Leptospirosis virulence control proteins and uses thereof

Abstract

The present invention provides a method for the preparation of a leptospiral virulence-modifying (VM) protein, or a fragment thereof comprising the DNase domain, and uses thereof as vaccines and therapeutics. Also provided is a pan-vaccine comprising multiple VM proteins.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grants AI115658, AI108276, and AI064466 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 230,244, filed August 6, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0003] 2. Background of the Invention The PF07598 gene family was identified as belonging exclusively to pathogenic Leptospira (Fouts et al., 2016, PLoS Negl Trop Dis. 10(2): e0004403; Lehmann et al., 2013, PLoS Negl Trop Dis, 7(10): e2468). Members of this gene family were previously known to be upregulated by osmolality, but their gene functions remain unknown (Matsunaga et al., 2007; Infect Immun, 75(6): 2864-2874). In vivo upregulation of PF07598 gene family members has been reported in a hamster model (Lehmann et al, 2013; PLoS Negl Trop Dis, 7(10): e2468), and a human antibody response against one of the members of this gene family has been reported in vivo (Lessa-Aquino, 2017, PLoS Negl Trop Dis, 11(1):e0005349). Furthermore, random transposon mutagenesis of Leptospira interrogans serovar Manilae has been reported (Marcsisin et al, 2013, J Med Microbiol, 62(Pt 10):1601-1608), although its function remains unclear.

[0004] Human leptospirosis is common in developing countries and its incidence is increasing in developed countries. Limited progress has been made towards implementing effective public health controls, and no vaccine is registered for human use. Summary of the Invention [Problem to be solved by the invention]

[0005] There remains a need in the art for novel compositions with vaccine potential against pathogenic Leptospira. The present invention fulfills this unmet need. [Means for solving the problem]

[0006] Summary of the Invention In one embodiment, the present invention relates to a composition comprising at least one Leptospira virulence modified (VM) protein or a fragment thereof comprising a DNase domain. In one embodiment, the at least one VM protein is selected from the group consisting of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_12851, LIC_12852, LIC_12853, LIC_12854, LIC_12855, LIC_12856, LIC_12857, LIC_12858, LIC_12859, LIC_12860, LIC_12861, LIC_12862, LIC_12863, LIC_12864, LIC_12865, LIC_12866, LIC_12867, LIC_12868, LIC_12869, LIC_12870, LIC_12871, LIC_12872, LIC_12871 ... C_11358, LIC_10639, LIC_12963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMAN V2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, or LMANV2_170091.

[0007] In one embodiment, the present invention relates to a fusion protein comprising a Leptospiral VM protein or a Leptospiral VM protein DNase domain and a targeting domain specific for binding to a target molecule. In one embodiment, the at least one VM protein is selected from the group consisting of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_12851, LIC_12852, LIC_12853, LIC_12854, LIC_12855, LIC_12856, LIC_12857, LIC_12858, LIC_12859, LIC_12860, LIC_12861, LIC_12862, LIC_12863, LIC_12864, LIC_12865, LIC_12866, LIC_12867, LIC_12868, LIC_12869, LIC_12870, LIC_12871, LIC_12872, LIC_12871, LIC_12871, LIC_12872, LIC_12871, LIC_12871, LIC_12871, LIC_12871, LIC_128 C_11358, LIC_10639, LIC_12963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, or LMANV2_170091. In one embodiment, the target molecule is a bacterial antigen, a viral antigen, a parasitic antigen, a cancer antigen, a tumor-associated antigen, and a tumor-specific antigen.

[0008] In one embodiment, the composition comprises a combination of two or more Leptospira VM proteins. In one embodiment, the composition comprises a combination of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_1 0639, LIC_12963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091. In one embodiment, the composition comprises a combination of LIC_12340 and LIC_12985. In one embodiment, the composition comprises a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

[0009] In one embodiment, the composition comprises at least one Leptospira VM protein comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. In one embodiment, the composition comprises a combination of VM proteins comprising the sequences set forth in SEQ ID NO:10 and SEQ ID NO:12. In one embodiment, the composition comprises a combination of VM proteins comprising the sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12.

[0010] In one embodiment, the composition comprises at least one lipid nanoparticle (LNP) comprising at least one VM protein or a fragment of a VM protein that contains a DNase domain, hi one embodiment, the composition comprises a combination of at least two LNPs comprising at least two VM proteins or a fragment of a VM protein that contains a DNase domain.

[0011] In one embodiment, the present invention relates to a composition comprising at least one nucleic acid molecule encoding at least one Leptospira virulence modified (VM) protein or a fragment thereof comprising the DNase domain of a VM protein. In one embodiment, the VM protein is selected from the group consisting of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11 358, LIC_10639, LIC_12963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2 _210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, or LMANV2_170091.

[0012] In one embodiment, the nucleic acid molecule encodes a fusion protein comprising a Leptospira VM protein or a fragment comprising the DNase domain of the VM protein fused to a targeting domain specific for binding to a target molecule. In one embodiment, the target molecule is a bacterial antigen, a viral antigen, a parasitic antigen, a cancer antigen, a tumor-associated antigen, or a tumor-specific antigen.

[0013] In one embodiment, the nucleic acid molecule encodes at least one amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. In one embodiment, the nucleic acid molecule comprises at least one nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18.

[0014] In one embodiment, the composition comprises one or more nucleic acid molecules encoding a combination of LIC_12340 and LIC_12985. In one embodiment, the composition comprises one or more nucleic acid molecules encoding a combination of SEQ ID NO: 10 and SEQ ID NO: 12. In one embodiment, the composition comprises one or more nucleic acid molecules comprising a combination of SEQ ID NO: 9 and SEQ ID NO: 11.

[0015] In one embodiment, the composition comprises one or more nucleic acid molecules encoding a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402. In one embodiment, the composition comprises one or more nucleic acid molecules encoding a combination of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. In one embodiment, the composition comprises one or more nucleic acid molecules comprising a combination of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11.

[0016] 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 or a fragment thereof comprising a DNase domain, hi one embodiment, the nucleic acid molecule comprises an mRNA molecule encoding at least one VM protein or a fragment thereof comprising a DNase domain.

[0017] In one embodiment, the composition comprises a vaccine.

[0018] In one embodiment, the composition comprises an adjuvant. In one embodiment, the adjuvant is glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water nanoemulsion (SE).

[0019] In one embodiment, the present invention relates to a method of inducing an immune response in a subject, comprising administering to the subject a composition comprising at least one modified virulence of Leptospira (VM) protein or a fragment thereof comprising a DNase domain, or a composition comprising at least one nucleic acid molecule encoding at least one modified virulence of Leptospira (VM) protein or a fragment thereof comprising the DNase domain of a VM protein, hi one embodiment, the subject is currently infected with Leptospira and the composition induces an immune response against Leptospira.

[0020] In one embodiment, the present invention relates to a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition comprising at least one modified virulence of Leptospira (VM) protein or a fragment thereof comprising a DNase domain, or a composition comprising at least one nucleic acid molecule encoding at least one modified virulence of Leptospira (VM) protein or a fragment thereof comprising the DNase domain of a VM protein, hi one embodiment, the disease or disorder is cancer, a bacterial infection, a viral infection, or a parasitic infection.

[0021] In one embodiment, the present invention relates to a method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition comprising at least one modified virulence (VM) antibody or a composition comprising at least one nucleic acid molecule encoding at least one modified virulence (VM) antibody, hi one embodiment, the disease or disorder is leptospirosis. [Brief description of the drawings]

[0022] The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0023] [Figure 1] FIG. 1 shows the design of the pan-vaccine challenge study. [Diagram 2] FIG. 2 shows the experimental schedule of the pan-vaccine challenge. [Diagram 3] Figure 3 shows exemplary experimental data demonstrating that immunized C3H / HeJ mice are protected from death / weight loss following lethal challenge (low passage L. interrogans serovar Canicola) by a pan-vaccine containing a mixture of five antigens (full-length mCherry fusions of LA3490, LA0620, and LA1402, and full-length LIC12340 and LIC12985); and a mixture of two antigens (full-length LIC12340 and LIC12985). Comparative genomic analysis shows a high level of conservation of these proteins among all pathogenic leptospires, including (but not exclusively) L. interrogans, making it likely that these protein homologs will have similar function and susceptibility to preventive and therapeutic interventions, including vaccines, drugs, and biologics developed against these and homologous proteins. [Figure 4] FIG. 4 shows the statistical analysis of mortality / weight loss in mice immunized with the pan-leptospira vaccine following lethal challenge. [Diagram 5]Figure 5 shows exemplary experimental data demonstrating that VM protein vaccine reduces bacterial burden in the kidney compared to PBS negative control. Data were statistically analyzed by Kruskal-Wallis test with Dunn's multiple comparison post-test, comparing all conditions to PBS-treated mice and comparing multiple groups (**, p<0.01; ***, p<0.001). There was a difference in kidney bacterial burden between the different vaccine groups (Kruskal-Wallis test, P=0.0003***). [Figure 6] FIG. 6 shows the statistical analysis of bacterial load in the kidney after immunization with VM protein vaccine. [Figure 7] FIG. 7 shows exemplary experimental data demonstrating that the VM protein vaccine reduced bacterial burden in the lungs compared to the PBS negative control. [Figure 8] FIG. 8 shows the statistical analysis of bacterial burden in the lungs after immunization with the VM protein vaccine. [Figure 9-1] Figure 9 shows exemplary experimental data demonstrating detection of cross-reactive VM protein antibodies pre-challenge. t-test, non-parametric test, unpaired two-tailed test, Mann-Whitney test, p<0.05, p<0.0001. [Figure 9-2] Figure 9 shows exemplary experimental data demonstrating detection of cross-reactive VM protein antibodies pre-challenge. t-test, non-parametric test, unpaired two-tailed test, Mann-Whitney test, p<0.05, p<0.0001. [Figure 9-3] Figure 9 shows exemplary experimental data demonstrating detection of cross-reactive VM protein antibodies pre-challenge. t-test, non-parametric test, unpaired two-tailed test, Mann-Whitney test, p<0.05, p<0.0001. [Figure 10]Figure 10A and Figure 10B show the evaluation and computational validation of the AlphaFold-derived 3D structure of VM protein. Figure 10A shows the Ramachandran plot analysis evaluating the artificial intelligence-based derived 3D structure of VM protein using the Zlab (zlab.umassmed.edu / bu / rama / ) online server. It provides an overview of the accepted and unaccepted regions of torsion angle values ​​that serve as a key indicator of the quality of the protein structure and the stability of the 3D conformation. Percentage of residues 98.211 (LA3490), 96.59 (LA0620), 98.59 (LA1400), 95.11 (LA1402), and 96.32 (LA0591) were favored regions (core beta), 1.78 (LA3490), 2.50 (LA0620), 1.20 (LA1400), 4.70 (LA1402), and 2.57 (LA0591) were allowed regions (core alpha), and 0.00 (LA3490), 0.896 (LA0620), 0.20 (LA1400), 0.18 (LA1402), and 1.13 (LA0591) were outliers (core left-handed alpha). FIG. 10B shows the Z-score mean, Z-score stddev, and Z-score RMS calculated using the online PROVE analysis server. [Figure 11A] Figure 11A and Figure 11B show experimental results demonstrating the structure and sequence representation of the QxW motif in the VM protein in L. interrogans serovar Lei. Figure 11A shows the high-resolution 3D structure of the LA3490VM protein generated by AlphaFold, showing that the amino acids code for the surface aromatic patches (red: tyrosine, phenylalanine, and tryptophan). Blue represents the QxW motif in the N-terminal RBL1 domain. [Figure 11B]Figure 11A and Figure 11B show experimental results demonstrating the structure and sequence representation of the QxW motif in the VM protein in L. interrogans serovar Rhee. Figure 11B shows the RBL1 domain displaying three conserved QxW motifs (blue: 40QKP42, 134QRW136, and 78QCW80), where the 134QRW136 motif is also conserved in the ricin B chain. The aromatic motif 158YGY160 is highly conserved in the VM protein and the ricin B chain. [Figure 12A] Figures 12A and 12B show experimental results demonstrating the structural and functional similarity between RBL2 of VM protein and CARDs toxin (D3 domain). Figure 12A shows the RBL2 domain (green: 196aa to 335aa) of LA3490VM protein structurally superimposed with the C-terminus of CARDs toxin (PDB: 4TLV_A chain, pink) with RMSD 1.218 Å. [Figure 12B] Figures 12A and 12B show experimental results demonstrating the structural and functional similarity between the RBL2 of the VM protein and the CARDs toxin (D3 domain). Figure 12B shows that the C-terminus (D3 domain) of the CARDs toxin encodes eight tryptophans, while the LA3490VM protein encodes nine tryptophans. Six of these tryptophans are structurally overlapped by both the C-terminus and the RBL2 domain of the CARDs toxin. [Figure 13]Figures 13A-E show experimental results demonstrating the representation and similarity of disulfide bonds in LA3490VM protein and ricin toxin. Figure 13A shows the 3D ribbon structure of LA3490 generated by the AlphaFold algorithm showing five disulfide bonds by pairing ten cysteine ​​residues. Figure 13B shows ricin toxin (PDB: 2AAI) showing five disulfide bonds by pairing ten cysteine ​​residues. Figure 13C shows the superposition of the ricin B chain with the CBR (RBL1) of LA3490 along with the disulfide bonds, but the ricin A chain does not overlap with the C-terminal domain of LA3490. FIG. 13D shows a similar pattern of disulfide bond superposition between LA3490 (magenta: Cys62aa-Cys79aa, Cys105aa-Cys127aa, Cys244aa-Cys262aa, Cys353aa-Cys608aa, and Cys630aa-Cys635aa) and ricin toxin (purple: Cys4aa-Cys259aa, Cys151aa-Cys164aa, Cys20aa-Cys39aa, Cys62aa-Cys79aa, Cys63aa-Cys80aa, Cys105aa-Cys127aa, Cys244aa-Cys262aa). FIG. 13E shows the presence of a single disulfide bond in LA0591, shown at position Cys303-Cys308. [Figure 14A] Figures 14A-D show the evaluation of hotspots and ligand binding residues in the CTD (carboxy-terminal domain) of LA3490VM protein. Figure 14A shows data demonstrating that the FTMap machine learning based algorithm indicates the number of interactions with hotspot residues and clusters based on high binding energy affinity. [Figure 14B] Figures 14A-14D show the hotspot and ligand-binding residues in the CTD (carboxy-terminal domain) of the LA3490VM protein. Figure 14B shows three-dimensional data demonstrating that the hotspot residues (Arg615, His533, Cys403, Gln486, Thr549, and Gln523) bind to ligands. [Figure 14C]Figures 14A-D show an evaluation of hot spots and ligand binding residues in the CTD (carboxy-terminal domain) of the LA3490VM protein. Figure 14C shows data demonstrating that the surface view of the CTD of LA3490 shows ligand binding to hot spot residues in a deep pocket. [Figure 14D] Figures 14A-D show the evaluation of hot spots and ligand binding residues in the CTD (carboxy-terminal domain) of the LA3490VM protein. Figure 14D shows data demonstrating that structural superposition of the CTD of LA3490 with bovine DNase (3DNI) shows an overlap of His533 (LA3490) with the catalytic residue His134 of bovine DNase. [Figure 15] Figures 15A-C show comparative evidence of hotspot residues based on the full-length FTMap and the C-terminal domains of VM proteins. Figure 15A shows data demonstrating that the AlphaFold-generated structures of full-length VM proteins (LA3490, LA0620, LA1402, and LA1400) show amino acids with high binding energy and multiple interactions with clusters. Figure 15B shows a histogram showing the carboxy-terminal domains (CTDs) of VM proteins (LA0620, LA1400, LA1402, and LA0591) with high binding energy amino acids with multiple interactions with clusters. Figure 15C shows a 3D view of the CTDs of VM proteins showing the ligand binding sites. [Figure 16]Figures 16A-D show PrankWeb and Deepsite-based evaluation of the ligand-binding site of the LA3490VM protein. Figure 16A shows the full-length LA3490 PDB file based on the AlphaFold algorithm submitted to PrankWeb. Fourteen deep ligand-binding pockets were identified by the machine learning-based tool, with pocket 1 with the highest score (18.39) shown in blue with its solvent-accessible surface (SAS) and the location of the evolutionarily conserved pockets shown in the lower panel. Figure 16B shows LA0591 showing five pockets with the highest score of 15.64, with pocket 1 shown in blue with its solvent-accessible surface (SAS) and the location of the evolutionarily conserved pockets shown in the lower panel. The Deepsite machine learning-based algorithm showing the His533 residue in the deep pocket as an interacting amino acid shows the surface view of LA3490 (Figure 16C) and LA0591 (Figure 16D). [Figure 17] FIG. 17 shows a comparative evaluation of hotspot residues and ligand binding sites in LA3490 and bovine DNase. [Figure 18]Figures 18A-F demonstrate the effect of divalent cations on the DNase activity of VM proteins. HeLa DNA (150 ng) was incubated for 30 min with 30 nM purified soluble recombinant VM proteins (t3490, LA3490, LA0620, LA1402, LA1400, and LA0591) in TM buffer (10 mM Tris, pH 7.4) with (Figure 18A), without (divalent cation) MgCl2 (Figure 18B), in the presence of 2 mM ZnCl2 (Figure 18C), in the presence of 3 mM CaCl2 (Figure 18D), or in the presence of CaCl2 + 3 mM MgCl2 (Figure 18E), and samples were subjected to 1% agarose gel electrophoresis. DNase activity by VM protein is indicated by smearing and disappearance of DNA; t3490, t0620 had no such effect (Figure 18F). Docking studies using MGLTools 1.5.7 showed that phosphate and magnesium ions interact with Gln412 (binding energy 0.95 kCal / mol) and Arg615 (binding energy 2.58 kCal / mol). [Figure 19]Figures 19A-C show the DeepMind AlphaFold algorithm derived structure, strategy for cloning, purification, and antigenicity of recombinant His-tagged VM proteins. Figure 19A shows artificial intelligence-based high-resolution structural modeling of (LA3490, LA0620, LA1402, LA1400, and LA0591) using the AlphaFold algorithm. Figure 19B shows a schematic diagram showing the organization of the recombinant mCherry (mC)-fused VM proteins used in this study: t3490, amino acid positions 40aa-147aa (no signal sequence); LA3490 (19aa-639aa), LA0620 (32aa-637aa), LA1402 (28aa-641aa), LA1400 (1aa-573aa), and LA0591 (23aa-313aa). Clones were designed without a signal sequence. LA1400 naturally lacks a signal sequence. The recombinant fusion contains a glycine-serine (Gly4S)3 linker (for flexibility), N- and C-terminal His6 tags (for purification), and an N-terminal thioredoxin. Figure 19C shows that AKTA-purified soluble His-tagged VM proteins (LA3490, t3490, LA0620, LA1402, LA0591, and LA1400) were analyzed by 4±12% SDS-PAGE followed by Coomassie staining. Immunoblot analysis was performed on multiplex gels. Proteins were transferred to nitrocellulose membranes and blots were probed with mouse anti-His monoclonal ALP conjugate (1:2,000 dilution; Santa Cruz Biotechnology, USA). M represents molecular weight marker. [Figure 20] Figure 20 shows the mouse immunization schedule and sample collection. C3H / HeJ mice were immunized with 25 μg of total antigen along with adjuvant (5 μg of GLA-squalene-oil-in-water emulsion) by intramuscular route on days 0, 21, and 42, respectively. Blood was collected before each immunization and before challenge infection, and on the day of necropsy. Control mice were immunized with PBS buffer and adjuvant. After immunization on day 52, mice were infected with live L. interrogans serovar canicola (approximately 1×105 leptospira, LD50<100) by intraperitoneal route. Blood and organs were collected after the following infections. [Figure 21] Figures 21A-C show data demonstrating body weight change, bacterial burden, and inflammatory cytokine response in mice challenged with L. interrogans serovar canicola. Figure 21A shows the body weight (% change) of mice recorded from 0 to 13 days post-infection. Concurrent assessment of clinical status (grooming, eating, drinking water, energy levels) was also observed. GI and G-II mice were sacrificed on days 6 and 5 (‡ and †). Statistical analysis was performed using unpaired two-tailed Mann-Whitney T-test to determine statistical significance of body weight between PBS control and vaccinated groups. p-values: VM mix vs PBS, p=0.0152*: unlabeled VM vs PBS, p=0.0005*: unlabeled VM vs VM mix, p<0.0001****: t3490 vs PBS, p=0.3869, not significant difference. Error bars indicate standard error. Total genomic DNA was extracted from kidney (FIG. 21B) and liver (FIG. 21C) and analyzed by qPCR performed in duplicate using lipL32 primers and SYBER Green probe to quantify Leptospira tissue burden. Statistical analysis was performed using Kruskal-Wallis test and Dunn's multiple comparison test. p<0.0001 was considered significant. Figure 21D shows the inflammatory cytokine response in pooled serum samples from each group: pre- and post-challenge GI (PBS control), G-II (t3490), G-III (mixture of five VM proteins), and G-IV mice (mixture of two VM proteins) were used to measure levels of IL-1β, IL-6, IL-5, IL-10, IFN-γ, TNF-α, KC / GRO by V-PLEX Pro-inflammatory Panel 1 Mouse Kit (Meso Scale Discovery, MD, USA), which is an electrochemiluminescence-based immunoassay. PIB means pre-immune blood. [Figure 22]Figures 22A and 22B show data demonstrating IgG responses to recombinant VM protein immunization. Figure 22A shows data demonstrating that antibody titers against individual VM proteins were measured in triplicate using ELISA in each test group pre-challenge and post-challenge. Each data line represents the mean IgG response for each animal (n=10). Box and Whisker Plots represent antibody titers against t3490, LA3490, LA0620, LA1402, LA1400, and LA0591, respectively. The four test groups include GI: PBS, G-II: t3490, G-III: VM mix, and G-IV: unlabeled VM. Box boundaries indicate median and interquartile range, and whiskers indicate maximum and minimum values. Statistical analysis was performed by t-tests and nonparametric tests, unpaired two-tailed Mann-Whitney test. Values ​​of p<0.0001 were considered significant. Figure 22B shows data from immunized recombinant purified VM proteins run on 4±12% SDS-PAGE followed by transfer to nitrocellulose membrane for Western blot analysis. Membranes were probed with 1:500 pooled sera collected after challenge. PIB means pre-immune blood and served as control. VM proteins were recognized by G-II, G-III, and G-IV sera. Lane 1 shows VM mixture proteins (LA3490, LA0620, LA1402, LA1400, and LA0591) and lane 2 shows unlabeled VM proteins (LA1400 and LA0591). Arrows indicate the expected size of VM proteins. M stands for molecular weight marker. [Diagram 23]Figures 23A-C show data demonstrating in vitro and in vivo recognition of VM protein in Leptospira cell-free lysates by sera from immunized mouse groups. Pathogenic L. interrogans serovars canicola, Lai, Copenhagni, and nonpathogenic L. biflexa serovar Pathoc were grown in log phase in conditioned and nonconditioned EMJH medium induced with 120 mM NaCl for 4 h, and cells were harvested. Cell-free lysates were analyzed by 4-12% SDS-PAGE, followed by transfer to nitrocellulose membranes for Western blot analysis. Figure 23A shows data demonstrating that the membranes were probed with polyclonal LA3490 antibody (1:2,000 dilution) and LipL32 monoclonal antibody (1:10,000), which served as a loading control. Figure 23B shows data demonstrating that another set of membranes was probed with pooled sera (1:100 dilution) collected pre-immune (before bleed) and after challenge with groups I (PBS + adjuvant), II (t3490), III (VM mix), and IV (VM unlabeled). Figures 23A and 23B show that leptospires grown in EMJH medium without added NaCl are represented by negative (-) and leptospires grown in EMJH medium to log phase at which point 120 nM NaCl was added are represented by positive (+). Arrows indicate expression of the 70.29 kDa native VM protein. Figure 23C shows data demonstrating that anti-leptospiral immunoglobulins were generated against serovar canicola after experimental infection of suspected C3H / HeJ mice. Whole cell IgG ELISA was performed using sera from immunized mice pre-bleed and after challenge. Serovar Patoc was used as a negative control. [Figure 24] FIG. 24 shows a table of orthologs and percent amino acid similarity of PF07598 gene family members in group I pathogenic Leptospires. [Diagram 25] FIG. 25 shows data demonstrating the reactivity of a monoclonal supernatant (YUSM001B) with recombinant VM protein. [Figure 26]FIG. 26 shows a table depicting the results of a search performed with five clones from YUMS1B against the target antigen LA0591 at a concentration of 500 nM. [Figure 27] FIG. 27 shows an overview of the reactivity of five clones from YUMS1B. [Figure 28] FIG. 28 shows data demonstrating the reactivity of monoclonal supernatants (YUSM001A, LA1400) with recombinant VM protein. [Figure 29] FIG. 29 shows a table of screening data confirmation. [Diagram 30] FIG. 30 shows a table of IgG quantification data for YUSM001A and YUSM001B mice. [Diagram 31] Figures 31A-C show data demonstrating that the Leptospira PF07598 gene family member, represented here as LA3490, is predicted with high confidence to have two tandemly repeated N-terminal ricin B-like (RBL) lectin domains. Figure 31A shows the visualization of AlphaFold 3D-generated models of full-length LA3490 (Callaway, 2020; Jumper et al., 2020; Senior et al., 2020) and shows that the four globular domains of residues from N-terminus to C-terminus (blue to red) visualized in PyMOL 2.4.0 pymol.org / 2 / . Phyre2 (Protein Fold Prediction Server; sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index) initially predicted with high (>94%) confidence that LA3490, as well as all other virulence modifier (VM) proteins encoded by the PF07598 gene family, contain an N-terminal b-trefoil structure identified as the ricin B domain. Figure 31B shows the ricin B domain (PBD; 2AAI-B, 7aa-129aa). FIG. 31C shows the superposition of 2AAI-B with the N-terminal region of LA3490 (i.e., amino acid positions 40-150) performed using PyMOL™ 2.4.0, showing the structural conservation of RBL1 and the B chain of ricin (RMSD=1.796 Å). [Diagram 32]Figures 32A-D show data demonstrating three-dimensional metric multidimensional scaling (3DMMDS / Galaxy) plots showing (orthologous) VM protein clusters. Among 940 PF07598 family VM proteins analyzed using bios2mds (Pele et al., 2012), clusters were identified and visualized using principal component analysis in R. In addition to typical PF07598 paralogs, 42 naturally occurring deletion mutants lacking the ricin B-like lectin, RBL, subdomain (i.e., containing only the amino-terminal signal sequence and toxin domain) were included. Figure 32A shows the carbohydrate-binding region (CBR), which contains two non-identical tandem RBL subdomains. Figure 32B shows the carboxy-terminal toxin domain (CTD), which encompasses separate transport and DNase subdomains. In both cases, the initial rendering was edited (only superficial changes) to enhance the 3D effect and aid visualization. The coordinates were not altered. Clusters containing VM protein variants found in L. interrogans are highlighted (large spheres) and named using the reference L. interrogans serovar Copenhagenii strain (PMID15028702), L1-130 (UniProtKB) protein ID. Based on percent identity (PID), the orthologous clusters were grouped into three superclusters containing VM protein paralogs (A, n=2; B, n=7; and C, n=4). The following conventions are used for the color key: for species, L. interrogans (ins), L. kirschneri (kri), L. noguchii (nii), etc.; for serotypes, e.g., canicola (CLA), rai (LAI), hard jaw (HJO), etc.; and for strains originating from Sri Lanka, e.g., unknown strain of L. interrogans serotype KW1 (KW1).Figure 32C shows a schematic diagram depicting the theoretical evolutionary history of VM proteins, including lateral transfer (LGT), gene duplication (purple arrow, II) and erosion (solid black arrow), and recombination (blue arrow = donor acquired via lateral gene transfer, I; dashed arrow indicates intragenomic donor from closely related paralogs). Circles represent VM proteins theoretically evolving over time; squares represent the final evolved form at present. Figure 32D shows the domain organization and binding of chimeric leptospiral VM proteins resulting from CBR and CTD domain fusions of paralogs belonging to closely related CBR clusters, such as those related to Q72NW3 (e.g., WP.017856587.1) and Q72TZ4 (e.g., QHH71994.1) (~99.1% PID). These natural VM protein variants are rare (~2%) in L. interrogans and its sister species L. kirschneri and L. noguchii. Chimeric VM proteins generally share a common junction, regardless of the paralog represented. [Diagram 33]Figures 33A-D show data demonstrating that VM protein LA3490 is a bona fide R-type lectin. Figure 33A shows a schematic showing the construction of the recombinant mCherry (mC) fusion proteins used in this study: t3490, amino acid positions 40-147aa (SS, excluding signal sequence); and rLA3490, 19-639aa, also lacking SS. The recombinant fusion also contains a glycine-serine linker, a C-terminal His6 tag (purified), and an N-terminal thioredoxin. RBL and CTD refer to ricin B-like lectin and carboxy-terminal domain, respectively. Figure 33B shows an asialofetuin binding assay demonstrating that truncated (t3490) and full-length (rLA3490) VM proteins bind asialofetuin in a dose-dependent manner, similar to the commercially available ricin B chain. Figure 33C shows a competition assay demonstrating that truncated (t3490), the ricin B domain of another VM protein, LA0620 (t0620), and full-length (rLA3490) compete for the same binding site with recombinant ricin B chain (25 nM and 50 nM). The assay was performed in microtiter plates using an ELISA format. Mouse polyclonal anti-LA3490 and anti-LA0620 antibodies (1:1,000 dilution) were used as primary antibodies, and anti-mouse IgG was used as secondary antibody (used alone as specificity control, labeled 2Ab control). Figure 33D shows that native LA3490 (70.29 kDa) secreted into EMJH culture supernatant by L. interrogans serovar lei in the presence of 120 mM NaCl binds to asialofetuin-conjugated Sepharose beads (AFS). Protein was eluted with 0.5 M lactose. Unbound Sepharose beads incubated with L. interrogans serovar Lye conditioned medium and AFS beads with PBS were used as controls. Assays were performed in triplicate and experiments were repeated twice. Mean absorbance (± SEM) was visualized in GraphPad Prism 8 and p < 0.05 was considered statistically significant. [Diagram 34]Figures 34A-C show data demonstrating Western immunoblots and Limulus amebocyte lysate assays confirming the identity and purity of recombinant protein preparations. Figures 34A and 34B show Western blots of recombinant protein preparations confirming the presence of a single band of the expected size. A, t3490, B. rLA3490. Membranes were probed with anti-His6 antibody (lane 2) and polyclonal anti-LA3490 antibody (lane 3). M-Molecular weight marker. Figure 34C shows that the Limulus amebocyte lysate assay using E. coli LPS as a positive control has no appreciable endotoxin contamination. Data was visualized in GraphPad Prism v8. [Diagram 35]Figures 35A-F show data demonstrating the cytotoxic effect of rLA3490. Figure 35A shows dose-dependent HeLa cell killing induced by r3490 as assessed by trypan blue dye exclusion. Negative controls, t3490, BSA, and no treatment had no such effect. Cell monolayers were treated for 4 hours with graded molar ratio doses (0-904 nM) of LA3490, t3490, and BSA. Data represent the mean ± SD of two independent experiments performed in triplicate for each condition (paired t-test, *p<0.005). Figure 35B shows time-lapse phase contrast images (40 frames, 5 s intervals) demonstrating the cytotoxic effect of HeLa cells after exposure to 45 nM rLA3490 and control. Only in the case of rLA3490 was cell blebbing evident from 1 hour onwards [seen in magnified view (top left and right panels, black arrows)]. Time-lapse images were captured using a Leica DMi8 inverted microscope using a ×40 objective. Scale bar, 10 mm. FIG. 35C shows that actin depolymerization occurs early after rLA3490 treatment. HeLa cell monolayers were incubated with 45 nM rLA3490, t3490, and BSA for up to 1 h. Monolayers were fixed with 4% paraformaldehyde followed by 0.1% Triton X-100 in PBS permeabilization. Monolayers were incubated with phalloidin-Alexa Fluor-488 nm conjugate, washed, and then mounted with ProLongTM Gold Antifade mounting medium containing DAPI. Images were captured at ×40 magnification using a Leica DMi8 confocal microscope [Alexa_488 nm (green), DAPI (blue)]. Untreated HeLa cells were used as control. Scale bar, 20 mm. Figure 35D shows rLA3490-induced HeLa cell death assessed by fluorescent live / dead staining. Negative controls (t3490, BSA, and no treatment) had no such effect. Live / dead staining of HeLa cell monolayers was performed after 4 h exposure to 45 nM rLA3490 (top left panel) and t3490 (bottom left panel). A dramatic reduction in attached cells and a concomitant accumulation of dead cells was observed upon treatment with rLA3490, but not with t3490 or BSA.Images were captured at 10x magnification using a Leica DMi8 inverted microscope. Scale bar, 100 mm. Figure 35E shows quantification of LA3490-induced detachment of HeLa cells from the monolayer after 4 h exposure compared to negative control exposure (t3490, BSA, and no treatment). After 1 h exposure to rLA3490, cells were clearly dissociated from the monolayer. Figure 35F shows quantification of time-dependent HeLa cell death by lactate dehydrogenase release after treatment with rLA3490 compared to the negative control. Groups were compared using one-way t-tests in GraphPad Prism 8 and considered statistically significant at p<0.05; ns, not significant. ** means statistically significant at p=0.0054. [Diagram 36]Figures 36A-C show data demonstrating caspase activation following rLA3490 treatment of HeLa cells. Super-resolution confocal fluorescence microscopy showed that addition of rLA3490-mCherry fusion protein to HeLa cells resulted in activation of caspase 3 associated with internalization of the recombinant protein, as evidenced by cleavage of the caspase 3 recognition sequence / substrate (DEVD) generating green fluorescence. This colocalization also showed prominent morphological changes in the nucleus, unlike the negative control t3490 or untreated HeLa cells (Figures 36A-B). Briefly, cell monolayers were treated with 45 nM recombinant fusion protein for 4 hours. Cells were washed and stained with PBS containing 10 μM NucView® 488 substrate, then mounted with ProLong™ Gold Antifade Mount plus DAPI. Images were captured using a 63x oil immersion objective using appropriate filters (blue, DAPI; green, caspase-3 active cells; red, mCherry fusion). Figure 36B shows a magnified view demonstrating the colocalization of rLA3490 in the nucleus and caspase-3 activation leading to cell apoptosis, unlike t3490. Figure 36C shows data demonstrating the effect of caspase-3 inhibitors and active caspase-3 fluorescence read at 488 / 520 nm (excitation / emission) on a spectrophotometer plate reader. Pretreatment of HeLa cells with caspase-3 inhibitors mitigated the effect of rLA3490 on apoptotic cells. Different treatments were evaluated by t-test in GraphPad Prism 8 and were considered significant if p<0.05, ns=not significant. [Figure 37]Figures 37A-C show data demonstrating surface binding and nuclear localization of rLA3490 in HeLa cells. Fluorescence confocal microscopy demonstrating the binding kinetics of mCherry-rLA3490 and mCherry-t3490 fusion proteins to HeLa cells. Figure 37A shows a 2D view at 60 min, where t3490 is only visible on the cell surface (red). rLA3490 is internalized (red / pink) by 60 min. Figure 37B shows 3D Z-stack and orthogonal images obtained by high-resolution fluorescence confocal microscopy, demonstrating internalization of the mCherry-rLA3490 fusion from 30 min onwards, with nuclear translocation and chromosomal disassembly (indicated by patchy DAPI staining, bottom right) evident within 60 min. t3490 remained on the cell surface at 30 and 60 min. Visualization of treated cells was performed after staining with CellMask™ green plasma membrane stain and mounting using ProLong™ Gold Antifade mounting medium + DAPI nuclear stain. Images were captured using an oil immersion ×100 objective with appropriate filters (blue, DAPI; green, plasma membrane; red, mCherry fusion). Figure 37C shows the time-dependent interaction of mCherry-tagged rLA3490 and t3490 proteins with HeLa cells (surface binding and internalization). Recombinant fusion proteins were quantified using HeLa cell monolayers using fluorescence confocal microscopy (using ImageJ version 1.53 software). Monolayers were exposed to 45 nM recombinant fusion proteins or controls for up to 60 min. Fluorescence intensity of mCherry-t3490 and -LA3490 fusion proteins was measured at 10 min intervals from 0 to 60 min. Data were visualized with GraphPrism 8. [Figure 38]Figures 38A-F show data demonstrating DNase activity of Leptospiral VM proteins. Figure 38A shows the DNase activity of rLA3490 observed upon incubation of 150 ng DNA from HeLa cells in TM buffer containing 3 mM Mg2+ at the indicated doses and times (no DNA degradation occurred in the absence of Mg2+ in the reaction). Samples were subjected to 1% agarose gel electrophoresis. DNase activity of rLA3490 is indicated by smearing and loss of DNA. t3490 had no such effect. Figure 38B shows data demonstrating that other recombinant VM proteins (LA0620, LA1400, LA1402, and LA0591) all have similar DNase activity. FIG. 38C shows DNase activity of rLA3490 on 400 ng of undigested plasmid pET28, showing partial degradation with uncoiling, linearization, and partial degradation, unaffected by t3490 as indicated by white arrows. FIG. 38D shows DNase activity of rLA3490 on linearized plasmid, showing complete loss of linearized and relaxed plasmid, with dose- and time-dependent smearing. L, DNA ladder. FIG. 38E shows quantification of rLA3490 DNase activity using real-time PCR and FAM fluorescent probe. Bovine DNase, 0.02 U / ml, was used as a positive control. Data represent the mean ± SD of three independent experiments. FIG. 38F shows an overlay of AlphaFold-generated CTDs of LA3490 and LA0591, respectively. As shown in Figure 1A, LA3490 represents most of the VM protein with two RBLs, the CTD, and the intervening functional sequence, whereas LA0591 lacks RBL1 and RBL2 but contains the remaining functional sequence. This paralog, represented by LA0591, is present entirely only in the L. interrogans species but absent in other pathogenic group I leptospira. The CTDs of LA3490 and LA0591 are predicted to be highly conserved at the structural level despite the amino acid sequence differences, as indicated by an RMSD value of 0.532 Å. Figures 39A and 39B show that the CTD of LA3490 has conserved active site residues identical to bovine DNaseI.Figure 39A shows a phylogenetic tree based on amino acid sequence alignments from the CTDs of LA3490, bovine DNaseI (Uniport ID: P00639), mouse DNase1 (P49183), rat DNase1 P21704, human DNaseI (P24855), E. coli_CdtB (Q46669), and human endonuclease (P27695) generated using phylogeny.fr. Scale bar, one substitution per amino acid site. Numbers indicate statistical reliability of branching order determined by bootstrap analysis of 100 alternative trees. Figure 39B shows the superposition of the CTDs of LA3490 (368-639aa) and bovine DNase (PDB: 3DNI) predicted structural similarity in the active site of bovine DNaseI with an RMSD of 9.012 Å. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description The present invention relates to Leptospira virulence-modified (VM) proteins, variants and fragments thereof, and vaccine compositions comprising them. It is demonstrated herein that the Leptospira VM protein is immunogenic and therefore can be used as a vaccine or immunogenic composition to treat or prevent leptospirosis in a subject in need thereof.

