A virulence modifying protein-based recombinant vaccine and rapid diagnostic assay for leptospira

Abstract

The invention provides a virulence modifying (VM) protein-based composition or vaccine for administration to a subject, comprising a recombinant VM protein antigen or a recombinant viral vector expressing a VM protein antigen and a pharmaceutically or veterinarily acceptable adjuvant. The invention further provides a method for vaccinating a subject susceptible to leptospirosis infection or eliciting an immune response in the subject against leptospirosis comprising administering the vaccine to the subject. The invention also provides a method for diagnosing leptospirosis in a subject by quantifying the presence of a secreted, leptospiral VM protein antigen that mediates disease pathogenesis in the subject.

Description

A VIRULENCE MODIFYING PROTEIN-BASED RECOMBINANT VACCINE AND RAPID DIAGNOSTIC ASSAY FOR LEPTOSPIRA ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under 1R41AI174377 awarded by the National Institutes of Health. The government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present application claims the benefit or priority to United States Provisional Application No.63 / 663,980, filed on June 25, 2024, the contents of which are incorporated herein by reference. FIELD

[0003] This invention relates generally to recombinant protein-based vaccines and diagnostics. In particular, the invention provides a recombinant protein-based, subunit human leptospirosis vaccine based on L. interrogans-encoded Virulence Modifying proteins. The invention also provides rapid, point-of-care, specific leptospirosis antigen- detection diagnostic tests in body fluids. BACKGROUND

[0004] Leptospirosis, a zoonotic bacterial disease of worldwide importance, affects more than 1 million humans annually with a conservatively estimated 5-20% case fatality rate. Multiple factors are expected to globally increase the incidence of leptospirosis in coming years, including climate change, increased urbanization, and worsening sanitation and water security challenges. Therefore, the clinical and public health significance of leptospirosis is high and growing. The most important agents of human leptospirosis are the strains of the bacterial spirochetal species, L. interrogans.

[0005] No widely available, safe and effective leptospirosis vaccine is available for humans. While bacterin (i.e., whole killed Leptospira-based) leptospirosis vaccines are available for dogs and livestock, they are not suitable for humans due to insufficient efficacy (not protective against many L. interrogans serovars) and are not safe because of reactogenicity. The inventors recently discovered the Virulence Modifying (VM) protein family, their biological function as secreted exotoxins and that vaccination of mice with as few as 2 VM proteins conferred protection against lethal challenge infection and near elimination of qPCR-determined bacterial load. Provided herein is data showing that oneor more recombinant Virulence Modifying (VM) proteins will protect hamsters from lethal leptospiral challenge; such data are important for advancing new vaccine candidates into human clinical development.

[0006] This invention provides a novel, recombinant protein-based, subunit human leptospirosis vaccine based on L. interrogans-encoded Virulence Modifying proteins. The vaccine protects against human leptospirosis and will have a global public health impact: on 100s of millions of people in low and middle income (LMIC) countries, where the burden of disease is high (comparable to cholera and typhoid); on 10s of millions in high income countries, e.g. those in at-risk occupations, travelers and military personnel.

[0007] The ambiguous clinical presentation of leptospirosis poses challenges in the accurate assessment of global burden of this emerging disease. As a result, leptospirosis hasn’t yet been recognized for inclusion in the WHO’s neglected tropical diseases list. This underscores the urgent need for a reliable diagnostic biomarker for early detection of illness, and well-defined estimation of disease burden in both urban and low-income settings.

[0008] Clinically actionable, specific, and rapid diagnostic tests for leptospirosis remain commercially unavailable and a key gap in the field.110Improved diagnostics must provide rapid and low-cost diagnosis of acute leptospirosis in humans. Also of high priority, better leptospirosis diagnostic tests must enable accurate estimates of leptospirosis disease burden in affected populations. Both outcomes are essential for improving clinical care of this neglected tropical disease and will directly lead to and justify clinical development of leptospirosis vaccines and novel adjunct therapeutics.

[0009] This invention further provides inexpensive, rapid, actionable, low-tech antigen-detection diagnostic tests for leptospirosis, available both at point-of-care and at reference laboratories for higher throughput. SUMMARY

[0010] In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a virulence modifying (VM) protein-based composition or vaccine for administration to a subject in need thereof comprising a recombinant VM protein antigen or a recombinant viral vector expressing a VM protein antigen and a pharmaceutically or veterinarily acceptable adjuvant, wherein the VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13..

[0011] In one embodiment, the VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 12, 8, 2 or 9. In another embodiment, the VM protein is tagless.

[0012] In another embodiment, the viral vector is an orthopox viral vector.

[0013] In one other embodiment, the composition or vaccine further comprises a pharmaceutically or veterinarily acceptable carrier, vehicle or, excipient. The pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant, or vehicle may be selected from the group consisting of polyacrylic acid, LF2 emulsion, LR6 emulsion, TS6 emulsion, LR4 emulsion, carbomer, aluminum hydroxide, aluminum phosphate, saponin, CpG, water-in-oil emulsion, oil-in-water emulsion, carbomer-based adjuvant, and adjuvant composition comprising a lipophile, a polymer of acrylic or methacrylic acid, saline, cholesterol, a saponin, and sodium hydroxide.

