of Use as a Vaccine

[0198] Thus, the present invention also encompasses a method of inducing anti-Leptospiral immunity using one or more of the compositions described herein. Anti -Leptospiral immunity can be induced by administering a composition of the invention, and the induction of anti-Leptospiral immunity enables treatment and prevention of pathologies associated with Leptospiral infection. Thus, the invention provides a method for treating, or preventing infection by genus Leptospira.

[0199] When a certain composition induces a Leptospiral immune response upon inoculation into an animal, the composition is determined to have an immunity inducing effect. The induction of immunity by a composition can be detected by observing in vivo or in vitro the response of the immune system in the host against the composition.

[0200] In another embodiment, the methods of the invention comprise administering to the subject a bacterium or virus comprising a nucleic acid sequence encoding a nucleic acid molecule encoding a Leptospiral VM protein, variant thereof, or fragment thereof. In another embodiment, the methods of the invention comprise administering to the subject a bacterium or virus, wherein a Leptospiral VM protein is absent. For example, in certain embodiments, administering a toxin-deficient mutant bacterium or virus, where a Leptospiral VM protein is absent, is unable to cause disease but is able to induce an adaptive immune response.

[0201] The therapeutic compounds or compositions of the invention may be administered prophylactically or therapeutically to subjects suffering from, or at risk of, or susceptible to, developing an infection, disease, or disorder associated with the antigen. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term “prevent” encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

[0202] The polypeptide or combination of polypeptides of the invention having immunological activity, or a polynucleotide or vector encoding such a polypeptide or combination of polypeptides, may optionally be combined with an adjuvant. An adjuvant refersto a compound that enhances the immune response against the polypeptide or combination of polypeptides when administered together (or successively) with the polypeptide having immunological activity. Examples of suitable adjuvants include a synthetic TLR4-agonist adjuvant, GLA-SE, cholera toxin, salmonella toxin, alum and such, but are not limited thereto. Furthermore, a vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such. Furthermore, the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants and such. The vaccine is administered systemically or locally. Vaccine administration may be performed by single administration or boosted by multiple administrations.

[0203] In one embodiment, the methods of the present invention comprise administering a composition comprising a) at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof; or (b) a nucleic acid molecule encoding at least one Leptospiral VM protein epitope, variant thereof, or fragment thereof, to a subject. In some embodiments, the fragment of the VM protein comprises the DNase domain of the VM protein. Administration of the composition can comprise, for example, intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

[0204] The actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular cell line utilized (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector employed for gene transfer to replicate in that cell line). Furthermore, the amount of vector to be added per cell will likely vary with the length and stability of the therapeutic gene inserted in the vector, as well as also the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and can be altered due to factors not inherent to the methods of the present invention (for instance, the cost associated with synthesis). One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

[0205] These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover,the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.Antibodies

[0206] In some embodiments, the invention provides compositions that bind to the VM protein epitope of the invention, or a fragment or variant thereof. In some embodiments, the composition that binds to the VM protein epitope of the invention is an antibody.

[0207] The instant invention relates to the design and development of anti-VM protein antibodies and use thereof for immunotherapy of Leptospiral infection, or Leptospirosis. Anti- VM protein antibodies can function as an immune-prophylaxis strategy for Leptospiral infection, or Leptospirosis.

[0208] The anti-VM antibody can bind a target antigen (i.e., a VM protein epitope) present in the subject. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, for example, a protein or nucleic acid, and elicit or induce an immune response to the antigen.

[0209] In one embodiment, the composition comprises at least one nucleic acid molecule encoding a synthetic antibody, or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a variable heavy chain region and a nucleotide sequence encoding a variable light chain region of an anti-VM protein antibody. In one embodiment, the invention provides a composition comprising a first nucleic acid molecule comprising a nucleotide sequence encoding a variable heavy chain region of an anti-VM protein antibody and a second nucleic acid molecule comprising a nucleotide sequence encoding a variable light chain region of an anti-VM protein antibody.

[0210] Antibodies, including anti-VM protein antibody fragments, of the present invention include, in certain embodiments, antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or formulated antibody. Further, antibodies of the present disclosure comprise antibodies having the structural and / or functional features of an anti-VM protein antibody described herein. In one embodiment, the anti-VM protein antibody binds a Leptospiral VM protein and, thereby partially or substantially alters at least one biological activity of the Leptospiral VM protein.

[0211] In one embodiment, anti-VM protein antibody of the invention specifically bind atleast one epitope specific to the VM protein and do not specifically bind to other polypeptides. The at least one epitope can comprise at least one antibody binding region that comprises at least one portion of the full-length VM protein. The term “epitope” as used herein refers to a protein determinant capable of binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0212] In some embodiments, the invention includes compositions comprising an antibody that specifically binds to a VM protein eptiope (e.g., binding portion of an antibody). In one embodiment, the anti-VM protein antibody is a polyclonal antibody. In another embodiment, the anti-VM protein antibody is a monoclonal antibody. In some embodiments, the anti-VM protein antibody is a chimeric antibody. In further embodiments, the anti-VM protein antibody is a humanized antibody.

[0213] The binding portion of an antibody comprises one or more fragments of an antibody that retain the ability to specifically bind to binding partner molecule (e.g., Leptospiral VM protein). It has been shown that the binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known tothose with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0214] An antibody that binds to a Leptospiral VM protein of the invention is an antibody that inhibits, blocks, or interferes with at least one Leptospiral VM protein activity in vitro, in situ and / or in vivo.

[0215] In one embodiment, the Leptospiral VM protein antibody binds to an epitope of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NOTO, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID N0:14, SEQ ID NO 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO 26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NOT 1, SEQ ID NO:32, SEQ ID NO 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40.

[0216] In one embodiment, the invention relates to a nucleotide sequence encoding an Leptospiral VM protein antibody or a fragment thereof. In one embodiment, the nucleotide sequence encoding an Leptospiral VM protein antibody comprises an RNA sequence encoding the Leptospiral VM protein antibody. In one embodiment, the nucleotide sequence encoding an Leptospiral VM protein antibody comprises a DNA sequence encoding the Leptospiral VM protein antibody.

[0217] The composition of the invention can treat, prevent and / or protect against a disease, disorder, or condition associated with Leptospiral infection.

[0218] The composition of the present invention can have features required of effective compositions such as being safe so that the composition does not cause illness or death; being protective against illness; and providing ease of administration, few side effects, biological stability and low cost per dose.

[0219] In some embodiments, the Leptospiral VM protein binding molecules (e.g., antibodies, etc.) of the present invention, exhibit a high capacity to detect and bind a Leptospiral VM protein in a complex mixture of salts, compounds and other polypeptides, e.g., as assessed by any one of several in vitro and in vivo assays known in the art. The skilled artisan will understand that the Leptospiral VM protein binding molecules (e.g., antibodies, etc.) describedherein as useful in the methods of diagnosis and treatment and prevention of disease, are also useful in procedures and methods of the invention that include, but are not limited to, an immunochromatography assay, an immunodot assay, a Luminex assay, an ELISA assay, an ELISPOT assay, a protein microarray assay, a Western blot assay, a mass spectrophotometry assay, a radioimmunoassay (RIA), a radioimmunodiffusion assay, a liquid chromatographytandem mass spectrometry assay, an ouchterlony immunodiffusion assay, reverse phase protein microarray, a rocket immunoelectrophoresis assay, an immunohistostaining assay, an immunoprecipitation assay, a complement fixation assay, FACS, a protein chip assay, separation and purification processes, and affinity chromatography (see also, 2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook, Gulf Professional Publishing; 1996, Diamandis and Christopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007).

[0220] In some embodiments, the Leptospiral VM protein binding molecules (e.g., antibodies, etc.) of the present invention, exhibit a high capacity to reduce or to neutralize Leptospiral VM protein activity as assessed by any one of several in vitro and in vivo assays known in the art.

[0221] In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For example, the heavy chain constant region is an IgGl heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. For example, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.Dosage and Formulation

[0222] The present invention envisions treating a disease, for example, diseases associated with a Leptospiral pathogen, in a subject by the administration of one or more of the therapeutic agents of the present invention (e.g., the VM-domain fusion constructs of the invention; toxoid vaccine, anti-VM antibodies or nucleic acid molecules encoding anti-VM antibodies).

[0223] Administration of the composition in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. In one embodiment, the cytokine composition, the antigen receptor composition, and the integration composition of the invention are administered locally to the same site. The amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art.

[0224] One or more suitable unit dosage forms having the therapeutic agent(s) of the invention, which, as discussed below, may optionally be formulated for sustained release (for example using microencapsulation, see WO 94 / 07529, and U.S. Pat. No. 4,962,091 the disclosures of which are incorporated by reference herein), can be administered by a variety of routes including parenteral, including by intravenous and intramuscular routes, as well as by direct injection into the diseased tissue. For example, the therapeutic agent may be directly injected into a tumor. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

[0225] Lipid nanoparticles (LNPs), liposomes or lipoplexes are effective drug delivery systems for biologically active compounds such as therapeutic proteins, peptides or nucleic acidbased therapeutics, which are otherwise cell impermeable. Therefore, in some embodiments, the invention relates to compositions comprising one or more lipid nanoparticles (LNPs), liposomes or lipoplexes comprising at least one VM protein of the invention, or a nucleic acid molecule encoding the same.

[0226] In certain embodiments, the therapeutic agent is combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9%by weight of the formulation. A “pharmaceutically acceptable” is a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.

[0227] Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients. The therapeutic agents of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.

[0228] The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

[0229] Thus, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-fdled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0230] It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

[0231] The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non-limiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the presentinvention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions pH 7.0-8.0.

[0232] The expression vectors, transduced cells, polynucleotides and polypeptides (active ingredients) of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the organism. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0233] In general, water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium Ethylenediaminetetraacetic acid (EDTA). In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field.

[0234] The active ingredients of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs that are described in U.S. Patent Nos. 4,082,735; 4,082,736; 4,101,536; 4,185,089; 4,235,771; and 4,406,890. Other adjuvants, which are useful, include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate and dimethyldioctadecylammonium bromide (DDA), Freund’s adjuvant, and IL-12. Other components may include a polyoxypropylenepolyoxyethylene block polymer (Pluronic®), a non-ionic surfactant, and a metabolizable oil such as squalene (U.S. Patent No. 4,606,918).

[0235] Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids,polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

[0236] Accordingly, the composition of the present invention may be delivered via various routes and to various sites in a mammal body to achieve a particular effect (see, e.g., Rosenfeld et al., 1991; Rosenfeld et al., 1991a; Jaffe et al., supra; Berkner, supra). One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. In one embodiment, the composition described above is administered to the subject by intratumoral injection. Other forms of administration that may be useful in the methods described herein include, but are not limited to, direct delivery to a desired organ, intramuscular, subcutaneous, intradermal, and other parental routes of administration.

[0237] The active ingredients of the present invention can be provided in unit dosage form wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term “unit dosage form” as used herein refers to physically discrete units suitable as unitary dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the composition in the particular host.

[0238] These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.Methods of Diagnosing

[0239] In some embodiments, the present invention includes methods for diagnosing infection of at least one pathogen in a human subject. In some embodiments, a method comprises detecting the levels of IgG and / or IgM antibody produced after infection in the subject and comparing the levels of IgG and / or IgM antibody in the subject with the levels of IgG and / or IgM antibody in a comparator control.