[0025] In one embodiment, the present invention provides a composition comprising at least one virulence-modifying (VM) protein or a fragment or variant thereof, hi one embodiment, the at least one VM protein is derived from L. interrogans serovar lei, L. interrogans serovar copenhagenii, L. interrogans serovar manilae, or a combination thereof. In one embodiment, the at least one VM protein is selected from the group consisting of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358 , LIC_10639, LIC_12963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or fragments or variants thereof.

[0026] In one embodiment, the invention relates to a vaccine comprising a combination of at least two, three, four, five, or more than five VM proteins. In one embodiment, the vaccine is selected from the group consisting of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_12963, LIC_12970, LIC_12975, LIC_12976, LIC_12977, LIC_12978 ... , LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or a fragment or variant thereof. In one embodiment, the vaccine comprises a combination of LIC_12340 and LIC_12985. In one embodiment, the vaccine comprises a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

[0027] In another embodiment, the composition of the invention comprises a nucleic acid sequence encoding at least one VM protein, fragment, or variant thereof. In another embodiment, the composition of the invention comprises a nucleic acid sequence encoding at least one VM protein, fragment, or variant thereof, such as LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_129 63, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or a fragment or variant thereof.In one embodiment, the composition of the invention comprises any one of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_12963, LIC_1 0695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or fragments or variants thereof. In one embodiment, the nucleotide sequence encodes a combination of LIC_12340 and LIC_12985. In one embodiment, the nucleotide sequence encodes a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

[0028] 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 cellular and / or humoral immunity against a desired antigen.

[0029] In one embodiment, the compositions of the invention function as antigens to induce immunity against Leptospira species bacteria. In certain embodiments, the compositions and methods are used to prevent, treat, and diagnose infections caused by Leptospira. In certain embodiments, the compositions and methods are used to prevent or treat diseases or disorders associated with infections caused by Leptospira, including but not limited to leptospirosis, kidney damage, meningitis, liver failure, respiratory distress, and even death. In one embodiment, the compositions of the invention are vaccines that induce cellular and / or humoral immunity against at least one Leptospira species protein.

[0030] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which 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 preferred methods and materials are described.

[0031] 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.

[0032] 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.

[0033] As used herein, when referring to a measurable value, such as an amount, duration, and the like, "about" is meant to encompass variations of ±20%, or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed methods.

[0034] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. 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).

[0035] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunocompetent cells, or both. Those skilled in the art will appreciate that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Thus, those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response will encode an "antigen" as the term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the 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 two or more genes, and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It is apparent that an antigen can be synthetically generated or derived from a biological sample.

[0036] The term "autologous" as used herein is meant to refer to any material derived from an individual that is later reintroduced into the same individual.

[0037] The term "adjuvant" as used herein is defined as any molecule that enhances the antigen-specific adaptive immune response.

[0038] The term "agent" includes any substance, metabolite, molecule, element, compound, or combination thereof. This includes, but is not limited to, for example, proteins, oligopeptides, small organic molecules, glycans, polysaccharides, polynucleotides, etc. It may be a natural product, a synthetic compound, a compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" can be used interchangeably. Furthermore, a "test agent" or a "candidate agent" is generally a subject agent for use in the assays of the present invention.

[0039] The term "bonded" refers to a direct association between at least two molecules, for example, by covalent bonds, electrostatic interactions, hydrophobic interactions, ionic bonding interactions, and / or hydrogen bonding interactions.

[0040] "CDR" is defined as the complementarity determining region amino acid sequences of an antibody that are the hypervariable regions of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987). There are three heavy and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs, or all three light chain CDRs (or all heavy and all light chain CDRs, as appropriate). The structure and protein folding of an antibody may mean that other residues are considered to be part of the antigen binding region, and will be so understood by those skilled in the art. See, for example, Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

[0041] "Chimeric antibody" refers to a type of engineered antibody that contains naturally occurring variable regions (light and heavy chains) from a donor antibody combined with light and heavy chain constant regions from an acceptor antibody.

[0042] "Contacting" refers to the process of bringing two or more molecules, or two or more components of the same or different molecules, into physical proximity such that they can interact. Molecules or components thereof may be contacted by combining two or more different components that contain the molecules, for example, by mixing two or more solution components, by preparing a solution containing two or more molecules, such as target molecules, candidate molecules, or competitive binding reference molecules, and / or by combining two or more flowing components.

[0043] As used herein, "combination therapy" means that a first agent is administered in combination with another agent. "In combination with" refers to the administration of a therapy in addition to another therapy. Thus, "in combination with" refers to the administration of a therapy before, during, or after an individual is administered another therapy. Such combinations are considered to be part of a single treatment plan or regime.

[0044] As used herein, the term "concurrent administration" means that the administration of a first therapy and a second therapy in a combination therapy overlap in time with each other.

[0045] A "disease" is a health condition in which an animal is unable to maintain homeostasis, and if the disease is not remedied, the animal's health will continue to deteriorate. In contrast, an animal's "disorder" is a health condition in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than it would be in the absence of the disorder. Leaving a disease untreated does not necessarily result in a further deterioration of the animal's health state.

[0046] The term "donor antibody" refers to an antibody (monoclonal and / or recombinant) that contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs to a first immunoglobulin partner, thus providing an altered immunoglobulin coding region and the resulting expressed donor antibody with the antigen specificity and neutralizing activity characteristics characteristic of the donor antibody.

[0047] The term "acceptor antibody" refers to an antibody (monoclonal and / or recombinant) heterologous to the donor antibody, which provides 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 initial immunoglobulin partner. In certain embodiments, a human antibody is the acceptor antibody.

[0048] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0049] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0050] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains 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 vectors 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.

[0051] The term "heavy chain antibody" or "multiple heavy chain antibodies" as used herein includes immunoglobulin molecules obtained from Camelid species by immunization with a peptide and subsequent isolation of serum, or by cloning and expression of nucleic acid sequences encoding such antibodies. The term "heavy chain antibody" or "multiple heavy chain antibodies" further encompasses immunoglobulin molecules isolated from animals with heavy chain disease or prepared by cloning and expression of a VH (variable heavy chain immunoglobulin) gene from an animal.

[0052] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If both positions of two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage 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 out of 10 positions of two sequences are matching or homologous, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, the comparison is performed when the two sequences are aligned to obtain maximum homology.

[0053] "Humanized antibody" refers to a type of engineered antibody that has CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule derived from one (or more) human immunoglobulins. In addition, framework support residues may be modified to maintain 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 selected from conventional databases, such as the KABAT database, the Los Alamos database, and the Swiss Protein Database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by homology to the framework regions (amino acid basis) of the donor antibody may be suitable to provide heavy chain constant regions and / or heavy chain variable framework regions for insertion of the donor CDRs. A suitable acceptor antibody capable of donating the light chain constant region or the variable framework region can be selected in a similar manner. It should be noted that the heavy and light chains of the acceptor antibody do not have to be derived from the same acceptor antibody. The prior art describes several methods for producing such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951).

[0054] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory tract and genitourinary system. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response of 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 an immunoglobulin with no known antibody function, but which may function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0055] The term "immune response" as used herein includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cytotoxicity, and B cell responses, e.g., antibody production. Additionally, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral response) and activation of cytokine-responsive cells (e.g., macrophages). Immune cells involved in immune responses include lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 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, e.g., macrophages, eosinophils, mast cells, basophils, granulocytes.

[0056] As used herein, an "inhibitory effective amount" is a detectable (e.g., measurable) amount of inhibition of activity. In some cases, the activity is the ability to bind to another component.

[0057] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide is "isolated" if it is partially or completely separated from the coexisting materials of the natural state. An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0058] As used herein, "mutation" refers to a change in a nucleic acid or polypeptide sequence compared to a reference sequence (preferably a naturally occurring normal or "wild type" sequence), and refers to translocations, deletions, insertions, and substitutions / point mutations. As used herein, "mutant" refers to either a nucleic acid or protein that contains a mutation.

[0059] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intradermal (id) injection, or infusion techniques.

[0060] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof that are amenable to the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0061] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a particular 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 that antigen from one or more species. However, such cross-species reactivity does not, in itself, change the specific classification of the antibody. 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, in itself, change the specific classification of the antibody. In some cases, the terms "specific binding" or "specifically bind" are used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody recognizes and binds to a particular protein structure, rather than proteins in general. If an antibody is specific for epitope "X", then in a reaction involving labeled "X" and the antibody, the presence of a molecule containing epitope X (or unlabeled free A) will reduce the amount of labeled X that binds to the antibody.

[0062] The term "synthetic antibody" as used herein refers to an antibody produced using recombinant DNA techniques, such as an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0063] The term "treatment" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppressing, reducing, ameliorating, or eradicating the disease symptoms.

[0064] The term "therapeutically effective amount" refers to an amount of the compound of interest that elicits the biological or clinical response of a tissue, system, or subject that is desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of the compound that is sufficient when administered to prevent or reduce to some extent the onset of one or more 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, as well as the age, weight, etc., of the subject being treated.

[0065] As the term is used herein, "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0066] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0067] Ranges: Throughout this disclosure, various aspects of the invention can be expressed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, 6). This applies regardless of the breadth of the range.

[0068] explanation The present invention relates to the Leptospira virulence-modifying (VM) protein encoded by the PF07598 Leptospira gene family, as well as variants and fragments thereof.

[0069] In some embodiments, the present invention provides compositions comprising a Leptospiral VM protein, a variant thereof, or a fragment thereof. In some embodiments, the composition comprises a fragment of a Leptospiral VM protein. For example, in one embodiment, the composition comprises a fragment of a Leptospiral VM protein, the fragment comprising the C-terminal domain (herein referred to as the DNase domain) of the Leptospiral VM protein that contains the nuclease activity.

[0070] In one embodiment, the composition comprises a fusion protein comprising a first domain comprising a Leptospira VM protein, a variant thereof, or a fragment thereof. In one embodiment, the fusion protein comprises a second domain. In one embodiment, the second domain is a targeting domain, which directs the fusion protein to a specific cell or tissue of interest. For example, in one embodiment, the targeting domain comprises an antibody, an antibody fragment, or a peptide that specifically binds to an antigen (e.g., a 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 visualization of the fusion protein.

[0071] In one embodiment, the present invention provides an isolated nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof. In some embodiments, the isolated nucleic acid molecule comprises a DNA, cDNA, RNA, or mRNA encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof. In one embodiment, the isolated nucleic acid molecule encodes a fusion protein comprising a Leptospiral VM protein, a variant thereof, or a fragment thereof.

[0072] In one embodiment, the composition comprises an immunological composition comprising (a) a Leptospiral VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof. As demonstrated herein, in certain embodiments, the Leptospiral VM protein, a variant thereof, or a fragment thereof induces a protective immune response capable of treating or preventing 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 the genus Leptospira) modified to express a Leptospiral VM protein, a variant thereof, or a fragment thereof. In one embodiment, the bacterium is attenuated in that it has reduced virulence, but is still capable of inducing a protective immune response. The composition is useful not only as a prophylactic therapeutic for immune protection, but also as a therapeutic for the treatment of ongoing infections, diseases, or disorders.

[0073] In one embodiment, the invention relates to a method of inducing cell death or damage, comprising administering to a cell a composition comprising: (a) a Leptospiral VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof. For example, as demonstrated herein, a Leptospiral VM protein is a cytotoxic protein. In one embodiment, the method comprises administering the composition to a tumor, thereby inducing tumor cell death or damage.

[0074] The present invention also provides a method of preventing, inhibiting, and treating infections caused by bacteria of the genus Leptospira in a subject in need thereof. In one embodiment, the method of the present invention induces immunity against Leptospira in a subject by generating an immune response against Leptospira VM protein in the subject. In a particular embodiment, the method induces broad immunity across the genus Leptospira. In one embodiment, the method of the present invention induces production of VM protein-specific antibodies in a subject. In one embodiment, the method of the present invention prevents Leptospira-associated pathologies, such as leptospirosis (also known as Weil's disease), in a subject in need thereof. In one embodiment, the method of the present invention comprises administering to a subject a composition comprising: (a) a Leptospira VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospira VM protein, a variant thereof, or a fragment thereof.

[0075] composition The present invention provides compositions comprising or encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof.

[0076] In one embodiment, the composition comprises a leptospiral VM protein from L. interrogans serovar lei, L. interrogans serovar copenhagenii, L. interrogans serovar manilae, or a combination thereof. In one embodiment, the at least one VM protein is selected from the group consisting of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_12715, LIC_1284 ... , LIC_10639, LIC_12963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or fragments or variants thereof.

[0077] In one embodiment, the invention relates to a vaccine comprising a combination of at least two, three, four, five, or more than five VM proteins. In one embodiment, the vaccine is selected from the group consisting of LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_12963, LIC_12970, LIC_12975, LIC_12976, LIC_12977, LIC_12978 ... , LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or a fragment or variant thereof. In one embodiment, the vaccine comprises a combination of LIC_12340 and LIC_12985. In one embodiment, the vaccine comprises a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

[0078] In one embodiment, the present invention relates to a toxoid vaccine comprising at least one, two, three, four, five, or more than five VM proteins. LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC _12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_12963, LIC_1 0695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or fragments or variants thereof. In one embodiment, the toxoid vaccine comprises a combination of LIC_12340 and LIC_12985. In one embodiment, the toxoid vaccine comprises a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

[0079] In one embodiment, a composition or vaccine of the invention comprises a VM protein comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, or a fragment or variant thereof. In one embodiment, a composition or vaccine of the invention comprises a combination of VM proteins comprising the amino acid sequences of SEQ ID NO:10 and SEQ ID NO:12. In one embodiment, a composition or vaccine of the invention comprises a combination of VM proteins comprising the amino acid sequences of SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:8.

[0080] In certain embodiments, the composition comprises a fragment of a Leptospiral VM protein. For example, in one embodiment, the composition comprises the DNase domain of a Leptospiral VM protein.

[0081] In various embodiments, the present invention provides proteins described elsewhere herein, or fragments, homologs, mutants, variants, derivatives, or salts of the proteins, wherein the activity of various domains of the Leptospiral VM protein (e.g., immunogenic activity, cytotoxic activity, or activity related to the mechanism of action of the Leptospiral VM protein) is retained.

[0082] The proteins or peptides of the present invention can be prepared using known techniques. For example, the proteins can be synthetically prepared using either recombinant DNA technology or chemical synthesis. The proteins of the present invention can be synthesized individually or as a longer protein composed of two or more proteins. The proteins of the present invention can be isolated, i.e., substantially free of other native host cell proteins and fragments thereof.

[0083] The proteins of the invention may contain modifications such as glycosylation, non-glycosylation, side chain oxidation, or phosphorylation, so long as the modifications do not destroy the immune activity of the protein. Other modifications include, for example, the incorporation of D-amino acids or other amino acid mimetics that can be used to extend the serum half-life of the protein.

[0084] The proteins of the invention can be modified by replacing an amino acid with another amino acid that conserves the properties of the amino acid side chain (a process known as conservative amino acid substitution). Examples of amino acid side chain properties 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 that share the following common functional groups or features: aliphatic side chains (G, A, V, L, I, P); hydroxyl-containing side chains (S, T, Y); sulfur atom-containing side chains (C, M); carboxylic acid and amide containing side chains (D, N, E, Q); base containing side chains (R, K, H); aromatic containing side chains (H, F, Y, W). Note that the letters in parentheses indicate the one-letter code of the amino acid. As used herein, X represents any amino acid.

[0085] The present invention should also be construed to encompass "mutants," "derivatives," and "variants" of the proteins of the invention (or the DNA encoding same), which are polypeptides altered at one or more amino acids (or, when referring to nucleotide sequences encoding same, altered at one or more base pairs) and the resulting proteins (or DNA) are not identical to the sequences set forth herein, but have the same biological properties as the proteins disclosed herein.

[0086] The present invention should also be construed to include any form of protein variant having substantial homology to the amino acid sequences disclosed herein. In one embodiment, the 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 the amino acid sequences disclosed herein.

[0087] The present invention should also be construed to include any form of fragment having a substantial length of the amino acid sequence disclosed herein, in one embodiment, the 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 the amino acid sequence disclosed herein.

[0088] The present invention should also be construed to include any form of fragment of a protein variant having both substantial homology and substantial length with the 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 the 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 the amino acid sequence disclosed herein.

[0089] Alternatively, the protein may be produced by recombinant means, or by cleavage from a longer protein. The protein can be confirmed by amino acid analysis or sequencing.

[0090] Variants of the proteins according to the invention may be (i) those in which one or more amino acid residues have been replaced with a conserved or non-conserved amino acid residue (e.g., a conserved amino acid residue), such that the replaced amino acid residue may or may not be encoded by the genetic code, (ii) those in which one or more modified amino acid residues are present, e.g., those residues modified by attachment of a substituent group, (iii) those in which the protein comprises an alternative splice variant of a protein or domain described herein, (iv) a fragment of a protein or domain described herein, and / or (v) those in which the protein has been fused to a leader or secretion sequence, or a sequence used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include proteins or peptides generated by proteolytic cleavage (including multiple site proteolysis) of the original sequence. Variants may be subject to post-translational or chemical modifications. Such variants are considered to be within the scope of one of skill in the art from the teachings herein.