[0014] In yet another embodiment, the subject is a human or an animal. The animal may be selected from a group consisting of equine, canine, feline, ovine, bovine, porcine, caprine, avian, primate, fish and the like. Additionally, the animal is susceptible to or has leptospirosis.

[0015] In another aspect, the invention relates to method for vaccinating a subject susceptible to leptospirosis infection or eliciting an immune response in the subject against leptospirosis comprising administering to the subject a vaccine as described herein. In one embodiment, the method further comprises administering to the subject boost-vaccine, wherein at the boost-vaccine is the vaccine as described herein. In another embodiment, the vaccine comprises a VM protein antigen, a recombinant viral vector that expresses, in vivo, a VM protein antigen, or both, and wherein the method protects the subject from leptospirosis infection, prevents leptospirosis disease progression in the subject, or the combination thereof. In another embodiment, the vaccine comprises a VM protein antigen, a recombinant viral vector that expresses, in vivo, a VM protein antigen, or both, wherein the boost-vaccine is the vaccine as described herein, and the method protects the animal from leptospirosis infection, prevents leptospirosis disease progression in the subject, or the combination thereof.

[0016] In yet another aspect, the invention relates to a method for diagnosing leptospirosis in a subject in need thereof comprising obtaining a sample from the subject, detecting leptospiral VM proteins antigen the sample, and quantifying leptospiral VM proteins antigen, wherein the method quantifies the presence of a secreted, leptospiral VM protein antigen that mediates disease pathogenesis in the subject. wherein theleptospiral VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

[0017] In one embodiment, the leptospiral VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 12, 8, 2 or 9.

[0018] In another embodiment, the subject is a human or an animal. The animal may be selected from a group consisting of equine, canine, feline, ovine, bovine, porcine, caprine, avian, primate, and fish. Additionally, the animal is susceptible to or has leptospirosis.

[0019] In yet another embodiment, the sample is selected from bodily fluids selected from saliva, blood, including serum and plasma, or urine.

[0020] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. BRIEF DESCRIPTIONS OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate (one) several embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

[0022] Figures 1A-1D show Genetic inactivation of recombinant wild type WT LA0591 yielding △LA0591 lacking DNase and HeLa cell toxicity (following our published assays11). Bioinformatics approaches (FTMAP,12, 13PrankWeb / P2Rank,14Deepsite27) were used to identify likely active site residues of the C-terminal VM protein toxin activity; predicted active site residues (Glu, His and Asp) were mutated to Ala. WT LA0591 and △LA0591 were expressed in pET32b and purified using column chromatography. FIG.1A shows affinity and size exclusion chromatography-purified WT LA0591 (upper left) and △LA0591 (lower left) yielded single bands of expected size (WT LA0591 without mCherry tag predicted at 44 kDa, △LA0591 mCherry fusion protein is predicted at 78 kDa; other recombinant VM proteins expressed mCherry fusions retain DNase and cytotoxicity activity (and data not shown).11, 30FIG. 1B shows WT LA0591 retains DNase activity and △LA0591 loses DNase activity when incubatedwith HeLa cell genomic DNA. Figures 1C and 1D show that a quantitative trypan blue assay (measured and imaged using a Countess 3 Automated Cell Counter (Invitrogen)), WT LA0591 was shown to retain HeLa cell cytotoxicity while △LA0591 is not cytotoxic. The experiment was repeated 3 times in duplicate.

[0023] Figure 2A-2F are Western blots showing Native L. interrogans and L. borgpetersenii VM proteins recognized by mAbs 5F8, 5G10 and 6A5 against conserved linear epitopes (Western blot). Notably, L. interrogans serovar Copenhageni upregulates VM protein expression under high salt conditions (+) that mimic in vivo conditions and pathogenesis-related gene expression.1-5Loading control at bottom left; Patoc,a non- pathogenic Leptospira lacking VMPs is a negative control.

[0024] Figures 3A-3D shows the detection of VM protein antigen in L. interrogans serovar Copenhagen-infected hamsters. Hamsters were inoculated IP (N=5 / group) with different numbers of Leptospira (as indicated). (FIG. 4A) Sandwich ELISA standard curve with positive (LA0591) and negative (RBL1 / 2) controls. mAb 6A5 (IgG2b), capture, and 5F8 (IgG1), detecting, with HRP-labeled, anti-IgG1 antibody against mAb5F8 used for color detection.. (FIG. 4B) Regression curve of spiked hamster serum (background subtracted). The log10 transformed [ VMP] yielded the regression equation, which was linear at the lower end of detection. (FIG. 4C) VMP in hamster serum in relation to infectious inoculum. (FIG.4D) Depiction of the capture ELISA protocol.

[0025] Figure 4 shows a possible prototype leptospirosis lateral flow rapid diagnostic test.