[0240] In some embodiments, detecting the level of IgG and / or IgM antibody in the subject comprises isolating a biological sample from the subject, mixing the biological sample from the subject with a first reagent, wherein the first reagent comprises at least one antigen and anti-human IgG antibody or anti-human IgM antibody conjugated to a label, wherein the antigen, IgG and / or IgM antibody from the sample, and the anti-human IgG antibody or anti-human IgM antibody conjugated to a label form a complex if the sample comprises IgG and / or IgM antibody that bind to the at least one antigen. In some embodiments, the at least one antigen is coated on a solid phase support. In some embodiments, the method comprises washing the complex after the mixing step. In some embodiments, the method further comprises incubating the complex with a second reagent, wherein the second reagent comprises a detection agent which produces a detectable signal when exposed to the label. In some embodiments, the method further comprises acquiring the detectable signal. In some embodiments, the method further comprises obtaining the level of the IgG or IgM antibody in the sample based upon the detectable signal. In some embodiments, the method comprises diagnosing the subject as infected when the level of IgG or IgM antibody in the subject is elevated compared with the level of IgG or IgM antibody in the comparator control.

[0241] In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection immediately following occurrence of the infection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 1-2 days post-infection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 2-3 days postinfection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 3-4 days post-infection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 4-5 days postinfection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 5-6 days post-infection. In some embodiments, the method fordiagnosing infection of at least one pathogen comprises diagnosing an infection 6-7 days postinfection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 1-2 weeks post-infection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 2-3 weeks post-infection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 3-4 weeks post-infection. In some embodiments, the method for diagnosing infection of at least one pathogen comprises diagnosing an infection 4-5 weeks post-infection.

[0242] In some embodiments the at least one pathogen comprises at least one pathogen selected from the group consisting of L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri, and L. alexanderi.

[0243] In some embodiments the biological sample from the subject plasma or serum from the subject. In some embodiments the biological sample from the subject comprises any sample from the subject which comprises antibodies.

[0244] In some embodiments the at least one antigen comprises at least one Leptospiral VM protein antigen selected from the group consisting 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, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO 27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or a fragment or variant thereof.

[0245] In some embodiments the first reagent comprises anti-human IgG antibody or anti-human IgM antibody conjugated to a label, wherein the label comprises horse radish peroxidase (HRP) or alkaline phosphatase (AP). In one embodiment, the label comprises horse radish peroxidase (HRP). In some embodiments the label comprises any chemical known to a skilled artisan to be usable to label an anti-IgG and / or anti-IgM antibody for subsequent detection as described herein.

[0246] In some embodiments the second reagent comprises a detection agent, wherein the detection agent which produces a detectable signal when exposed to the label. In some embodiments, the detection agent comprises 3 ’,5, 5’ tetramethylbenzidine (TMB), o- phenylenediamine dihydrochloride (OPD), 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), or p-Nitrophenyl Phosphate, Disodium Salt (PNPP). In one embodiment, the detection agent comprises 3, 3’, 5, 5’ tetramethylbenzidine (TMB) substrate. In some embodiments, the detection agent is correspondingly selected based upon the choice of label to which the anti-human IgG antibody or anti-human IgM antibody is conjugated. In some embodiments the detection agent comprises any chemical known to a skilled artisan to be usable to detect the label to which the anti-human IgG antibody or anti-human IgM antibody is conjugated.

[0247] In some embodiments, acquiring the detectable signal comprises measuring the detectable signal. In some embodiments, acquiring the detectable signal comprises obtaining optical density (OD) measurements. In some embodiments, acquiring the detectable signal comprises obtaining optical density (OD) measurements at 450nm.

[0248] In some embodiments the comparator control comprises plasma or serum sample from an uninfected human subject. In some embodiments the comparator control comprises a biological sample from an uninfected human subject that corresponds to the biological samples isolated from the human subject to be diagnosed.

[0249] In other various embodiments of the methods of the invention, the level of IgG and / or IgM in the human subject is determined to be increased when the level of IgG and / or IgM in the human subject is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10-20%, by at least 20-30%, by at least 30-40%, by at least 40-50%, by at least 50-60%, by at least 60-70%, by at least 70-80%, by at least 80-90%, by at least 90-100%, or by more than 100%, when compared to the comparator control.

[0250] In some embodiments, IgG and / or IgM antibody produced after infection in the subject can be measured and detected through a variety of assays, methods and detection systems known to one of skill in the art. Various methods include but are not limited to refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), fluorescence analysis, electrochemical analysis, radiochemical analysis, near-infrared spectroscopy (near-IR), infrared (IR)spectroscopy, nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyrolysis mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography, liquid chromatography, gas chromatography combined with mass spectrometry, liquid chromatography combined with mass spectrometry, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) combined with mass spectrometry, ion spray spectroscopy combined with mass spectrometry, capillary electrophoresis, colorimetry and surface plasmon resonance (such as according to systems provided by Biacore Life Sciences). See also PCT Publications WO / 2004 / 056456 and WO / 2004 / 088309. In this regard, IgG and / or IgM antibody produced after infection in the subject can be measured using the above-mentioned detection methods, or other methods known to the skilled artisan.

[0251] In some embodiments, IgG and / or IgM antibody produced after infection in the subject are first immunoprecipitated from a biological sample isolated from the subject and then detected by one or more of the aforementioned assays / methods for detection. In some embodiments, the immunoprecipitation is performed using at least one antigen. In some embodiments, the at least one antigen used to perform the immunoprecipitation is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID N0:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO 29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or a fragment or variant thereof.

[0252] In some embodiments, when the subject is diagnosed as infected, the subject is treated.Methods of Detecting

[0253] In some embodiments, the present invention includes methods for detecting at least one pathogen in a human subject. In some embodiments, the method comprises detecting the levels of IgG and / or IgM antibody produced after infection in the subject and comparing thelevels of IgG and / or IgM antibody in the subject with the levels of IgG and / or IgM antibody in a comparator control.

[0254] In some embodiments, detecting the level of IgG and / or IgM antibody in the subject comprises isolating a biological sample from the subject, mixing the biological sample from the subject with a first reagent, wherein the first reagent comprises at least one antigen and anti-human IgG antibody or anti-human IgM antibody conjugated to a label, wherein the antigen, IgG and / or IgM antibody from the sample, and the anti-human IgG antibody or anti-human IgM antibody conjugated to a label form a complex if the sample comprises IgG and / or IgM antibody that bind to the at least one antigen. In some embodiments, the at least one antigen is coated on a solid phase support. In some embodiments, the method comprises washing the complex after the mixing step. In some embodiments, the method further comprises incubating the complex with a second reagent, wherein the second reagent comprises a detection agent which produces a detectable signal when exposed to the label. In some embodiments, the method further comprises acquiring the detectable signal. In some embodiments, the method further comprises obtaining the level of the IgG or IgM antibody in the sample based upon the detectable signal. In some embodiments, at least one pathogen is detected when the level of IgG or IgM antibody in the subject is elevated compared with the level of IgG or IgM antibody in the comparator control.

[0255] In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection immediately following occurrence of the infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 1-2 days post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 2-3 days post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 3-4 days post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 4-5 days post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 5-6 days post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 6-7 days post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 1-2 weeks post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 2-3 weeks post-infection. In someembodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 3-4 weeks post-infection. In some embodiments, the method for detecting infection of at least one pathogen comprises detecting an infection 4-5 weeks post-infection.

[0256] In some embodiments the at least one pathogen comprises at least one pathogen selected from the group consisting of L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri, and L. alexanderi.

[0257] In some embodiments the biological sample from the subject plasma or serum from the subject. In some embodiments the biological sample from the subject comprises any sample from the subject which comprises antibodies.

[0258] In some embodiments the at least one antigen comprises at least one Leptospiral VM protein antigen selected from the group consisting 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, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO 27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or a fragment or variant thereof.

[0259] In some embodiments the first reagent comprises anti-human IgG antibody or anti-human IgM antibody conjugated to a label, wherein the label comprises horse radish peroxidase (HRP) or alkaline phosphatase (AP). In one embodiment, the label comprises horse radish peroxidase (HRP). In some embodiments the label comprises any chemical known to a skilled artisan to be usable to label an anti-IgG and / or anti-IgM antibody for subsequent detection as described herein.

[0260] In some embodiments the second reagent comprises a detection agent, wherein the detection agent which produces a detectable signal when exposed to the label. In some embodiments, the detection agent comprises 3 ’,5, 5’ tetramethylbenzidine (TMB), o- phenylenediamine dihydrochloride (OPD), 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), or p-Nitrophenyl Phosphate, Disodium Salt (PNPP). In one embodiment, the detection agent comprises 3, 3’, 5, 5’ tetramethylbenzidine (TMB) substrate. Insome embodiments, the detection agent is correspondingly selected based upon the choice of label to which the anti-human IgG antibody or anti-human IgM antibody is conjugated. In some embodiments the detection agent comprises any chemical known to a skilled artisan to be usable to detect the label to which the anti-human IgG antibody or anti-human IgM antibody is conjugated.

[0261] In some embodiments, acquiring the detectable signal comprises measuring the detectable signal. In some embodiments, acquiring the detectable signal comprises obtaining optical density (OD) measurements. In some embodiments, acquiring the detectable signal comprises obtaining optical density (OD) measurements at 450nm.

[0262] In some embodiments the comparator control comprises plasma or serum sample from an uninfected human subject. In some embodiments the comparator control comprises a biological sample from an uninfected human subject that corresponds to the biological samples isolated from the human subject in whom an infection is to be detected.

[0263] In other various embodiments of the methods of the invention, the level of IgG and / or IgM in the human subject is determined to be increased when the level of IgG and / or IgM in the human subject is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10-20%, by at least 20-30%, by at least 30-40%, by at least 40-50%, by at least 50-60%, by at least 60-70%, by at least 70-80%, by at least 80-90%, by at least 90-100%, or by more than 100%, when compared to the comparator control.

[0264] In some embodiments, IgG and / or IgM antibody produced after infection in the subject can be measured and detected through a variety of assays, methods and detection systems known to one of skill in the art. Various methods include but are not limited to refractive index spectroscopy (RI), ultra-violet spectroscopy (UV), fluorescence analysis, electrochemical analysis, radiochemical analysis, near-infrared spectroscopy (near-IR), infrared (IR) spectroscopy, nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, pyrolysis mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography, liquid chromatography, gas chromatography combined with mass spectrometry, liquid chromatography combined with mass spectrometry, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) combined with mass spectrometry, ion spray spectroscopy combined with mass spectrometry, capillary electrophoresis, colorimetry andsurface plasmon resonance (such as according to systems provided by Biacore Life Sciences). See also PCT Publications WO / 2004 / 056456 and WO / 2004 / 088309. In this regard, IgG and / or IgM antibody produced after infection in the subject can be measured using the above-mentioned detection methods, or other methods known to the skilled artisan.

[0265] In some embodiments, IgG and / or IgM antibody produced after infection in the subject are first immunoprecipitated from a biological sample isolated from the subject and then detected by one or more of the aforementioned assays / methods for detection. In some embodiments, the immunoprecipitation is performed using at least one antigen. In some embodiments, the at least one antigen used to perform the immunoprecipitation is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NON 1, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO 36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or a fragment or variant thereof.

[0266] In some embodiments, when at least one pathogen is detected in the human subject, the subject is treated.Methods of Treatment

[0267] In one embodiment, the invention includes methods of treating an immune response in a subject in need thereof comprising administering a Leptospiral VM protein, a toxoid vaccine, an anti-VM antibody or a nucleic acid molecule encoding an anti-VM antibody of the invention.

[0268] In one embodiment, the invention includes methods of inducing an immune response in a subject in need thereof comprising administering a Leptospiral VM protein, a toxoid vaccine, an anti-VM antibody or a nucleic acid molecule encoding an anti-VM antibody of the invention.