[0091] As known in the art, the "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide and its conserved amino acid substitutes with the sequence of a second peptide. A variant is defined to include peptide sequences that differ from the original sequence, for example, that differ from the original sequence by less than 40% of the residues per target segment, that differ from the original sequence by less than 25% of the residues per target segment, that differ from the original sequence by less than 10% of the residues per target segment, or that differ from the original sequence by only a few residues per target segment, and at the same time, that are sufficiently homologous to the original sequence and that the function of the original sequence is preserved. 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 can be determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences can also be determined 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)).

[0092] The proteins of the present invention may or may not be post-translationally modified. For example, post-translational modifications within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding, and proteolytic processing. Some modifications or processing events require the introduction of additional biological machinery. For example, processing events such as signal peptide cleavage and core glycosylation are examined by adding dog microsomal membranes or Xenopus egg extracts (US Pat. No. 6,103,489) to a standard translation reaction. The polypeptides or proteins of the present invention may be phosphorylated using conventional methods, such as those described in Reedijk et al. (The EMBO Journal 11(4):1365, 1992).

[0093] Proteins of the invention may include unnatural amino acids formed by post-translational modification or by the introduction of unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during translation of a polypeptide.

[0094] The proteins of the present invention may be conjugated to other molecules, such as polyethylene glycol (PEG). This is accomplished by inserting cysteine ​​mutations or unnatural amino acids that can be modified with chemically reactive PEG derivatives. In one embodiment, the protein is conjugated to another protein to prepare a fusion protein. This can be accomplished, for example, by synthesis of N- or C-terminal fusion proteins, so long as the resulting fusion protein retains the function of the protein described herein.

[0095] Cyclic derivatives of the proteins of the invention are also part of the invention. Cyclization allows proteins to bind to other molecules and adopt a more favorable conformation. Cyclization can be achieved using techniques known in the art. For example, a disulfide bond can be formed between two appropriately spaced components with free sulfhydryl groups, or an amide bond can be formed between an amino group of one component and a carboxyl group of another component. Cyclization can also be achieved using azobenzene-containing amino acids as described in Ulysse, L., et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The bond-forming component can be the side chain of an amino acid, a non-amino acid component, or a combination of the two. In one embodiment of the invention, the cyclic peptide can include a beta turn at the correct position. A beta turn can be introduced into the peptides of the invention by adding the amino acid Pro-Gly at the correct position.

[0096] It may be desirable to generate cyclic proteins that are more flexible than those containing peptide bonds as described above. More flexible proteins can be prepared by introducing cysteines at the right and left positions of the polypeptide and forming disulfide bridges between the two cysteines. The two cysteines are positioned so as not to distort the beta sheets and turns. The protein is more flexible due to the length of the disulfide bonds and the fewer number of hydrogen bonds in the beta sheet portion. The relative flexibility of cyclic proteins can be determined by molecular dynamics simulations.

[0097] The present invention also relates to fusion proteins. For example, in one embodiment, the fusion protein comprises a first domain comprising Leptospira VM protein, its variant, or its fragment. In one embodiment, the fusion protein comprises a second domain. In one embodiment, the second domain is a targeting domain, which 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., a 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 visualization of the fusion protein.

[0098] In one embodiment, the fusion protein comprises a targeting domain that can direct the resulting protein to a desired cellular component or cell type or tissue. Chimeric or fusion proteins can also comprise additional amino acid sequences or domains. Chimeric or fusion proteins are recombinant in the sense that the various components are derived from different sources and therefore are not found together in nature (i.e., heterologous).

[0099] In one embodiment, the targeting domain can be a transmembrane domain, a membrane-binding domain, or a sequence that directs the protein to bind to, for example, vesicles or cell surfaces.In one embodiment, the targeting domain can target the protein to a specific cell type or tissue.For example, the targeting domain can be a cell surface ligand, or an antibody against a cell surface antigen of the target tissue.The targeting domain can target the protein of the present invention to cellular components.

[0100] In one embodiment, the targeting domain may comprise an antibody or an antibody fragment thereof. Antibodies may exist in various forms in which the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, single domain antibody fragments (sdAb), single chain antibodies (scFv), 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). In one embodiment, the targeting domain of the composition of the present invention comprises an antibody fragment. In one embodiment, the targeting domain comprises an antibody fragment comprising scFv.

[0101] The VM domain-containing fusion molecules of the present invention can be generated to react with any desired antigen of interest or fragment thereof, including, but not limited to, tumor antigens, bacterial antigens, viral antigens, or autoantigens. In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder, such as cancer. In certain embodiments, 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, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0102] The antigens discussed herein are included merely as examples: this list is not intended to be exhaustive, and further examples will be readily apparent to one of skill in the art.

[0103] The proteins of the invention can be synthesized by conventional techniques. For example, the proteins can be synthesized by chemical synthesis using solid phase peptide synthesis. These methods use either solid phase or liquid phase synthesis methods (for example, for solid phase synthesis techniques, see JM Stewart, and JD Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford Ill. (1984) and G. Barany and RB Merrifield, The Peptides: Analysis Synthesis, Biology editors E. Gross and J. Meienhofer Vol. 2 Academic Press, New York, 1980, pp. 3-254; and for classical solution synthesis, see 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). As an example, the polypeptides of the invention can be synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) solid phase chemistry to directly incorporate phosphothreonine as the N-fluorenylmethoxy-carbonyl-O-benzyl-L-phosphothreonine derivative.

[0104] N- or C-terminal fusion proteins comprising the peptides or proteins of the invention linked to at least one other molecule can be prepared by recombinant techniques by fusing the N- or C-terminus of the peptide or protein and the sequence of a selected protein or selectable marker having a desired biological function. The resulting fusion protein contains a Leptospira VM protein, a variant thereof, or a fragment thereof fused to a selected protein or marker protein as described herein. Examples of proteins that can be used to prepare fusion proteins include immunoglobulins and regions thereof, glutathione-S-transferase (GST), hemagglutinin (HA), and truncated myc.

[0105] The proteins of the invention can be developed using biological expression systems. These systems allow the generation of large libraries of random sequences and screening of these libraries for sequences that bind to specific proteins. Libraries can be made by cloning synthetic DNA encoding 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 can also be constructed by simultaneous synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).

[0106] The proteins of the present invention can be converted into pharmaceutical salts by reaction with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, and the like, 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, benzenesulfonic acid, toluenesulfonic acid, and the like.

[0107] The present invention further encompasses fusion proteins produced by recombinantly fusion or chemically linking (including both covalent and non-covalent linkages) a protein of the invention or a fragment thereof to a heterologous protein (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 a polypeptide). The fusion need not necessarily be direct, but may occur through a linker sequence.

[0108] In one example, the protein of the present invention or a fragment thereof is a fusion protein that can be fused to sequences derived from various types of immunoglobulins. For example, the polypeptide of the present invention can be fused to the constant region (e.g., hinge, CH2, and CH3 domains) of human IgG or IgM molecules, for example, as described herein, to make the fusion protein or a fragment thereof more soluble and stable in vivo. In another embodiment, such a fusion protein can be administered to a subject to inhibit the interaction between a ligand and its receptor in vivo. Inhibition of such interaction blocks or suppresses signal transduction that causes a specific cellular response.

[0109] In one embodiment, a fusion protein comprises a polypeptide of the invention fused at its N-terminus to a heterologous signal sequence. For example, the signal sequence naturally found in the protein of the invention can be replaced by a signal sequence from a heterologous source. A variety of 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. Examples of prokaryotic heterologous signal sequences include 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, NJ). 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).

[0110] In another embodiment, the proteins of the invention can be fused to a tag sequence, such as a hexahistidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. Hexahistidine provides for convenient purification of the fusion protein, as described, for example, in Gentz, et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824. 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 particularly useful for purification of recombinantly produced proteins of the invention.

[0111] In one embodiment, the protein of the present 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. The fluorescent tag can also be photoconvertible, such as kindling red fluorescent protein (KFP-Red), PS-CFP2, Dendra2, CoralHue Kaede, and CoralHue Kikume. However, the present invention should not be limited to a particular label. Rather, any detectable label can be used to tag the expressed protein.

[0112] In some embodiments, the present invention provides compositions comprising lipid nanoparticles (LNPs) or liposomes conjugated or encapsulated with at least one VM protein or peptide of the present invention. In one embodiment, the composition comprises a combination of two or more LNPs that encapsulate a combination of two or more VM proteins. In some cases, the LNPs enhance cellular uptake of the VM proteins.

[0113] In some embodiments, the composition comprises a scaffold, such as a tissue engineering scaffold, that comprises a nucleic acid molecule that encodes growth factor.For example, in one embodiment, the scaffold comprises LNP that encapsulates a nucleic acid molecule that encodes growth factor.In one embodiment, the scaffold comprises a cell or cell population that comprises a nucleic acid molecule that encodes growth factor.In some embodiments, the scaffold comprises a hydrogel, an electrospun scaffold, etc., that comprises a biopolymer, a synthetic polymer, or a combination thereof.

[0114] The present invention also provides isolated nucleic acid molecules encoding the proteins described herein. Thus, in one embodiment, a composition of the invention comprises an isolated nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof.

[0115] In one embodiment, the isolated nucleic acid molecule encodes a protein or toxoid having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, or a fragment or variant thereof. In one embodiment, the isolated nucleic acid molecule encodes a fragment comprising the DNase domain of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12. In one embodiment, the isolated nucleic acid molecule encodes at least two, at least three, at least four, at least five, or more than five VM proteins. In one embodiment, the isolated nucleic acid molecule encodes LIC_12340 and LIC_12985. In one embodiment, the isolated nucleic acid molecule encodes SEQ ID NO:10 and SEQ ID NO:12. In one embodiment, the isolated nucleic acid molecule comprises SEQ ID NO:9 and SEQ ID NO:11. In one embodiment, the isolated nucleic acid molecule encodes LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402. In one embodiment, the isolated nucleic acid molecule encodes SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. In one embodiment, the isolated nucleic acid molecule comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11.

[0116] In one embodiment, the present invention relates to a combination of at least two isolated nucleic acid molecules encoding a combination of at least two, at least three, at least four, at least five, or more than five VM proteins or toxoids. In one embodiment, the at least two isolated nucleic acid molecules encode LIC_12340 and LIC_12985. In one embodiment, the composition comprises a combination of at least two isolated nucleic acid molecules encoding a combination of SEQ ID NO:10 and SEQ ID NO:12. In one embodiment, the composition comprises a combination of at least two isolated nucleic acid molecules comprising SEQ ID NO:9 and SEQ ID NO:11. In one embodiment, the at least two isolated nucleic acid molecules encode LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402. In one embodiment, the composition comprises a combination of at least two isolated nucleic acid molecules encoding a combination of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:12. In one embodiment, the composition comprises a combination of at least two isolated nucleic acid molecules comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11.

[0117] The one or more isolated nucleic acid molecules can include any type of nucleic acid, including, but not limited to, DNA, cDNA, and RNA.For example, in one embodiment, the composition includes, for example, an isolated cDNA molecule that encodes a protein or a functional fragment thereof.In one embodiment, the composition includes an isolated RNA molecule that encodes a protein or a functional fragment thereof.

[0118] Nucleic acid sequences include both DNA sequences that are transcribed into RNA and RNA sequences that are translated into proteins. In another embodiment, the nucleic acid sequences of the present invention are deduced from the amino acid sequences of the proteins of the present 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 protein.

[0119] Additionally, the present invention encompasses isolated nucleic acid molecules encoding proteins having substantial homology to the VM proteins disclosed herein, hi some embodiments, the present invention encompasses isolated nucleic acid molecules encoding proteins 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 identity to the amino acid sequence of a protein disclosed herein. In some embodiments, the nucleic acid sequences encoding the proteins of the present invention are "substantially homologous" to the nucleic acid sequences described herein, i.e., 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.

[0120] It should be clearly understood that the scope of the present invention encompasses shorter and longer protein and nucleic acid molecules, as well as protein and nucleic acid molecule analogs having one or more amino acid or nucleic acid substitutions, as well as homologs, analogs, variants, fragments, derivatives, and salts, including amino acid or nucleic acid derivatives known in the art, unnatural amino acids or nucleic acids, and synthetic amino acids or nucleic acids, provided that these modifications retain the activity of the original molecule.Specifically included are any active fragments, as well as extensions, conjugates, and mixtures of active protein and nucleic acid molecules and are disclosed herein in accordance with the principles of the present invention.

[0121] The present invention includes any isolated nucleic acid sequences that are homologous to the nucleic acid sequences described and referenced herein, provided that these homologous nucleic acid sequences encode proteins having the biological activity of the proteins disclosed herein.

[0122] Those skilled in the art will understand that the nucleic acid sequences of the present invention encompass RNA or DNA sequences encoding the proteins of the present invention and any modified forms thereof, including chemical modifications of DNA or RNA that make the sequences more stable when unbound or bound to a cell. Chemical modifications of nucleotides can also be used to increase the efficiency with which a nucleic acid sequence is taken up into a cell or expressed in a cell. Any combination of modifications of nucleic acid sequences is contemplated in the present invention.

[0123] Additionally, any number of methods can be used for the production of mutant, derivative, or variant forms of the proteins of the invention by using recombinant DNA methods known in the art, for example, as described in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York). Methods for introducing amino acid changes into a polypeptide by modifying a DNA sequence encoding one or more polypeptides are known in the art and described in these and other publications.

[0124] The nucleic acid molecules of the invention can be modified to improve their stability in serum or growth medium for cell culture. Modifications can be made to enhance the stability, functionality, and / or specificity of the nucleic acid molecules of the invention and minimize their immunostimulatory properties. For example, to enhance stability, the 3' residues can be stabilized against degradation, e.g., selected so that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogs (e.g., substitution of uridine by 2'-deoxythymidine) is tolerated and does not affect the function of the molecule.

[0125] In one embodiment of the invention, the nucleic acid molecule may comprise at least one modified nucleotide analogue, for example, the termini may be stabilized by incorporating modified nucleotide analogues.

[0126] Non-limiting examples of nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester groups connected to adjacent ribonucleotides are replaced by modified groups, such as, for example, phosphothioate groups. 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, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0127] Another example of modification is nucleobase-modified ribonucleotide, i.e., ribonucleotide containing at least one non-natural nucleobase instead of a natural nucleobase. Bases can also 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, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine. The above modifications may be combined.

[0128] In some cases, 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, the nucleic acid molecule of the present invention can be enhanced in resistance to nucleases. To increase nuclease resistance, the nucleic acid molecule can comprise, for example, 2'-modified ribose units and / or phosphorothioate linkages. For example, the 2' hydroxyl group (OH) can be modified or replaced with many different "oxy" or "deoxy" substituents. To increase nuclease resistance, the nucleic acid molecule of the present invention can comprise 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. Locked nucleic acids (LNA), ethylene nucleic acids (ENA), such as 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 targets.

[0129] In one embodiment, the nucleic acid molecule comprises a 2'-modified nucleotide, such as 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'-ON-methylacetamide (2'-O-NMA). In one embodiment, the nucleic acid molecule comprises at least one 2'-O-methyl modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule comprise a 2'-O-methyl modification.

[0130] Nucleic acid agents discussed herein include otherwise unmodified RNA and DNA, as well as RNA and DNA modified, for example to improve efficacy, and polymers of nucleoside substitutes. Unmodified RNA refers to molecules in which the building blocks of nucleic acid, i.e., sugar, base, and phosphate moieties, are essentially the same as those found in nature, e.g., naturally occurring in the human body. In the art, rare or unusual but naturally occurring RNA has been referred to as modified RNA. See, for example, Limbach et al. (Nucleic Acids Res., 1994, 22:2183-2196). Such rare or unusual RNAs are often referred to as modified RNAs, typically the result of post-transcriptional modifications, and are included within the scope of the term unmodified RNA as used herein. Modified RNA, as used herein, refers to molecules in which one or more of the building blocks of nucleic acid, i.e., sugar, base, and phosphate moieties, are different from those found in nature, e.g., different from those found in the human body. These are referred to as "modified RNAs," but of course, because they are modified, they also include molecules that are not strictly RNA. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct, thereby allowing the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with a ribophosphate backbone (e.g., an uncharged mimic of a ribophosphate backbone).

[0131] Modifications of the nucleic acids of the invention can be at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobase.

[0132] The invention also includes vectors into which an isolated nucleic acid of the invention has been inserted. The art is replete with suitable vectors useful in the present invention.

[0133] Briefly summarized, expression of a natural or synthetic nucleic acid encoding a protein is typically achieved by operably linking the nucleic acid encoding the protein or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector used is suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors include transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0134] The vector of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0135] The isolated nucleic acids of the present invention can be cloned into many types of vectors. For example, the nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0136] Furthermore, vectors can be provided to cells in the form of viral vectors. Viral vector technology is known in the art and described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors include an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0137] Numerous virus-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 into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0138] For example, vectors derived from retroviruses, such as lentiviruses, are suitable tools to achieve long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of being less immunogenic. In one embodiment, the composition comprises a vector derived from an adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become a powerful gene delivery tool for the treatment of various diseases. AAV vectors have many characteristics that make them ideally suited for gene therapy, including lack of toxicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. By selecting the appropriate combination of AAV serotype, promoter, and delivery method, the expression of a particular gene contained within the AAV vector can be specifically targeted to one or more types of cells.

[0139] In some embodiments, the vector also includes conventional control elements operably linked to the transgene in a manner that allows transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences include both expression control sequences adjacent to 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 and termination sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and, if necessary, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0140] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0141] 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 operably linked to it. Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences can also be used, including but not limited to Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0142] Enhancer sequences found on vectors also regulate the expression of genes contained in the vector. Enhancers usually bind to protein factors to enhance the transcription of genes. Enhancers may be located upstream or downstream of the gene they regulate. Enhancers may also be tissue-specific to enhance transcription in certain cell or tissue types. In one embodiment, the vector of the present invention comprises one or more enhancers to promote the transcription of genes present in the vector.

[0143] To assess protein expression, the expression vector introduced into the cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the cell population that is desired to be transfected or infected via the viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0144] Reporter genes are used to identify potentially transfected cells or to evaluate the function of regulatory sequences. Generally, reporter genes are genes that encode a polypeptide that is not present or expressed in the recipient organism or tissue and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are known and may be prepared using known techniques or may be commercially available. Generally, the construct with the minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to the reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0145] Methods for introducing and expressing genes into cells are known in the art. In the case of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0146] Physical methods for introducing peptides or proteins into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0147] Biological methods for introducing peptides or proteins of interest into host cells include the use of DNA and RNA vectors. Viral vectors, 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 lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patents Nos. 5,350,674 and 5,585,362.

[0148] Chemical means for introducing peptides or proteins into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0149] When 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 nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. A lipid-associated nucleic acid 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 attached to both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained as a suspension in lipids, contained or complexed in micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles, or in "collapsed" structures. They may also simply be dispersed in a solution and form aggregates that are not uniform in size or shape. Lipids are fatty substances and may be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0150] Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO. Dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY). Cholesterol ("Choi") is available from Calbiochem-Behring. Dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available 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 because it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes are characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as a heterogeneous collection of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0151] Regardless of the method used to introduce exogenous nucleic acid into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays known to those skilled in the art, such as, for example, Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays that detect the presence or absence of specific polypeptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein, to identify agents that fall within the scope of the invention.

[0152] In one embodiment, the present invention provides a delivery vehicle comprising a protein or a nucleic acid molecule encoding a protein. Exemplary delivery vehicles include, but are not limited to, microspheres, microparticles, nanoparticles, polymersomes, liposomes, and micelles. For example, in some embodiments, the delivery vehicle is loaded with a protein or a nucleic acid molecule encoding a protein. In some embodiments, the delivery vehicle provides controlled, delayed, or continuous release of the loaded cargo. In some embodiments, the delivery vehicle comprises a targeting moiety that targets the delivery vehicle to a treatment site.

[0153] In one embodiment, the present invention provides an implantable scaffold or device comprising a protein or a nucleic acid molecule encoding the protein. For example, in some embodiments, the present invention provides tissue engineering scaffolds, including but not limited to hydrogels, electrospun scaffolds, polymer matrices, etc., comprising a protein or a nucleic acid molecule encoding the protein within or on the scaffold.

[0154] In some embodiments, the present invention provides compositions comprising lipid nanoparticles (LNPs) or liposomes that bind to or encapsulate at least one nucleic acid molecule encoding at least one VM protein or peptide of the present invention. In one embodiment, the composition comprises a combination of two or more LNPs that encapsulate a combination of two or more nucleic acid molecules encoding at least two or more VM proteins or peptides of the present invention. In one embodiment, the nucleic acid molecule comprises an mRNA molecule encoding at least one VM protein or peptide of the present invention. Thus, in some embodiments, the present invention provides at least one LNP or liposome that binds to or encapsulates at least one mRNA molecule encoding at least one VM protein. In one embodiment, the present invention provides a combination of LNPs or liposomes that bind to or encapsulate mRNA molecules encoding LIC_12340 and LIC_12985. In one embodiment, the present invention provides a combination of LNPs or liposomes that bind to or encapsulate mRNA molecules encoding LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

[0155] In certain embodiments, the present invention encompasses compositions comprising polypeptides, nucleotides, vectors, bacteria, and vaccines that induce or enhance an immune response when administered to a subject. In certain examples, the compositions induce an immune response against bacteria of the genus Leptospira, such as an immune response against Leptospira VM proteins. Furthermore, when the compositions are administered to a subject, they induce an immune response that serves to protect the inoculated subject against conditions associated with Leptospira infection.

[0156] In one embodiment, the present invention provides compositions useful as immunomodulators, for example, to stimulate immune responses and prevent Leptospira-associated pathologies. In various embodiments, the immunomodulator comprises (a) a Leptospira VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospira VM protein, a variant thereof, or a fragment thereof. In one embodiment, the immune response is not harmful to the host, so that the compositions of the present invention are useful as vaccines. In one embodiment, the immunomodulator is administered in combination with an adjuvant. In one embodiment, the adjuvant is glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water nanoemulsion (SE), referred to as GLA-SE adjuvant. In another embodiment, the immunomodulator is administered in the absence of an adjuvant.

[0157] In some embodiments, the compositions are used as immunostimulants to induce or enhance the production of specific antibodies, hi certain aspects, the immunostimulants protect against Leptospira-induced pathologies.

[0158] In one embodiment, the composition comprises a bacterium comprising (a) a Leptospiral VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof. For example, in one embodiment, the composition comprises a bacterium of the genus Leptospira comprising: (a) a Leptospiral VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof. In one embodiment, the composition comprises a bacterium that is not of the genus Leptospira, the bacterium comprising (a) a Leptospiral VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof.

[0159] Bacteria containing a nucleotide sequence encoding a Leptospiral VM protein, variants thereof, or fragments thereof can be generated using any method known in the art, including, but not limited to, allelic exchange and site-directed mutagenesis.

[0160] Any bacterium or bacterial strain having at least one nucleotide sequence encoding a Leptospiral VM protein, its variants, or its fragments can be selected and used according to the present invention. In one embodiment, naturally occurring mutants or variants, or naturally occurring 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 a non-permissive host. Screening in a differential growth system can be used to select mutants with mutations in Leptospiral VM proteins.

[0161] In another embodiment, a "reverse genetics" approach can be used to introduce mutations into bacteria, such as Leptospira bacteria. In this way, naturally occurring other mutations that confer inactivated or attenuated phenotypes can be engineered into bacterial strains. For example, deletions, insertions, or replacements of the coding region of the gene responsible for the Leptospira VM protein can be engineered. Deletions, replacements, or insertions in non-coding regions of the gene responsible for the Leptospira VM protein are also contemplated. To this end, mutations of signals involved in transcription, replication, polyadenylation, and / or packaging of the gene responsible for the Leptospira VM protein can be engineered.

[0162] In one embodiment, the bacteria are engineered to be deficient in a Leptospiral VM protein. For example, in certain embodiments, toxin-deficient mutant bacteria or viruses lacking one or more Leptospiral VM proteins are unable to cause disease but are capable of inducing an adaptive immune response against Leptospira.

[0163] The bacteria produced by the approaches described herein can be used in the vaccines and pharmaceutical formulations described herein. Using reverse genetics techniques, additional mutations can also be engineered into other genes important for vaccine production. That is, epitopes of useful vaccine strain variants can be engineered into the bacteria. Alternatively, completely foreign epitopes, including antigens from other pathogens, can be engineered into inactivated or attenuated strains.

[0164] The inactivated or attenuated bacteria of the present invention can be used as the active ingredient of a vaccine or pharmaceutical formulation itself. In a particular embodiment, the bacteria can be used as a vector or "backbone" of a recombinantly produced vaccine. To this end, "reverse genetics" techniques can be used to engineer mutations or introduce foreign epitopes into the bacteria that serve as the "parent" strain. In this way, vaccines can be designed for immunity against strain variants or against completely different infectious agents or disease antigens.

[0165] For example, in one embodiment, the immunological compositions of the invention include bacteria engineered to express one or more epitopes or antigens of a given pathogen. For example, the bacteria can be engineered to express neutralizing epitopes of other preselected strains. Alternatively, epitopes of other pathogens can be incorporated into the mutant bacteria.

[0166] In one embodiment, the bacterium can induce strong immune response in the host.This characteristic contributes to the generation of strong immune response when used as a vaccine, and other biological results make the bacterium useful as a medicine for preventing and / or treating infectious diseases, diseases or disorders related to antigen.For example, in certain embodiments, the bacterium induces anti-leptospira immune response.

[0167] For an antigenic composition to be useful as a vaccine, the antigenic composition must induce an immune response against an antigen in a cell, tissue, or subject (e.g., a human). In certain embodiments, the vaccine induces a protective immune response in the subject. An "immunological composition" as used herein may include, by way of example, an antigen (e.g., a protein), a nucleic acid molecule encoding the antigen (e.g., an antigen expression vector), or a cell expressing or presenting the antigen. In certain embodiments, the antigenic composition includes or encodes all or a portion of any protein antigen described herein, or an immunologically functional equivalent thereof. In other embodiments, the antigenic composition is in a mixture that includes an additional immunostimulatory agent or a nucleic acid encoding such an agent. The immunostimulatory agent includes, but is not limited to, an additional antigen, an immunomodulatory agent, an antigen-presenting cell, or an adjuvant. In other embodiments, one or more additional agents are covalently bound to the antigen or immunostimulatory agent in any combination. In certain embodiments, the antigenic composition is bound to or includes an HLA anchor motif amino acid.

[0168] In the context of the present invention, the term "vaccine" (also called immunogenic composition) refers to a substance that induces immunity when administered to an animal. In one embodiment, the vaccine induces anti-leptospiral immunity. In various embodiments, the vaccine of the present invention comprises:

[0169] 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.

[0170] The vaccines of the present invention may differ in the composition of their nucleic acid and / or cellular components. In a non-limiting example, the nucleic acid encoding the antigen may also be formulated with an adjuvant. Of course, it will be understood that the various compositions described herein may further comprise additional components. For example, one or more vaccine components may be contained in lipids or liposomes. In another non-limiting example, the vaccine may comprise one or more adjuvants. The vaccines of the present invention and their various components may be prepared and / or administered by any method disclosed herein or by methods known to those of skill in the art in view of the present disclosure.

[0171] In one embodiment, the protein vaccine of the present invention includes, but is not limited to, at least one Leptospira VM protein, variants thereof, or fragments thereof, optionally mixed with an adjuvant substance. In some embodiments, the protein is introduced together with antigen-presenting cells (APCs). The most common cells used in the latter type of vaccines are bone marrow-derived dendritic cells and peripheral blood-derived dendritic cells, because these cells express costimulatory molecules that aid in the activation of T cells. WO00 / 06723 discloses a cellular vaccine composition comprising an APC presenting a tumor-associated antigenic polypeptide. Presentation of the protein can be achieved by introducing a polynucleotide (e.g., DNA, RNA) encoding the protein into the APC, or by introducing the protein itself into the APC.

[0172] For example, methods for detecting induction of cytotoxic T lymphocytes are well known. Foreign substances that enter the body are presented to T cells and B cells by the action of APCs. T cells that respond specifically to the antigens presented by APCs are differentiated into cytotoxic T cells (also called cytotoxic T lymphocytes or CTLs) by stimulation with the antigen. These antigen-stimulated cells then proliferate. This process is referred to herein as "activation" of T cells. Thus, CTL induction by a particular polypeptide or combination of polypeptides of the present invention can be evaluated by presenting the polypeptides to T cells by APCs and detecting induction of CTLs. Furthermore, APCs have the effect of activating CD4+ T cells, CD8+ T cells, macrophages, eosinophils, and NK cells.