[0026] Figures 5A-5D are blots showing SDS-PAGE periodate silver stain and Western immunoblot analysis comparing our new method with a published method of purifying L. interrogans serovar Copenhageni and serovar Lai lipopolysaccharide (LPS). FIG. 5A shows a periodate silver stain of an SDS-PAGE gel. FIG. 5B shows a Western immunoblot. FIGs. 5A and 5B: lane 1 is E. coli LPS (Sigma), lanes 2 are serovar Copenhageni LPS, and lane 3 is a serovar Lai LPS. Our new method yields high MW LPS that ladders with repeat units of at least 20kDa to a MW >250 kDa (FIG.5A). These repeat units are immunoreactive with polyclonal rabbit antisera raised against L. interrogans serovar Copenhageni and Lai (Lai not shown but similar to the antisera against Copenhageni indicating they strongly cross-react). FIGs. 5C and 5D (modified from Nally JE, et al. Infect Immun.2005 Jun:73(6)3251-60, Fig.3); Figure C, lanes 3 and 4 show LPS extraction from two different Copenhagni strains analyzed by Western blot using polyclonal rabbit antisera raised against L. interogans serovar Copenhageni as inFIG. 5B. Note that as seen in previous publications, the Copenhageni LPS is found in a band from 20-25 kDa but not in the high molecular weight laddering rom typical of LPS, as demonstrated in Figures 5A and 5B.

[0027] Figures 6A-6B show the Schematic organization and immunoreactivity of VMPs. FIG.6A shows the domain architecture of full-length LA3490, N-terminal RBLs, and C-terminal domains. RBLs are the N-terminal ricin binding like the lectin domain (41 aa - 335 aa) and LA0591 is a natural variant that encodes the C-terminal domain and lacks RBLs. FIG. 6B represents the immunoreactivity of RBLs, LA3490 and LA0591. RBLs were developed with polyclonal antibodies generated against full-length VMP, however, LA3490 and LA0591 reacted with monoclonal 5G10 antibody generated against LA0591. The arrow represents the reactivity and molecular weight of VMPs. M shows the molecular weight.

[0028] Figures 7A-7I show the distribution of mean OD values by qPCR and MAT results and final disease confirmation among suspected cases of leptospirosis and healthy controls.

[0029] Figures 8A-8C show the distribution of VM antibody OD values by MAT results among patients with paired serum samples.

[0030] Figures 9A-9C show the distribution of VM antibody OD values by the duration of illness.

[0031] Figure 10 shows a ROC Curve for the diagnostic accuracy of VM Protein antibodies to detect Leptospira infection.

[0032] Figure 11 shows the orthologs of Leptospira interrogans serovar Copenhageni PF07698 gene family protein sequences, from UniProt, paired with Leptospira interrogans serovar Lai. DETAILED DESCRIPTION

[0033] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description. I. Definitions

[0034] To facilitate an understanding of the principles and features of the various embodiments of the disclosure, various illustrative embodiments are explained herein. Although exemplary embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended thatthe disclosure is limited in its scope to the details of construction and arrangement of components set forth in the description or examples. The disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0035] In describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a composition containing “a” constituent is intended to include other constituents in addition to the one named.

[0036] Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particular value.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Likewise, "or" is intended to include "and" unless the context clearly indicates otherwise.

[0038] As used herein, the term "animal" refers to mammals, birds, and the like. Animals or hosts include mammals and humans. The animal can be selected from the group consisting of equine (e.g., horse), canine (e.g., dog, wolf, fox, coyote, jackal), feline (e.g., lion, tiger, domestic cat, wild cat, other big cats, and other felines, including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g., cow), porcine (e.g., pig), caprine (e.g., goat), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finch, hawk, crow, ostrich, emu, and turkey), primate (e.g., monkey of the order Prosiuma, tarsal monkey, gibbon, ape), and fish. The term "animal" also includes individual animals at all stages of development, including embryonic and fetal stages.

[0039] The term "antigen" or "immunogen" refers to a substance that induces a specific immune response in a host animal. The antigen may comprise the whole organism, killed, attenuated or live; a subunit or a portion of an organism; a recombinant vector containing an insert having immunogenicity; a DNA or DNA fragment capable of inducing an immune response when presented to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof. Alternatively, the immunogen or antigen may comprise a toxin or an antitoxin.

[0040] The term "immunogenic protein, polypeptide or peptide" as used herein includes polypeptides that are immunologically active in the sense that, once administered to a host, they are capable of eliciting an immune response against the humoral and / or cellular types of the protein. Preferably, the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises or essentially consists of or consists of at least one epitope or antigenic determinant. An "immunogenic" protein or polypeptide as used herein includes the full-length sequence of the protein, an analog thereof, or an immunogenic fragment thereof. By "immunogenic fragment" is meant a fragment of a protein that includes one or more epitopes, and thus elicits the immune response described above. Such fragments may be identified using any number of epitope mapping techniques well known in the art. See, for example, Epipope Mapping Protocols in Methods in Molecular Biology, Vol.66 (Glenn E. Morris, Ed., 1996). For example, a linear epitope can be produced by, for example, simultaneously synthesizing a plurality of peptides on a solid support, the peptides corresponding to a portion of a protein molecule, and reacting the peptides with an antibody while the peptides are still attached to the support. Such techniques are known in the art and are described, for example, in U.S. Pat. Nos. 4,708,871; Geysen et al, 1984, PNAS USA, 81(13) 3998-; Geysen et al, 1985, PNAS USA, 82(1): 178-82. Similarly, conformational epitopes are readily identified by determining the spatial conformation of amino acids, for example by x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.