[0269] In one embodiment, a Leptospiral VM protein or peptide or a vector comprising anucleotide sequence encoding for a Leptospiral VM protein or peptide, serves a toxoid vaccine. Also provided herein is a method of treating, protecting against, and / or preventing disease in a subject in need thereof by administering the toxoid vaccine to the subject. Administration of the vaccine to the subject can induce or elicit an immune response in the subject. The induced immune response can be used to treat, prevent, and / or protect against disease, for example, an infectious disease, including but not limited to pathologies relating to Leptospira infection.

[0270] In one embodiment, a fusion protein comprising a Leptospiral VM protein or peptide fused to an antigenic peptide or a vector comprising a nucleotide sequence encoding for fusion protein comprising a Leptospiral VM protein or peptide fused to an antigenic peptide, serves as a therapeutic agent for the treatment of a disease or disorder associated with the antigenic peptide.

[0271] In some embodiments, the antigenic peptide is a tumor associated peptide. Therefore, in some embodiments, the induced immune response can be used to treat, prevent, and / or protect against cancer. The following are non-limiting examples of cancers that can be treated by the disclosed methods and compositions: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, central nervous system atypical teratoid / rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cerebral astrocytoma / malignant glioma, cervical cancer, childhood visual pathway tumor, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family of tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoides, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular (eye) cancer, intraocular melanoma, islet cell tumors, Kaposi sarcoma, kidney(renal cell) cancer, Langerhans cell cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (nonmelanoma), skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, supratentorial primitive neuroectodermal tumors and pineoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0272] In one embodiment, the methods of the invention include administering an antigenic protein, peptide to a subject, wherein the antigenic protein, peptide promotes the generation of an immune response against the antigen. In one embodiment, the methods of the invention include administering a nucleic acid molecule to a subject wherein the nucleic acidmolecule comprises an expression construct for expression of at least one antigenic protein or peptide, wherein the antigenic protein or peptide promotes the generation of an immune response against the encoded antigenic protein or peptide. In one embodiment, the methods of the invention include administering an antibody to a subject wherein the antibody targets a disease- associated antigen. In one embodiment, the methods of the invention include administering at least one nucleic acid molecule encoding an antibody, or fragment thereof, to a subject wherein the encoded antibody targets a disease-associated antigen.

[0273] In some embodiments, an induced immune response can include an induced humoral immune response and / or an induced cellular immune response. The humoral immune response can be induced by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3 -fold to about 10-fold. The induced humoral immune response can include IgG antibodies and / or neutralizing antibodies. The induced cellular immune response can include a CD8+ T cell response, which is induced by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold.

[0274] The vaccine dose can be between 1 pg to 10 mg active component / kg body weight / time, and can be 20 pg to 10 mg component / kg body weight / time. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0275] In one embodiment, the vaccine or antibody of the invention can be administered alone. In one embodiment, the vaccine or antibody of the invention can be administered in combination with another treatment for a disease or disorder.

[0276] In one embodiment the vaccine or antibody of the invention is administered in combination with an additional vaccine composition as a prime or a boost vaccine. In one embodiment, a subject who has been immunized with a vaccine (as a priming vaccine) is then administered a vaccine of the invention as a boosting vaccine to increase the immune response.

[0277] In one embodiment a vector of the invention may expresses at least two antigenic polypeptides, wherein at least one antigenic polypeptide is a Leptospiral VM protein of the invention.

[0278] In one embodiment, the invention includes methods of treating Leptospiral infection in a subject in need thereof. In some embodiments, the treatment comprisesadministering at least one therapeutic drug to the subject. In some embodiments, the at least one therapeutic drug comprises an antibiotic. In one embodiment, the antibiotic comprises doxycycline, azithromycin, amoxicillin, benzylpenicillin, tetracycline, ceftriaxone, cefotaxime, ampicillin, oxytetracycline, penicillin G, fluoroquinolone such as ciprofloxacin or levofloxacin, or erythromycin.

[0279] In some embodiments, the at least one therapeutic drug comprises an antiinflammatory drug. In one embodiment, the anti-inflammatory drug comprises a corticosteroid, wherein the corticosteroid comprises cortisone, hydrocortisone, or prednisone.

[0280] In some embodiments, the treatment comprises mechanical ventilation, plasmapheresis, intravenous hydration, administration of potassium supplements, hemodialysis or peritoneal dialysis, or tracheal intubation.

[0281] In some embodiments, the invention comprises methods of administering a Leptospiral VM protein, a toxoid vaccine, an anti-VM antibody or a nucleic acid molecule encoding an anti-VM antibody of the present invention to a patient in need thereof.Administration

[0282] The compositions of the invention can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The subject can be a mammal, such as a human, a horse, a cow, a pig, a sheep, a cat, a dog, a rat, or a mouse.

[0283] The composition can be administered prophylactically or therapeutically. In prophylactic administration, the compositions can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the compositions are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the treatment regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the subject, and the judgment of the prescribing physician.

[0284] The composition can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of all of which are incorporated herein by reference in their entirety. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

[0285] The composition can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular, intratumoral or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the composition in particular, the composition can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The composition can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the composition can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).

[0286] In one embodiment, the proteins, nucleic acid molecules or antibodies of the present invention can be administered to cells of a mammal including a human.

[0287] In some embodiments, the proteins, nucleic acid molecules or antibodies of the present invention can be administered as an injection (subcutaneous, intradermal, or intramuscular injection) to cells of a mammal including a human. The injection can be prepared by a standard method. For example, a culture supernatant containing the virus vector is concentrated, if necessary, and suspended together with an appropriate carrier or excipient in a buffer solution such as PBS or saline. Then, the suspension can be sterilized by fdtration through a fdter or the like according to the need and subsequently charged into an aseptic container to prepare the injection. The injection may be supplemented with a stabilizer, a preservative, and the like, according to the need. The expression vector thus obtained can be administered as the injection to a subject.

[0288] In some embodiments, the proteins, nucleic acid molecules or antibodies can beformulated for administration by way of intradermal (ID) vaccination (e.g., ID injection by the Mantoux technique, use of a hollow microneedle, using a gene gun, using scarification or by other methods for ID delivery). The formulation for ID vaccination can be prepared by a standard method. For example, a culture supernatant containing the proteins, nucleic acid molecules or antibodies is concentrated, if necessary, and suspended together with an appropriate carrier or excipient in a buffer solution such as PBS, a nucleic acid molecule-stabilizing solution, or saline. Then, the suspension can be sterilized by filtration through a filter or the like according to the need and subsequently charged into an aseptic container to prepare the formulation for ID vaccination. The formulation for ID vaccination may be supplemented with a stabilizer, a preservative, and the like, according to the need. The composition thus obtained can be administered intradermally to a subject.

[0289] The invention also provides a method for generating an immune response in an animal comprising administering any of the proteins, peptides, nucleic acid molecules or compositions described above to an animal in an amount effective to stimulate the immune response. In one embodiment, the immune response comprises one or more of the production of memory CD8+ T cells specific for an expressed target antigen, the production of memory CD4+ T cells specific for an expressed target antigen, and the production of antibodies specific for an expressed target antigen. In one embodiment, at least some of the antibodies are neutralizing antibodies.

[0290] The invention further provides pharmaceutical compositions (e.g., vaccines) comprising the Leptospiral VM-proteins of the invention. In one embodiment, the composition comprises a pharmaceutically acceptable diluent, carrier, or excipient carrier. The composition may also contain an aqueous medium or a water-containing suspension, to increase the activity and / or the shelf life of the composition. The medium / suspension can include salt, glucose, pH buffers, stabilizers, emulsifiers, and preservatives.

[0291] In some embodiments, the composition further comprises an adjuvant, e.g., including, but not limited to: muramyl dipeptide; aluminum hydroxide; saponin; polyanions; anamphipatic substances; bacillus Calmette-Guerin (BCG); endotoxin lipopolysaccharides; keyhole limpet hemocyanin (GKLH); and cytoxan.

[0292] In one aspect, the invention provides a method of administering a therapeutically effective compositions according to the invention. The desired therapeutic effect comprises oneor more of: reducing or eliminating bacterial load, increasing numbers of CD4+ and / or CD8+ T cells or antibodies which recognize the encoded antigen; increasing overall levels of CD4+ T cells; increasing levels of neutralizing antibodies which recognize the antigen; decreasing the number of or severity of symptoms of a disease; decreasing the expression of a cancer specific marker; decreasing size or rate of growth of a tumor; preventing metastasis of a tumor; preventing infection by a pathogenic organism; and the like. The therapeutic effect may be monitored by evaluating biological markers and / or abnormal physiological responses. Generally, an effective dose of a composition according to the invention comprises a titer that can modulate an immune response against the encoded antigen such that memory T cells are generated which are specific for the encoded antigen.

[0293] Both the dose and the administration means can be determined based on the condition of the subject (e.g., age, weight, general health), risk for developing a disease, or the state of progression of a disease.

[0294] In one embodiment, an effective amount of recombinant virus ranges from about 10 pl to about 25 pl of saline solution containing concentrations, of from about 1 x 1010 to 1 x 1011 plaque forming units (pfa) virus / ml.

[0295] In one embodiment of the invention, a priming immunization is performed, followed, optionally, by a booster immunization at about 3-4 weeks after the priming immunization. However, subsequent immunizations need not be provided until at least about 4 months, about 6 months, about 8 months, about 12 months, about 10 months, about 16 months, about 18 months, or about 24 months after the priming boost. In one aspect, the composition is a prophylactic vaccine, administered to a subject who has not tested positive for the vaccine antigen, e.g., such as to an individual who is at risk of exposure to Leptospira bacteria. In another aspect, the vaccine is administered therapeutically, to a person who is seropositive for the vaccine antigen (although not necessarily displaying symptoms) (i.e., such as to a Leptospria positive individual).Kits

[0296] The invention also includes a kit comprising one or more of the compositions described herein. For example, in one embodiment, the kit comprises a Leptospiral VM protein, a variant or fragment thereof, a nucleic acid molecule encoding a Leptospiral VM protein, avariant or fragment thereof or a fusion construction comprising a Leptospiral VM domain. In one embodiment, the kit comprises an anti-Leptospiral VM antibody or nucleic acid molecule encoding the same. In one embodiment, the kit comprises instructional material which describes the use of the composition. For instance, in some embodiments, the instructional material describes administering the composition(s), to a subject as a therapeutic treatment or a nontreatment use as described elsewhere herein. In an embodiment, the kit further comprises one or more additional reagents for use in an assay, for example in an immunoassay of the invention.EXPERIMENTAL EXAMPLES

[0297] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.Example 1 : Discovery of a Novel Leptospirosis-Secreted Exotoxin that Mediates Pathogenesis: Clinical Translation to a Pan-Leptospirosis Vaccine

[0298] To demonstrate that anti-VM protein immunity is mediated by antibody, given the premise that Leptospira, an extracellular pathogen, causes systemic disease via circulation of secreted VM protein exotoxins, polyclonal and monoclonal antibodies were tested for their ability to protect against lethal challenge infection.

[0299] Leptospira-secreted VM exotoxins mediate cellular pathogenesis of leptospirosis. Vaccination of VM proteins protected mice from lethal infection and reduced bacterial load in key target organs (liver, kidney) by >4 orders of magnitude. Ongoing work is examining the potential for anti-VM protein monoclonal antibodies to neutralize VM protein cytotoxicity and confer protection against challenge infection. The defined epitope and mAbs — protection study will underlie the development of human therapeutic and preventive measures to ameliorate the global impact of this devastating disease. These new data are compelling to support placing leptospirosis on the WHO’s Neglected Tropical Disease list to accelerate the development of new leptospirosis vaccines and therapeutics.The materials and methods are now described:

[0300] To generate polyclonal anti-serum, 4 rabbits were vaccinated with two VM proteins (LA1402, LA0591) and hyperimmune serum harvested.