[0173] Methods for evaluating the induction of CTLs using dendritic cells (DCs) as APCs are well known in the art. DCs are a representative APC with the strongest CTL induction activity among APCs. In this method, a polypeptide or a combination of polypeptides is first contacted with DCs, and then the DCs are contacted with T cells. Detection of T cells that have a cytotoxic effect on target cells after contact with DCs indicates that the polypeptide or combination of polypeptides has the activity of inducing cytotoxic T cells. In addition, the induced immune response can also be examined by measuring the IFN-gamma produced and released by CTLs in the presence of antigen-presenting cells carrying immobilized polypeptides or combinations of polypeptides, by visualizing using anti-IFN-gamma antibodies in an ELISPOT assay.

[0174] Apart from DCs, peripheral blood mononuclear cells (PBMCs) can also be used as APCs. It has been reported that the induction of CTLs is enhanced by culturing PBMCs in the presence of GM-CSF and IL-4. Similarly, it has been shown that CTLs are induced by culturing PBMCs in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0175] A polypeptide or a combination of polypeptides confirmed to have CTL-inducing activity by these methods is a polypeptide having DC activation activity and subsequent CTL-inducing activity. Thus, a polypeptide or a combination of polypeptides that induces CTLs against Leptospira VM proteins is useful as a vaccine against Leptospira-associated pathologies. Furthermore, CTLs that have acquired cytotoxicity through presentation of a polypeptide or a combination of polypeptides by APCs can also be used as a vaccine against Leptospira infection.

[0176] In general, when using polypeptides for cellular immunotherapy, the efficiency of CTL induction can be increased by contacting DCs with a combination of multiple polypeptides with different structures. Therefore, when stimulating DCs with protein fragments, it is advantageous to use a mixture of multiple types of fragments.

[0177] The induction of immunity by a polypeptide or a combination of polypeptides can be further confirmed by observing the induction of antibody production against a particular antigen. For example, if antibodies against a polypeptide or a combination of polypeptides are induced in an experimental animal immunized with the polypeptide or combination of polypeptides and Leptospira-associated pathology is suppressed by these antibodies, the polypeptide or combination of polypeptides is determined to induce anti-Leptospira immunity.

[0178] method In various embodiments, the compositions of the invention can be used in biological assays, including methods to detect proteins (eg, asialofetuin). Exemplary biological assays include, but are not limited to, immunochromatographic assays, immunodot assays, Luminex assays, ELISA assays, ELISPOT assays, protein microarray assays, Western blot assays, mass spectrophotometric assays, radioimmunoassays (RIA), radial immunodiffusion assays, liquid chromatography-tandem mass spectrometry assays, Ouchterlony immunodiffusion assays, reversed-phase protein microarrays, rocket immunoelectrophoresis assays, immunohistochemistry assays, immunoprecipitation assays, complement fixation assays, FACS, enzyme-substrate binding assays, enzyme assays, enzyme assays using a detectable molecule such as a chromophore, fluorophore, or radioactive substrate, substrate binding assays using such substrates, substrate displacement assays using such substrates, and protein chip assays (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; see also Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007). In some embodiments, the level of asialofetuin in a biological sample is measured in an assay that uses at least one Leptospiral VM protein, variant, or fragment thereof; or a nucleic acid molecule encoding a Leptospiral VM protein, variant, or fragment thereof of the invention as described elsewhere herein.

[0179] In various embodiments, the present invention provides a method comprising administering a composition described herein to a subject in need thereof. For example, in one embodiment, the method comprises administering to the subject a composition comprising: (a) a Leptospiral VM protein, a variant thereof, or a fragment thereof; or (b) a nucleic acid molecule encoding a Leptospiral VM protein, a variant thereof, or a fragment thereof. In various embodiments, the composition of the present invention can be used as an agent for inducing Leptospiral immunity or as a cytotoxic agent for treating a disease or disorder.

[0180] The composition of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the condition of the patient, the type and severity of the patient's disease, and the appropriate dosage may be determined by clinical trials. When an "effective amount" or a "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, progression of the disease, and individual differences in the condition of the patient (subject). The optimal dosage and treatment regime for a particular patient can be readily determined by one of ordinary skill in the art of medicine by following the subject for signs of disease and adjusting the treatment accordingly.

[0181] Administration of the subject compositions can be performed in any convenient manner, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to a subject subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally.

[0182] The administration forms that may be useful in the methods described herein include, but are not limited to, direct delivery to desired organ, oral, inhalation, intranasal, intratracheal, intravenous, intramuscular, intratumoral, subcutaneous, intradermal, and other parenteral administration routes.Furthermore, administration routes may be combined as necessary.In one embodiment, administration route is intradermal injection or intratumoral injection.In one embodiment, one or more compositions are administered to the treatment site during a surgical procedure, such as surgical removal of all or part of a tumor.

[0183] How it is used as a vaccine Thus, the present invention also encompasses methods 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 present invention, and induction of anti-leptospiral immunity allows for the treatment and prevention of pathologies associated with Leptospiral infection. Thus, the present invention provides methods of treating or preventing infection by Leptospira genus.

[0184] A particular composition is judged to have an immunity-inducing effect if it induces a Leptospira immune response when inoculated into an animal. The induction of immunity by a composition can be detected by observing the response of the host's immune system to the composition in vivo or in vitro.

[0185] In another embodiment, the method of the invention comprises administering to a subject a bacterium or virus comprising a nucleic acid sequence encoding a nucleic acid molecule encoding a Leptospiral VM protein, variant, or fragment thereof. In another embodiment, the method of the invention comprises administering to a subject a bacterium or virus that is absent from a Leptospiral VM protein. For example, in certain embodiments, administration of a toxin-deficient mutant bacterium or virus that is absent from a Leptospiral VM protein cannot cause disease, but can induce an adaptive immune response.

[0186] The therapeutic compounds or compositions of the present invention can be administered prophylactically or therapeutically to subjects suffering from, at risk of, or susceptible to developing an infection, disease, or disorder associated with the antigen. Such subjects can be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs before overt clinical symptoms of the disease appear, thus preventing or slowing the progression of the disease or disorder. In the context of the medical field, the term "preventing" includes any activity that reduces the burden of mortality or morbidity due to a disease. Prevention occurs at primary, secondary, and tertiary prevention levels. While primary prevention avoids the onset of a disease, secondary and tertiary levels of prevention include activities aimed at preventing the progression and manifestation of the disease by restoring function and reducing disease-related complications, as well as reducing the adverse effects of an already established disease.

[0187] The immunologically active polypeptide or combination of polypeptides of the present invention, or the polynucleotide or vector encoding such polypeptide or combination of polypeptides, can be optionally combined with an adjuvant. An adjuvant refers to a compound that enhances the immune response to the polypeptide or combination of polypeptides when administered together (or sequentially) with an immunologically active polypeptide. Examples of suitable adjuvants include, but are not limited to, synthetic TLR4 agonist adjuvant, GLA-SE, cholera toxin, Salmonella toxin, alum, and the like. Furthermore, the vaccine of the present invention can be appropriately combined with a pharma-ceutically acceptable carrier. Examples of such carriers include sterile water, physiological saline, phosphate buffer, culture fluid, and the like. Furthermore, the vaccine can contain stabilizers, suspending agents, preservatives, surfactants, and the like, as necessary. The vaccine is administered systemically or locally. The vaccine may be administered by a single administration, or may be boosted by multiple administrations.

[0188] In one embodiment, the method of the present invention comprises administering to a subject a) at least one Leptospiral VM protein, variant, or fragment thereof, or (b) a nucleic acid molecule encoding at least one Leptospiral VM protein, variant, or fragment thereof. In some embodiments, the fragment of the VM protein comprises the DNase domain of the VM protein. Administration of the composition can include, for example, intramuscular, intravenous, peritoneal, subcutaneous, intradermal, and topical administration.

[0189] The actual dose and schedule may vary depending on whether the composition is administered in combination with other pharmaceutical compositions, or depending on individual differences in pharmacokinetics, drug properties, and metabolism. Similarly, for in vitro applications, the amount will vary depending on the particular cell line used (e.g., based on the number of vector receptors present on the cell surface, or the ability of the particular vector used for gene transfer to replicate in that cell line). Furthermore, the amount of vector added per cell will likely vary depending on the length and stability of the therapeutic gene inserted into the vector, as well as the nature of the sequence, parameters that must be determined empirically, among others, and may vary depending on factors that are not inherent to the method of the present invention (e.g., costs associated with synthesis). Those skilled in the art can easily make the necessary adjustments depending on the exigencies of a particular situation.

[0190] These methods described herein are by no means comprehensive, and further methods suitable for particular applications will be apparent to those of skill in the art. Moreover, the effective amount of the composition can further be estimated by analogy with compounds known to exert the desired effect.

[0191] antibody In some embodiments, the present invention provides a method for the treatment of a pulmonary edema including, but not limited to, LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, L Compositions are provided that bind to the VM protein of the antigen of the invention, including IC_12963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091, or fragments or variants thereof. In some embodiments, the compositions that bind to the VM protein of the invention are antibodies.

[0192] The present invention relates to the design and development of anti-VM protein antibodies and their use for the immunotherapy of leptospirosis or infection with Leptospirosis. Anti-VM protein antibodies function as an immunoprophylactic strategy for leptospirosis or infection with Leptospirosis.

[0193] Anti-VM antibodies are capable of binding to a target antigen (i.e., a VM protein) present in a subject. Such binding can neutralize the antigen, block recognition of the antigen by another molecule, e.g., a protein or nucleic acid, and induce or induce an immune response against the antigen.

[0194] 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.

[0195] Antibodies, including anti-VM protein antibody fragments, of the present invention include, in certain embodiments, the antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or formulated antibody. Furthermore, the antibodies of the present disclosure include antibodies having structural and / or functional characteristics of the anti-VM protein antibodies described herein. In one embodiment, the anti-VM protein antibody binds to Leptospiral VM protein, thereby partially or substantially altering at least one biological activity of Leptospiral VM protein.

[0196] In one embodiment, the anti-VM protein antibody of the invention immunospecifically binds to at least one epitope specific for VM protein and does not specifically bind to other polypeptides. The at least one epitope can include at least one antibody binding region that includes at least a 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 are usually composed of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and 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.

[0197] In some embodiments, the present invention includes a composition comprising an antibody (e.g., a binding portion of an antibody) that specifically binds to a VM protein. 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 a further embodiment, the anti-VM protein antibody is a humanized antibody.

[0198] The binding portion of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to a binding partner molecule (e.g., Leptospira 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 included in the term "binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant techniques, making it possible to produce a single protein chain in which VL and VH pair to form a monovalent molecule (known as a 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 to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0199] Antibodies that bind to a Leptospiral VM protein of the present invention are antibodies that inhibit, block, or interfere with at least one Leptospiral VM protein activity in vitro, in situ, and / or in vivo.

[0200] In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three HCDR sequences set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23. In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three HCDR sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47. In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three HCDR sequences set forth in SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63. In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three HCDR sequences set forth in SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79.

[0201] In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three LCDR sequences as set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three LCDR sequences as set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three LCDR sequences as set forth in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three LCDR sequences as set forth in SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In one embodiment, the Leptospiral VM protein antibody comprises at least one, two or three LCDR sequences as set forth in SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82.

[0202] In one embodiment, the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and the LCDR sequences set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. In one embodiment, the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and the LCDR sequences set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. In one embodiment, the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, and the LCDR sequences set forth in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. In one embodiment, the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, and the LCDR sequences set forth in SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In one embodiment, the Leptospira VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79, and the LCDR sequences set forth in SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82.

[0203] In one embodiment, the Leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 27 and the LC sequence set forth in SEQ ID NO: 28. In one embodiment, the Leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 27 and the LC sequence set forth in SEQ ID NO: 43. In one embodiment, the Leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 51 and the LC sequence set forth in SEQ ID NO: 52. In one embodiment, the Leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 67 and the LC sequence set forth in SEQ ID NO: 68. In one embodiment, the Leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 83 and the LC sequence set forth in SEQ ID NO: 84. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0204] In one embodiment, the present invention relates to a nucleotide sequence encoding a Leptospiral VM protein antibody or a fragment thereof. In one embodiment, the nucleotide sequence encoding a Leptospiral VM protein antibody comprises an RNA sequence encoding a Leptospiral VM protein antibody. In one embodiment, the nucleotide sequence encoding a Leptospiral VM protein antibody comprises a DNA sequence encoding a Leptospiral VM protein antibody.

[0205] In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three HCDR sequences as set forth in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three HCDR sequences as set forth in SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three HCDR sequences as set forth in SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three HCDR sequences as set forth in SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79.

[0206] In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three LCDR sequences as set forth in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three LCDR sequences as set forth in SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three LCDR sequences as set forth in SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three LCDR sequences as set forth in SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising at least one, two or three LCDR sequences as set forth in SEQ ID NO:80, SEQ ID NO:81 and SEQ ID NO:82.

[0207] In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HCDR sequences set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and the LCDR sequences set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HCDR sequences set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and the LCDR sequences set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HCDR sequences set forth in SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47, and the LCDR sequences set forth in SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HCDR sequences set forth in SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63, and the LCDR sequences set forth in SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In one embodiment, the nucleic acid molecule encodes a Leptospira VM protein antibody comprising the HCDR sequences set forth in SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79, and the LCDR sequences set forth in SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:82.

[0208] In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HC sequence set forth in SEQ ID NO: 27 and the LC sequence set forth in SEQ ID NO: 28. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HC sequence set forth in SEQ ID NO: 27 and the LC sequence set forth in SEQ ID NO: 43. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HC sequence set forth in SEQ ID NO: 51 and the LC sequence set forth in SEQ ID NO: 52. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HC sequence set forth in SEQ ID NO: 67 and the LC sequence set forth in SEQ ID NO: 68. In one embodiment, the nucleic acid molecule encodes a Leptospiral VM protein antibody comprising the HC sequence set forth in SEQ ID NO: 83 and the LC sequence set forth in SEQ ID NO: 84.

[0209] In one embodiment, a nucleic acid molecule encoding a leptospiral VM protein antibody comprises at least one, two, or three HCDR sequences set forth in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In one embodiment, a nucleic acid molecule encoding a leptospiral VM protein antibody comprises at least one, two, or three HCDR sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55. In one embodiment, a nucleic acid molecule encoding a leptospiral VM protein antibody comprises at least one, two, or three HCDR sequences set forth in SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71. In one embodiment, a nucleic acid molecule encoding a leptospiral VM protein antibody comprises at least one, two, or three HCDR sequences set forth in SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87.

[0210] In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises at least one, two, or three LCDR sequences set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises at least one, two, or three LCDR sequences set forth in SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises at least one, two, or three LCDR sequences set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises at least one, two, or three LCDR sequences set forth in SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises at least one, two, or three LCDR sequences set forth in SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90.

[0211] In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, and the LCDR sequences set forth in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31, and the LCDR sequences set forth in SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55, and the LCDR sequences set forth in SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, and the LCDR sequences set forth in SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74. In one embodiment, a nucleic acid molecule encoding a Leptospira VM protein antibody comprises the HCDR sequences set forth in SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87, and the LCDR sequences set forth in SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90.

[0212] In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 35 and the LC sequence set forth in SEQ ID NO: 36. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 35 and the LC sequence set forth in SEQ ID NO: 44. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 59 and the LC sequence set forth in SEQ ID NO: 60. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 75 and the LC sequence set forth in SEQ ID NO: 76. In one embodiment, the nucleic acid molecule encoding the leptospiral VM protein antibody comprises the HC sequence set forth in SEQ ID NO: 91 and the LC sequence set forth in SEQ ID NO: 92.

[0213] The compositions of the present invention are capable of treating, preventing, and / or protecting against diseases, disorders, or conditions associated with Leptospira infection.

[0214] The compositions of the present invention can be safe, so as not to cause illness or death; provide protection against illness; and possess the characteristics desired for an effective composition, including ease of administration, few side effects, biological stability, and low cost per dose.

[0215] In some embodiments, the Leptospiral VM protein binding molecules (e.g., antibodies, etc.) of the present invention exhibit a high ability to detect and bind to Leptospiral VM protein in complex mixtures 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. Those skilled in the art will appreciate that the Leptospiral VM protein binding molecules (e.g., antibodies, etc.) described herein as useful in methods of diagnosing and treating and preventing disease will also be useful in procedures and methods of the invention, including, but not limited to, immunochromatographic assays, immunodot assays, Luminex assays, ELISA assays, ELISPOT assays, protein microarray assays, Western blot assays, mass spectrophotometric assays, radioimmunoassays (RIA), radial immunodiffusion assays, liquid chromatography-tandem mass spectrometry assays, Ouchterlony immunodiffusion assays, reversed-phase protein microarrays, rocket immunoelectrophoresis assays, immunohistochemical staining assays, immunoprecipitation assays, complement fixation assays, FACS, ProteinChip assays, separation and purification processes, and affinity chromatography (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).

[0216] In some embodiments, the Leptospiral VM protein binding molecules (e.g., antibodies) of the present invention exhibit high ability to reduce or neutralize Leptospiral VM protein activity as assessed by any one of several in vitro and in vivo assays known in the art.

[0217] In certain embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In addition, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. Preferably, 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.

[0218] Dosage and Formulation The present invention contemplates treating a disease in a subject, such as a disease associated with a Leptospira pathogen, by administration of one or more therapeutic agents of the invention (e.g., a VM domain fusion construct of the invention; a toxoid vaccine, an anti-VM antibody, or a nucleic acid molecule encoding an anti-VM antibody).

[0219] The administration of the composition according to the invention can be continuous or intermittent, whether the purpose of administration is therapeutic or prophylactic, depending, for example, on the physiological state of the recipient, and other factors known to those skilled in the art. The administration of the agent of the invention can be essentially continuous over a preselected period of time, or can be a series of spaced doses. In one embodiment, the cytokine composition, antigen receptor composition, and embedded composition of the invention are administered locally at the same site. The dosage will vary depending on a variety of factors, including, but not limited to, the composition selected, the particular disease of the mammal, the weight, physical condition, age, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician using animal models or other test systems well known in the art.

[0220] One or more suitable unit dosage forms having the therapeutic agent of the present invention can be optionally formulated for sustained release (e.g., using microencapsulation, see WO 94 / 07529 and U.S. Pat. No. 4,962,091, the disclosures of which are incorporated herein by reference) as discussed below, and can be administered by a variety of routes, including parenterally, including intravenous and intramuscular routes, and by direct injection into the affected tissue. For example, the therapeutic agent may be injected directly into a tumor. The formulations can, where appropriate, be conveniently provided in discrete unit dosage forms and can be prepared by any of the methods well known in pharmacy. Such methods can include the steps of combining the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers, or combinations thereof, and then, where appropriate, introducing or shaping the product into the desired delivery system.

[0221] Lipid nanoparticles (LNPs), liposomes, or lipoplexes are effective drug delivery systems for biologically active compounds that are inherently cell impermeable, such as therapeutic proteins, peptides, or nucleic acid-based therapeutics. Thus, in some embodiments, the present invention relates to compositions comprising one or more lipid nanoparticles (LNPs), liposomes, or lipoplexes comprising at least one VM protein of the present invention, or a nucleic acid molecule encoding same.

[0222] In certain embodiments, the therapeutic agent is combined with a pharma- ceutically acceptable carrier, diluent, or excipient to form a pharmaceutical formulation or unit dosage form. The total active ingredient in such a formulation comprises 0.1-99.9% by weight of the formulation. "Pharmaceutically acceptable" refers to 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 granules, as a solution, suspension, or emulsion.

[0223] Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using known and readily available ingredients. The therapeutic agents of the invention can also be formulated as solutions suitable for parenteral administration, for example, by intramuscular, subcutaneous or intravenous routes.

[0224] Pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or the form of an emulsion or suspension.

[0225] Thus, the therapeutic agent can be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and can be provided in ampoules, prefilled syringes, small volume infusion containers, or in unit dosage forms in multiple doses with added preservatives. The active ingredient can take such forms as a suspension, solution, or emulsion in an oily or aqueous vehicle and can contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient can be in powder form, obtained by aseptic isolation of a sterile solid or by lyophilization from solution, for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0226] It will be understood that the unit content of active ingredient contained in an individual aerosol dose of each dosage form need not itself constitute an effective amount for treating a particular indication or disease, since the required effective amount may be achieved by administration of multiple dosage units, and furthermore, an effective amount may be achieved using less than the dose in the dosage form, either individually or in a series of administrations.

[0227] The pharmaceutical formulations of the present invention may contain, as optional ingredients, pharma- ceutically acceptable carriers, diluents, solubilizers or emulsifiers, and salts of the type known in the art. Non-limiting examples of carriers and / or diluents useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline, such as phosphate buffered saline at pH 7.0-8.0.

[0228] The expression vectors, transduced cells, polynucleotides, and polypeptides (active ingredients) of the present invention can be formulated and administered to treat various disease states by any means that results in contact of the active ingredients with the drug's site of action in the organism. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutically active ingredients or as a combination of therapeutically active ingredients. They can be administered alone, but will generally be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0229] In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions, as well as glycols, such as propylene glycol or polyethylene glycol, are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents, and buffer substances, if necessary. Antioxidants such as sodium hydrogen sulfate, sodium sulfite, or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Also used are citric acid and its salts, and sodium ethylenediaminetetraacetate (EDTA). In addition, parenteral solutions may contain preservatives, such as benzalkonium chloride, methylparaben, propylparaben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference in this field.

[0230] The active ingredient of the present invention may be formulated to be suspended in a pharma- ceutical acceptable composition suitable for use in mammals, particularly humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs described in U.S. Patents 4,082,735, 4,082,736, 4,101,536, 4,185,089, 4,235,771, and 4,406,890. Other adjuvants that are useful include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate, and dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12. Other ingredients may include polyoxypropylene-polyoxyethylene block polymers (Pluronic®), non-ionic surfactants, and metabolizable oils such as squalene (U.S. Patent 4,606,918).

[0231] Furthermore, standard pharmaceutical methods can be used to control the duration of action. These are known in the art and include controlled release formulations and can include suitable polymers, such as polymers, polyesters, polyamino acids, polyvinylpyrrolidone, ethylene vinyl acetate, methyl cellulose, carboxymethyl cellulose, or protamine sulfate. The concentration and method of incorporation of the polymer can be adjusted to control the release. Furthermore, the drug can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylene vinyl acetate copolymers. These materials can be used to trap the compound in microcapsules as well as being incorporated.

[0232] Thus, the compositions of the present invention can be delivered to various sites in the mammalian body via various routes to achieve a particular effect (see, for example, Rosenfeld et al., 1991; Rosenfeld et al., 1991a; Jaffe et al., supra; Berkner, supra). Those skilled in the art will recognize that, although multiple routes of administration can be used, certain routes can provide more immediate and effective responses than other routes. In one embodiment, the compositions are administered to a 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 the desired organ, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration.

[0233] The active ingredient of the present invention can be provided in unit dosage form, with each dosage unit, such as a teaspoon, tablet, solution, or suppository, containing a predetermined amount of the composition alone or in appropriate combination with other active substances. As used herein, the term "unit dosage form" refers to a physically discrete unit suitable as a unit dosage form for human and mammalian subjects, each unit containing a predetermined amount of the composition of the present invention alone or in combination with other active agents, in an amount calculated to be sufficient to produce the desired effect, optionally in combination with a pharma- ceutically acceptable diluent, carrier, or vehicle. The specifications of the unit dosage form of the present invention depend on the particular effect to be achieved and the particular pharmacokinetics associated with the composition in a particular host.

[0234] These methods described herein are by no means comprehensive, and further methods suitable for particular applications will be apparent to those of skill in the art. Moreover, the effective amount of the composition can be further approximated by analogy with compounds known to exert the desired effect.

[0235] Treatment method In one embodiment, the invention includes a method of inducing an immune response in a subject in need thereof comprising administering a Leptospira VM protein, a toxoid vaccine, an anti-VM antibody, or a nucleic acid molecule encoding an anti-VM antibody of the invention.

[0236] In one embodiment, a vector comprising a leptospiral VM protein or peptide, or a nucleotide sequence encoding a leptospiral VM protein or peptide, functions as a toxoid vaccine. Also provided herein is a method for treating, protecting against, and / or preventing disease in a subject in need thereof by administering a 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, infectious diseases, including but not limited to pathologies associated with leptospiral infection.

[0237] 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 a fusion protein comprising a Leptospiral VM protein or peptide fused to an antigenic peptide, functions as a therapeutic agent for the treatment of a disease or disorder associated with the antigenic peptide.

[0238] In some embodiments, the antigenic peptide is a tumor-associated peptide.Thus, 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 cancer and spinal cord tumors, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system atypical teratoma / rhabdoid tumor, central nervous system embryonic tumor, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant tumor glioma, cerebral astrocytoma / malignant tumor glioma, cervical cancer, pediatric visual pathway tumor, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing family tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer, extrahepatic cancer, eye cancer, fungoid, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, pregnancy cancer, gestational chorionic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin's disease Lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumors, intraocular (eye) cancer, intraocular melanoma, islet cell tumors, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell carcinoma, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia, bone malignant fibrous histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous cell carcinoma of the neck, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycoses, myelodysplastic syndromes, myelodysplasia / myeloproliferative disorders, myeloid leukemia, myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovary, 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, intermediately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor,Plasma cell neoplasms / 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, airway cancer involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue cancer, soft tissue sarcoma, squamous epithelial Cancer, cervical squamous cell carcinoma, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor and pineoblastoma, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0239] In one embodiment, the method of the present invention comprises administering to a subject an antigenic protein, peptide, where the antigenic protein, peptide promotes the generation of an immune response against the antigen. In one embodiment, the method of the present invention comprises administering to a subject a nucleic acid molecule, where the nucleic acid molecule comprises an expression construct for the expression of at least one antigenic protein or peptide, where the antigenic protein or peptide promotes the generation of an immune response against the encoded antigenic protein or peptide. In one embodiment, the method of the present invention comprises administering to a subject an antibody that targets a disease-associated antigen. In one embodiment, the method of the present invention comprises administering to a subject at least one nucleic acid molecule that encodes an antibody or fragment thereof, where the encoded antibody targets a disease-associated antigen.

[0240] In some embodiments, the induced immune response may include an induced humoral immune response and / or an induced cellular immune response. The humoral immune response may be induced 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 may include IgG antibodies and / or neutralizing antibodies. The induced cellular immune response may include induced CD8 +This can include a 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.

[0241] The vaccine dose may be 1 μg to 10 mg active ingredient / kg body weight / dose, and may be 20 μg to 10 mg ingredient / kg body weight / dose. The vaccine may 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 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0242] 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.

[0243] In one embodiment, the vaccine or antibody of the invention is administered in combination with additional vaccine compositions as a prime or boost vaccine. In one embodiment, a subject immunized with a vaccine (as a prime vaccine) is administered a vaccine of the invention as a boost vaccine to enhance the immune response.

[0244] In one embodiment, a vector of the invention is capable of expressing at least two antigenic polypeptides, where at least one antigenic polypeptide is a Leptospira VM protein of the invention.

[0245] Administration The compositions of the present invention can be formulated according to standard techniques known to those skilled in the pharmaceutical arts. Such compositions can be administered in doses and by techniques known to those skilled in the medical arts, taking into account factors such as the age, sex, weight, and condition of the specific subject, as well as the route of administration. The subject can be a mammal, such as a human, horse, cow, pig, sheep, cat, dog, rat, or mouse.

[0246] The composition can be administered prophylactically or therapeutically. In prophylactic administration, the composition can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the composition is administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount sufficient to accomplish this is defined as a "therapeutically effective amount." Amounts effective for this use will depend, for example, on the particular composition of the treatment regimen administered, the method of administration, the stage and severity of the disease, the patient's general health, and the judgment of the prescribing physician.

[0247] The compositions can be administered by methods known in the art, as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Felgner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Felgner (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), all of which are incorporated herein by reference in their entireties. One of ordinary skill in the art will know that the choice of a pharma- ceutically acceptable carrier, including a physiologically acceptable compound, will depend, for example, on the route of administration of the expression vector.