[0041] As discussed, the present invention includes active fragments and variants of antigenic polypeptides. Thus, the term "immunogenic protein, polypeptide or peptide" further contemplates deletions, additions and substitutions of sequences, so long as the polypeptide functions to generate an immune response as defined herein. The term "conservative variation" denotes the replacement of an amino acid residue by another, biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue is not changed, or is another, biologically similar residue. In this regard, particularly preferred substitutions are generally conservative in nature, i.e., substitutions that occur within a family of amino acids. For example, amino acids are generally divided into four families: (1) the acidic amino acids aspartic acid and glutamic acid; (2) basic amino acids-lysine, arginine, histidine; (3) nonpolar amino acids alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) amino acids without electrical polarity-glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the replacement of one polar residue for another, such as the replacement of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like; or similar conservative substitutions by replacing an amino acid with a structurally related amino acid, which does not have a significant effect on biological activity. Thus, proteins having substantially the same amino acid sequence as a reference molecule but having minor amino acid substitutions that do not substantially affect the immunogenicity of the protein are within the definition of a reference polypeptide. All polypeptides produced by these modifications are included herein. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, so long as the antibody produced from the substituted polypeptide also immunoreacts with the unsubstituted polypeptide.

[0042] The term “epitope” refers to the site on an antigen or hapten to which specific B cells and / or T cells respond. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site”. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

[0043] An “immunological response” to a composition or vaccine is the development in the host of a cellular and / or antibody-mediated immune response to a composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and / or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and / or a lowered viral titer in the infected host.

[0044] Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al., 1993; Bergmann et al., 1996; Suhrbier, 1997; Gardner et al., 1998. Immunogenic fragments, for purposes of the present invention, will usually includeat least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or about 15-25 amino acids or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full- length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

[0045] The term “nucleic acid” and “polynucleotide” refers to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thiolate, and nucleotide branches. The sequence of nucleotides may be further modified after polymerization, such as by conjugation, with a labeling component. Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides or solid support. The polynucleotides can be obtained by chemical synthesis or derived from a microorganism.

[0046] The term “gene” is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs and / or the regulatory sequences required for their expression. For example, gene also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

[0047] The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment” are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer can be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0048] An “isolated” biological component (such as a nucleic acid or protein or organelle) refers to a component that has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, for instance, other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant technology as well as chemical synthesis.

[0049] The term “purified” as used herein does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified polypeptide preparation is one in which the polypeptide is more enriched than the polypeptide is in its natural environment. That is the polypeptide is separated from cellular components. By “substantially purified” it is intended that such that the polypeptide represents several embodiments at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%, or more of the cellular components or materials have been removed. Likewise, the polypeptide may be partially purified. By “partially purified” is intended that less than 60% of the cellular components or material is removed. The same applies to polynucleotides. The polypeptides disclosed herein can be purified by any of the means known in the art.

[0050] As noted above, the antigenic polypeptides or fragments or variants thereof are VMP antigenic polypeptides that are produced viral vector in vivo. Fragments and variants of the disclosed polynucleotides and polypeptides encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the antigenic amino acid sequence encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence have immunogenic activity as noted elsewhere herein. Fragments of the polypeptide sequence retain the ability to induce a protective immune response in an animal.

[0051] As used herein, the term “derivative” or “variant” refers to a polypeptide, or a nucleic acid encoding a polypeptide, that has one or more conservative amino acid variations or other minor modifications such that (1) the corresponding polypeptide has substantially equivalent function when compared to the wild type polypeptide or (2) an antibody raised against the polypeptide is immunoreactive with the wild-type polypeptide. These variants or derivatives include polypeptides having minormodifications of the VMP polypeptide primary amino acid sequences that may result in peptides which have substantially equivalent activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. The term “variant” further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.

[0052] The term “conservative variation” denotes the replacement of an amino acid residue by another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence such that the encoded amino acid residue does not change or is another biologically similar residue. In this regard, particularly preferred substitutions will generally be conservative in nature, as described above.

[0053] The polynucleotides of the disclosure include sequences that are degenerate as a result of the genetic code, e.g., optimized codon usage for a specific host. As used herein, “optimized” refers to a polynucleotide that is genetically engineered to increase its expression in a given species. To provide optimized polynucleotides coding for VMP polypeptides, the DNA sequence of the VM protein gene can be modified to 1) comprise codons preferred by highly expressed genes in a particular species; 2) comprise an A+T or G+C content in nucleotide base composition to that substantially found in said species; 3) form an initiation sequence of said species; or 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA, or that form secondary structure hairpins or RNA splice sites. Increased expression of VM protein in said species can be achieved by utilizing the distribution frequency of codon usage in eukaryotes and prokaryotes, or in a particular species. The term “frequency of preferred codon usage” refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the disclosure as long as the amino acid sequence of the VMP polypeptide encoded by the nucleotide sequence is functionally unchanged.

[0054] The sequence identity between two amino acid sequences may be established by the NCBI (National Center for Biotechnology Information) pairwise blast and the blosum62 matrix, using the standard parameters (see, e.g., the BLAST or BLASTX algorithm available on the “National Center for Biotechnology Information” (NCBI, Bethesda, Md., USA) server, as well as in Altschul et al.; and thus, this document speaksof using the algorithm or the BLAST or BLASTX and BLOSUM62 matrix by the term “blasts”).