[0301] Hybridomas were tested on recombinant and native VM proteins by ELISA. Affinities, avidities, and epitope binning were evaluated by biolayer interferometry (Octet). Epitope mapping was performed using synthetic overlapping peptides.The results are now described:

[0302] The anti-LA0591 mAbs 5F8 (KD1.41E-09), 6A5 (KD<1.0E-12) and 5G10 (KD<1.0E-12) reacted with homologous antigens and cross-reacted with other recombinant and native VM proteins. Isotyping and pairing analysis revealed that 6A5 (IgG2b) pair with 5F8 (IgGl) and 5G10 (IgGl), however 5F8 pair with 5G10 and 6A5. The study reinforces by demonstration of epitope mapping in which, 5F8 and 5G10 recognizes linear (95-106aa and 99- 1 lOaa) and 6A5 recognizes confirmational (287- 299aa) epitopes. These sequences are highly >85% conserved in pathogenic strains. Low-passage L. interrogans serovar Copenhageni. Strain FioCruz Ll-130 reacted with 5F8 and showed upregulation of multiple VM proteins. Capture- ELISA detected VM antigens in serum of infected hamsters. Insights into the anti-VM antibody mediated neutralization of VM activity will be a breakthrough to design a novel vaccine candidate. The epitopes that have been mapped can be used to support vaccine development.ELISA-based detection of VM antigens in the serum of hamster at day 4 of infection with pathogenic Leptospira.

[0303] A challenge study was performed with four groups of hamsters (n-10), and three groups were challenged with serovar Copenhageni stain Ll-130 at IxlO4, IxlO6, and IxlO8bacterial cells respectively. PBS group served as a control. The blood was collected on day 4 and serum was separated by centrifugation at 2,000g for 10 minutes, at 4°C. Figure 11A depicts a standard graph generated by coating the plate with (1000 ng — 1.9 ng / mL) rLA0591 or RBLs (control) VM antigens. The signal was detected using anti-LA0591 (5F8 mAbs; 1 :2000 dilution) monoclonal antibodies at 450 nm. The data represents the log transform of the concentration of antigens. The Y-intercept and slope were plotted, P<0001 for LA0591 and non-significant forRBLs. Figure 1 IB depicts the subtracted background using the sum of mean (OD-O.179) value form the control PBS group. The log transforms concentration of VM protein in severely infected hamsters (n-5, each group) was interpolated using the standard curve. The circle bar shows the intersection of OD and the concentration of VM proteins in the serum of the infected hamsters. The lime color show hamster challenged with IxlO4bacterial cells, and pink and purple shows challenge with IxlO6and IxlO8bacterial cells. The simple linear regression model was generated, and the equation was plotted. R2 shows the goodness fit of the model. Figure 11C depicts the concentration of VM protein detected in the serum of each hamster was plotted in relation to challenge dose. The error bar shows the standard deviation in the experiment.Full length LA_0591 VM protein (SEQ ID NO: 1)MGRWIVLLLVLLISLGVGYSYGVNPDQYCPASKKENHNIRIKRTLPPDFQLTEEWLRRL YDIATSASLTEGQIHGICGVCLLQTFQMLAELQEYNSHGPLQGGGYFFNTAPDTDPFDSF RQRYPELDTMLTDAATAYGPAYNTTRLLTLVSAMTMMPQYEWTPSREFTTRSDMHSHI RSLIASPPGSIWLGLIQRRESDETLRWHALPILRTSQGLIVIQTRVSTMSFELYRLYLTPSTS IVQIINDYLEEADRTLTVLVTIQLEQAYQNLFDFMVSNMNCTGEGENRRGSGGYPTSAT VNQCSGGRCALPNWLA_0591 Epitopes:Example 2: Clinical assessment of leptospirosis illness by early detection of Virulence- Modifying biomarker or early serological diagnosis of leptospirosis using Virulence-Modifying protein as antigens.

[0304] Conservative estimations of leptospirosis impact over a million individuals, resulting in approximately 60,000 fatalities worldwide, with a 5-20% case fatality rate, comparable to other tropical diseases like dengue, cholera, typhoid, and leishmaniasis (Costa et al., PLoS Negl Trop Dis 2015, 9(9): e0003898; Torgerson et al., PLoS Negl Trop Dis 2015, 9(10): e0004122; Rudd et al., Lancet 2020, 395(10219): 200-11). The disease burden is estimated to have $29.3 billion annual global productivity loss contributing significantly to poverty in already deprived communities (Agampodi et al., PLoS Negl Trop Dis 2023, 17(8): eOOl 1291). In a recent field study conducted over two and a half years, 25 strains of leptospirosis were isolated in 1,192 patients across 15 districts in Sri Lanka (Jayasundara et al., PLoS Negl Trop Dis 2021, 15(5): e0009471). Genome sequencing revealed 25 new core genome sequence types in 15 clonal groups, including 12 new clonal groups. These isolated belonged to four pathogenic Leptospira species identified as L. interrogans, L. borgpetersenii, L. weilii, and L. kirschneri, dominated by six serogroups Autumnalis, Pyrogenes, Icterohaemorrhagiae, Celledoni, Grippotyphosa, and Bataviae (Jayasundara et al., PLoS Negl Trop Dis 2021, 15(5): e0009471). L. interrogans infections were associated with acute kidney injury and cardiovascular involvement, while both L. interrogans and L. borgpetersenii infections were linked to thrombocytopenia and liver impairment (Jayasundara et al., PLoS Negl Trop Dis 2021, 15(5): e0009471). Three L. borgpetersenii isolates were found in male patients from the dry zone, indicating exposure during outdoor activities common in rice paddy farming areas with cattle and buffalo. These findings provide insights into strain-level factors, environmental responsiveness, and species-specific host interactions.

[0305] Despite identifying potential virulence factors, the clinical pathogenesis of leptospirosis remains poorly understood. For over a century, there has been speculation about the presence of a leptospiral toxin responsible for the clinical progression of the disease (Sun et al., Biomed J 2020; 43(1): 24-31; Guerra et al., J Am Vet Med Assoc 2009; 234(4): 472-8, 30; Bharti et al., Lancet Infect Dis 2003; 3: 757-71; Picardeau et al., Nat Rev Microbiol 2017; 15(5): 297-307; Noguchi et al., J Exp Med 1917; 25(5): 755-63; Noguchi et al., J Exp Med 1919; 30(2): 87-93; Noguchi et al., J Exp Med 1919; 30(1): 13-29; Inada et al., The Journal of Experimental Medicine 1915; XXIII: 377-402). The recent discovery of the PF07598 gene family, unique to pathogenic Leptospira group I, encodes virulence modifying (VM) proteins that likely play a crucial role in the disease's clinical progression, addressing a long-standing knowledge gap(Fouts et al., PLoS Negl Trop Dis 2016; 10(2): e0004403; Lehmann et al., PLoS Negl Trop Dis 2013; 7(10): e2468). These VM proteins, comprising 12 / 13 conserved paralogs in L. interrogans serovars, are massively transcriptionally upregulated in vivo and are secreted exotoxins (Lehmann et al., PLoS Negl Trop Dis 2013; 7(10): e2468). They are novel R-type lectins containing N-terminal ricin B chain-like lectin domains (RBL1 and RBL2) and C-terminal toxin domain (Chaurasia et al., Frontiers in Microbiology 2022; 12: 859680; Chaurasia et al., Front Mol Biosci 2022; 9: 1092197). LA0591, a variant lacking RBLs, may function differently in pathogenesis, possibly originating intracellularly (Chaurasia et al., Front Mol Biosci 2022; 9: 1092197). Furthermore, vaccination with two VM proteins - LA1400 (LIC12340 orthologs in Copenhageni) and LA0591 (LIC12985) - has shown efficacy in protecting mice from death and reducing bacterial load in key target organs, liver, and kidney (Chaurasia et al., Front Cell Infect Microbiol 2022; 12: 926994). Given the observations that VM proteins mediate the pathogenesis of leptospirosis, in the current study, the hypothesis that leptospiral anti-VM proteins antibodies could serve as a potential diagnostic maker in the early screening of leptospirosis was tested. The current study evaluated the potential of recombinant VM proteins to detect anti-VM PROTEIN leptospiral antibodies in patients’ sera collected in Sri Lanka (Jayasundara et al., PLoS Negl Trop Dis 2021, 15(5): e0009471). The efficacy of VM proteins was assessed by comparing the diagnostic potential of full-length LA3490 (one of the highly upregulated in vivo and cytotoxic to HeLa cells (Lehmann et al., PLoS Negl Trop Dis 2013; 7(10): e2468; Chaurasia et al., Front Mol Biosci 2022; 9: 1092197)) (SEQ ID NO: 1), N-terminal encoding ricin-like lectin domain (RBLs) (SEQ ID NO:2), and C-terminal encoding natural variant LA0591 (SEQ ID NO:3). This study has developed VM proteins-based leptospiral prognostic and diagnosis markers to address the under-reporting of this neglected disease and to enable early intervention to prevent disease progression.

[0306] The recently discovered PF07598 gene family encoding Virulence-Modifying exotoxins, especially full-length LA3490 (SEQ ID NO:41), its truncated N-terminal ricin binding lectin domain (RBLs) (SEQ ID NO:42), and LA0591 (SEQ ID NO: 1), natural variant encodes only the C-terminal domain, were evaluated for their potential to detect anti-VM proteins specific-IgG-antibodies in early infection. The study was conducted on Microscopic Agglutination Test (MAT) and qPCR-screened serologically defined leptospirosis / febrile patients’ serum, which was collected in Sri Lanka, a hot spot of leptospirosis.

[0307] Among 222 patient samples and 41 healthy controls, 26.6% were qPCR-positive and 16.0% were probable cases. MAT confirmed 21.7% positivity, with 19.0% reactive (titer < 400) and 41.4% non-reactive. LA0591 (SEQ ID NO: 1) antigens in IgG-specific ELISA effectively distinguished positive cases (AUC-0.947) from non-reactive / reactive ones from day 2 onset of symptom. Similarly, LA3490 (SEQ ID NO:41) showed good discrimination (AUC- 0.930) in identifying samples with nonreactive results from the rest, but not RBLs (SEQ ID NO:42).