[0248] 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. In particular for the DNA of the composition, the composition can be delivered to the interstitial space of tissues of an individual (Felgner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, all of which are incorporated herein by reference in their entirety). The composition can be administered into muscle, or transdermally, such as via intradermal or subcutaneous injection, or by iontophoresis. Epidermal administration of the composition can also be used. Epidermal administration can include mechanically or chemically irritating the outermost layer of the epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, incorporated herein by reference in its entirety).

[0249] In one embodiment, the proteins, nucleic acid molecules, or antibodies of the invention can be administered to cells of a mammal, including a human.

[0250] In some embodiments, the protein, nucleic acid molecule, or antibody 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 standard methods. For example, if necessary, the culture supernatant containing the viral vector is concentrated and suspended in a buffer solution such as PBS or physiological saline together with a suitable carrier or excipient. Next, if necessary, the suspension can be sterilized by filtration using a filter or the like, and then filled into a sterile container to prepare the injection. If necessary, a stabilizer, a preservative, etc. may be added to the injection. The expression vector thus obtained can be administered to a subject as an injection.

[0251] In some embodiments, the protein, nucleic acid molecule, or antibody may be formulated for administration by intradermal (ID) vaccination (e.g., ID injection by Mantoux technique, using hollow microneedles, using a gene gun, using scarification, or other methods for ID delivery). The formulation for ID vaccination may be prepared by standard methods. For example, the culture supernatant containing the protein, nucleic acid molecule, or antibody is concentrated as necessary, and suspended in a buffer such as PBS, a nucleic acid molecule stabilizing liquid, physiological saline, or the like, together with a suitable carrier or excipient. Next, if necessary, the suspension may be sterilized by filtration using a filter, etc., and then filled into a sterile container to prepare the formulation for ID vaccination. Stabilizers, preservatives, etc. may be added to the formulation for ID vaccination as necessary. The composition obtained in this way may be administered intradermally to the subject.

[0252] The present invention also provides a method of generating an immune response in an animal, comprising administering to the animal any of the above-mentioned proteins, peptides, nucleic acid molecules, or compositions in an amount effective to stimulate an immune response. In one embodiment, the immune response comprises one or more of the following: production of memory CD8+ T cells specific for the expressed target antigen, production of memory CD4+ T cells specific for the expressed target antigen, and production of antibodies specific for the expressed target. In one embodiment, at least some of the antibodies are neutralizing antibodies.

[0253] The present invention further provides a pharmaceutical composition (e.g., a vaccine) comprising the Leptospira VM protein of the present invention. In one embodiment, the composition comprises a pharma- ceutical acceptable diluent, carrier, or excipient carrier. The composition may also comprise an aqueous medium or a suspension containing water to extend the activity and / or shelf life of the composition. The medium / suspension may comprise salts, glucose, pH buffers, stabilizers, emulsifiers, and preservatives.

[0254] In some embodiments, the composition further comprises an adjuvant, including, but not limited to, muramyl dipeptide, aluminum hydroxide, saponin, polyanions, amphiphiles, bacillus Calmette-Guerin (BCG), endotoxin lipopolysaccharide, keyhole limpet hemocyanin (GKLH), and cytoxan.

[0255] In one embodiment, the present invention provides a method of administering a therapeutically effective composition according to the present invention. The desired therapeutic effect includes one or more of the following: reduction or elimination of bacterial load; increase in the number of CD4+ and / or CD8+ T cells or antibodies that recognize the encoded antigen; increase in the overall level of CD4+ T cells; increase in the level of neutralizing antibodies that recognize the antigen; reduction in the number or severity of disease symptoms; reduction in expression of cancer-specific markers; reduction in size or growth rate of tumors; prevention of tumor metastasis; prevention of infection by pathogens, etc. The therapeutic effect can be tracked by evaluating biological markers and / or abnormal physiological responses. In general, an effective dose of a composition according to the present invention includes a titer that can modulate the immune response to the encoded antigen such that memory T cells specific for the encoded antigen are generated.

[0256] Both the dose and the means of administration can be determined based on the condition of the patient (eg, age, weight, general health), the risk of developing the disease, or the progression of the disease.

[0257] In one embodiment, an effective amount of the recombinant virus is about 1×10 10 ~1×10 11 A saline solution ranging from about 10 μl to about 25 μl containing a concentration of plaque forming units (pfa) virus / ml.

[0258] In one embodiment of the invention, a primary immunization is administered, followed by a booster immunization, if desired, about 3-4 weeks after the primary immunization. However, the next immunization need not be administered 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 primary-boost immunization. In one embodiment, the composition is a prophylactic vaccine and is administered to patients who do not test positive for the vaccine antigen, e.g., individuals at risk of exposure to Leptospira bacteria. In another embodiment, the vaccine is administered therapeutically to individuals who are seropositive (but not necessarily symptomatic) for the vaccine antigen (i.e., Leptospira positive individuals, etc.).

[0259] kit The present invention also includes kits 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, a variant or fragment thereof, or a fusion construct comprising a Leptospiral VM domain. In one embodiment, the kit comprises an anti-Leptospiral VM antibody, or a nucleic acid molecule encoding same. In one embodiment, the kit comprises instructional material describing the use of the composition. For example, in some embodiments, the instructional material describes administering the composition to a subject as a therapeutic treatment or non-therapeutic use, as described elsewhere herein. In one embodiment, the kit further comprises one or more additional reagents for use in an assay, such as an immunoassay of the present invention. EXAMPLES

[0260] The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any variations that become evident as a result of the teachings provided herein.

[0261] Example 1: Leptospiral VM ("virulence modified") protein Leptospiral VM protein vaccines containing one or more VM proteins have been designed and tested for their ability to protect mice from Leptospiral challenge, and a pan-vaccine containing multiple VM proteins shows complete protection from death.

[0262] Leptospira VM proteins were expressed in two ways. In part II, L. interrogans serovar L. proteins, LA3490, tLA3490, LA0620, and LA1402, were generated as mCherry fusions. In part II, L. interrogans serovar Copenhagenii proteins LIC12340 (the conserved ortholog of LA1400) and LIC12985 (the conserved ortholog of LA0591) were not generated as mCherry fusions.

[0263] Figures 1 and 2 show the design and experimental timeline of the pan-vaccine challenge study.

[0264] Immunized C3H / HeJ mice are protected from death / weight loss by the pan-vaccine (low passage L. interrogans serovar canicola) after lethal challenge (Figures 3 and 4).

[0265] The VM protein vaccine reduced the bacterial burden in the kidneys (Figures 5 and 6) and lungs (Figures 7 and 8) compared to the PBS negative control.

[0266] Cross-reactive VM protein antibodies were detected prior to challenge (FIG. 9).

[0267] VM protein-mCherry fusion protein vaccine components Expression of full length LA3490 (amino acids 40-639), truncated tLA3490 (short, ricin B domain only, amino acids 40-174), full length LA0620 (amino acids 41-637), and full length LA1402 (amino acids 28-641) into the XhoI / NcoI restriction sites of pET32b. These proteins are expressed as fusions with thioredoxin (TrxA), S-tag, His6 affinity / epitope tag, and enterokinase cleavage site at the amino terminus, and with an additional enterokinase cleavage site, mCherry fusion, and His6 affinity / epitope tag at the carboxy terminus. The linker is (Gly4Ser)5 as a hinge.

[0268] Challenge studies: Results for protection and reduction of bacterial burden Challenge bacterial inoculum: Leptospira interrogans serovar canicola

[0269] Group 1: PBS and glucopyranosyl lipid A (GLA), formulated in a stable oil-in-water nanoemulsion (SE), (GLA-SE) adjuvant. No protection, approximately 8.5 log10 bacterial copies per gram of tissue.

[0270] Group 2: t3490 (ricin B domain of LA3490) plus adjuvant. Compared to Group 1, bacterial load was reduced by approximately 2.5 log10 magnitude in the kidney and approximately 4 log10 magnitude in the liver.

[0271] Group 3: (5 proteins) recombinant full length LA3490, LA0620, LA1402 with LIC12340 (LA1400) and LIC12985 (LA0591 with GLA-SE adjuvant) with GLA-SE adjuvant. Compared to Group 1, there was complete protection from death, a reduction in bacterial burden in the kidney by approximately 3.5 log10 magnitude, and a reduction in bacterial burden in the liver by approximately 3.9 log10 magnitude.

[0272] Group 4: (two proteins) recombinant full-length LIC12340 (LA1400) and LIC12985 (LA0591) with GLA-SE adjuvant, compared to Group 1, provided complete protection from death and reduced bacterial burden in the kidney by approximately 4.0 log10 magnitude and in the liver by approximately 4.1 log10 magnitude.

[0273] LA3490-mCherry expressed in pET32b Full length LA3490 DNA sequence (E. coli preferred codons) of the construct cloned into pET32b (SEQ ID NO:1)

[0274] Full-length LA3490 amino acid sequence Name: LA_3490_mCherry, Sequence: protein, 5' sequence:, 3' sequence:, Sequence length: 877 (SEQ ID NO: 2) FEYGVNHTHIHALSKIEYSVIQKPTDPPKDKPIKVIVSDGGKFCYGPNFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPEKVIKAEKNWDYVHLRPC TINDPLQRWIIKNNSFWTANGFYRLKDYNWYGYISRNSGDRYNHTLDSSMNDWVNTIATPGNISIQTSIAWNLQTTEGQERYFIRWGGSDKNTTPLYYNPENGHLAQYDP ISGSLYCMYSQVDNYQWNWVKWKWCSDLLESKSKGNPTFWNVFFETDQGGMITDYKGNALRVTRYGSNWGSAYTAKPSYLEKDTTNSPTSLFVVNKDLLDWTRYTASNL GKTGQYCPAGKRENIVHRRVKRELPPDFQLTEAWIRRLYEIATSVSAESETRVSGICGPCALHSFQMLAELLEYHSREPLQSGGYFFDTAPNTDPFISFGQRYPHLERLL EDIPKKYAPYPHYSTQSFLSFASIDSMLPQYFWSASTEFTNRDEILSHISSLINSPAGSIWLGVMEQQHPDGTITGHAAPILRISQGLVVIPTNVHLWTLEEFRRFLIPTTELSQIVANLEGSNTLIRFTTIQSLGMLTTNMFDSMVSNRNCTGEGEDRRGSGEYPTSTSVNQCPSGRCALPFGGGGSGGGGSGGGGSDDDDKMVSKGEEDNMAIIKEF MRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYK VKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0275] LA0620-mCherry expressed in pET32b Full length DNA sequence of the construct cloned into pET32b (E. coli preferred codons) (SEQ ID NO:3)

[0276] Amino acid sequence Name: Full_LA_0620_mCherry, Sequence: Protein, 5' sequence:, 3' sequence:, Sequence length: 862 (SEQ ID NO: 4) SSKIEYSVIQKPTDPPKDKPIKVIVSDGGKFCYGPNFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPEKVIKAEETWDYVHLRPCTINDPLQRWI IKNNSFWTANGFYRLKDYNWYGYISRNSGDRYNHTLDPSMNDWVNTIATPGNISIQTSIAWNLQTTEGQERYFIRWGSSNKNTTPLYYNPENGHLAQYDPISGSLYCM YSQVDNYQWNWVKWKWCSDSLESKSKGNPTFWNVFFETDQGGMITDYKGNALRVTRYGSNWGVAYTAKPDFVKTDTKNSPTSLFVVDKSLLDWTRYTSSNLGKTEQYC PAGNKESVVHKKAKRTLPPDFQLTEAWIRRLYEIARTDPSSRTSRGVCGVCMLQALQMIAELQEYHSQGPLQSGGYFFNTAPNTNPFISFGQRYPHLDRLLVDIYRVF DHFFDTSHTLGYLSAMNLLPQYEWGRTREFSTMSEIRSHIRSLITSPPGNIWLVLMTMIYPDGTRGGHAVPILRTPQGLVVIETTMATATFEEYRAALRPTTDFEQIIRNLRGPNNILIGLSTLQLGRFYHNPLDSMISNRNCTGEGSDRRGTGGYPASTSVNQCSSKSSRCSLQGGGGSGGGGSGGGGSDDDKMVSKGEEDNMAIIKEFMRFK VHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVK LRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0277] t3490 (LA3490, amino acids 40–174), mCherry fusion, expressed in prSET; DNA sequence of the construct cloned into pRSET (E. coli preferred codons) (SEQ ID NO:5)

[0278] Amino acid sequence (SEQ ID NO:6) QKPTDPPKDKPIKVIVSDGGKFCYGPNFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPEKVIKAEKNWDYVHLRPCTINDPLQRWIIKNNSFWTANGFYRLKDYNWYGYISRNSGDRYNHTLDGGGGSGGGGSGGGGSDDDDKMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEG RPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0279] LA1402-mCherry expressed in pET32b DNA sequence of the construct cloned into pET32b (E. coli preferred codons) (SEQ ID NO: 7)

[0280] Amino acid sequence Name: LA_1402_mCherry, Sequence: protein, 5' sequence:, 3' sequence:, Sequence length: 870 (SEQ ID NO: 8) SSSKANYSIAQKPTDPPPKDKPINIVTHDGKTYCYSPVFSKGEGYVWIEKCGDNTAKARYDVFQRISYNINNTWLCITAPEPVVKGNARWGYVNLRPCTINDPLQRWIVKENSFWTADGKYRLKDTNWYGYISKTSGDNYNHTLNSSMDNWVKTVATPGNISIRTSISWNSGWGDGIWDINMAPSAYFIHSKGSSKEDIIPLYYNPESGHIAQYDPSS GLLSCMYSKMTDKYDWNWVQWGKCSDAPIKKENPAFWNVYFVANAGGMITDYKGNILRVTKEGPNWGVAYTAKPSYLEKDTTHSPTSVFTVDVDLLKWIRYTTSNGLKTDQYCPAGKKESRIYQRVKRNLPSDFQLSVAWVQRLYDIARSATFESANPGAIPQRHGACGVCLLHSFQMIAELMEYHSREPLTSGGYFFNTASNRDPFLSQRYPELD RLVTNVPVDYANRGRVLAFASAMIMLPQYEWESSSPLTTRSDIQSHIRSLINSPPGSIWLGLLRRQRANGSISGHAVPILRTSEGLVVIPTNMPTASLNTYIQSLAPTMDPNEVINRLENGRTLTTLTTIRPVGTYETPFSLTVSSRDCTGDGDDRRGSGRYPISSLINQCSGGRCILQGGGSGGGSGGGSDDDDKMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0281] VM protein without mCherry fusion Genes encoding L. interrogans serovar copenhageni proteins encoding amino acids 31-627 of LIC12340 (>99% conserved orthologue of LA1400) and amino acids 23-314 of LIC12985 (>99% conserved orthologue of LA0591) were synthesized with E. coli preferred codons and cloned into the XhoI / NcoI restriction sites of pET32b. These proteins were expressed as fusions with thioredoxin (TrxA), S-tag, His6 affinity / epitope tag, and enterokinase cleavage site at the amino terminus, and with His6 affinity / epitope tag at the carboxy terminus, with all fusion partners encoded by the pET32b vector.

[0282] LIC12340 (conserved ortholog of LA1400) DNA sequence of the construct cloned into pET32b (E. coli preferred codons) (SEQ ID NO:9)

[0283] Amino acid sequence Name: LIC_12340 (LA_1400 ortholog), sequence: protein, 5' sequence:, 3' sequence:, sequence length: 597 (SEQ ID NO: 10) SSKSDYSIAQKPADQPKDKSIQVVMHGGSNYCYSPVFTKGEGYIWIDYCSDNTAKARYDVFQRISYNINNTWLCITAPETVKGEETWNYVNLRPCTINDPLQRWIVKDNSFWTANGRYRLKDTNWYGYISRNSGDRYDHTLNSSMDDW IKTVAIPGNISIQTSIAWDLQTTEGNERYFIRWGSSNKNTTPLYYNPESGHIAQYDPSSGLLNCMYSKMTDKYDWNWVKWGKCSDAPIKKDNPAFWNVFFETDKEGAITDYKGNVLRVTRYGLNWGVAYTVKPSYLEKDTTHSPTSLFV IDKDLLDWTRYTYSNLGKTDQYCPAGNKESLVRKRVKRNLNLPSDFQLTREWIQRLYEIARSSISRAIPCRGVCGVCMLHSYQMIAELLEYHSRGPLTGGGYFFDTAPNRDPFISFNQRYPQLNALLTNVPSYANRPGFGSTLVLMLPQY EWTSSDTITTRSGRLLHARSLINSPPGSIWLGLLRGRDADGSTWGHAVPILRTSQGIVVIPTNSPTMSLNTYIRSLAPTMDPNEVINRLENGSTLTELTTIQPVRIYDIPFSLTVSTRDCTGDGDGRRGSGRYPTSSLINQCSGGRCILQ

[0284] LIC12985 (highly conserved ortholog of LA0591) DNA sequence of the construct cloned into pET32b (E. coli preferred codons) (SEQ ID NO:11) GTTAACCCGGATCAGTATTGTCCGGCAAGTAAAAAAGAAAATCATAATATCCGCATCAAGCGCACCCTGCCGCCGGATTTTCAGCTGACCGAAGAATGGCTGCGTCGCCTGTATGATATTGCAACCAGTGCCAGTCTGACCGAAGGTCAGATTCATGGCATTTGTGGCGTTTGTCTGCTGCAGACCTTTCAGATGCTGCAGAACTGCAGGAATATCAT AGTCATGGCCCGCTGCAGGGTGGCGGCTATTTCTTTAATACCGCCACCGGATACCGATCCGTTTGATAGTTTTCGTCAGCGCTATCCGGAACTGGATACCATGCTGACCGATGCAGCCACCGCATATGGTCCGGCATATAATACCACCCGTCTGCTGACCCTGGTGAGCGCCATGACCATGATGCCGCAGTATGAATGGACCCCGAGCGTGAATTACT ACCCGTAGCGATATGCATAGCCATATTCGCAGTCTGATTGATAGCCCGCCGGGTAGTATTTGGCTGGGTCTGATGCAGCGTCGCGAAAGTGAATGAAACCCTGCGCTGGCATGCCCTGCCGATTCTGCGCACCAGTCAGGGTCTGTTATTCAGACCCGTGTTTAGCACCATGAGCTTTGAACTGTATCGCCTGTATCTGACCCCGAGCACCAGTATT GTGCAGATTATTAATGATTACCTGGAAAGCAGATCGCACCCTGACCGTTCTGGTTACCATTCAGCTGGAACAGGCATATCAGAATCTGTTTGATTTTATGGTTAGCAATATGAACTGCACCGGGCGAAGGTGAAAATCGCCGTGGCAGTGCGGCTATCCGACCAGCGCAACCGTGAATCAGTGTAGCGGTGGTCGCTGCGACTGCCGAATTGGtaa

[0285] amino acid sequence Name: LIC_12985 (LA_0591 ortholog), sequence: protein, 5' sequence:, 3' sequence:, sequence length: 291 (SEQ ID NO: 12) VNPDQYCPASKKENHNIRIKRTLPPDFQLTEEWLRRLYDIATSASLTEGQIHGICGVCLLQTFQMLAELQEYHSHGPLQGGGYFFNTAPDTTDPFDSFRQRYPELDTMLTDAATAYGPAYNTTRLLTLVSAMTMMPQYEWTPSREF TTRSDMSHIRSLIDSPPGSIWLGLMQRRESDETLRWHALPILRTSQGLIVIQTRVSTMSFELYRLYLTPSTSIVQIINDYLEEADRTLTVLVTIQLEQAYQNLFDFMVSNMNCTGEGENRRGSGGYPTSATVNQCSGGRCALPNW

[0286] Q8F6A9_LEPIN Ricin type B lectin domain-containing protein OS=Leptospira interrogans serovar Icterohaemorrhagiae serovar lepidium (strain 56601) OX=189518 GN=LA_1400 PE=4 SV=2 (SEQ ID NO: 13) MHGGSNYCYSPVFTKGEGYIWIDYCRDNTAKARYDVFQRISYNINNTWLCITAPETVVKGEETWNYVNLRPCTINDPLQRWIVKDNSFWTANGRYRLKDTNWYGYISRNSGDRYDHTLDSSMDDWIETVAIPGNISIQTSIAW DLQTTEGNERYFIRWGSSNKNTTPLYYNPESGHIAQYDPSSGLLNCMYSKMTDKYDWNWVKWGKCSDAPIKKDNPAFWNVFFETDKEGAITDYKGNVLRVTRYGLNWGVAYTVKPSYLEKDTTHSPTSLFVIDKDLLDWTRYT YSNLGKTDQYCPAGNKESLGRKRVKRNLNLPSDFQLTREWIQRFYEIARSNISGTIPRRGVCGVCMLHSYQMIAELLEYHSRGPLTSGGYFFDTAPNRDPFISFNQRYPQLNTLLTDVPNYANRLGFGSTLVMLPQYEWTSSD TITTRSGRLLHARTLINSPPGSIWLGLLRGRNANGSTWGHAVPILRTSQGIVVIPTNVLTMSLNTYIRSLAPTMDPNEVINRLENGNTLTELTTIQPVRIYDIPFSLTVSTRDCTGDGDGRRGSGRYPTSSLINQCSGGRCILQ

[0287] Q72PX7_LEPIC Uncharacterized protein OS=Leptospira interrogans serovar cterohaemorrhagiae serovar copenhageni (strain FiocruzL1-130) GN=LIC_12340 PE=4 SV=1 (SEQ ID NO:14) The signal sequence (bold) is not included in the recombinant protein construct. MGRWIVLRVSLLVLIGIGFEYGINHTSINASSKSDYSIAQKPADQPKDKSIQVVMHGGSNYCYSPVFTKGEGYIWIDYCSDNTAKARYDVFQRISYNINNTWLCITAPETVVKGEETWNYVNLRPCTINDPLQRWIVKDNSFWTANGRYRLKDTNWYGYISRNSGDRYDHTLNSSMDDWIKTVAIPGNISIQTSIAWDLQTTEGNERYFIRWGSSNKNTTPLYYNPESGHIAQYDPSSGLLNCMYSKMTDKYDWNWVKWGKCSDAPIKKDNPAFWNVFFETDKEGAITDYKGNVLRVTRYGLNWGVAYTVKPSYLEKDTTHSPTSLFVIDKDLLDWTRYTYSNLGKTDQYCPAGNKESLVRKRVKRNLNLPSDFQLTREWIQRLYEIARSSISRAIPCRGVCGVCMLHSYQMIAELLEYHSRGPLTGGGYFFDTAPNRDPFISFNQRYPQLNALLTNVPSYANRPGFGSTLVMLPQYEWTSSDTITTRSGRLLHARSLINSPPGSIWLGLLRGRDADGSTWGHAVPILRTSQGIVVIPTNSPTMSLNTYIRSLAPTMDPNEVINRLENGSTLTELTTIQPVRIYDIPFSLTVSTRDCTGDGDGRRGSGRYPTSSLINQCSGGRCILQ

[0288] Example 2: Computational Intelligence-Based Reevaluation of the Leptospiral Novel PF07598VM Gene Family of AB Toxins to Infer Pathogenic Mechanisms Despite the identification of several leptospirosis virulence factors and useful in vitro and small animal models, the pathogenesis and biology of Leptospira remain challenging. Vascular instability, liver and kidney dysfunction, and pulmonary hemorrhage are predominantly seen in severe leptospirosis, which is thought to be caused by circulating toxins secreted by pathogenic Leptospira bacteria. The virulence modifying (VM) protein (PF07598) is a typical feature of group I virulent Leptospira and is an experimentally validated bona fide R-type lectin domain-containing exotoxin. The VM protein contains tandem N-terminal ricin B chain-like β-trefoil domains and a C-terminal DNase activity, which rapidly induces chromosome fragmentation and cell death.

[0289] Here, using computational tools and an artificial intelligence-derived high-resolution 3D structure of the VM protein using the DeepMind AlphaFold algorithm, the mechanism of action of novel leptospiral toxins is explored at the sequence, atomic, and structural levels. Our findings show that the PF07598 protein family shares a remarkable degree of characteristic conserved sequence motifs and structural similarities with plant-derived ricin B chain [a unique (QxW)3 motif at the N-terminus], bacterial CARDs toxins [D3 domain, aromatic patch], and mammalian DNases [C-terminal catalytic residues], all exclusively encoded by one gene and one protein. The evolutionary divergent origin of the unique VM protein provides important insights into virulence and host tropism. Structure-function validation of VM proteins by mutagenesis approaches provides a unique opportunity to understand the pathogenic mechanism and develop novel therapeutic and preventive measures for leptospirosis.

[0290] A novel R-type lectin from Leptospira: A distinctive carbohydrate-binding aromatic patch and sequence motif (QXW) 3 The presence of these proteins confirms that VM proteins are bona fide R-type lectins. R-type lectins are members of a superfamily of proteins, all of which contain a carbohydrate recognition domain (CRD) and binding functions for complex carbohydrates (glycoproteins, proteoglycans / glycosaminoglycans, and glycolipids) such as ricin B chain (Cummings et al., 2017, Cold Spring Harbor Laboratory Press). Ricin, a toxic protein from castor bean (Ricinus Communis), was the first lectin discovered in plants (Olsnes et al., 1974, Nature, 249(458):627-31). The R-type lectin domain is related to the binding domain (B chain) of AB toxins (Varki et al., 2015, Cold Spring Harbor). The greater heterogeneity in the composition and structure of B chains has most likely evolved to recognize a broad range of target cells (DiRienzo et al., 2014, New Journal of Science, 26).

[0291] The high-resolution three-dimensional (3D) structural framework of VM protein obtained by AlphaFold algorithm has enabled us to understand the importance of structure-function relationships in multiglobular proteins (in press) (Callaway et al., 2020, Nature, 588(7837):203-4; Jumper et al., 2020, predictioncenterorg / casp14 / doc / CASP14_Abstracts; Senior et al., 2020, Nature, 577(7792):706-10). Virulent Leptospira encodes just over a dozen paralogous VM proteins with molecular mass of approximately 640 aa, which encode a single polypeptide transcribed from a single gene locus, different from most other bacterial AB toxins. The 3D structure of the VM protein was verified by Ramachandran plot followed by Verify3D (doe-mbi.ucla.edu / verify3d / ) [residues LA3490: 89.20%, LA0620: 92.15%, LA1402: 90.02%, LA1400: 98.60%, and LA0591: 84.98% showed a mean 3D-1D score ≥ 0.2 and passed the score] (Pontius et al., 1996, J Mol Biol, 264(1):121-36). This structure was further validated by the program PROVE (PROtein Volume Evaluation) (saves.mbi.ucla.edu / Jobs / 1016444 / prove / PROVE_PLOT.ps), and the Z-score mean, Z-score stddev, and Z-score RMS were calculated (Figure 10).