[0055] The “identity” with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm. The sequence identity or sequence similarity of two amino acid sequences, or the sequence identity between two nucleotide sequences can be determined using Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, Calif.). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.

[0056] Hybridization reactions can be performed under conditions of different “stringency.” Conditions that increase stringency of a hybridization reaction are well known. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989).

[0057] The invention further encompasses the VMP polynucleotides contained in a vector molecule or an expression vector and operably linked to a promoter element and optionally to an enhancer.

[0058] A “vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered to a target cell, either in vitro or in vivo. The heterologous polynucleotide may comprise a sequence of interest for purposes of prevention or therapy, and may optionally be in the form of an expression cassette. As used herein, a vector needs not be capable of replication in the ultimate target cell or subject. The term includes cloning vectors and viral vectors.

[0059] The term “recombinant” means a polynucleotide semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in an arrangement not found in nature.

[0060] “Heterologous” means derived from a genetically distinct entity from the rest of the entity to which it is being compared. For example, a polynucleotide may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter. II. COMPOSITIONS

[0061] Compositions or vaccines comprising recombinant viral vectors expressing VMP antigens that elicit an immunogenic response in an animal are provided. The recombinant viral vectors may be an orthopox vector expressing VMP antigens. The recombinant viral vectors expressing the antigens may be formulated into vaccines or pharmaceutical compositions and used to elicit or stimulate a protective response in an animal.

[0062] It is recognized that the antigenic polypeptides of the disclosure may be full length polypeptides or active fragments or variants thereof. By “active fragments” or “active variants” is intended that the fragments or variants retain the antigenic nature of the polypeptide. Thus, the present disclosure encompasses any VMP polypeptide, antigen, epitope or immunogen that elicits an immunogenic response in an animal. The VMP polypeptide, antigen, epitope or immunogen may be any VMP polypeptide, antigen, epitope or immunogen, such as, but not limited to, a protein, peptide or fragment or variant thereof, that elicits, induces or stimulates a response in an animal. A. Leptospirosis

[0063] Currently there is no safe and effective vaccine to prevent human leptospirosis. Provided herein are compositions for an effective human leptospirosis vaccine. Further provided are rapid assays using leptospiral proteins of the present invention to accurately diagnose leptospiral infection.

[0064] Leptospirosis is a globally important neglected disease caused by pathogenic diverse spirochetes of the genus Leptospira1. Leptospirosis affects humans in diverse epidemiological settings.1-4It is estimated to cause more than 1 million global cases annually with a 5-20% case-fatality rate, significant morbidity, and important public health consequences7, 8. Recognized as an important emerging pathogen by NIAID and in the literature,1, 5, 6. Urban slum dwellers, agricultural and slaughterhouse workers, sewer workers, military personnel, adventure travelers3, 4, 9, 10and generally residents of warm, humid regions are at highest risk of acquiring the disease,2with climate change likely to cause increased frequency and severity of leptospirosis epidemics in the future.9,10, 15-17Hence there is a global need for a vaccine to prevent severe human leptospirosis, which the National Institute of Allergy and Infectious Diseases (NIAID) prioritizes as an emerging infectious disease.6

[0065] The genus Leptospira is a complex group of spirochetes, with the most clinically important being serovars of L. interrogans.18-21 22, 23Serovars within the genus L. interrogans are the most pathogenic and are almost exclusively responsible for the severe morbidity and mortality of human leptospirosis.24-26In recent field study conducted over two and a half years by the inventor’s laboratory, we isolated 25 strains of leptospirosis in 1,192 patients across 15 districts in Sri Lanka19. Genome sequencing revealed 25 new core genome sequence types in 15 clonal groups, including 12 new clonal groups. These isolated belonged to four pathogenic Leptospira species identified as L. interrogans, L. borgpetersenii, L. weilii, and L. kirschneri, dominated by six serogroups Autumnalis, Pyrogenes, Icterohaemorrhagiae, Celledoni, Grippotyphosa, and Bataviae19.

[0066] Pathogenic Leptospira are extracellular organisms, but mechanisms by which they exert their pathogenetic effects were unclear until the inventors’ discovery of the leptospiral Virulence Modifying (VM) proteins’ cytotoxin function, followed by their demonstration of the potential for VM proteins to be the antigen components of a panleptospirosis vaccine. Severe human leptospirosis has almost exclusively been reported to be due to strains of L. interrogans. This invention presents a L. interrogans VM protein-based leptospirosis vaccine, although cross species protection may be possible. Recently published animal model data demonstrated that as few as two recombinant VM proteins provide robust cross-serovar protection from disease / death after lethal L. interrogans challenge in mice. These data indicate strong potential for VM proteins as pan- L. interrogans protective antigens. Presented herein are compositions and methods to optimize dose, delivery and composition of recombinant E. coli produced VM proteins in combination with human-compatible adjuvant to determine protective efficacy in the lethal hamster challenge model of leptospirosis. Provided herein are recombinant tagless VM protein immunogens, analysis of their immunogenicity and, in the standard hamster model, comparison of their protective efficacy to standard, commercially available bacterin vaccines; contingency experiments will also assess chemically inactivated wild type proteins and proteins purified from inclusion bodies as alternative forms of the VM protein immunogen; structural modeling suggests that VM protein disulfide bond-dependent conformation may not be critical for antigenicity.