[0308] The study was reinforced by isolating pathogenic strains of Leptospira from four patients. The substantial in vivo expression of VM proteins and detection of anti-VM proteins antibodies in early infection suggest its potential as a reliable diagnostic marker for early diagnosis of leptospirosis.The materials and methods are now described:Patient samples

[0309] A subset of samples that were systematically collected as part of a comprehensive investigation into the leptospirosis disease burden in Sri Lanka was used (Jayasundara et al., PLoS Negl Trop Dis 2021, 15(5): e0009471). These samples were derived from a prospective fever surveillance study that implemented consecutive sampling to minimize selection bias. The study enrolled patients presenting with acute febrile illness lasting less than 14 days, who lacked a definitive diagnosis at the time of hospital admission. For the analysis, only the acute-stage samples were employed. The diagnosis of leptospirosis 95 was determined using validated quantitative PCR (qPCR) techniques, employing both lipl32 and 16s rrs primer pairs, and a Microscopic Agglutination Test (MAT) with a wide-ranging panel of serovars (Niloofa et al., PLoS One 2015; 10(6): eO 129236) using acute as well as convalescent sera.Cloning

[0310] VM proteins were produced using the protocol previously described (Chaurasia et al., Frontiers in Microbiology 2022; 12: 859680). Briefly, E. coli codon optimized VM genes, specifically LA3490 (Uniprot ID: Q8F0K3) (SEQ ID NO:41), LA0591 (Q8F8G6) (SEQ ID NO:1), and truncated N-terminal ricin binding domain, RBLs (SEQ ID NO:42) were synthetically synthesized, and linked to mCherry (X5DSL3) via a glycine-serine hinge (G4S)3 then cloned into pET32b (+) (Gene Universal Inc., USA) between enterokinase cleavage sites,'llfacilitating the easy removal of the mCherry fluorescent tag. Dual histidine (His6) tags were introduced at the N and C-terminals to enable nickel-NTA purification. To ensure accuracy, the gene sequences and their orientations within the constructs were confirmed through restriction digestion and sequencing before utilization (Chaurasia et al., Protein J 2023; 42(6): 792-801). It is worth mentioning that LA0591 was produced without mCherry tag. Earlier studies showed that mCherry tag does not interfere with the functional studies (Chaurasia et al., Frontiers in Microbiology 2022; 12: 859680; Chaurasia et al., Protein J 2023; 42(6): 792-801).Expression and purification of soluble VM proteins

[0311] Cysteine-rich VM proteins LA3490 (SEQ ID NO:41), LA0591 (SEQ ID NO: 1), or RBLs (SEQ ID NO:42) were expressed in Shuffle®T7 competent E. coli cells (New England Biolabs, USA), leveraging their cytoplasmic disulfide bond-forming capabilities for proper protein folding (Chaurasia et al., Frontiers in Microbiology 2022; 12: 859680; Chaurasia et al., Protein J 2023; 42(6): 792-801). Expression and purification were performed by following the previous published protocol (Chaurasia et al., Frontiers in Microbiology 2022; 12: 859680; Chaurasia et al., Protein J 2023; 42(6): 792-801). Transformants were cultured in Luria-Bertani (LB) medium with 100 pg / mL ampicillin. Upon reaching an OD of 0.6, expression was induced at 16°C and 250 rpm for 24 h with 1 mM isopropyl-P-D-thiogalactoside (IPTG; Sigma- Aldrich, USA). Post-induction, cells were pelleted, lysed in CelLytic™ B (Sigma-Aldrich, USA) with benzonase nuclease (Sigma-Aldrich, USA), lysozyme, protease inhibitor cocktail (Roche, USA), and 1 mM PMSF, and centrifuged 4°C and 18,514 g for 10 min.

[0312] Supernatants and pellets were analyzed on 4-12% bis-tris SDS-PAGE, and protein concentrations were determined using a BCA assay (Thermo Fisher Scientific, USA). Recombinant proteins were purified using a 1 mL pre-packed Ni-Sepharose AKTA Hi-TRAP column (GE Healthcare, USA) equilibrated with a buffer containing 100 mM NaH2PO4, 10 mM Tris-HCl, and 25 mM imidazole (pH 8.0). The bound fusion protein was eluted with 500mM imidazole (pH 8.0), pooled, concentrated using a 10 kDa Amicon® Ultra centrifugal filter, and subjected to a high-capacity endotoxin-removal spin column (Thermo Fisher Scientific, USA) for lipopolysaccharide elimination. The recombinant purified protein was dialyzed overnight in IX PBS (pH 7.4) with gentle stirring (350 rpm) at 4°C using a 10 kDa cutoff Slide-A-Lyzer (Thermo Scientific™, USA) and further desalted using 7 kDa Zeba™ desalting spin column (Thermo Fisher Scientific, USA).Protein Analysis and Western blot

[0313] SDS-PAGE analyzed purified proteins according to the method of Laemmli (Laemmli et al., Nature 1970; 227(5259): 680-5), then proteins were transferred to nitrocellulose membrane and blocked for 2 h with 5% non-fat dry milk dissolved in IX TBST buffer (AmericanBio, USA). The membrane with LA3490 (SEQ ID NO:41) and LA0591 (SEQ ID NO: 1) was probed with mouse anti-LA0591 monoclonal antibodies (1 :2,000 dilution) at 4°C overnight on the rocker. It is noteworthy that monoclonal antibodies (5G10) targeting the C- terminal domain of VM proteins are generated against the natural variant LA0591 (SEQ ID NO: 1), which lacks RBLs.

[0314] Therefore, 5G10 mAbs exhibit cross-reactivity with the entire protein but not with RBLs alone. The membrane with RBLs (SEQ ID NO:42) was probed with a laboratory source, rabbit polyclonal antibodies (1:2,000 dilution) generated against full-length VM PROTEIN (LA1402) (SEQ ID NO: 44). After three washes, membranes were incubated for 2.5 h with alkaline phosphatase-conjugated goat anti-mouse (for monoclonal) and anti-rabbit (for polyclonal) IgG (H+L) (dilution 1 :5000, KPL, USA). Blots were developed in 5-bromo-4- chl oro-3 -indolyl phosphate and nitroblue tetrazolium 143 solution (BCIP / NBT; KPL, USA).Demonstration of anti-VM proteins antibodies in patient’s serum by Indirect-ELISA

[0315] Serologically defined patient serums were screened for anti-VM proteins antibodies. ELISA was performed following the previously published protocol (Chaurasia et al., Front Cell Infect Microbiol 2022; 12: 926994). Briefly, a 96-well microtiter ELISA plate (Corning, USA) was coated with 100 ng / wells of each recombinant VM proteins (LA3490 (SEQ ID NO:41), LA0591 (SEQ ID NO: 1), and RBL1+RBL2; RBLs (SEQ ID NO:42), in equimolar ratio), in 100 pL of bicarbonate / carbonate coating buffer. Subsequently, 100 pL of the patient's serum sample was diluted at 1 :50 and added into the wells followed by the addition of 1 : 5000 dilution of goat anti-human IgG (Fc specific) HRP-conjugate (KPL, USA), and further procedures were carried out as mentioned earlier. The reaction was stopped with 2 M H2SO4 and absorbance was read at 450 nm using a SpectraMax® M2e Microplate Reader (Molecular Devices, USA). Antigen and antibody blanks were served as a control.The results are now described:

[0316] The study sample comprised 222 serum samples from individuals suspected of having leptospirosis or presenting with fever, along with 41 healthy controls. Among the 222 patient samples, 70 (26.6%) confirmed positive, and 42 (16.0%) were categorized as probable cases of leptospirosis, showing a positive result in at least one of two or more replicates on the qPCR plate. An additional 93 (35.4%) were categorized as Leptospira antigen ‘not detected’ in qPCR. For 17 cases, qPCR results were not available. As for the MAT results, 57 (21.7%) were positive, 50 (19.0%) showed a reactive response (titer < 400), and 109 cases (41.4%) were non- reactive. MAT results were not available for 6 samples. These samples underwent screening using AKTA-purified soluble VM proteins encoding full length LA3490 (SEQ ID NO:41), the N-terminal ricin binding domain (RBLs) (SEQ ID NO:42), and the C-terminal toxin domain (SEQ ID NO: 1) individually (Figure 13), aiming to assess their ability to detect anti-VM PROTEIN antibodies in acute leptospirosis. Previously, the secondary structure of VM proteins (helix and P-sheet) was evaluated using CD-spectroscopy and assessed their stability through molecular dynamics simulation (Chaurasia et al., Front Mol Biosci 2022; 9: 1092197; Chaurasia et al., Protein J 2023; 42(6): 792-801). VM proteins-based IgG-specific ELISA suggested that anti-LA0591 and anti-LA3490 antibodies were significantly higher in MAT-confirmed / reactive and qPCR-confirmed / probable cases compared to healthy controls. Remarkably, this pattern was seen in over 97.5% of cases, compared to healthy control. The results for the N-terminal ricin binding domain (RBLs) (SEQ ID NO:42) followed a similar pattern, albeit with some overlap and notably higher OD values for suspected cases compared to confirmed or probable cases. When comparing the collective final diagnosis of confirmed and probable cases versus healthy controls, it is evident that the OD values of LA0591 (SEQ ID NO: 1) and LA3490 (SEQ ID NO:41) are distinctly different between confirmed and suspected cases (Figure 14). ANOVA, after excluding test-negative cases, showed significant differences in OD values between healthy subjects and both confirmed and probable / reactive cases (Table 1). Follow-up post hoc analysis using Tukey’s HSD test identified distinct groupings within the data. Specifically, LA0591 (SEQ ID NO:3) levels were distinct enough to categorize each group into separate homogeneous subsets. In contrast, the other two markers distinguished healthy controls from confirmed / probable cases, forming two homogeneous subsets. Antibodies to the VM PROTEIN were tested only in acute-phase samples. Of the 222 samples tested, 118 were confirmed to have leptospirosis using the MAT with paired serum samples. Among these 118 patients, 33 had apositive MAT titer (>400) in the acute sample, while an additional 24 were confirmed retrospectively using convalescent serum samples (>400, seroconversion, or a fourfold rise). None of these patients were confirmed as laboratory confirmed leptospirosis cases during their hospital stay. Figure 15 shows the OD values of anti-VM PROTEIN antibodies during the acute stage, grouped by paired sample MAT results. However, RBLs (SEQ ID NO:2) detect anti-RBLs antibodies in reactive and non-reactive cases but are unable to distinguish confirmed / reactive and confirmed / probable cases compared to healthy controls (Figure 15, Table 1).Table 1. Descriptive and ANOVA table for OD values by leptospirosis confirmatory test resultsAbbreviations:

[0317] These findings indicate that individuals with diagnostic MAT titers during the acute stage had notably higher mean OD values compared to healthy subjects. Moreover, individuals who tested negative during the acute stage but had diagnostic titers in convalescent samples (indicating they were diagnosed later) also exhibited significantly high levels of VM protein antibodies in the early stages of the illness (Table 2). The VM protein LA0591 (SEQ ID NO:1) differentiated positive from non-reactive / reactive results, while LA3490 (SEQ ID NO:41) differentiated samples with non-reactive results from the rest, but not RBLs (SEQ ID NO:42).Table 2. ANOVA and Tukey HSD results for OD values by paired sera MAT results ANOVA Tukey HSDSum of Mean FSquares df Square (p) MAT Results N 1 2LA0591Between Groups 18.31 4 4.578 19.996 Both non-reactive 41 0.437Within Groups 32.51 142 0.229 (< 001) Acute NR, Con R 9 0.728Total 50.82 146 Acute reactive 43 0.764Acute >400 33 1.267Acute NR, Con + 21 1.347LA3490Between Groups 4.87 4 1.218 13.721 Both non-reactive 41 0.473Within Groups 12.60 142 0.089 (< 001) Acute NR, Con + 21 0.699 0.699Total 17.47 146 Acute reactive 43 0.826Acute NR, Con R 9 0.867Acute >400 33 0.949RBLsBetween Groups 0.57 4 0.142 4.926 Acute NR, Con R 9 0.422Within Groups 3.99 139 0.029 (< 001) Acute NR, Con + 20 0.483Total 4.56 143 Acute >400 31 0.523 0.523Acute reactive 43 0.532 0.532Both non-reactive 41 0.635Abbreviations:

[0318] To determine the optimal day for the sample collection, the mean OD values distribution based on symptom duration at the sample collection was analyzed (Figure 16). The results show that VM protein antibodies were detectable as early as day 2 since the first patient- reported symptom.