[0292] Paralogs of VM proteins containing highly conserved tandem N-terminal trefoil-like lectin domains (RBL1 and RBL2) and variable C-terminal domains (Chaurasia et al., 2022, Front Microbiol, 13:859680). Notably, L. interrogans contains a naturally occurring CBR deletion variant (~313aa) that contains a predicted signal sequence. Computational and in vitro experimental validation confirmed that only the RBL1 domain (N-terminal region of LA3490, 40aa-150aa) structurally overlaps with ricin B chain (PBD; 2AAI-B: 7aa-129aa) with RMSD of 1.796 and is involved in binding to the N-terminal galactosyl glycoprotein moiety present on the host cell receptor (Chaurasia et al., 2022, Front Microbiol, 13:859680). RBL1 and RBL2 are enriched in aromatic patches due to the presence of surface-exposed aromatic (tyrosine) and heterocyclic (phenylalanine and tryptophan) amino acids (Figure 11A). These aromatic patches appear to play an important role in host receptor / carbohydrate recognition. In addition to the aromatic patches, the RBL1 domain contains three sequences of conserved QxW motifs within the β-trefoil domain, similar to the ricin B chain ( 40 QKP 42 , 78 QCW 80 , and 134 QRW 136) (Figure 11B). Interestingly, multiple sequence alignment of RBL1 and ricin B chain shows that only the 134QRW136 motif is conserved in both RBL1 and ricin B chain, and notably, the first QxW motif (40QKP42) in the RBL1 domain has a tryptophan to proline substitution. The conserved motif of the QxW sequence plays an important role in receptor recognition and contributes to the stabilization of the structure by hydrogen bonds within the carbohydrate-binding motif (Hatakeyama et al., 2007, J Biol Chem, 282(52):37826-35; Hazes et al., 1995, Nat Struct Biol, 2(5):358-9; Hazes et al., 1996, Protein Sci, 5(8):1490-501). In the RBL1 domain, the sequence motif 158 YGY 160 is highly conserved in ricin B chain and is thought to have a functional carbohydrate-binding ability similar to that of ricin B chain. LA1402 and LA1400 are ancestral VM proteins of virulent Leptospira of group I and belong to the same cluster A. Computational analysis predicts that these two proteins (LA1402 and LA1400) have 78 QCW 80 The lack of a motif suggests that evolution of VM proteins by successive gene duplications has led to the development of a novel mechanism for binding to the host cell surface / host tropism. 78 QCW 80These results suggest that the essentiality of the motif may explain the acquisition of essentiality (Chaurasia et al., 2022, Front Microbiol, 13:859680; Fouts et al., 2016, PLoS Negl Trop Dis, 10(2):e0004403). The essentiality of binding of VM proteins to host receptors is an important step in understanding the mechanisms of host-pathogen interactions. The well-studied ricin B chain (RTB) is a galactose-specific lectin that contains two identical sugar-binding sites, preferably oligosaccharides (Frankel et al., 1996, Biochemistry, 35(47):14749-56). One terminal galactose binds to binding site 1 (W37) of RTB, while the other terminal galactose can bind to binding site 2 (Y248) of another molecule of RTB without steric hindrance, and forms strong hydrophobic interactions to stabilize the protein-sugar complex (Sphyris et al., 1995, J Biol Chem, 270(35):20292-7). Thus, without being bound by theory, it is speculated that (QxW)3 or (QxW)4 of VM protein is involved in the ricin B chain. 158 YGY 160 It was hypothesized that the deletion of aromatic patches or the substitution of tyrosine and tryptophan in the motifs would likely destabilize the basic structure of the β-trefoil fold, which would most likely inhibit the binding of VM protein to the host cell surface, inhibit toxin assembly, and block prominent virulence factors. Fundamental studies of these motifs or carbohydrate-binding domains could be achieved by mutagenesis and glycan microarrays, which are excellent tools for investigating glycan-protein (host-pathogen) interactions and identifying host receptors / innate immune receptors (Geissner et al., 2019, Proc Natl Acad Sci USA, 116(6):1958-67).

[0293] Sequence and structural similarities between the RBL2 domain of VM protein and CARDS toxin rationalize functional similarities in binding and internalization To elucidate the structure and function of the VM protein RBL2, the amino acid sequence of VM protein was submitted to the high-throughput Predictprotein online server (predictprotein.org) (Bernhofer et al., 2021, Nucleic Acids Res, 49(W1):W535-W40). This software uses machine learning algorithms with evolutionary information to predict protein structure and function. When 32 proteins were aligned by Predictprotein, 31 matches belonged to the PF07598 protein family, and the other hit was CARDs toxins (PDB:4TLV_A chain), which showed a significant match with identity 0.55, expectation value: 2e-94, and match length (310aa). The full length of VM protein and CARDs toxins (PDB:4TLV) were structurally superimposed and visualized by PyMOL2.4.0 (pymol.org / 2 / ). Only RBL2 (196a-335aa) of VM protein overlapped with the C-terminus of CARDs toxins (PDB: 4TLV, D3 domain: 447aa-591aa) with RMSD of 1.218 Å (Figure 12A,B). The amino acid sequences of RBL2 (196aa-335aa) and CARDs toxins (PDB: 4TLV, 447aa-591aa) of the PF07598 protein family were aligned using MAFFT (multiple alignment using fast Fourier transform) with L-INS-i (accuracy-oriented) algorithm and visualized with Jalview v2.11.5 (jalview.org). The D3 domain of CARDs toxins contains eight tryptophans, and RBL2 contains nine tryptophans, and interestingly, six tryptophans were conserved at the sequence and structure levels in both RBL2 and CARDs toxins. The CARDs toxins (D2+D3 trefoil) lack a galactose-binding site, suggesting that the VM protein RBL1 is likely to be the sole carbohydrate-binding partner.Mutagenesis of residues 571aa-591aa of CARDs toxins, which are essential for proper folding of D3 and formation of its aromatic patch, lacked internalization by HeLa cells, suggesting that CARDs toxins enter host cells via the D3 domain (Becker et al., 2015, Proc Natl Acad Sci USA, 112(16):5165-70; Ramasamy et al., 2018, mBio, 9(1)). Superposition of RBL2 with the CARD (D3 domain) of VM protein with RMSD=1.218Å and the six conserved tryptophans within the aromatic patch reinforces the function of RBL2 as a translocation domain of VM protein that internalizes VM protein into host cells. From this information, without being bound by theory, it was hypothesized that mutations in these conserved tryptophan / aromatic patches abolished internalization of the protein into host cells. Taken together, mutagenesis, glycan microarray, and surface plasma resonance (SPR) will open the door to characterize the functions of RBL domains and identify their carbohydrate binding partners and translocation of VM proteins in host cells.

[0294] The intramolecular disulfide bond structure similar to that of ricin toxin confirms that the VM protein is a bona fide AB toxin. The literature review identified diphtheria toxin [Corynebacterium diphtheriae: AB (Murphy et al., 2011, Toxins (Basel), 3(3):294-308)], pertussis toxin [Bordetella pertussis: A(S1)-B(S2-S5) (Stein et al., 1994, Structure, 2(1):45-57)], Shiga toxin [Shigella dysenteriae: AB5 (Johannes, 2017, Toxins (Basel), 9(11))], exotoxin A [Pseudomonas aeruginosa AB (Ogata et al., 1990, J Biol Chem, 265(33):20678-85)], and plant-derived ricin toxin [Ricinus communis):AB (Lord et al., 2011, Toxins (Basel), 3(7):787-801)] have been shown to belong to the class of AB-type toxins (Cherubin et al., 2018, Sci Rep, 8(1):2494). They are known as AB toxins because they contain at least one subunit or polypeptide (B chain) that recognizes a specific receptor on the cell surface and one subunit or polypeptide (A chain) that enters the cell and accesses the target site. These AB toxins specifically modify the host target by ADP-ribosylation, glycosylation, deamidation, deadenylation, proteolysis, acetylation, etc. These modifications often result in target inactivation, which can alter cellular physiology and lead to necrotic or apoptotic cell death (Odumosu et al., 2010, Toxins (Basel), 2(7):1612-45; Biernbaum et al., 2022, Toxins (Basel), 14(1)).The formation of disulfide bonds in the AB toxin affects its structural and functional properties, as it modulates the folding and stability needs required for the activation of its functional role (Hogg, 2003, Trends Biochem Sci, 28(4):210-4). Reduction of disulfide bonds in the A and B chains of ricin reduces its toxicity in mice and also reduces its ability to inhibit protein synthesis in HeLa cells (Lappi et al., 1978, Proc Natl Acad Sci USA, 75(3):1096-100).

[0295] The structural architecture of VM proteins derived from the AlphaFold algorithm indicates that they contain 12 cysteine ​​residues, 10 of which are involved in the formation of five disulfide bridges (5-disulfide bonds) similar to ricin toxin (Chaurasia et al., 2022, Front Microbiol, 13:859680; Lappi et al., 1978, Proc Natl Acad Sci USA, 75(3):1096-100) (Figure 13A, B, C). Interestingly, the RBL1 domain of the VM protein contains two disulfide bonds (62aa-79aa, 105aa-127aa), the RBL2 domain contains one disulfide bond (244aa-262aa), and the C-terminal globular domain encodes two disulfide bonds (353aa-608aa and 630aa-635aa) (Figure 13A-D). Because the VM gene encodes a single polypeptide chain, unlike ricin toxin, proteolytic cleavage of disulfide bonds plays an important role in processing RBL1, RBL2, and the C-terminus into their functional domains. Ricin A chain (RTA) contains two cysteine ​​residues (Cys171 and Cys259), and Cys259 forms an interchain disulfide bond with Cys4 of ricin B chain (RTB) in ricin holotoxin. Interestingly, disruption of this disulfide bond by site-directed mutagenesis in the A chain (substitution of alanine for cysteine ​​at amino acid position 259 of ricin) reduces cytotoxicity (Mohanraj et al., 1995, Biochim Biophys Acta, 1243(3):399-406) or introduction of a new disulfide bond into the ricin A chain reduces the cytotoxicity of ricin (Argent et al., 1994, J Biol Chem, 269(43):26705-10). Based on the structural superposition of ricin with VM protein, the disulfide bond at positions 353aa-608aa (4aa-259aa in ricin) is thought to be crucial for C-terminal proteolysis or hydrolysis in endosomes, which then releases the biologically active C-terminal domain into the cytosol and translocates to the nucleus.Interestingly, LA0591(313aa) naturally lacks RBLs (RBL1 and RBL2), suggesting that this naturally occurring mutant variant does not require host cell binding and internalization, thus inferring their intracellular role in pathogenesis. Two cysteines in LA0591 form a disulfide bond at the C-terminus (303aa-308aa), and this mutagenesis study may be informative about the functional role of disulfide bonds in VM protein and its orthologues (Figure S13E).

[0296] CARDs toxins encode six cysteine ​​residues at amino acid positions 230, 247, 324, 406, 425, and 548, and disulfide bond formation essential for their cytotoxicity occurs between residues C230 and C247. Mutagenesis studies revealed that the disulfide bond protects the ADPRT(D1) domain of CARDS toxins from proteases, and disrupted disulfide bonds do not affect cell binding, internalization, and intracellular trafficking (Balasubramanian et al., 2019, Cell Microbiol, 21(8):e13032). In the A chain of Shiga toxin, disulfide bonds stabilize the toxin subunit after protease cleavage in endosomes or the trans-Golgi network (Garred et al., 1995, J Biol Chem, 270(18):10817-21; Tam et al., 2007, Microbiology (Reading), 153(Pt 8):2700-10), but disulfide bond-deficient variants of Shiga toxin were susceptible to protease degradation and had low cytotoxicity to cells (Garred et al., 1997, J Biol Chem, 272(17):11414-9). Similarly, in pertussis toxin, reduction of disulfide bonds alters the conformation required for the toxin to exhibit NAD glycohydrolase and ADPRT activities (Moss et al., 1983, J Biol Chem, 258(19):11879-82; Burns et al., 1989, J Biol Chem, 264(1):564-8). In diphtheria and cholera toxins, reduction of disulfide bonds leads to release of active fragments from endosomes into the cytosol (Falnes et al., 1994, J Biol Chem, 269(11):8402-7; Collier, 2001, Toxicon, 39(11):1793-803; Tsai et al., 2001, Cell, 104(6):937-48; Sandvig et al., 2002, FEBS Lett, 529(1):49-53).

[0297] The importance of disulfide bonds in bacterial toxins and their role in pathogenesis reinforces the computational information of RBL and disulfide bond structures of the PF07598 protein family (Figure 13). Approaches directed to site-directed mutagenesis of cysteine ​​residues or engineering of new disulfide bonds in VM proteins could potentially be used to reduce cytotoxicity and therefore be used as vaccine candidates. Taken together, these data lead to the hypothesis of a crucial role of disulfide bonds in VM toxin activation and subsequent cytopathological events.

[0298] Comparative computational analysis elucidating hotspot residues and active sites in the C-terminus of the PF07598 protein family This study was carried out to identify functionally important regions of amino acids of VM protein that are actively involved in substrate or ligand binding. To predict the ligand binding site based on the structure and to determine the relatively conserved hotspot residues that were actively involved in ligand binding, the 3D structure of VM protein (full length and C-terminal domain) generated by AlphaFold algorithm was subjected to online machine learning based servers such as FTMap server (ftmap.bu.edu) (Kozakov et al., 2015, Nat Protoc, 10(5):733-55), PrankWeb (prankweb.cz) (Jendele et al., 2019, Nucleic Acids Res, 47(W1):W345-W9), and Deepsite (playmolecule.com / deepsite / ) (Jimenez et al., 2017, Bioinformatics, 33(19):3036-42). Ligand-binding site analysis is often used for function identification and 3D structure-based drug discovery. The FTMap server uses 16 small molecules (ethanol, isopropanol, isobutanol, acetone, acetaldehyde, dimethyl ether, cyclohexane, ethane, acetonitrile, urea, methylamine, phenol, benzaldehyde, benzene, acetamide, and N,N-dimethylformamide) as probes to identify hot-spot regions, which are areas that contribute significantly to the binding free energy and are therefore important for ligand binding (Kozakov et al., 2015, Nat Protoc, 10(5):733-55; Ngan et al., 2012, Nucleic Acids Res, 40:W271-5).FTMap analysis revealed that amino acids Cys403, His533, and Ser482 were hotspot residues in the full-length LA3490 protein, showing 2111, 1457, and 1128 interactions with the cluster, whereas the C-terminal domain of LA3490 (368aa-369aa) showed a higher number of interactions with hotspot residues and clusters (Arg615-3109, His533-2510, Cys403-2400, Gln486-1890, Thr549-1622, and Gln523-1357) (Figures 14 and 15). Interestingly, for LA3490, His533 showed high binding energy as the best hotspot residue, as well as in other VM proteins (LA0620:His530, LA1400:His469, LA1402:His537, LA0591:His205) (Figure 15B). This study suggests that His533 (LA3490) is a crucial amino acid and its functional role in catalysis may be elucidated by mutagenesis approaches.

[0299] PrankWeb and Deepsite are two separate template-free online machine learning-based algorithms for structure-based ligand-binding site prediction (Jendele et al., 2019, Nucleic Acids Res, 47(W1):W345-W9; Jimenez et al., 2017, Bioinformatics, 33(19):3036-42; Krivak et al., 2018, J Cheminform, 10(1):39). PrankWeb identified 14 pockets in the full-length LA3490 and ranked them from 1 to 14 based on their probability and solvent-accessible surface (SAS points). Pocket 1 had a score of 18.30 with the highest probability of 0.817 and exhibited 106 solvent-accessible surface (SAS points) among the remaining pockets (Figure 16A, 16C). In particular, the highest scoring pocket 1 was located in the C-terminal groove and importantly contained amino acids Cys403, Gln523, His533, and Thr549, which were also screened by the FTMap server (Figure 17). Two deep pockets were identified by the Deepsite machine learning-based algorithm, with His533, Thr549, and Gln523 located at the C-terminus of pocket 1 and His451, Tyr621 located in pocket 2. Amino acids in pocket 1 were also identified by both FTMap and PrankWeb. In particular, amino acids Cys406, His525, Thr531, Pro548, Asn550, Trp554, and Asn580 were identified and shared by both PrankWeb and Deepsite, but not by the FTMap server (Figure 17). This comparative study suggests that His533, Thr549, and Gln523 are highly conserved, reliable, and actively involved in ligand binding, and therefore these amino acids may be useful for functional studies by site-directed mutagenesis. The natural mutant variant LA0591 lacking RBL indicates that His205, identified by all three online servers (FTMAp, PrankWeb, and Deepsite), is involved in ligand binding (Figures 16B, 16D, and 17).

[0300] Comparative studies to identify the ligand-binding site of VM protein for DNase activity were verified using the 3D structure of bovine DNase (PDB:3DNI), and its active site was superimposed with VM protein (Figure 17). Notably, in bovine DNase, Asn7, Glu39, Tyr76, Arg111, Asp251, His134, Asp168, Asn170, and His252 were identified as hotspot residues by FPMap, and these amino acids were shared by PrankWeb and Deepsite (Figure 17). The crystal structures of bovine DNase (PDB:3DNI) and human DNase (4AWN) suggest that Arg9, Arg41, Tyr76, Glu78, His134, Asp168, Asp212, and His252 are the amino acids present in the active site (Suck et al., 1984, EMBO J, 3(10):2423-30; Parsiegla et al., 2012, Biochemistry, 51(51):10250-8). The list of shared amino acids in VM proteins by three independent servers FTMap, PrankWeb, and Deepsite and the identification of ligand-binding sites of bovine and human DNases that overlap with the active site suggest that these machine learning based algorithms are reliable to screen hotspot residues / ligand-binding sites / active sites of proteins. In bovine and human, His134 and His252 and their hydrogen-bonding pairs Glu78 and Asp212 are crucial for functional DNase I activity (Pan et al., 1998, Protein Sci, 7(3):628-36), and mutation of any of the four catalytic amino acids (His134, His252, Glu78, and Asp212) dramatically reduced the hydrolytic activity of DNase I (Pan et al., 1998, Protein Sci, 7(3):628-36).The superposition of heterocyclic His134 of bovine DNase and His533 (LA3490) of PF07598 gene family, which reinforces the catalytic site of VM protein, is mediated by the highly conserved His533 (LA0620:His530, LA1400:His469, LA1402:His537, LA0591:His205) at the C-terminus of VM protein (Figure 14D, Figure 15). Approaches to site-directed mutagenesis will help to reveal the functional catalytic residues at the C-terminus of VM protein. Also, Predictprotein (predictprotein.org) online server further provided useful structure-function knowledge by showing the DNA binding domain at the C-terminus of VM protein, enhancing the computational analysis (Figure 15).

[0301] The DNase activity of VM protein was +2 Depending on the ion, Zn +2 , Ca +2 Presence of Mg +2 The lack of ions leads to loss of catalytic activity of VM protein (Figure 18). Using MGLTools 1.5.7, docking studies were performed with phosphate and magnesium ions against the C-terminus of LA3490. The magnesium ion interacted with the hotspot residue Gln412 and showed a binding energy of 0.95 kCal / mol, whereas the phosphate ion interacted with the hotspot residue Arg615 and showed a binding energy of 2.58 kCal / mol (Figure 18). Hotspot residues or ligand-binding residues are optimal targets for site-directed mutagenesis approaches leading to functional characterization of the active sites of the PF07598 protein family.

[0302] Example 3: Vaccination with virulence-modifying proteins encoded by the Leptospira interrogans PF07598 gene family protects mice from severe leptospirosis and reduces bacterial burden in the liver and kidney Since the pathogenesis of leptospirosis was first described, the mechanism by which pathogenic leptospira cause severe disease has remained unclear (Noguchi et al., 1917, J Exp Med, 25(5):755-63; Inada et al., 1915, The Journal of Experimental Medicine, XXIII:377-402). Historically, the nomenclature of leptospira has been complex, but recent genomic and molecular approaches have clarified the relationship between species and serotypes. Of particular importance is the discovery that the PF07598 gene family is present only in pathogenic group 1 leptospira and extends to the most pathogenic species, L. interrogans, L. kirschneri, and L. noguchii. Severe disease in humans is thought to result primarily from infection with serotypes belonging to L. interrogans. Such data are limited due to insufficient isolates obtained from severe leptospirosis cases that can reliably identify the infecting Leptospira species in such cases. Because current gene knockout approaches against Leptospira are still limited, especially when applied to multiple gene families, we used an immunological approach to establish whether the VM protein, encoded by the Leptospiral PF07598 gene family, could be a virulence factor contributing to the manifestation of severe leptospirosis in a mouse model. The data presented here support the hypothesis that the VM protein has central importance as a virulence factor in the development of severe leptospirosis.

[0303] Vaccination of C3H / HeJ mice with as few as two L. interrogans serovar Lepidoptera VM proteins (G-IV, LA1400, and LA0591) but as many as five L. interrogans serovar Lepidoptera VM proteins (G-III, LA1400, and LA0591, plus LA3490, LA0620, and LA1402) protected the mice from all clinical symptoms of disease and reduced bacterial burdens by approximately 3-4 log10 in the liver and kidney, two key organs in leptospirosis pathogenesis and transmission of leptospira, respectively. Previous data have shown that all PF07598 gene family members are variably upregulated in a hamster model of acute severe leptospirosis (Lehmann et al., 2013, PLoS Negl Trop Dis, 7(10): e2468). VM protein antigens specific for groups G-III and G-IV were selected based on previous data showing the highest and lowest expression in vivo (Lehmann et al., 2013, PLoS Negl Trop Dis, 7(10): e2468). The present findings suggest that vaccination with VM proteins with a minimal complement of cross-reactive VM proteins may confer protective immunity, although at this time it remains unclear whether both LA1400 and LA0591 are required as immunogens. Interestingly, post-immunization / pre-challenge sera from G-IV heterologously cross-reacted with the highest titers to LA1402 and LA3490, despite low titers to the homologous proteins. LA1400 is the ancestral VM protein of group 1 pathogenic leptospira belonging to cluster A (Fouts et al., 2016, PLoS Negl Trop Dis. 10(2):e0004403) and contains two N-terminal tandemly repeated ricin B-like lectin domains (RBLs) and a C-terminal toxin domain (CTD). LA0591 contains the CTD but lacks the RBLs. Although these domains might be predicted a priori to be most strongly cross-reactive with homologous VM proteins, experimental data indicate that rather heterologous cross-reactivity was strongest.Further experiments are underway to further determine whether immunization with either these proteins alone, concatenated or isolated subdomains of the various VM proteins, or another full-length VM protein such as LA1402 or LA3490, could confer pan-leptospiral immunity. A possible scenario is that the general cross-reactivity against VM proteins induced by vaccination with LA1400 and LA0591 mediates protection against lethal challenge infection and tissue colonization. This possibility is suggested by bioinformatics analyses showing that VM proteins are highly conserved at the amino acid level within L. interrogans, and is also supported experimentally (Chaurasia et al., 2022, Frontiers in Microbiology, 13:859680).

[0304] Immunization of leptospiral disease-susceptible C3H / HeJ mice with full-length leptospiral VM protein (Viriyakosol et al., 2006, Infect Immun. 74(2):887-95) protected against severe disease, whereas isolated RBL, t3490, a recombinant protein containing only the N-terminal ricin B domain (G-II), enhanced disease but reduced bacterial burden in the liver and kidney. Multiplex cytokine analysis of serum showed that group II mice showed a unique elevation of inflammatory cytokine markers (IL-β, IL-6, IL-10, IFN-γ, TNF-α, and KC / GRO (Wolpe et al., 1989, Proc Natl Acad Sci USA. 86(2):612-6)), a neutrophil chemoattractant related to rodent IL-8, suggesting that these cytokine storms may have led to the death of the mice. The mechanism by which RBD domain-induced immune enhancement leads to severe disease is unclear. Without wishing to be bound by theory, it was speculated that one potential mechanism by which t3490 immunization leads to enhanced disease could be the induction of antibodies against the N-terminal RBL of leptospiral secreted VM proteins that deliver full-length proteins to the proinflammatory pathways of Fc receptor-containing cells in vivo, although this hypothesis requires experimental validation. Nevertheless, cross-reactive antibodies generated against the RBD in G-II-immunized mice did not protect against severe disease. These observations suggest that the N-terminal RBD alone should not be used in VM protein-based leptospirosis vaccine research. Further studies examining RBD-mediated immune enhancement are warranted.

[0305] In this study, ELISA and Western blot analysis of sera after vaccination with recombinant VM protein and osmotically induced in vitro cultured L. interrogans serovar lei indicate that vaccination resulted in both homologous and heterologous VM protein recognition associated with protective immunity. These experimental results confirm the bioinformatic prediction of cross-reactivity of polyclonal antisera against VM proteins within the L. interrogans genus. Further studies are essential to confirm protection by other L. interrogans serovars against challenge infection in rodent models. Cross-species protection experiments against challenge with virulent isolates of the closely related L. kirschneri and L. noguchii, other group 1 highly pathogenic Leptospira species, after VM protein vaccination (Lehmann et al, 2013, PLoS Negl Trop Dis, 7(10): e2468; Fouts et al., 2012, J Hepatol, 56(6):1283-92) are planned. Cross-species protection experiments following vaccination with homologous or heterologous VM proteins followed by challenge with virulent isolates of other group 1 pathogens, such as L. borgpetersenii, which have few PF07598 paralogs in their genomes, will contribute to determining which VM proteins are suitable for further development of a pan-leptospira vaccine.

[0306] A serotype-independent pan-leptospira vaccine that confers protection against leptospirosis is a major priority in the leptospirosis field (Wunder et al., 2021, Elife., 10; Beutler et al., 2000, Eur Cytokine Netw, 11(2):143-52). Various inactivated whole bacterial cell-based vaccines (bacterins) are serotype specific and are limited to use in animals, and this conventional technology remains incompletely effective. Subunit vaccines, and more recently a naturally occurring attenuated variant of L. interrogans serovar copenhageni (Wunder et al., 2021, Elife., 10), have been proposed in the search for pan-leptospira vaccine candidates (Haake et al., 1999, Infect Immun, 67(12):6572-8223; Conrad et al., 2017, PLoS Negl Trop Dis, 11(3):e0005441; Techawiwattanaboon et al., 2019, Vaccines (Basel), 7(3); Govindan et al., 2021, Appl Nanosci, 1-15; Phoka et al., 2021, Vet Microbiol, 262:109220; de Oliveira et al., 2021, Vaccine, 39(39):5626-34; Haake et al., Front Immunol, 11:579907; Teixeira et al., 2020, Front Immunol, 11:568694; Coutinho et al., 2011, PLoS Negl Trop Dis, 5(12):e1422).Bacterins have limited widespread use due to side effects and suboptimal efficacy, including a lack of sustained protective and sterilizing immunity (Felix et al., 2020, Expert Opin Drug Discov. 15(2):179-88; Techawiwattanaboon et al., 2019, Vaccines (Basel), 7(3); Levett, 2001, Clin Microbiol Rev, 14(2):296-326; Zaugg et al., 2021, Schweiz Arch Tierheilkd, 163(9):545-52).

[0307] This report demonstrates protective immunity induced by vaccination with a subset of L. interrogans VM proteins against lethal challenge infection. Immunization strategies to induce anti-VM protein antibodies validate the role of VM proteins in mediating leptospirosis pathogenesis.

[0308] The materials and methods used in the experiments are described below.

[0309] bacterial culture Leptospira interrogans serovar canicola strain LOCaS46 was grown at 30°C in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH, BD Biosciences, USA) (Ellinghausen et al., 1965, Am J Vet Res, 26:39-44). Leptospira were grown under conditions mimicking the in vivo host environment known to induce virulence gene expression in vitro (Matsunaga et al., 2005, Infect Immun, 73(1):70-8). Briefly, mid-logarithmic cultures in unmodified EMJH medium were harvested by centrifugation at 18,514 g. Pelleted cells were washed twice with 1x phosphate-buffered saline, resuspended in liquid EMJH medium supplemented with 120 mM NaCl, and incubated for 4 h at 37°C (Sigma Aldrich, USA). The LD50 of the LOCaS46 strain is a median lethal dose LD50<100 (Salinas et al., 2020, Vaccines (Basel), 8(4)).

[0310] Chemically competent E. coli strain DH5α (New England Biolabs, Ipswich, MA) was used for gene cloning, and SHuffle® T7 competent E. coli cells (New England Biolabs, USA) were used for protein expression and purification. E. coli was grown in Luria-Bertani (LB) medium (BD Biosciences, Sparks, MD) supplemented with 100 μg / mL ampicillin (Sigma-Aldrich, St. Louis, MO).

[0311] L. interrogans serovars rae, canicola, copenhagenii, and non-pathogenic L. biflexa serovar patoc were grown in liquid EMJH medium and harvested by centrifugation at 18,514 g for 10 min. Cells were washed twice with 1x PBS (pH 7.4) and pellets were resuspended in 5 mL / gram of BugBuster® Protein Extraction Reagent (Sigma-Aldrich, St. Louis, MO) containing "Protease Inhibitor Cocktail with EDTA" (Roche, USA). Cell lysates were incubated for 15 min at room temperature on a rotating mixer. Insoluble cell debris was removed by centrifugation at 18,514 g for 20 min at 4 °C. Supernatants were stored at -20 °C until analysis.

[0312] computational biology The N- and C-terminal amino acid sequences of the PF07598 family (LA3490, LA0620, LA1402, LA1400, and LA0591) were aligned using MAFFT (Multiple Alignment Using Fast Fourier Transform) with L-INS-i (Accuracy Emphasis) and visualized using Jalview v2.11.5 (jalview.org). The originally deposited LA1400 sequence was found to be incomplete in that it lacked sequence encoding the first 54 amino acids of the fully encoded protein. This conclusion was based on the use of cluster analysis comparing the amino acid sequence of L. interrogans serovar Lei LA1400 to LIC12340, the LA1400 orthologue of L. interrogans serovar Copenhagenii strain FioCruzL1-130 (Supplementary Information). The recombinant protein, designated LA1400 in this study, is composed of amino acids 31 to 54 from LIC12340 followed by amino acids 55 to the end from LA1400.