[0067] The expansion of the VM protein family and >90-95% amino acid identity within the most important leptospiral species, L. interrogans,30provides a strong rationale for exploiting these proteins in vaccine development.

[0068] A human leptospirosis vaccine to meet this important clinical need is not currently in development. There is little prospect for adapting bacterins31(whole killed cell vaccines) or attenuated live spirochetal vaccines from veterinary to human vaccine use because of lack of safety, efficacy and practicality.32, 33 34-37Current vaccines for leptospirosis are bacterins (whole killed cell bacteria) exclusively in veterinary use. They depend on serovar- and LPS-dependent immunity; the main exception is the live attenuated spirochetal vaccine approach which has been reported to be serovar- transcending but does not induce a high degree of cross-serovar sterile immunity.38The regulatory pathway for any leptospirosis vaccine for human use remains dependent on initial demonstration of efficacy in animal models, followed by progressive assessment of safety and efficacy in human clinical trials.38A recombinant protein subunit vaccine remains promising for humans, but depends first and foremost on identifying and validating a leptospirosis vaccine candidate. The compositions and methods provided herein prioritize a vaccine that protects against the most clinically important strains of Leptospira species that cause severe human disease; such strains are diverse serovars of the species, Leptospira interrogans.1, 3, 4, 39Therefore, the inventors present herein a novel pan-L. interrogans vaccine as proof-of-principle, having recently identified protective leptospiral VM proteins as immunogens in an animal model.30

[0069] The invention provides a comparison of wild type and genetically mutated toxoid-forms of tagless, recombinant E. coli-produced VM proteins in the lethal hamster model. Protective immune responses for presented herein are assessed by protection from clinical disease and death and by determination of bacterial load and viability in liver and kidney at various time points. Biomarkers of protective immunity (i.e., antibody titers against the prototype vaccine antigens and cross-reactivity among serovars and between different Leptospira species’ VM proteins) are measured; the putative antibody-mediated protective mechanism of immunity is tested by passive transfer experiments.

[0070] The compositions provided herein present pan- L. interrogans VM-protein- based vaccine prototypes for further animal testing. Following Phase I, this prototype is to be tested in dog clinical trials; the success of which , which will lead to a first commercial product for companion and production animals. Successful animal vaccine development will lead towards regulatory submission for a human leptospirosis vaccine candidate. B. Leptospirosis vaccine

[0071] To date, other than the work presented herein,11, 30experimental support for a protein subunit vaccine as the basis of pan-L. interrogans vaccine has not beendemonstrated, despite calls for exploitation of leptospiral genomics to do so.50-52The discovery of the spirochete Leptospira as the etiological agent of leptospirosis was first reported in 1915 and 1917,53, 54and even then, was recognized as a disease of global importance. While veterinary vaccines are commercially available for livestock and dogs, there is no such vaccine widely available and acceptable for human use.24, 55-58The lack of a human vaccine is due to several factors, including reactogenicity of current bacterin- based vaccines, limitations of serovar coverage, and, importantly, a lack of knowledge regarding specific vaccine antigens targeting conserved pathogenetic mechanisms.59-62Previous work with live attenuated Leptospira,63bacterins,31, 64, 65LPS66-69, recombinant protein subunit vaccine (OmpL1, LipL4, LipL32, LigAvar, GspD),25, 70-73recombinant subunit vaccine, LigA (LigA7'-13), LigB (LigB0-7),74LigB(131-645)75, and LigA DNA76vaccines in animals has not led to definitive vaccine candidates; indeed, as reflected by experts in the field, currently there is no viable human leptospirosis vaccine candidate.31Recently, an attenuated live Leptospira vaccine and fcpA- mutants conferred partial cross- protective immunity against heterologous leptospiral serovars in an animal model38, 77but is not being developed for humans. 1. VM protein-based leptospirosis vaccine

[0072] Presented herein are VM protein-based leptospirosis vaccine candidates to be advance to human clinical development via dog vaccination experiments and for further development in a Phase II study.

[0073] NIAID prioritizes vaccine development against emerging infectious diseases that are on NIAID’s list.6Leptospiral VM proteins have been demonstrated to be secreted exotoxins that not only mediate cytotoxicity78and target endothelial cell dysfunction (data not shown) but also via vaccination induce protective immunity and prevent death in a lethal mouse model of leptospirosis.30The potential impact of a leptospirosis vaccine for humans is global, applicable to LMIC countries where severe and fatal leptospirosis due predominantly to L. interrogans are endemic, and will be applicable to high income countries targeted at travelers, at-risk workers, and military personnel.