[0319] Among the analyzed serum samples, four samples yielded isolation of Leptospira, these patients comprised two cases of L. interrogans and two of L. borgpetersenii. For L. interrogans infections, the OD values for the LA0591 antibodies ranged from 0.7183 to 1.1937, while those for the LA03490 antibodies ranged from 0.759 to 0.855. Regarding L. borgpetersenii infections, the OD values for LA0591 were recorded as 0.2243 to 0.887, while those for LA03490 were recorded as 0.775 and 1.115. ROC analysis was performed to assess the diagnostic accuracy of LA0591 (SEQ ID NO:1), LA3490 (SEQ ID NO:41), and RBLs (SEQ ID NO:42) (Figure 17), using only confirmed cases and healthy adults. The AUC for LA0591 (SEQ ID NO:1) was 0.947 (SE = 0.017, p < 0.001), indicating excellent discriminatory power in distinguishing positive and negative cases. Similarly, the AUC for LA3490 (SEQ ID NOL 41) was 0.930 (SE = 0.020, p < 0.001), suggesting its potential as a reliable diagnostic marker for leptospirosis illness. The Gini Index values were 0.893 for LA0591 (SEQ ID NO: 1) and 0.860for LA3490 (SEQ ID NO:41), confirming their strong discri inatory abilities. The optimal cutoff values for classification, determined by maximum K-S metrics, were 0.505333 for LA0591 (SEQ ID NO: 1) and 0.42317 for LA3490 (SEQ ID NO:41).

[0320] The diagnostic accuracy of the VM proteins to detect anti-VM proteins antibodies presented here surpasses that of reported qPCR or MAT tests. Furthermore, among confirmed cases, there was no discernible difference in the distribution of anti-VM proteins antibodies between complicated and uncomplicated cases, including those with mild, moderate, or severe renal / liver impairment.

[0321] Here, the hypothesis that VM proteins antigens could predict and diagnose early anti-VM proteins antibodies in serologically confirmed acute leptospirosis / febrile patient serum in an endemic region in Sri Lanka was tested and assessed. The serum samples were previously evaluated by qPCR and MAT systematically using paired serum samples without selection bias. Out of 222 patient samples, 26.6% and 21.7% of cases were definite positive by qPCR and MAT respectively. However, 16% and 19% of cases were suspected / probable cases by qPCR and MAT. The current study, for the first time, evaluated the potential of both recombinant full- length VM proteins (LA3490) (SEQ ID NO:41) and their domains (RBLs (SEQ ID NO:42) and C-terminals; LA0591 (SEQ ID NO: 1)) for their ability to detect leptospiral IgG-specific anti-VM proteins antibodies in definite and suspected patient serum and eventually differentiate healthy controls. LA3490 (full-length) (SEQ ID NO:41) and LA0591 (C-terminal domain) (SEQ ID NO:1) showed remarkable distinguished and statistically significant potential to detect anti-VM proteins antibodies in confirmed and probable cases versus healthy controls. LA0591 (SEQ ID NO:1) is a superior antigen because it was able to well-differentiate the groups into distinct subsets, and substantially distinguishes positive sera from non-reactive / reactive serum; however, LA3490 (SEQ ID NO:41) and RBLs (SEQ ID NO:42) separated healthy controls from confirmed / probable cases. VM proteins were able to detect antibodies in serum samples that tested negative in the MAT, indicating the potential omission of certain serovars necessary for accurately detecting agglutinating antibodies. Nonetheless, the use of VM proteins has significantly improved the detection of antibodies and has helped reduce false negative results.

[0322] Earlier studies show that ELISA-based diagnosis of leptospirosis offers several advantages over the conventional methods such as MAT, culture, and serological detection ofantibodies. MAT, considered the gold standard, detects agglutinating (IgM and IgG) antibodies and is serovar-specific. However, it can produce false negatives if a serovar is omitted and cannot distinguish early / late infections, limiting its utility in acute illness settings. MAT also requires technical expertise, a broad range of live pathogenic Leptospira cultures, and paired sera collected at specific intervals for accurate interpretation of results. On the other hand, qPCR detects bacterial load but does not provide information on disease severity. ELISA, in contrast, is rapid, simpler than MAT, and offers high sensitivity and specificity (Niloofa et al., PLoS One 2015; 10(6): e0129236; Chaurasia et al., Eur J Clin Microbiol Infect Dis 2018; 37(3): 423-33).

[0323] Leptospirosis, an acute undifferentiated illness, can be life-threatening, if severe and undiagnosed, leading to Weil's disease with multi-organ involvement, including jaundice (liver injury), renal failure (kidney injury), and potentially fatal pulmonary hemorrhage (Ko et al., Nat Rev Microbiol 2009; 7(10): 736-47; Haake et al., Curr Top Microbiol Immunol 2015; 387: 65-97; Levett et al., Clin Microbiol Rev 2001; 14(2): 296-326; Sehgal et al., Indian J Med Res 1995; 102: 9-12). The absence of reliable diagnostic markers increases morbidity and mortality in humans, livestock, and companion animals, impacting veterinary health and economics significantly. The recently discovered PF07598 gene family encoding VM proteins belong to the classical secreted toxin paradigm but have a unique ABC domain architecture, unlike classical AB toxins such as diphtheria toxin, pertussis toxin, shiga toxin, or ricin toxin which are typically encoded by two or more genes and self-assembled into a multi-domain holotoxin (Chaurasia et al., Front Mol Biosci 2022; 9: 1092197). VM proteins possess secretory signal peptides and are secreted exotoxins with discrete N-terminal ricin B-like domains involved in host cell surface binding and a C-terminal DNase / toxin domain, mediating cell death (Chaurasia et al., Frontiers in Microbiology 2022; 12: 859680; Chaurasia et al., Front Mol Biosci 2022; 9: 1092197). The current study underscores the potential VM proteins antigens (LA3490 (SEQ ID NO:41) and LA0591 (SEQ ID NO: 1)) to detect antibody onset on day 2 since the patient showed the first symptom. LA0591 (SEQ ID NO: 1) is superior antigen in detecting early anti VM proteins antibodies. LA0591 (SEQ ID NO: 1) is exclusively a natural variant that encodes only the C-terminal domain and lacks N-terminal RBLs (RBL1 and RBL2) (SEQ ID NO:42), suggesting that this natural variant does not require binding and internalization into the host cell, rather it may produce intracellularly and imply a unique role in pathogenesis. L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri, and L.alexanderi pathogens are the leading causes of the disease (Haake et al., Curr Top Microbiol Immunol 2015; 387: 65-97; Evangelista et al., Future Microbiol 2010; 5(9): 1413-25). Interestingly, both L. interrogans and L. borgpetersenii were cultured from two patient serum samples each. These four patients also exhibited detectable in vivo expression of anti-VM proteins antibodies (LA3490 (SEQ ID NO:41) and LA0591 (SEQ ID NO: 1)) early in the course of infection. The expression of anti-LA0591 antibodies was relatively higher in L. interrogans, a species that infects humans, compared to the animal infecting L. borgpetersenii, while the opposite was observed for anti-LA3490 antibodies.

[0324] Recently, Putz et al. conducted a study that found the serologically identical L. borgpetersenii serovar Hardjo strains JB197 and HB203 exhibit varying levels of disease severity in the hamster model (Fouts et al., PLoS Negl Trop Dis 2016; 10(2): e0004403). Despite their genomic and nucleotide level similarities, JB197 causes a severe acute infection, while HB203 results in a persistent chronic infection. Proteome analysis of these two strains, isolated from experimentally challenged hamsters at 29°C and 37°C, revealed that the VM protein Q04V07 (LIC12339 orthologs) a member of the PF07598 gene family, was significantly upregulated at 29°C and 37 °C (3.26 and 6.43 log2-fold, respectively) in acute disease-causing strain FBI 97 compared to chronic strain HB203. This finding enhances the understanding of VM protein- mediated host-pathogen interactions and contributes to potential improvements in vaccine and diagnostic strategies (Fouts et al., PLoS Negl Trop Dis 2016; 10(2): e0004403).

[0325] Previously, in the field, various recombinant leptospiral proteins, including rLipL21 (Seenichamy et al., Biomed Res Int 2014; 2014: 592858), rLipL32 (Croda et al., J Clin Microbiol 2007; 45(5): 1528-34; Boonyod et al., Asian Pac J Allergy Immunol 2005; 23(2-3): 133-41; Flannery et al., Microbiol 2001; 39(9): 3303-10), rLipL41 (Flannery et al., Microbiol 2001; 39(9): 3303-10; Mariya et al., Comp Immunol Microbiol Infect Dis 2006; 29(5-6): 269- 77), the D5-CBD fragment of LigA (Sharapova et al., Zh Mikrobiol Epidemiol Immunobiol 2010; (2): 79-84), and rGroEL (Flannery et al., Microbiol 2001; 39(9): 3303-10; Park et al., DNA Cell Biol 1999; 18(12): 903-10), were evaluated for detecting anti-leptospiral antibodies but lacked adequate sensitivity for identifying acute-phase leptospirosis. Lig proteins are sensitive markers for acute infection, although their usefulness is mainly evaluated in urban Brazilian outbreaks (Croda et al., J Clin Microbiol 2007; 45(5): 1528-34; Koizumi et al., Vaccine 2004; 22(11-12): 1545-52). Additionally, rLipL21, rLoa22, rLipL32, and rLigACon4-8 wereevaluated in equine and canine samples (Ye et al., Clin Vaccine Immunol 2014; 21(4): 478-83; Ye et al., PLoS One 2014; 9(12): el 11367). The LIC13341 antigen, capable of binding to various host matrices, showed diagnostic potential in human and bovine leptospirosis serum (Ghosh et al., Microbiology 2018; 164(8): 1023-37). A recent study highlighted rChi2, a multi epitope chimeric protein that recognizes antibodies in both onset (MAT-, 75%) and convalescent (MAT+, 82%) phases (Fernandes et al., Trop Med Infect Dis 2022; 7(11)), suggesting as a promising candidate for an early, broad, and cross-reactive diagnostic test. Eight recombinant fragments of LigA, LigB, and LipL32, ranging from 31-70 kDa and encoded by various serovars were assessed for canine leptospirosis diagnosis, using the Multi-antigen Print Immunoassay (MAPIA) platform, demonstrating promising accuracy (AUC = 0.826 to 0.869) with specificity at 70% and sensitivity ranging from 89% to 95% (Thome et al., BMC Vet Res 2014; 10: 288). Additionally, studies have explored urine screening for anti-leptospiral antibodies (KCT et al., International Journal of Research in Medical Sciences 2017; 5(2), 646- 652) and specific antigens such as LipL32, LipL41, HbpA, and sphingomyelinase (Chaurasia et al., Eur J Clin Microbiol Infect Dis 2018; 37(3): 423-33), aiding in the differentiation of patients with dengue who tested negative for these leptospiral antigens (Chaurasia et al., Eur J Clin Microbiol Infect Dis 2018; 37(3): 423-33).