[0313] animal Three-week-old specific pathogen-free female C3H / HeJ mice (The Jackson Laboratory, ME, USA) were purchased from Jackson Laboratories (ME, USA) and maintained in a specific pathogen-free environment at the Yale Animal Resources Center. Mice were housed in individually ventilated microisolator cages with sterile absorbent bedding that was changed twice weekly. Animals were provided with food and water throughout the experiment. After challenge with L. interrogans serovar canicola, mice were weighed and followed twice daily until the terminal endpoint. They were observed for anorexia, severe lethargy, labored breathing, weakness, disheveled fur, and 10% weight loss. Mice that exhibited these symptoms were euthanized by CO2 according to AAALAC / AVMA-approved procedures and were considered to have met the endpoint of severe / fatal leptospirosis.

[0314] Plasmid construction and cloning The complete PF07598 gene encoding NCBI locus tags LA3490 (Uniprot: Q8F0K3), LA0620 (Q8F8D7), and LA1402 (Q8F6A7) from serovar Lai, locus tags LIC12340 (Q72PX7) (Lai ortholog: LA1400), and LIC12985 (Q72N53) (Lai ortholog: LA0591) from serovar Copenhageni, coding sequence excluding the predicted signal peptide, or synthetic E. coli codon-optimized genes consisting of truncated 3490, the N-terminal domain, were constructed and synthesized by Gene Universal (geneuniversal.com). The LA3490, LA0620, LA1402, and t3490 were linked to mCherry (AST15061.1) via a glycine-serine hinge (Gly4Ser)3 and cloned into pET32b(+) (Gene Universal Inc., USA) between the enterokinase cleavage sites for convenient removal of the mCherry fluorescent tag. Full-length LA1400 and LA0591 constructs without mCherry fusion were generated (Figure 19A). Prior to use, the sequence and orientation of the genes in the constructs were verified by restriction digestion and sequencing.

[0315] Expression and purification of recombinant soluble PF07598 antigen Recombinant PF07598 protein constructs were expressed in SHuffle® T7 competent E. coli cells (New England Biolabs, USA). Transformants were subcultured in Luria-Bertani (LB) medium containing 100 μg / mL ampicillin. PF07598 protein expression was induced at OD 0.6 by addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG; Sigma-Aldrich, USA) and incubated at 16°C and 250 rpm for 24 hours. After induction, cells were harvested and pellets were lysed with CelLytic™ B (cell lysis reagent; Sigma-Aldrich, USA) containing 50 units of benzonase nuclease (Sigma-Aldrich, USA), 0.2 μg / mL lysozyme, non-EDTA protease inhibitor cocktail (Roche, USA) and 100 mM PMSF (Sigma-Aldrich, USA) for 30 minutes at 37°C. The supernatant and pellet were separated and then analyzed by 4–12% bis-tris sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was measured by BCA assay (Bio-Rad, Hercules, CA).

[0316] Recombinant PF07598 fusion and fusion-free constructs were purified using a 5 mL pre-packed Ni-Sepharose AKTA Hi-TRAP column (GE Healthcare, USA) pre-equilibrated with a buffer containing 100 mM NaH2PO4, 10 mM Tris-HCl, 25 mM imidazole (pH 8.0). The PF07598 protein bound to the Hi-TRAP column was then eluted in the presence of 500 mM imidazole (pH 8.0). The eluates were pooled, concentrated with a 10 kDa Amicon® ultracentrifugal filter, and further dialyzed overnight against 1×PBS (pH 7.4) with gentle agitation (350 rpm) at 4° C. (10 kDa cutoff, Slide-A-Lyzer, Thermo Scientific™, USA). Purified recombinant PF07598 protein was resolved by SDS-PAGE and verified by immunoblotting with mouse anti-His monoclonal-ALP conjugate (1:2,000 dilution, Santa Cruz Biotechnology, USA). Booster and SDS-PAGE aliquots were prepared from a single preparation and stored at -80°C to prevent repeated freeze-thaw cycles.

[0317] Animal immunization, Leptospira challenge, and sample collection C3H / HeJ mice were immunized with recombinant PF07598 protein via the intramuscular (IM) route (Viriyakosol et al., 2006, Infect Immun, 74(2):887-95). GLA-squalene-oil-in-water emulsion adjuvant (0.25 mg / mL) was obtained from the Infectious Disease Research Institute (IDRI), Seattle, WA, USA (idri.org). Immediately prior to injection, the adjuvant was added to the recombinant protein or PBS to a final volume of 100 μL and vortex mixed briefly (Patra et al., 2015, Infect Immun, 83(5):1799-1808).

[0318] Mice were divided into four groups; GI was injected with 1× phosphate buffered saline (PBS) mixed with adjuvant (EM082; 5 μg GLA-squalene-oil-in-water emulsion) as a negative control. Similarly, mice were immunized with G-II (t3490), G-III [VM mix, (LA3490, LA0620, LA1400, LA1402, and LA0591], and G-IV [VM unlabeled, (LA1400 and LA0591)] at an equimolar ratio of 25 μg total antigen with adjuvant (5 μg GLA-squalene-oil-in-water emulsion) and then immunized with two injections of 25 μg total antigen at 3-week intervals (Figure 20). Two weeks after the last immunization, immunized mice were bled and serum samples were pooled to level out individual differences between groups, and anti-VM antibodies in the serum were measured, known as pre-challenge bleeds. All groups received 1×10 5 Mice were experimentally infected by intraperitoneal (IP) injection with an isolate of the virulent low-passage microorganism L. interrogans serovar canicola strain LOCaS46. Mice that survived infection were euthanized 13 days after infection challenge. Blood was collected by terminal cardiac puncture and serum was isolated from whole blood. Serum was allowed to clot at room temperature and stored overnight at 4°C. Samples were then centrifuged at 11,292g for 15 min at 4°C. Serum was collected and stored at -80°C. Organs were collected and stored in RNALater at 4°C. Kidney and liver tissues were used to quantify L. interrogans by quantitative PCR (qPCR).

[0319] Assessment of PF07598 protein-induced immunity by ELISA Serum antibody responses against recombinant PF07598 protein in immunized groups were quantified by ELISA (61). Briefly, PF07598 antigens (LA3490, LA0620, LA1402, LA1400, and LA0598, respectively) in 100 μL of bicarbonate / carbonate coating buffer were coated (250 ng) onto 96-well microtiter ELISA plates (Corning, USA) and incubated overnight at 4 °C. Each antigen set was incubated with pre- and post-immune serum groups (groups I-IV, 1:1000) for 1 h, followed by incubation with goat anti-mouse IgG (Fc specific)-alkaline phosphatase conjugate (1:5000; KPL, USA) for 1 h, washed three times with TBST, and developed with p-nitrophenyl phosphate (1-Step™ PNPP substrate solution; KPL, USA). The reaction was stopped with 2M NaOH and absorbance was read at 405 nm using a SpectraMax® M2e microplate reader (Molecular Devices, USA). For whole cell ELISA, plates were coated with 500 ng / well of cell-free lysate. Controls included pre-bleed, pre-immune serum samples, antigen, and antibody blanks.

[0320] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis SDS-PAGE was performed according to the method of Laemmli (Laemmli, 1970, Nature, 227(5259):680-5). Immunoblot analysis was performed to determine whether sera from immunized animals recognized recombinant or native Leptospiral PF07598 protein. Purified recombinant PF07598 protein or Leptospiral whole cell lysates (induced and uninduced with 120 mM NaCl) were transferred to nitrocellulose membranes and blocked with 5% nonfat dry milk in 1×TBST buffer (AmericanBio, USA) for 2 h. The membranes were incubated overnight at 4°C on a rocker with pooled sera from immunized groups (groups I–IV, 1:100) and pre-bleeds and pre-immune bleeds as controls. They were probed with goat anti-mouse IgG (Fc specific)-alkaline phosphatase conjugate (1:5000; KPL, USA) for 2.5 h, washed three times with TBST, and developed with p-nitrophenyl phosphate (1-Step™ PNPP substrate solution). Monoclonal LipL32 antibody served as a loading control (diluted 1:10,000).

[0321] quantitative PCR DNA was extracted by dicing 40 ± 50 mg of kidney and liver tissue and suspending in 500 μL of 1x PBS. To reduce the risk of cross-contamination, all operations were performed under positive pressure in an area separate from other handling of leptospira and PCR products. After homogenization of the tissue, total genomic DNA was extracted from 25 mg equivalent of tissue using the DNeasy Blood and Tissue kit (Qiagen, USA) according to the manufacturer's instructions and eluted in 50 μL of elution buffer. L. interrogans serovar canicola was cultured at 2 × 10 7 Leptospira were grown in 5 mL of EMJH medium at a density of 1000 leptospira / mL. Cells were harvested and DNA was extracted for standard curves using the same DNeasy Blood and Tissue kit (Qiagen, USA).

[0322] The concentration of eluted DNA was measured using a NanoDrop spectrophotometer ND-1000 (NanoDrop Technologies, DE, USA). All DNA samples were stored at -80 °C until use. Serial dilutions of DNA (1 × 10 to 1 × 10) were 7 A genome equivalent (GEq) / 5 μL was prepared and L. interrogans serovar canicola genome was quantified by qPCR using 2XiQ5 SYBR Green supermix (Bio-Rad, CA, USA) containing 5 pmol of forward LipL32 primer (5'-TCTGTGATCAACTATTACGGATAC-3'; SEQ ID NO:19) and reverse LipL32 primer (5'-ATCCAAGTATCAAACCAATGTGG-3'; SEQ ID NO:20). Four microliters of standard or sample DNA was added to 10 μL PCR mix and reactions were amplified on a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) using the following program: 95°C for 3 min, 44 cycles of 95°C for 0.10 min, 62°C for 0.30 min, followed by 72°C for 1.00 min, then a final extension at 72°C for 7 min. A standard curve was generated using Bio-Rad iQCycle5 software, and the number of GEq was extrapolated from the threshold cycle (CT) values. A negative result was assigned if no amplification occurred or if the CT value exceeded 3SD+Ct. Data are presented as the number of L. interrogans GEq per gram of tissue.

[0323] statistical analysis All experiments were repeated twice in triplicate. Kruskal-Wallis tests were used to determine significant differences in bacterial counts in kidneys or livers between survivors from different immunization groups. Results were analyzed by nonparametric Mann-Whitney tests to determine significant differences between individual groups and were considered statistically significant when p<0.05, p<0.001. All analyses and graphs were generated using Graph Prism version 8 (GraphPad Software, Inc., La Jolla, CA).

[0324] Here, experimental results are provided.

[0325] Conservation of the PF07598 protein family and its orthologues in pathogenic Leptospira The VM paralog protein family encoded by PF07598 has an expanded repertoire within L. interrogans, with at least 12 distinct paralogs in serovars Rai, Copenhageni, and Canicola. The orthologs share >90% amino acid identity (Figure 24). Most VM proteins consist of approximately 640 amino acids, with an AB domain structure consisting of two tandemly arranged β-trefoils, an N-terminal ricin B-like lectin domain, and a C-terminal toxin domain with DNase activity. L. interrogans serovars also encode a single unique ortholog that lacks the N-terminal ricin B-like domain (represented by LA0591 at approximately 313 aa) but contains a signal sequence.

[0326] Immunization with full-length VM protein prevented severe leptospirosis in mice Full-length recombinant VM proteins LA3490, LA0620, LA1402, LA1400, and LA0591 (following the nomenclature of L. interrogans serovar Lei) were expressed in E. coli as N-terminal fusions with a thioredoxin (TRX)-His6 affinity tag to promote solubility and affinity purification, and as C-terminal fusions with mCherry-His6 to facilitate protein affinity purification and visualization by fluorescence microscopy, respectively (Figure 19A). The homogeneity of the recombinant VM proteins was verified by SDS-PAGE and Western immunoblot (Figure 19B).

[0327] Mice were injected intramuscularly with recombinant protein mixed with glucopyranosyl lipid A / squalene oil-in-water (GLA-SE) adjuvant or PBS control (schematically shown in FIG. 20). This adjuvant was chosen for this experiment because it is compatible with human use and is useful for testing in animal models toward eventual vaccine development for humans. The GLA component, a synthetic, non-toxic moiety with six acyl chains and one phosphate group on a disaccharide backbone (Pantel et al., 2012, Eur J Immunol, 42(1):101-9), would not be expected to have the immunostimulatory effects of a TLR4 agonist in C3H / HeJ mice, which are genetically reduced in responsiveness to lipid A due to a mutation in the gene encoding a functional Toll-like receptor (TLR4) (Beutler et al., 2000, Eur Cytokine Netw, 11(2):143-52).

[0328] The main outcome of this immunological study was that mice were able to withstand lethal challenge infection (median lethal dose LD50 < 100) with low passage (P3) L. interrogans serovar canicola strain LOCaS46 at 10 ng / mL. 5 Mice were euthanized and considered to have reached a severe disease endpoint if they developed severe symptoms of leptospirosis after challenge infection, defined as a weight loss of >15% from the start of the experiment, or an inability to groom, eat or drink, or develop severe lethargy or hunched posture. Secondary outcomes were 1) quantitative bacterial burden in liver and kidney measured by quantitative real-time PCR, and 2) antibody responses measured by ELISA and Western immunoblot.

[0329] None of the mice developed severe disease after the immunization protocol. Mice receiving PBS (GI) plus adjuvant or ricin B domain RBL1 [t3490, (G-II)] plus adjuvant showed slight weight loss after challenge infection but had to be euthanized on days 6 and 5, respectively, due to severe illness manifested by lethargy and inability to eat and drink. Vaccination with full-length VM protein, either the five-species mixture (G-III) or the two-species mixture (G-IV), prevented all observable clinical disease (Figure 21A). This observation indicates that full-length VM protein is required for protection against severe leptospirosis.

[0330] Immunization with rVM protein significantly reduced bacterial burden in the liver and kidneys The leptospiral load in the liver and kidney of the four experimental groups was quantified by qPCR. After challenge infection, the three groups immunized with recombinant protein and adjuvant (G-II, G-III, and G-III) showed approximately 10 genome equivalents (Geq) per gram of tissue in the liver and kidney compared to the PBS control group (GI). 3 ~10 4 fold reduction (Kruskal-Wallis test, ANOVA results: liver p<0.0001, kidney p=0.0003) (Figures 21B and 21C). This statistically significant difference was confirmed by Dunn's multiple comparison statistical test (VM mixture (G-III), p=0.0054, and VM unlabeled protein (G-IV), p<0.0001) with the control group PBS (GI).

[0331] Immunization with t3490 significantly reduced bacterial burden in the liver and kidneys, yet still led to severe disease driven by inflammatory cytokines To determine whether immunization with the first highly conserved ricin B-like domain (RBL1) confers protection from lethal challenge, E. coli -produced recombinant RBL1 domain (truncated 3490, t3490) was produced as a control for the full-length VM protein LA3490, purified using the same procedure as full-length LA3490, and used in immunization studies. Surprisingly, mice immunized with t3490 (G-2) developed accelerated clinical disease after challenge infection, but with reduced bacterial burden in the liver and kidney (Figure 21B, Figure 21C). Enhanced disease in G-2 was associated with higher levels of TNF-alpha, IFN-gamma, IL-6, and IL-10, as well as the chemokine KC / GRO, compared to the PBS and full-length protein receptor groups (Figure 21D).

[0332] Antibody profile and cross-reactivity of mouse responses to PF07598 (VM) protein before and after challenge To determine whether mice immunized with VM proteins would develop IgG antibody responses, sera were collected from pre- and post-immunization mice and antibody profiles were examined by ELISA using all six antigens used in the study (Figure 22A). Control groups (G-I) and pre-immunization sera did not show detectable IgG antibodies to any of the VM antigens. Antibody responses to the t3490 antigen were observed in sera from t3490-immunized mice (G-II), and cross-reactivity was seen with the LA3490 (p=0.0010) and LA1402 (p=0.0010) antigens.

[0333] Sera from mice immunized with the VM mixture (G-III) reacted with all VM antigens tested [t3490, LA3490, LA0620, LA1402, LA1400, and LA0591, (p<0.0001)], with the highest titers observed for the LA3490 and LA1402 antigens. Antibody responses to each antigen in the VM mixture group [t3490 (p=0.0015), LA3490 (p<0.0001), LA0620 (p=0.0004), LA1402 (p<0.0001), LA1400 (p=0.0003), LA0591 (p<0.0001)] were observed in sera from the VM non-labeled group (G-IV), with the highest titers detected for the LA1400, LA0591, and LA0620 antigens. Antibody responses against t3490, LA3490, and LA1402 antigens were also observed in mice after immunization and before challenge. Prechallenge antibody titers against LA1400, LA0591, and LA0620 were lower than postinfection titers after challenge with live L. interrogans serovar canicola. Further experimental studies are needed to understand the direct action of VM proteins and their immune profiling in vivo. Despite having over 90% amino acid similarity, each VM protein exhibits unique reactivity to prechallenge and postchallenge sera and may have different in vivo functions. The difference in reactivity of VM proteins indicates differences in immunogenicity, and because of their high amino acid similarity, they cross-react with prechallenge and postchallenge sera. Generation of VM protein-specific monoclonal antibodies and identification of protective epitopes will help distinguish the roles and mechanisms by which various VM proteins contribute to the pathogenesis of leptospirosis.

[0334] Cross-reactivity was confirmed by Western immunoblot analysis using pooled sera from immunized animals probing recombinant VM proteins immobilized on nitrocellulose membranes (Figure 22B). Prebleed sera and PBS control (G-1) groups showed no reactivity with the VM mixture and the VM unlabeled recombinant antigen cocktail (five and two proteins, respectively). Sera from mice immunized with t3490 showed large antibody titers against the t3490 antigen (not shown), cross-reacting with full-length VM protein, but reacting weakly with LA1400 and not with LA0591, which lacks the N-terminal ricin B domain, suggesting that only t3490 cross-reacts with a shared epitope in the N-terminal region of the VM protein. Sera from the VM mixture group (G-III) cross-reacted with all five antigens, with reactivity patterns consistent with each other. The reactivity of LA1400 with sera from the VM non-labeled group (G-IV) was the highest among all VM proteins and in the same cocktail lot of VM antigens immunized in G-III and G-IV mice. The finding that high titer antibodies (measured by both ELISA and Western blot) against LA1400 antigen were induced in the VM non-labeled group sera (G-IV) suggests that LA1400 elicits the strongest humoral immune response in mice compared with other VM proteins and may be involved in mediating protective immunity. However, these data do not provide strong confidence that one or more of these VM proteins mediate the pathogenesis of this animal model. Future optimization of which VM proteins should be used for vaccination based on these observations will be supported by these data.

[0335] Expression of VM proteins in vitro and in vivo and cross-reactivity among pathogenic serotypes Protein extracts from L. interrogans serovars rae, canicola, and copenhageni, and the nonpathogenic strain L. biflexa serovar patok, induced with or without 120 mM NaCl, were examined by Western blot using the polyclonal anti-LA3490 antibody. Expression of the native VM protein was seen at the predicted size with a molecular weight of approximately 70 kDa in the pathogenic serovars rae, canicola, and copenhageni, but not in serovar patok (negative controls showed the absence of PF07598 gene family members in this saprophytic species) (Figure 23A).

[0336] To determine whether immunization with a limited set of VM proteins results in broadly cross-reactive anti-VM protein antibodies, in vivo VM protein expression and cross-reactive serotype immune profiles were estimated by Western immunoblot analysis using cell-free protein extracts from Leptospira interrogans serovar canicola and the noninfectious saprophytic bacterium L. biflexa serovar patok. Antibodies from the immunized groups showed that antibodies against serovar canicola recognized VM proteins of the predicted size (approximately 70 kDa), suggesting in vivo VM protein expression during challenge infection. This antibody reactivity also reacted with G-III and G-IV sera after challenge. However, no reactivity was seen with negative control serovar patok cell-free lysates (Figure 23B). Lower molecular weight reactive proteins were detected in sera from G-III and G-IV, suggesting that VM proteins may be undergoing proteolytic processing (Figure 23B). Further studies are needed to determine whether these low molecular weight proteins play a role in Leptospira pathogenesis.

[0337] IgG antibody profiles were quantified against homologous and heterologous VM proteins. Cell-free protein extracts from L. interrogans serovar canicola and the avirulent strain L. biflexa serovar patok were used as solid-phase antigens adsorbed to ELISA plates. ELISA confirmed the reactivity of serovar canicola with sera from VM mix (G-III) and VM unlabeled (G-IV) mouse groups after challenge. VM proteins encoded by L. interrogans serovar lei, used for immunization of G-III and G-IV groups, cross-reacted with lysates of serovar canicola. Notably, orthologs of VM proteins are highly conserved in serovar L. interrogans, consistent with this observed cross-reactivity. The avirulent serovar patok did not cross-react with sera from control and immunized mouse groups, either before or after challenge (Figure 23C).

[0338] Example 4: Mouse monoclonal studies Scouting was performed using five clones from YUMS1B against the target antigen LA0591 at 500 nM concentration. All five clones were scored as positive with affinities ranging from pM to double digit nM. In the table above, clones are ranked from highest to lowest affinity. Please note that KDs from scouting at a single analyte concentration are only estimates and can be up to 10-fold higher or lower compared to KDs determined by full kinetics over a range of 5-6 analyte concentrations.

[0339] Figures 25-27 show the reactivity of monoclonal supernatant (YUSM001B) with recombinant VM protein. FIG. 28 shows the reactivity of monoclonal supernatants (YUSM001A, LA1400) with recombinant VM protein. FIG. 29 provides confirmatory screening data. FIG. 30 provides a table of mouse IgG quantification data.

[0340] Example 5: Virulent Leptospira have evolved a unique gene family consisting of cytotoxins containing a ricin B-like lectin domain Here, we demonstrate that Leptospira virulence modified (VM) proteins, represented by LA3490 (Q8F0K3), are bona fide R-type lectin domain-containing cytotoxins, the first experimentally validated leptospiral exotoxins. rLA3490 binds to HeLa cells via its N-terminal R-type lectin domain with specificity for terminal galactosyl residues and is rapidly internalized into the cells. After binding / internalization, it translocates to the HeLa cell nucleus via a nuclear targeting signal and twin LxxLL motifs for nuclear receptor binding. Cell surface binding and internalization have been shown to be rapid, occurring within 30 min after exposure. rLA3490 induces pleiotropic effects on HeLa cells, including actin depolymerization, caspase 3 activation, nuclear fragmentation, and ultimately blebbing and cell death. One mechanism of cell death may result from genomic DNA degradation that occurs following nuclear localization of the VM protein. Supporting in vitro experiments using purified HeLa cell genomic DNA and supercoiled and linearized bacterial plasmid DNA indicate that rLA3490, and at least four other VM proteins tested to date, have endo- and exo-DNase activity.

[0341] With the exception of the CBR deletion variants, most VM proteins fit the classical AB toxin paradigm (Odumosu et al., 2010, Toxins (Basel), 2:1612-1645). The entire Leptospiral VM protein gene contains domains commonly encoded by two or three separate genes in other bacteria. There are at least two functionally distinct regions in the VM protein, the N-terminus which is partly involved in host cell targeting (binding and internalization) and the C-terminus which partly mediates cytotoxicity (intracellular trafficking / enzymatic activity). The N-terminal segment is fairly well conserved among Leptospiral serotypes (average identity of paired amino acids is about 78%) and contains a confirmed R-type lectin domain (amino acid positions 40-174) that shares binding specificity with the ricin B chain for terminal galactosyl residues of glycoproteins. In contrast, the C-terminal segment is less conserved (average identity of paired amino acids is about 63%) and is thought to mediate cytotoxicity. Since other VM proteins tested so far show DNase activity in vitro, this sequence diversity is likely to affect the cellular targeting specificity (i.e., successful binding / internalization and intracellular trafficking) of VM proteins, rather than catalytic activity. Despite the evidence that the family cluster of paralogs has expanded, the reason for the diversification of the PF07598 gene family remains unclear, but one leading hypothesis is that the expansion of paralogs has allowed adaptation of different leptospira to different hosts. Further experiments comparing the structure and function of VM proteins and in silico analysis are needed to determine sequence motifs that may indicate differences in virulence between PF07598 family members.

[0342] Comparison of intragenomic distances revealed that the expanded VM protein repertoire in virulent group I pathogenic Leptospira arose from a series of gene duplication events followed by autonomous evolution of the N- and C-terminal segments, the latter occurring more rapidly. Based on available data, the first duplication event appears to have generated LA1402 / / LA1400, which constitutes the only L. interrogans VM protein identified so far with a close orthologue in a less virulent group I pathogenic species (Fouts et al., 2016, PLoS Negl Trop Dis. 10(2):e0004403). This initial event was followed by successive duplications, presumably derived from LA1400, resulting in the formation of three distinct gene clusters [A, B, and C (Fouts et al., 2016, PLoS Negl Trop Dis. 10(2):e0004403)], the largest of which contains seven VM protein-encoding genes (including LA3490 and LA0620). Some serotypes appear to have lost specific VM protein genes (e.g., LICRS03300 from serotype Rai and LA3271 from Hardjo), while others contain various CBR deletion variants. The uneven distribution of VM proteins among Leptospira serotypes and these implicit differences in host cell targeting specificity are the first conclusive evidence that some serotypes are inherently more virulent than others, thus increasing clinical and public health importance. Most of the genes encoding VM proteins are found only in L. interrogans and its sister species. As some VM proteins, such as LA3490(Q8F0K3), have proven to be particularly toxic to human cells, their presence in serum may herald severe disease complications and provide prognostic information on which to base effective clinical risk assessment.

[0343] Site-specific expression of specific VM proteins, coupled with putative differences in host cell specificity (i.e., exposure and susceptibility) as well as pathway-dependent ricin, where virulence is clearly linked and pathogenesis is most lethal (inhalation and severe respiratory failure are the most lethal), may explain the various clinical manifestations of severe leptospirosis. Indeed, efforts to understand the molecular and cellular pathogenesis of leptospirosis are still in their infancy, and approaches to prevent or ameliorate leptospirosis are based on a mechanistic understanding of the biology of leptospiral-host interactions. For example, pulmonary hemorrhage and refractory shock are particularly important clinical manifestations of leptospirosis (Sehgal et al., 1995, Indian J. Med. Res, 102:9-12; Marotto et al., 1999, Clin. Infect. Dis, 29:1561-1563; Segura et al., 2005, Clin. Infect. Dis, 40:343-351; Gouveia et al., 2008, Emerg. Infect. Dis, 14:505-508; Truong and Coburn, 2011, Front. Cell Infect. Microbiol, 1:24; Helmerhorst et al., 2012, Neth. J. Med. 70:215-221; Ruwanpura et al., 2012, Med. J. Malaysia, 67:595-600). Indirect evidence that these severe symptoms improve with hemodialysis / hemofiltration (Andrade et al., 2007; Cleto et al., 2016) suggests that there may be a circulating soluble toxin in leptospirosis.Histopathological analysis of lung tissue in severe pulmonary leptospirosis syndromes does not detect intact leptospires (Nicodemo et al., 1997, Am. J. Trop. Med. Hyg, 56:181-187), but rather alveolar epithelial damage and endothelial cell activation, with secondary deposition of immunoglobulins and complement (Nally et al., 2004; Croda et al., 2010, Clin. Microbiol. Infect, 16:593-599; De Brito et al., 2013, PLoS One, 8:e71743). Nevertheless, several potential leptospiral toxins have been identified apart from various sphingomyelinases / hemolysins (Narayanavari et al., 2015, PLoS Negl. Trop. Dis. 9:e0003952; Chaurasia and Sritharan, 2020, Microbiology (Reading) 166, 1065-1073) and collagenases (Kassegne et al., 2014, Leptospira species. J. Infect. Dis, 209:1105-1115), but none can adequately explain the etiologic features of the diverse clinical spectrum of leptospirosis. Nevertheless, hemodialysis and hemofiltration remain life-saving interventions, but exceed clinical resources and / or capacities in the majority of leptospirosis-endemic areas.In contrast, monoclonal antibody (mAb)-based biologics or small molecule inhibitors (Benz and Barth, 2017, Curr. Top. Microbiol. Immunol., 2017, Toxins (Basel), 9:311) that disrupt cell surface binding / cell entry and / or intracellular trafficking / toxicity of VM proteins, as well as other AB toxins (Odumosu et al., 2010, Toxins (Basel), 2(7):1612-45), such as CARDS (Somarajan et al., 2014, mBio, 5:e01497-e01514; Becker et al., 2015, Proc Natl Acad Sci USA, 112(16):5165-70), and ricin (Yermakova et al., 2014, mBio, 5:e00995; Gal et al., 2017, Toxins (Basel), 9:311), have been developed to inhibit the cell surface binding / cell entry and / or intracellular trafficking / toxicity of VM proteins. 406:229-256) would constitute a more generally available alternative to reduce the toxicity of VM proteins.