[0074] The vaccine compositions presented herein will have an impact on populations in leptospirosis-endemic regions, e.g., those subject to flooding and poor sanitation, and workers in at-risk occupations, e.g. farming, sanitation / sewage. Subsequent market expansion would include populations / individuals in industrialized countries. Because leptospirosis is endemic in areas that also have higher rates of infections for cholera, we can estimate our potential market size based on that of existingvaccines against cholera. The global cholera vaccines market was valued at $80M in 2020 but is expected to grow at a CAGR of 8.6% through 2026.79It is expected that comparable numbers, or even greater, for a leptospirosis vaccine for humans, but precise numbers are not available and await definitive analysis. Leptospirosis vaccine development addresses a global burden of disease on par with other diseases of low- and middle-income countries, e.g., cholera and typhoid fever.7, 80The United Nations- chartered International Vaccine Institute (IVI) has been actively involved in commercialization of such vaccines. IVI strongly supports the need for a human leptospirosis vaccine (Letter of Support from Dr. Jerome Kim). LeptoX and IVI are actively discussing the business proposition and logistics to support vaccine commercialization, including addressing the question of how many would need to be vaccinated on a population level.

[0075] A multi-serogroup bacterin vaccine for humans is produced in Cuba and China,81and a single-serogroup (Icterohaemorrhagiae) bacterin leptospirosis vaccine is produced for use in France.62These vaccines are neither widely approved nor widely available for human use and limited by a narrow spectrum of efficacy (being serogroup- specific), short-term efficacy, and toxicity. The leptospiral VM protein-based vaccines presented herein are serogroup-transcending, hence generalizable, of higher efficacy, longer-term duration, and a notably lower rate of adverse events compared to bacterins.

[0076] This study is expected to yield one or more VM protein-based vaccine candidates to advance to human clinical development. Once a vaccine candidates are identified, they will be advanced to human clinical Phase 1, 2, and 3 testing.

[0077] The VM protein-based vaccine technologies will be applicable to the global animal health industry for livestock (cattle, horses, pigs, sheep, alpacas) and dogs, which has a global market size of $2.5 billion currently and is projected to grow at a CAGR of 3.6%.822. Leptospira Sequences

[0078] Table 1 and Figure 11 show orthologs of Leptospira interrogans serovar Copenhageni PF07698 gene family protein sequences, from UniProt, paired with Leptospira interrogans serovar Lai.

[0079] Table 1B LIC_10870 LA_3271 5 B LIC_12715 LA_0934 6 BLA_26287 A LIC_12340 LA_1400 8 A LIC_12339 (L. borg has 1) LA_1402 9 C LIC_12986 LA_0589 10 C LA_3388 11C LA_0835 12 C LIC_12985 LA_0591 13

[0080] The following are sequences for members of the OS=Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni family of proteins that are used as vaccine candidates. a. LIC_12963 / LA_0620

[0081] The amino acid sequence for LA_0620 is MRNRKKVIIVILLVATITYLKYGIDHTHIHASSKIEYSVIQKPTDPPKDKPIKVIVSD GGKFCYGPNFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPEKV IKAEETWDYVHLRPCTINDPLQRWIIKNNSFWTANGFYRLKDYNWYGYISRNSG DRYNHTLDPSMNDWVNTIATPGNISIQTSIAWDLQTTEGQERYFIRWGSSNKNTT PLYYNPENGHLAQYDPISGSLYCMYSQVDNYQWNWVKWKWCSDSLESKSKGN PTFWNVFFETDQGGMITDYKGNALRVTRYGSNWGVAYTAKPDFVKTDTKNSPT SLFVVDKSLLDWTRYTSSNLGKTEQYCPAGNKESVVHKKAKRTLPPDFQLTEAW IRRLYEIARTDPSSRTSRGVCGVCMLQALQMIAELQEYHSQGPLQSGGYFFNTAP NTNPFISFEQRYPHLDRLLVDIYRVFDHFFDTSHTLGYLSAMNLLPQYEWGRTRE FSTMSEIRSHIRSLITSPPGNIWLVLMTMIYPDGTRGGHAVPILRTPQGLVVIETTM ATATFEEYRAALRPTTDFEQIIRNLRGPNNILIGLSTLQLGRFYHNPLDSMISNRNC TGEGSDRRGTGGYPASTSVNQCSSKSSRCSLQ (SEQ ID NO: 1).

[0082] The UNIPROT number for LA_0620 is Q72N74_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_12963 PE=4 SV=1). b.LIC_10695 / LA_3490

[0083] The amino acid sequence for LA_3490 is MYKWKVLTAFFLISIGSGFEYGVNHTHIHALSKIEYSVIQKPTDPPKDKPIKVIVSD GGKFCYGPNFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPEKV IKAEKNWDYVHLRPCTINDPLQRWIIKNNSFWTANGFYRLKDYNWYGYISRNSG DRYNHTLDSSMNDWVNTIATPGNISIQTSIAWDLQTTEGQERYFIRWGGSDKNTT PLYYNPENGHLAQYDPISGSLYCMYSQVDNYQWNWVKWKWCSDLLESKSKGN PTFWNVFFETDQGGMITDYKGNALRVTRYGSNWGSAYTAKPSYLEKDTTNSPTS LFVVNKDLLDWTRYTASNLGKTGQYCPAGKRENIVHRRIKRELPPDFQLTEAWI RRLYEIATSVSAESETRVSGICGPCALHSFQMLAELLEYHSREPLQSGGYFFDTAP NTDPFISFGQRYPHLERLLEDIPKKYAPYPHYSTQSFLSFASIDSMLPQYFWSASTE FTNRDEILSHISSLINSPAGSIWLGVMEQQHPDGTITGHAAPILRISQGLVVIPTNVH LWTLEEFRRFLIPTTELSQIVANLEGSNTLIRFTTIQSLGMLTTNMFDSMVSNRNCT GEGEDRRGSGEYPTSTSVNQCPSGRCALPF (SEQ ID NO:2).