[0326] In conclusion, this study offers advantages in detecting IgG antibodies in early infection even on day 2, compared to earlier studies. The current study also highlights the diagnostic potential VM proteins, which can even detect the circulatory antibodies generated from previous exposure and reaffirm the diagnostic accuracy. The AUC for LA0591 (SEQ ID NO: 1) was 0.947 (SE = 0.017, p < 0.001), relatively superior to LA3490 (SEQ ID NO:41) 0.930 (SE = 0.020, p < 0.001) in distinguishing the potential positive and negative cases and suggesting they are a reliable diagnostic marker and further superior to qPCR or MAT tests for detecting leptospirosis illness.Example 3: Early detection of Virulence-Modifying biomarker or early serological diagnosis of leptospirosis using Virulence-Modifying protein as antigens in rhesus macaque.The materials and methods are now described:Culture, growth condition, and maintenance of low-passage strains

[0327] L. interrogans serovar Copenhagen! strains LI- 130 and LV3726 were maintained at 30°C in semi-solid Ellinghausen, McCullough, Johnson, and Harris medium (EMJH, BD Biosciences, USA) (Ellinghausen and McCullough, 1965). Growth was monitored regularly by observing under the darkfield microscope (Nikon Eclipse E600, Japan). Mid-logarithmic cultures in EMJH liquid medium were harvested by centrifugation at 18,514 g. Pelleted cells were washed twice with IX PBS and hamsters were routinely infected with 2.5xl08cells, which were prepared in 1 mb of IX PBS. LD50 was estimated for both the strains LI-130 and LV3726. Low- passage organisms were recovered from blood, liver, and kidney.Study design and subjects

[0328] Rhesus macaques (Macaca mulatta) namely Xena, Skeeter, and Melody who was 10-year-old females of body weight 9.6 kg, 7.9 kg, and 9.27 kg respectively. Xena has a longstanding history of vomiting that was managed on 5mg famotidine, lOmg omeprazole, and 100 mg sucralfate. Since 2020, all the breeding attempts did not result in pregnancy. A reproductive exam was unremarkable. Hematology consistently revealed thrombocytosis and hyperglycemia. Fecal exam results were consistently negative for parasites. NHP rooms were maintained on a 12-hour light / dark cycle with temperature and humidity conditions maintained between 64-84°F and between 30-70% humidity. NHPs were fed primate chow daily and supplemented with dietary enrichment (fruits and vegetables) at least 3 times per week. The amount of primate chow given to each NHP was proportional to their weight. NHPs were pair housed whenever possible, however after infection the NHPs were individually housed for biosafety / safety reasons. All NHPs received water through automatic watering systems attached to each cage rack. NHPs were infected with 5xl08cells / mL by right deltoid sub-cutaneous (Xena was infected with low-passage virulent L. interrogans serovar Copenhegani strain LV3726, however, Skeeter and Meldoy were infected with low-passage virulent L. interrogans serovar Copenhegani strain LI-130). Additionally, 50 pL cultures were inoculated in the right conjunctiva, and 200 pL cultures were inoculated through the right nostril respectively. Urine and blood were collected routinely. Serum was tested for anti-leptospiral antibodies by Gold Standard MAT. Serology was performed by Antech Diagnostics (USA). Prior to the collection of samples, NHPs were sedated at days 0, 4, 8, 14, and 21 with midazolam 0.1 mg / kg (0.16 mL), ketamine 5 mg / kg (0.4 mL), and dexmedetomidine 0.02 mg / kg (0.3 mL). The subjects wereeuthanized at the end of the experiment by intravenous injection of 3 mL of Euthasol euthanasia solution. A complete necropsy was performed on each animal under BSL-3 containment at the end of the study period.Processing of Serum, plasma, and urine samples

[0329] Blood was collected at the scheduled time point in a red-topped BD Vacutainer Plus Venous Blood Collection Tube; however, urine was collected daily or every two days. Whole blood was allowed to clot by incubating the tubes for 20 minutes at room temperature and serum was separated by centrifugation at 1,500 x g for 10 minutes at 4°C. Similarly, plasma was separated using EDTA-treated (lavender tops) tubes by centrifugation at l,000xg 10 minutes 4°C. Urine samples were collected and centrifuged at 28000 x g for 30 minutes. The pellet was resuspended in 1 mL lx PBS and stored as mentioned above. Samples were aliquoted to avoid freeze-thaw cycles and stored at -80°C until further use.Urine, serum, and tissue-RT-PCR

[0330] DNA extraction was performed by dicing 25±35 mg of kidney and liver tissues followed by homogenization in 200 pL of IX PBS using TissueLyser (Qiagen; Retsch, Haan, Germany) and homogenates were centrifuged. Concurrently DNA was extracted from 200 pL urine or serum. The entire procedure was carried out under positive pressure conditions in a designated area separate from other Leptospira and PCR handling to minimize the risk of crosscontamination. After homogenizing the tissues, genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, USA) following the manufacturer's instructions, with the final elution in 30 pL. For standard curves, DNA was extracted from infecting L. interrogans serovar Copenhagen strains LL130 and LV3726 at a density of 2xl08leptospires / mL, cultured in 5 mL of EMJH culture medium. Cells were harvested, and DNA was extracted for standard curves using the same DNeasy Blood and Tissue Kit (Qiagen, USA). The concentration of eluted DNA was assessed using a NanoDrop Spectrophotometer ND-1000 (NanoDrop Technologies, DE, USA). The qPCR was performed in 10 pL reaction volume Containing 4 pL of standard or DNA sample, 5 pL of PerfeCTa SYBR Green FastMix (Quanta bio, USA), 0.4 pL (10 picomole) of each LipL32, forward primer (5’-TCT GTG ATC AAC TAT. TAC GGA TAC-3’) and reverse primer (5’-ATC CAA GTA TCA AAC CAA TGT GG -3’) and 0.2 pL of nuclease-free water. The reaction was subjected to amplification in the CFX96 Real-time PCR Detection System (Bio-Rad, USA) using the following program: 3 min at 95°C, 0.10 min at 95°C, 0.30 min at62°C, followed by 44 cycles at 1 .00 min at 72°C then final extension 7 min at 72°C, followed by a melt curve from 65 °C to 90°C performed at an increment of 0.5 °C per cycle. The cut-off was determined from the linear standard curve. A negative result was assigned where no amplification occurred or if the CT value was greater than 3 SD+Ct.NTA-ELISA

[0331] The assay was performed in a commercially available pre-blocked 96-well NTA- ELISA plate coated with His6x tag following manufacturer instructions. Briefly, 100 ng of recombinant VM protein antigens (in equimolar ratio) per well in 100 pL of 0.05 M sodium carbonate buffer, pH 9.6, were added to each well and incubated for 1 h at 37°C. To quantify leptospiral antibodies targeting VM protein antigens, NHP serum was diluted (1 :50) in IxPBST and added to the wells followed by incubation for 1 h at 37°C. The plate was washed thrice with IxPBST and then 1 :5,000 dilution of anti-rhesus-IgG-HRP conjugate (KPL, USA) was added to the wells for 1 h at 37°C. The reaction was then developed with 100 pL of ready-to-use TMB substrate (Sigma, USA). To stop the reaction, 2 M H2SO4 was added, and absorbance was measured at 450nm using a SpectraMax® M2e Microplate Reader (Molecular Devices, USA). The recombinant LA0591 (SEQ ID NO:3) served as a positive control, while the secondary antibody acted as a negative control. The mean average (x) and standard deviation (SD) of OD 450 nm of all samples were determined. Results are expressed as mean and standard deviation. Statistical analysis and the graphs were generated using GraphPad Prism Version 9 (GraphPad Software, Inc., La lolla, CA). The results were analyzed by the ANOVA, and Tukey’s multiple comparisons to determine significant differences between individuals and were considered statistically significant when p < 0.05.Gross pathology: histopathology and Immunohistochemistry

[0332] At the end of the monitoring period, a necropsy was performed on all animals, involving the dissection of thoracic and abdominal cavities to examine major organs. The degree of leptospiral pathology consolidation was determined through visual estimation of the affected organs such as lung, liver, kidney, and spleen tissue percentage. All the same were obtained for subsequent histopathological examination and analysis using immunohistochemistry. These tissues underwent fixation in 10% neutral -buffered formalin, paraffin embedding, and staining with H&E for histopathological assessment. The H&E-stained tissue sections were then inspected under light microscopy (Zeiss Axioplan).Cytokine measurement by luminex multiplex bead array kits

[0333] The immune cell response to the challenge posed by pathogenic Leptospira was investigated by examining the release of cytokines in serum or plasma. This was achieved using the MILLIPLEX® non-human primate cytokine magnetic bead pre-mixed 23 plex panel (Millipore Sigma, USA). This panel could simultaneously detect 23 cytokines, including G-CSF, GM-CSF, IFN-y, IL-lra, IL-1 , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 / 23 (p40), IL-13, IL-15, IL-17, IL-18, MCP-1, MIP-la, MIP-10, sCD40L, TGF-a, TNF-a, and VEGF. The cytokine levels were measured using bead-based multiplexed sandwich immunoassays employing Luminex™ technology on a Luminex 200™ analyzer system. The assay was performed following the manufacturer’s instructions, with cytokine-specific antibodies pre-coated onto magnetic microparticles embedded with fluorophores. Briefly, 200 pL of assay buffer was added into the well and incubated on a plate rocker for 10 minutes at room temperature. 25 pL of standards and quality control into their respective wells, and then added 25 pL of serum matrix solution exclusively to the background, standards, and control wells. In test sample wells, 25 pL of serum or plasma samples were added, followed by 25 pL of assay buffers. Added 25 pL of mixed beads into each well. Sealed the plates with a plate sealer and incubated them with agitation on a plate shaker at 4°C overnight followed by two washes with 200 pL of wash buffer. Added 25 pL of detection antibody, sealed the plate, and incubated it with agitation on a plate shaker for 1 hour at room temperature. 25 pL of streptavidin-phycoerythrin conjugate (Streptavidin-PE) were added to each well and plates were sealed followed by covering it with foil, then incubated with agitation on a plate shaker for 30 minutes at room temperature. Washed the plate twice with wash buffer and, finally, added 150 pL of sheath fluid. Plates were agitated for 5 minutes on a plate shaker before proceeding to read. It is worth noting that the plate was sealed and incubated, followed by each washing step using a Luminex magnet plate stand base. Final parameters were detected using the Luminex 200 Multiplexing Instrument with xPONENT software (Luminex, Austin, TX, USA) and analyzed using Milliplex Analyst software (Millipore Sigma, Billerica, MA, USA) based on the manufacturer’s instructions

[0029] , Graphs and statistical analyses were performed in GraphPad Prism version 10 (GraphPad Software, Inc., San Diego, CA). Analyte concentrations are presented as pg / mL ± SD, range, and median fluorescence intensity units (MFI, the instruments’ raw data output). The comparison of the means wasperformed by one-way analysis of variance (ANOVA) for repeated measurements followed by Tukey’s multiple comparison test. Differences of p < 0.05 were considered significant.Assessment of physical and biochemical parameters

[0334] NHPs were observed at least twice daily for up to 21 days for clinical illness of leptospirosis. NHPs were monitored for body weight, respiratory oxygen rate, abnormal behavior, and body temperature at each time point. Clinical chemistry analyses were performed at ANTECH diagnostic.The results are now described:

[0335] As depicted in Tables 3-5 and Figures 18-27, VM protein antigens LA0591 (SEQ ID NO: 1), LA1400 (SEQ ID NO:43), LA1402 (SEQ ID NO:44), LA3490 (SEQ ID NO:41), and RBLs (SEQ ID NO:42) are successfully used for the early detection of Virulence-Modifying biomarkers or early serological diagnosis of leptospirosis in rhesus macaque. Accordingly, these data demonstrate the successful use of Virulence-Modifying proteins as antigens for the early detection of Virulence-Modifying biomarkers and / or early serological diagnosis of leptospirosis.Table 3. Serology evaluation of Leptospira spp. serovars that reacted in Microscopic Agglutination Test (MAT)XenaDay0 4 8 14 21Pomona 0 0 3200 400 200Icterohaemorrhagiae 0 0 0 800 800Canicola 0 0 0 50 0Grippo 0 0 0 50 0Hardjo 0 0 1600 50 0Autumnalis 0 0 400 200 100Bratislava 0 0 200 1600 800SkeeterDay0 4 8 14 21Pomona 0 0 1600 800 1600Icterohaemorrhagiae 0 400 6400 3200 6400Camcola 0 0 400 100 200Grippo 0 0 0 0 50Hardjo 0 0 0 0 50Autumnalis 0 50 3200 400 400Bratislava 0 200 6400 3200 6400Pomona 0 0 400 400 200Icterohaemorrhagiae 0 100 1600 1600 800Canicola 0 0 0 0 50Grippo 0 0 0 0 50Hardjo 0 0 0 0 50Autumnalis 0 0 800 400 100Bratislava 0 100 1600 800 800Table 4. qPCR based quantitative assessment of bacterial load in serumNA-No Amplification, Positive (+), Negative (-), Standard deviation (±)Table 5. qPCR based quantitative assessment of bacterial load in urineNA-No Amplification, Positive (+), Negative (-), Standard deviation (±)LA3490 VM protein (SEQ ID NO:41)FEYGVNHTHIHALSK1EYSV1QKPTDPPKDKPIKVIVSDGGKFCYGPNFSGGESYIIIEQCW QMHVMNARYDVFQRISYNINNTWLCITAPEKVIKAEKNWDYVHLRPCTINDPLQRWIIK NNSFWTANGFYRLKDYNWYGYISRNSGDRYNHTLDSSMNDWVNTIATPGNISIQTSIA WNLQTTEGQERYFIRWGGSDKNTTPLYYNPENGHLAQYDPISGSLYCMYSQVDNYQW NWVKWKWCSDLLESKSKGNPTFWNVFFETDQGGMITDYKGNALRVTRYGSNWGSAY TAKPSYLEKDTTNSPTSLFVVNKDLLDWTRYTASNLGKTGQYCPAGKRENIVHRRVKRELPPDFQLTEAWIRRLYEIATSVSAESETRVSGICGPCALHSFQMLAELLEYHSREPLQSG GYFFDTAPNTDPFISFGQRYPHLERLLEDIPKKYAPYPHYSTQSFLSFASIDSMLPQYFWS ASTEFTNRDEILSHISSLINSPAGSIWLGVMEQQHPDGTITGHAAPILRISQGLVVIPTNVH LWTLEEFRRFLIPTTELSQIVANLEGSNTLIRFTTIQSLGMLTTNMFDSMVSNRNCTGEGE DRRGSGEYPT ST S VNQCP SGRC ALPFRBLs VM protein (SEQ ID NO: 42)FEYGVNHTHIHALSKIEYSVIQKPTDPPKDKPIKVIVSDGGKFCYGPNFSGGESYIIIEQCW QMHVMNARYDVFQRISYNINNTWLCITAPEKVIKAEKNWDYVHLRPCTINDPLQRWIIK NNSFWTANGFYRLKDYNWYGYISRNSGDRYNHTLDSSMNDWVNTIATPGNISIQTSIA WNLQTTEGQERYFIRWGGSDKNTTPLYYNPENGHLAQYDPISGSLYCMYSQVDNYQW NWVKWKWCSDLLESKSKGNPTFWNVFFETDQGGMITDYKGNALRVTRYGSNWGSAY TAKPSYLEKDTTNSPTSLFVVNKDLLAI 400 VM protein (SEQ ID NO: 43)S SKSDYSIAQKPADQPKDKSIQVVMHGGSNYC YSPVFTKGEGYIWIDYC SDNTAKARYD VFQRISYNINNTWLCITAPETVVKGEETWNYVNLRPCTINDPLQRWIVKDNSFWTANGRYRLKDTNWYGYISRNSGDRYDHTLNSSMDDWIKTVAIPGNISIQTSIAWDLQTTEGNER YFIRWGSSNKNTTPLYYNPESGHIAQYDPSSGLLNCMYSKMTDKYDWNWVKWGKCSD APIKKDNPAFWNVFFETDKEGAITDYKGNVLRVTRYGLNWGVAYTVKPSYLEKDTTHS PTSLFVIDKDLLDWTRYTYSNLGKTDQYCPAGNKESLVRKRVKRNLNLPSDFQLTREWI QRLYEIARSSISRAIPCRGVCGVCMLHSYQMIAELLEYHSRGPLTGGGYFFDTAPNRDPFI SFNQRYPQLNALLTNVPSYANRPGFGSTLVMLPQYEWTSSDTITTRSGRLLHARSLINSP PGSIWLGLLRGRDADGSTWGHAVPILRTSQGIVVIPTNSPTMSLNTYIRSLAPTMDPNEVI NRLENGSTLTELTTIQPVRIYDIPFSLTVSTRDCTGDGDGRRGSGRYPTSSLINQCSGGRCI LQLA1402 VM protein (SEQ ID NO:44)S S SK ANYSI AQKPTDPPKDKPINI VTHDGKTYC YSP VF SKGEGYVWIEKC GDNTAKARY DVFQRISYNINNTWLCITAPEPVVKGNARWGYVNLRPCTINDPLQRWIVKENSFWTADG KYRLKDTNWYGYISKTSGDNYNHTLNSSMDNWVKTVATPGNISIRTSISWNSGWGDGI WDINMAP S AYFIHSKGS SKEDIIPL YYNPESGHIAQ YDP S SGLL SCMYSKMTDK YDWNW VQWGKCSDAPIKKENPAFWNVYFVANAGGMITDYKGNILRVTKEGPNWGVAYTAKPS YLEKDTTHSPTSVFTVDVDLLKWIRYTTSNLGKTDQYCPAGKKESRIYQRVKRNLPSDF QLSVAWVQRLYDIARSATFESANPGAIPQRHGACGVCLLHSFQMIAELMEYHSREPLTS GGYFFNTASNRDPFLSFSQRYPELDRLVTNVPVDYANRGRVLAFASAMIMLPQYEWESS SPLTTRSDIQSHIRSLINSPPGSIWLGLLRRQRANGSISGHAVPILRTSEGLVVIPTNMPTAS LNTYIQSLAPTMDPNEVINRLENGRTLTTLTTIRPVGTYETPFSLTVSSRDCTGDGDDRRG SGRYPISSLINQCSGGRCILQ

[0337] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS1. A composition comprising at least one Leptospiral virulence modifying (VM) protein epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID N0:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO 34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof.

2. The composition of claim 1, wherein the composition is a fusion protein comprising a Leptospiral VM protein epitope fused to a targeting domain specific for binding to a target molecule.

3. The composition of claim 2, wherein the target molecule is selected from the group consisting of a bacterial antigen, viral antigen, parasitic antigen, cancer antigen, tumor-associated antigen, and tumor-specific antigen.

4. The composition of claim 1, wherein the composition comprises a combination of two or more Leptospiral VM protein epitopes.

5. The composition of claim 1, wherein the composition comprises at least one lipid nanoparticle (LNP) comprising at least one Leptospiral VM protein epitope.

6. The composition of claim 1, wherein the composition comprises a combination of at least two LNP comprising at least two VM protein epitopes.

7. A composition comprising at least one nucleic acid molecule encoding at least one Leptospiral virulence modifying (VM) protein epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQIDNO:6, SEQIDNO:7, SEQIDNO:8, SEQ ID NOV, SEQ ID NO: 10, SEQ IDNO:11, SEQIDNO:12, SEQIDNO:13, SEQIDNO:14, SEQ IDNO:15, SEQ ID NO:16, SEQIDNO:17, SEQIDNO:18, SEQIDNO:19, SEQIDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NQ:30, SEQ ID NO:31, SEQIDNO32, SEQIDNO:33, SEQIDNO:34, SEQIDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof.

8. The composition of claim 7, wherein the nucleic acid molecule encodes a fusion protein comprising a Leptospiral VM protein epitope fused to a targeting domain specific for binding to a target molecule.

9. The composition of claim 8, wherein the target molecule is selected from the group consisting of a bacterial antigen, viral antigen, parasitic antigen, cancer antigen, tumor-associated antigen, and tumor-specific antigen.

10. The composition of claim 7, wherein the composition comprises at least one lipid nanoparticle (LNP) comprising at least one nucleic acid molecule encoding at least one VM protein epitope.

11. The composition of claim 7, wherein the nucleic acid molecule comprises an mRNA molecule encoding the at least one VM protein epitope.

12. The composition of any one of claims 1-11, wherein the composition comprises a vaccine.

13. The composition of any one of claims 1-12, wherein the composition comprises an adjuvant.

14. An anti-Leptospiral VM protein antibody specific for binding to an epitope selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO 35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 or SEQ ID NO:40, or a fragment or variant thereof.

15. The antibody of claim 14, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody and an scFv antibody fragment.

16. A composition comprising an antibody of any one of claims 14-15, or a fragment thereof.

17. The composition of claim 16, comprising polyclonal anti-serum.

18. A nucleic acid molecule encoding an antibody of any one of claims 14-15, or a fragment thereof.

19. A method of inducing an immune response in a subject, the method comprising administering the composition of any one of claims 1-13.

20. The method of claim 19, wherein the subject is currently infected with I.eplospira sp and the composition induces an immune response against Leptospira sp.

21. A method of treating or preventing a disease or disorder in a subject, comprising administering the antibody of any one of claims 14-15, the composition of any one of claims 16-17, or the nucleic acid molecule of claim 18 to the subject.

22. The method of claim 21, wherein the disease or disorder is at least one selected from the group consisting of cancer, a bacterial infection, a viral infection, and a parasitic infection.

23. A method for diagnosing infection of at least one pathogen in a human subject, comprising detecting IgG or IgM antibody produced after infection in the subject, comprising: a) determining the level of IgG and / or IgM antibody in the subject; wherein the determining comprises: i) mixing a biological sample from the subject with a first reagent; wherein the first reagent comprises at least one Leptospiral VM protein antigen coated on a solid phase support, and anti-human IgG antibody or anti-human IgM antibody conjugated to a label; wherein the antigen, IgG or IgM antibody from the sample, and the antihuman IgG antibody or anti-human IgM antibody conjugated to a label form a complex if the sample comprises IgG or IgM antibody that bind to the at least one antigen; ii) washing the complex after the mixing step; iii) incubating the complex with a second reagent, wherein the second reagent comprises a detection agent which produces a detectable signal when exposed to the label;iv) acquiring the detectable signal; and v) obtaining the level of the IgG or IgM antibody in the sample based upon the detectable signal; b) comparing the level of IgG or IgM antibody in the sample with a comparator control; c) diagnosing the subject as infected when the level of IgG or IgM antibody in the subject is elevated compared with the level of IgG or IgM antibody in the comparator control, wherein the at least one Leptospiral VM protein antigen comprises at least one Leptospiral VM protein antigen selected from the group consisting of SEQ ID NO: 1, SEQ ID NON, SEQ ID NO:3, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID N0:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO 19, SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NON 1, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO 38, SEQ ID NO:39 or SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, or a fragment or variant thereof.

24. The method of claim 23, wherein the at least one pathogen comprises at least one pathogen selected from the group consisting of L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri, and L. alexanderi.

25. The method of claim 23, wherein the label comprises Horse Radish Peroxidase (HRP).

26. The method of claim 23, wherein the detection agent comprises 3,3 ’,5,5’ tetramethylbenzidine (TMB) substrate.

27. The method of claim 23, wherein the acquiring comprises obtaining optical density (OD) measurements at 450nm.

28. The method of claim 23, wherein the diagnosing occurs immediately following infection.

29. The method of claim 23, wherein the diagnosing occurs at least 2 days following infection.

30. The method of claim 23, wherein the biological sample comprises plasma or serum from the human subject.

31. The method of claim 23, wherein the comparator control comprises the level of IgG and / or IgM in a plasma or serum sample from an uninfected human subject.

32. The method of claim 23, wherein when the subject is diagnosed as infected, the subject is treated.

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