[0344] The data presented here build on previously published observations (Matsunaga et al., 2007; Infect Immun, 75(6): 2864-2874; Lehmann et al., 2013, PLoS Negl Trop Dis, 7(10): e2468; Fouts et al., 2016, PLoS Negl Trop Dis. 10(2):e0004403) and indicate that the leptospiral VM protein is likely a major virulence factor involved in the molecular and cellular pathogenesis of leptospirosis. Since then, transposon mutagenesis screens have shown that multiple VM proteins, especially Q8F6G8 (gene ID, LA0589), contribute to the fatal disease in hamsters (Murray et al., 2009, Infect. Immun, 77:810-816; Truong and Coburn, 2011, Front. Cell Infect. Microbiol, 1:24). Nevertheless, VM proteins were previously classified as PF07598, a useless protein family of unknown function. Now, a Phyre2-based prediction identifies these proteins as belonging to a superfamily of proteins, R-type lectins, named after the ricin B chain, with structurally similar carbohydrate-binding activity, and present in plants, animals, and bacteria (Cummings et al., 2017, Cold Spring Harbor Laboratory Press).Ricin and its B chain (and other R-type lectins) bind to terminal galactose or other related glycans of various host cell surface glycoconjugates, which promotes the translocation and internalization of ricin A chain into target cells, leading to cell killing via inhibition of protein synthesis (Montanaro et al., 1973, Biochem. J, 136:677-683; Sperti et al., 1973, Biochem. J, 136:813-815; Lord et al., 2003, Toxicol. Rev, 22:53-64; Sowa-Rogozinska et al., 2019, Toxins (Basel), 11:350). Similarly, bacterial AB toxins such as Shiga toxin, pertussis toxin, and diphtheria toxin mediate cell killing either by ADP-ribosylation of 28S rRNA or the ai subunit of the heterotrimeric G protein, or by inactivation of elongation factor 2 (Brown et al., 1980, FEBS Lett. 117, 84-88; Cemal, 1999, Design And Construction Of Membrane-Acting Immunotoxins For Intracellular And Secreted Protein Expression In Pichia Pastoris. Ph.D. Thesis, University of Cambridge, Cambridge; Coutte and Locht, 2015, Future Microbiol, 10:241-254; Cherubin et al., 2018, Sci Rep, 8(1):2494). Genotoxins such as the Leptospiral VM protein (e.g., cytolethal distending toxin (CDT)) have been less studied. Characterization of the leptospiral VM protein revealed several general features of bacterial genotoxins.For example, they have pleiotropic effects on target cells, such as exhibiting DNase activity, translocating to the nucleus via a nuclear localization signal (McSweeney and Dreyfus, 2004, Cell Microbiol, 6:447-458), and inducing cell death via caspase-3-dependent and -independent mechanisms (Ohara et al., 2008, Infect. Immun, 76:4783-4791).

[0345] By recontextualizing the data presented here within the classical AB toxin paradigm, we suggest that, as reported for ricin B, which conferred overlapping VM protein-ricin B carbohydrate binding specificities (Simmons et al., 1986, J. Biol. Chem, 261:7912-7920; Lord et al., 1992, Biochem. Soc. Trans, 20:734-738; Newton et al., 1992, J. Biol. Chem, 267:11917-11922), after binding to the cell surface, presumably via the mannose receptor, ricin and most AB toxins, such as Shiga toxin (Arfilli et al., 2010, Biochem. J, 20:11917-11922), may be able to bind to the ribosomal protein carboxylase (Ricin) via a mannose receptor. 432:173-180), it has been proposed that VM proteins are endocytosed; released into the cytoplasm and then transported to the nucleus via internal nuclear localization signal-acquired entry after binding to the nuclear pore complex via amphipathic α-helices that contain one or more LxxLL motifs; then actively translocate through the pore into the nucleoplasm, triggering nuclear fragmentation via intrinsic exonuclease activity; and possibly, if free in the cytosol, may induce caspase-3 activation and degradation of the cytosolic backbone during transport via an unknown mechanism.

[0346] Although it has been demonstrated that the ricin B-like lectin domains of different VM proteins (rLA3490, t3490, and t0620) bind to immobilized asialofetuin (Wales et al., 1994, Glycoconj J, 11:274-281; ​​Frankel et al., 1996, Biochemistry, 35:14749-14756; Dawson et al., 1999, J. Appl. Toxicol, 19:307-312), the natural target ligands and cellular targets remain to be defined. Second, although the differences in cytotoxic potential between VM proteins, their host cell target specificity, and the molecular pathways by which they exert their pleiotropic effects are still to be fully elucidated, these initial experiments indicate that they are likely cytotoxic genotoxins of the AB type. Third, it is becoming increasingly clear that leptospiral VM proteins arose from successive gene duplication events, while the number of genes in L. interrogans, L. kirschneri, and L. noguchii (Lehmann et al., 2013, PLoS Negl Trop Dis, 7(10): e2468) is smaller than that in other pathogenic Leptospira species (Lehmann et al., 2013, PLoS Negl Trop Dis, 7(10): e2468; Fouts et al., 2016, PLoS Negl Trop Dis. 10(2):e0004403). The reasons for this expansion of the repertoire in are not understood, nor is the reason for the uneven distribution of VM paralogs among Leptospiral serovars, although an as yet unknown ecological niche specialization is very plausible, perhaps a defense against eukaryotic predation in soil / surface water, similar to Shiga toxin production in E. coli (Lainhart et al., 2009, J. Bacteriol, 191:5116-5122).

[0347] The materials and methods used in the experiments are described.

[0348] Computational analysis To identify functional subdomains, the amino acid sequences of Q8F0K3 and its closest paralog Q8F8D7, encoded by LA3490 and LA0620, respectively, of L. interrogans serovar Rai, were submitted separately to the Phyre2 Remote Homology Search Portal1 (Kelley et al., 2015). Short functional regions and motifs, such as amphipathic potentially membrane-binding α-helices, putative eukaryotic protein sorting signals, proteolytic cleavage, phosphorylation sites, and binding / docking motifs, were identified by HeliQuest2 (Gautier et al., 2008, Bioinformatics, 24:2101-2102) and the Eukaryotic Linear Motif (ELM) resource3 (Kumar et al., 2020, Nucleic Acids Res. 48, D296-D306). Approximately 3,000 PF07598|VM proteins representing all clinically relevant Leptospira species, as well as L. alexanderi and L. alstonii, were aligned against a custom-built HMM model based on the complete PF07598 reference alignment. After visual inspection of the aligned amino acid residues, all (VM protein) sequences containing ambiguous amino acids were partially annotated if derived from the draft genome; those that did not span at least one putative functional subdomain [i.e., either RBL1 / RBL2 / CTD (carboxy-terminal domain)] were excluded. For the clustering analysis, partial alignments including the CBR (carbohydrate-binding region) and CTD (i.e., amino acid positions 23–343 and 344–639, respectively, for Q8F0K3) were removed from the curated global alignment and used as input for the calculation of an all-discrete-all-pairs distance matrix, excluding “gapped” columns (i.e., containing >50% gaps), using the R package bio2mds (Pele et al., 2012, BMC Bioinformatics, 13:133).Pairwise distance matrices showing close amino acid relatedness of full-length alignments and CBR and CTD partial alignments are provided for six Leptospira serovars of public health importance (L. interrogans serovars copenhageni, canicola, hardjaw, rae, manilae, and pomona, and L. kirschneri pomona). Poorly aligned columns were manually improved by visual inspection in Jalview v2.11.4. Fragmented VM proteins (i.e., those that did not span at least one functional subdomain) were removed. Curated multiple sequence alignments were used as input for HMM profile-based remote homology searches against the PDB, SCOPe70, SMARTv6, and UniProt-SissProt-viral70 databases via HHpred4. Consensus secondary structures were predicted using Ali2D5 and 3D (protein) structures were predicted using AlphaFold (Callaway et al., 2020, Nature, 588(7837):203-4; Jumper et al., 2020, predictioncenterorg / casp14 / doc / CASP14_Abstracts; Senior et al., 2020, Nature, 577(7792):706-10). Separate distance matrices were generated for the amino-terminal half, containing the predicted lectin domain, and the C-terminal half, containing the putative toxin subdomain, to infer their evolutionary associations and clustering relationships.

[0349] In vitro Leptospira culture and virulence gene expression Low-passage virulent L. interrogans serovar leprosy strain 56,601, which had been passaged through hamsters to regain high virulence (LD50<100) (Lehmann et al, 2013, PLoS Negl Trop Dis, 7(10): e2468), was maintained at 30°C in semisolid Ellinghausen-McCullough-Johnson-Harris medium (EMJH, BD Biosciences, United States) (Ellinghausen et al., 1965, Am J Vet Res, 26:39-44). Because published data showed that VM protein is transcriptionally upregulated in vivo in a hamster model of acute leptospirosis (Lehmann et al, 2013, PLoS Negl Trop Dis, 7(10): e2468), and to maximize expression in vitro, leptospires were grown under conditions mimicking the in vivo host environment known to induce virulence gene expression in vitro (Matsunaga et al., 2005, Infect Immun, 73(1):70-8). Mid-logarithmic phase cultures (2 × 10 8 Leptospira / ml) were harvested by centrifugation at 18,514 g . Pelleted cells were washed twice with 1× PBS and resuspended in liquid EMJH medium supplemented with 120 mM NaCl, then incubated at 37°C for 4 h (Sigma Aldrich, United States).

[0350] After induction of virulence genes, culture supernatants were collected by centrifugation at 18,514×g for 20 min, clarified by filtration through a 0.22 mm membrane filter (Merck Millipore, Germany), and then concentrated through a 30 kDa Amicon® ultracentrifugal filter (Merck Millipore, Germany). Induced and uninduced culture supernatants (representing baseline in vitro expression) were analyzed by Western blot probed with rabbit anti-LA3490 polyclonal antiserum. Total protein was estimated by BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, United States).

[0351] mammalian cell culture HeLa cells, obtained from the American Type Culture Collection (United States), were grown as monolayers in tissue culture plates in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, United States) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (penicillin, 100 units / ml; streptomycin, 100 mg / ml; and amphotericin, 25 mg / ml; Invitrogen, United States) at 37°C in a humidified incubator containing 5% CO2. Before each experiment, antibiotic-containing medium was replaced with fresh antibiotic-free medium.

[0352] Plasmid construction and cloning E. coli codon-optimized gene fusions consisting of either the complete LA3490 coding sequence (NP_713670.1) minus the predicted signal peptide (i.e., corresponding to nucleotide positions 57 to 1,917 bp) or an N-terminal truncation including positions 40 to 174 bp, and containing the predicted ricin B-like lectin subdomain in Phyre2 linked to mCherry (AST15061.1) via a glycine-serine hinge (GGGGSGGGGSGGGGS; SEQ ID NO: 93), were synthesized and cloned into pET32b(+) (Gene Universal Inc., United States). Constructs were verified by sequencing prior to use.

[0353] Recombinant protein expression and purification As the PF07598 protein is rich in cysteines [LA3490 encodes 12 cysteines], due to its ability to promote disulfide bonds in the cytoplasm and ensure proper protein folding, recombinant proteins were expressed in SHuffleR T7 competent E. coli cells (New England Biolabs, United States). Transformants were subcultured in Luria-Bertani (LB) medium containing 100 mg / ml ampicillin. When the culture reached an OD of 0.6, expression was induced by adding 1 mM isopropyl-bD-thiogalactoside (IPTG; Sigma-Aldrich, United States) at 16°C and 250 rpm for 24 h.

[0354] After induction, cells were pelleted by centrifugation and lysed for 30 min at 37°C with CelLytic™ B (cell lysis reagent; Sigma-Aldrich, United States) containing 50 U benzonase nuclease (Sigma-Aldrich, United States), 0.2 mg / ml lysozyme, non-EDTA protease inhibitor cocktail (Roche, United States), and 1 mM PMSF (Sigma-Aldrich, United States). Lysates were centrifuged at 18,514 × g for 10 min at 4°C. Supernatants and pellets were separated and analyzed by 4–12% bis-sodium tris dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0355] Protein concentration was measured by BCA assay as described above. Recombinant thioredoxin (TRX)-His6-VM protein-(GGGGSGGGGSGGGGS; SEQ ID NO: 93)-mCherry-His6 fusion protein was isolated using a 5 ml pre-packed Ni-Sepharose AKTA Hi-TRAP column (GE Healthcare, United States) pre-equilibrated with a buffer containing 100 mM NaH2PO4, 10 mM Tris-HCl, and 25 mM imidazole (pH 8.0). The bound fusion protein was then eluted from the column in the presence of 500 mM imidazole (pH 8.0). The eluates were pooled and concentrated with a 30 kDa Amicon® ultracentrifugal filter, then centrifuged using a large capacity endotoxin removal spin column (Thermo Fisher Scientific, United States) to remove lipopolysaccharide contamination. Recombinant protein preparations were dialyzed overnight against 1× PBS (pH 7.4) with gentle agitation (350 rpm) at 4°C (30 kDa cutoff, Slide-A-Lyzer, Thermo Fisher Scientific, United States), then the imidazole was removed using a 40-kDa Zeba™ desalting spin column (Thermo Fisher Scientific, United States) and stored at −80°C until use.

[0356] Asialofetuin binding and ricin B chain competitive binding assays To confirm the binding specificity of the Phyre2 predicted ricin B-like lectin domain, we tested whether recombinant full-length and truncated Q8F0K3 (i.e., rLA3490 and t3490, respectively) produced in E. coli and Leptospira secreted VM protein bind to immobilized asialofetuin like ricin B. Binding assays using rLA3490 / t3490 were performed using Immulon® 2HB flat-bottom microtiter plates (Thermo Fisher Scientific, United States). Plates were precoated with asialofetuin (5 ng / ml in carbonate-bicarbonate buffer, pH 9.4) and incubated overnight at 4°C. Before use, plates were blocked with 5% nonfat dry milk in 1×TBST for 2 h at 37°C. After blocking, rLA3490, t3490, and recombinant ricin B chain (Vector Laboratories, Inc., United States) were added individually in triplicate at molar concentrations of 0.9, 4.50, and 9.05 nM in 1×TBST. Plates were incubated for 2 h, washed three times with 1×TBST, and then incubated for 1 h with anti-LA3490 polyclonal or anti-ricin B chain monoclonal antibodies at 1:1,000 in TBST (Invitrogen, United States). To quantify bound rLA3490 / t3490, plates were incubated for 1 h with goat anti-mouse IgG (1:5,000; KPL, United States), washed three times with TBST, and developed with p-nitrophenyl phosphate (1-Step™ PNPP substrate solution; KPL, United States). The reaction was stopped with 2 M NaOH and the absorbance was read at 405 nm using a SpectraMax® M2e microplate reader (Molecular Devices, United States). For competitive binding assays, plates precoated with asialofetuin (2.5 ng / ml) were preincubated with 25 or 50 nM recombinant ricin B chain (Vector Laboratories, United States) for 2 h, followed by the addition of 50 nM rLA3490 / t3490 for the final 2 h of incubation.Anti-LA3490 polyclonal antibody was used to quantitate bound recombinant protein. The ability of Leptospiral secreted VM proteins to bind asialofetuin was assessed using coated Sepharose R beads. Commercially available asialofetuin (1 mg / ml) (Sigma-Aldrich, United States) dissolved in 0.1 M NaHCO3 was coupled to NHS-activated Sepharose beads (GE Healthcare, United States) washed with PBS. The suspension was gently stirred for 1 h at room temperature and unoccupied NHS groups were blocked with 1 M ethanolamine (pH 9) for 1 h. The washed beads were incubated with 250 mg of clarified Leptospira culture supernatant containing secreted proteins for 1 h and then washed twice at 200 × g for 1 min with MEPBS (4 mM b-mercaptoethanol, 2 mM EDTA, and 20 mM sodium phosphate (pH 7.2) buffer. Bound proteins were eluted with 0.5 M lactose and analyzed by 4-10% Bis-Tris SDS-PAGE followed by Western blotting with mouse anti-LA3490 polyclonal antibody (1:2,000 dilution) as described above.

[0357] rLA3490-mediated HeLa cell cytotoxicity HeLa cells (35,000 cells / 200 ml) were seeded in 8-well chamber slides (LabTek, United States) and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. Cells were treated with a pre-optimized concentration of 45 nM rLA3490; t3490-treated, BSA-treated, and untreated HeLa cells were used as controls. Slides were incubated for up to 4 h and time-lapse images were taken with a ×40 objective using a Leica DMi8 inverted microscope (Leica Microsystems, Germany). Adherent cells before and after exposure to rLA3490, t3490, or BSA, or untreated HeLa cells were captured with a ×10 objective and cells were counted using LAS AF 2D quantitative image analysis software (Leica Application Suite X, LAS X; Leica Microsystems, Germany). A grayscale prefilter was applied to improve image clarity. Detachment was quantified as 100 × (cell number at 4 h / cell number at 0 h) and is reported as the average of two or more replicate experiments.

[0358] Live / dead and LDH assays, and F-actin staining HeLa cells were exposed to 45 nM rLA3490 or t3490 for 4 h. The monolayers were then washed twice with 1× PBS (pH 7.4). 200 ml of 2 mM calcein AM / 4 mM ethidium homodimer-1 in PBS (Live / Dead® Viability Kit, Invitrogen, United States) was added to each well and the plates were incubated in the dark for 30 min. The monolayers were washed with PBS (pH 7.4) to reduce nonspecific background fluorescence. BSA and untreated HeLa cells were used as controls. Images were taken using a Leica DMi8 microscope through a ×10 objective with appropriate excitation and emission filters for green (live cells) and red (dead cells) fluorescence. Cell lysis was quantified by assaying the concentration of lactate dehydrogenase in the culture supernatant (CyQUANT™ LDH Cytotoxicity Assay, Invitrogen, United States). For F-actin staining, cell monolayers were exposed for up to 1 h, washed twice with PBS (pH 7.4), and fixed with 4% paraformaldehyde (Sigma-Aldrich, United States) for 30 min at room temperature. After aspiration of the fixative, monolayers were washed twice with PBS, then 0.1% Triton X-100 in PBS was added to each well for 5 min, before repeating the wash with PBS. Monolayers were incubated with phalloidin Alexa_488nm conjugate (Invitrogen, United States) for 30 min at room temperature in the dark, according to the manufacturer's instructions. Nuclei were stained with 0.1 mg / ml ProLong™ Gold Antifade Mount with DAPI for 10 min. All imag...

Claims

1. A composition comprising at least one Leptospira virulence modified (VM) protein or fragment thereof selected from the group consisting of: a) LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_107 78, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_1 a VM protein selected from the group consisting of 2963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091; and b) LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LI C_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_12963, LIC_1 A fragment of a VM protein comprising the DNase domain of a VM protein selected from the group consisting of: 0695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091.

2. 2. The composition of claim 1, which is a fusion protein comprising a Leptospiral VM protein or a fragment of a VM protein containing a DNase domain 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, a viral antigen, a parasitic antigen, a cancer antigen, a tumor-associated antigen, and a tumor-specific antigen.

4. 2. The composition of claim 1, comprising a combination of two or more Leptospiral VM proteins or fragments of VM proteins containing the DNase domain.

5. 5. The composition of claim 4 comprising a combination of LIC_12340 and LIC_12985.

6. 5. The composition of claim 4, comprising a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

7. 2. The composition of claim 1, comprising at least one Leptospiral VM protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO:

12.

8. 6. The composition of claim 5, comprising a combination of VM proteins comprising the sequences set forth in SEQ ID NO: 10 and SEQ ID NO:

12.

9. 7. The composition of claim 6, comprising a combination of VM proteins comprising the sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:

12.

10. 2. The composition of claim 1, comprising at least one lipid nanoparticle (LNP) comprising at least one VM protein or a fragment of a VM protein comprising a DNase domain.

11. The composition of claim 10, comprising a combination of at least two VM proteins or at least two LNPs comprising fragments of VM proteins containing a DNase domain.

12. A composition comprising at least one nucleic acid molecule encoding at least one Leptospira virulence modified (VM) protein or fragment thereof selected from the group consisting of: a) LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_107 78, LIC_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_1 a VM protein selected from the group consisting of 2963, LIC_10695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091; and b) LA_3388, LA_0835, LA_0591, LA_0589(v), LA_1402, LA_1400, LA_3271, LA_0934, LA_0769, LA_2628, LA_0620, LA_3490, LIC_10778, LI C_12791, LIC_12985, LIC_12986, LIC_12339, LIC_12340, LIC_10870, LIC_12715, LIC_12844, LIC_11358, LIC_10639, LIC_12963, LIC_1 A fragment of a VM protein comprising the DNase domain of a VM protein selected from the group consisting of: 0695, LMANV2_260038, LMANV2_240079, LMANV2_70075, LMANV2_70078, LMANV2_210058, LMANV2_210056, LMANV2_80114, LMANV2_320010, LMANV2_240142, LMANV2_150103, LMANV2_170032, LMANV2_70050, and LMANV2_170091.

13. 13. The composition of claim 12, wherein the nucleic acid molecule encodes a fusion protein comprising a Leptospiral VM protein or a fragment thereof comprising the DNase domain, fused to a targeting domain specific for binding to a target molecule.

14. 14. The composition of claim 13, wherein the target molecule is selected from the group consisting of a bacterial antigen, a viral antigen, a parasitic antigen, a cancer antigen, a tumor-associated antigen, and a tumor-specific antigen.

15. 13. The composition of claim 12, comprising one or more nucleic acid molecules encoding a combination of LIC_12340 and LIC_12985.

16. 13. The composition of claim 12, comprising one or more nucleic acid molecules encoding a combination of LIC_12340, LIC_12985, LA_3490, LA_0620, and LA_1402.

17. The composition of claim 12, wherein the nucleic acid molecule encodes at least one amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:

12.

18. 13. The composition of claim 12, wherein the nucleic acid molecule comprises at least one nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:

18.

19. 16. The composition of claim 15, comprising one or more nucleic acid molecules encoding a combination of SEQ ID NO: 10 and SEQ ID NO:

12.

20. 20. The composition of claim 19, comprising one or more nucleic acid molecules comprising a combination of SEQ ID NO:9 and SEQ ID NO:

11.

21. 17. The composition of claim 16, comprising one or more nucleic acid molecules encoding a combination of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:

12.

22. 22. The composition of claim 21, comprising one or more nucleic acid molecules comprising a combination of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:

11.

23. 13. The composition of claim 12, comprising at least one lipid nanoparticle (LNP) comprising at least one nucleic acid molecule encoding at least one VM protein or fragment thereof comprising a DNase domain.

24. 24. The composition of claim 23, wherein the nucleic acid molecule comprises an mRNA molecule encoding at least one VM protein or a fragment thereof comprising a DNase domain.

25. The composition of claim 1 , comprising a vaccine.

26. The composition of claim 1 , comprising an adjuvant.

27. 27. The composition of claim 26, wherein the adjuvant is glucopyranosyl lipid A (GLA) formulated in a stable oil-in-water nanoemulsion (SE).

28. The composition of claim 1 , comprising a toxoid vaccine.

29. An anti-Leptospira VM protein antibody comprising a CDR sequence selected from the group consisting of: a) HCDR sequences set forth in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, and LCDR sequences set forth in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26; b) HCDR sequences set forth in SEQ ID NOs: 21, 22, and 23, and LCDR sequences set forth in SEQ ID NOs: 37, 38, and 39; c) HCDR sequences set forth in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, and LCDR sequences set forth in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50; d) the HCDR sequences set forth in SEQ ID NOs: 61, 62, and 63, and the LCDR sequences set forth in SEQ ID NOs: 64, 65, and 66; and e) HCDR sequences set forth in SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79, and LCDR sequences set forth in SEQ ID NO:80, SEQ ID NO:81, and SEQ ID NO:

82.

30. 30. The anti-Leptospira VM protein antibody of claim 29, comprising a heavy chain sequence and a light chain sequence selected from the group consisting of: a) the HC sequence set forth in SEQ ID NO: 27 and the LC sequence set forth in SEQ ID NO: 28; b) the HC sequence set forth in SEQ ID NO: 27 and the LC sequence set forth in SEQ ID NO: 43; c) the HC sequence set forth in SEQ ID NO: 51 and the LC sequence set forth in SEQ ID NO: 52; d) the HC sequence set forth in SEQ ID NO: 67 and the LC sequence set forth in SEQ ID NO: 68; and e) The HC sequence set forth in SEQ ID NO: 83 and the LC sequence set forth in SEQ ID NO:

84.

31. A nucleic acid molecule encoding the antibody or fragment thereof of claim 29.

32. 32. The nucleic acid molecule of claim 31, comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence comprising SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31 encoding HCDR; b) a nucleotide sequence comprising SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34 encoding the LCDR; c) a nucleotide sequence comprising SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42 encoding an LCDR; d) a nucleotide sequence comprising SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55 encoding HCDR; e) a nucleotide sequence comprising SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58 encoding an LCDR; f) a nucleotide sequence comprising SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71 encoding HCDR; g) a nucleotide sequence comprising SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74 encoding the LCDR; h) a nucleotide sequence comprising SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87 encoding HCDR; and i) Nucleotide sequences encoding LCDRs comprising SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:

90.

33. 33. The nucleic acid molecule of claim 32, comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:91, and SEQ ID NO:

92.

34. A composition comprising a combination of at least two nucleic acid molecules according to claim 32.

35. 35. The composition of claim 34, comprising a combination of nucleic acid molecules selected from the group consisting of: a) a first nucleic acid molecule comprising SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31 encoding an HCDR, and a second nucleotide sequence comprising SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34 encoding an LCDR; b) a first nucleic acid molecule comprising SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31 encoding an HCDR, and a second nucleotide sequence comprising SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42 encoding an LCDR; c) a first nucleic acid molecule comprising SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55 encoding an HCDR, and a second nucleotide sequence comprising SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58 encoding an LCDR; d) a first nucleic acid molecule comprising SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71 encoding an HCDR, and a second nucleotide sequence comprising SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74 encoding an LCDR; e) a first nucleic acid molecule comprising SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87 encoding an HCDR, and a second nucleotide sequence comprising SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90 encoding an LCDR.

36. 35. The composition of claim 34, comprising a combination of nucleic acid molecules selected from the group consisting of: a) a first nucleic acid molecule comprising SEQ ID NO:35 and a second nucleotide sequence comprising SEQ ID NO:36; b) a first nucleic acid molecule comprising SEQ ID NO: 35 and a second nucleotide sequence comprising SEQ ID NO: 44; c) a first nucleic acid molecule comprising SEQ ID NO:59 and a second nucleotide sequence comprising SEQ ID NO:60; d) a first nucleic acid molecule comprising SEQ ID NO:75 and a second nucleotide sequence comprising SEQ ID NO:76; and e) a first nucleic acid molecule comprising SEQ ID NO:91 and a second nucleotide sequence comprising SEQ ID NO:

92.

37. Use of the composition of claim 1 in the manufacture of a medicament for inducing an immune response to Leptospira in a subject or for treating or preventing a disease or disorder.

38. 38. The use of claim 37, wherein the disease or disorder is at least one selected from the group consisting of cancer, bacterial infection, viral infection, and parasitic infection.

39. Use of the antibody or nucleic acid molecule encoding same of claim 29 in the manufacture of a medicament for treating or preventing a disease or disorder in a subject.

40. The use described in claim 39, wherein the disease or disorder is at least one selected from the group consisting of cancer, bacterial infection, viral infection, and parasitic infection.

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