[0084] The UNIPROT number for LA_3490 is Q72UG2_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_10695 PE=4 SV=1. c. LIC_10639

[0085] The amino acid sequence for LIC_10639 is MKMYNWKKILIVVLLASIMVYLEYEMDHTLVHAASSSKTTNSIVQKPTDPPKDK PIKVNVSGGGTFCYGPNFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWL CITAPETVVQGEEIWDYVHLRPCTINDPLQRWIIKDNSFWTANGFYRLKDTNWY GYISRNSGDKYNHTLDSSMKDWMNTIATPGNISILTSIAWDLNHSWGNERYFIRL GGSDKNTTPLYYNPENGHLAQYDPISGSLYCMYSQVDSYQWNWVSWESCSDAA ISKDNPTYWNVSFETEEGGMITDYKGNALRVTRYGSNWGAAYAAKLSYLEKDT TNSPTSLFIVNKDLLDWTRYTTSNLGKTEQYCPAPGNQASTTHKRISRTLPPSFQL TEAWVQRLYEITRSTSGSDISSGVCGVCLLHGFQMIAELQEYHSREPLQSGGYFFD TNPNTDPFISFGQRYPNLNTSLRDIVSTYGPTVRSSRRLILISARTMLPQYEWSLSS ESSTLSDMLSHIQSLIDSPPGSIWLVIMRRWRPDGTAGKHSVPILRTSQGLVVIPTA TTNLTLDNFRQALTPTMDPQQVIRNLEARPDRDLARFSTIQLGSFYHNPFDSAVSN RNCTGEGEDRRGSGEFPTSASINQCVSGRCSLSQ (SEQ ID NO:3).

[0086] The UNIPROT number for LIC_10639 is Q72UL8_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_10639 PE=4 SV=1.d. LIC_12844 (1-3 in Borg) / LA_0769

[0087] The amino acid sequence for LA_0769 is MEANMRHSIVFIVVLLVLVTTSFEYSINNIHIHASSNTSNSIIQKPTDQPKDKPIKIVI HDGGKFCYGPVFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPE TVVYAKENWDYVHLRPCTINDPLQRWVIKDNSFWTANERYQLKDTNWYVYISK NSGDRYNHTLDSSMNDWLNTIATPGNISIQTSIAWDLQTTEGQERYFIRRRGSNK NTTPLYYNPESGHLAQYDPVSGSLYCMYSQVDDNQWNWVTWTLCSDIPISKDNP AFWNVFFEADQGGIITDYKGNLLRVTRYGSNWGVAYAAKPDFVKTDTKNSPTSL FVVDKSLLDWTRYTYSNLGKTDQYCPAGNKENILHKRAKRTLPPGFQLTEDWV RRLYAIATSTIPEAQSSGICATCLLHSFQMLAELQEYHSQRPLQSGGYFFNTAPNT DPFISFRQRYPLLDMLLSDVPTVYSSAGRTTRQLGLVSARTVLPQYNWIASSEFIT RSEIRSHITSLIASPSGSIWLAILRLRRTDGVSGWHAVPILRTSQGLVVIRTRASLTS LDNYRQSLTPTMDPDLVIDNYLERPDLSLERLTTIQLGEVYHNTFDFIISNRNCTG EGDDRRGTGEYPTSASVNQCSSRRNRCALQ (SEQ ID NO: 4).

[0088] The UNIPROT number for LA_0769 is Q72NJ0_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_12844 PE=4 SV=1. e. LIC_10870 / LA_3271

[0089] The amino acid sequence for LA_3271 is MRNWKKVSMIVLLVLIGFGFEYGMNPTPVDASSKIEYLVIQKPTDPPKDQPIKVIV SGEGKFCYGPDFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPE KVIKAEKNWDYVHLRPCTINDPLQRWTIKDNSFWTADGVYRLKNYNWYGYISR NSGDRYNHTLDPSMNDWIKTVATPGNISIQTSIAWNLQTTEGQERYFIRWGGSDK NTTPLYYNPESGHIAQYDPISGSLYCMYSQVGGNQWNWVTWALCSDAAISKDNP AFWNVFFKTEEGGIITDYKGNLLRVTRYGSNWGVAYAAKPDFVKTDTKNSPTSL FVLDKSLLDWTRYTYSNLGKTDQYCPAGKHGSIIHRRIKRTLPPDFQLTEEWIQRL YAIARSTTRQTQHSGICGVCLLQTFQMLAELQEYHSQGPLSAGGYFFDTAPNADP FISFRQRYPLLDNTLSDAINIFGPSYNTTWLLTLAYAITMLPQYEWTLSNTFNTRPE ILSYISSLINSPPGSIWLAILRWRRPDGTFIGHSVPILRTSQGLVVIPTNVSSSRTLEN FRQSLIPSTDPNHIITNLERPNVTLTRFTTIELGGLYQNTFDFLISNNNCTGEGEDRR GTGNYPSSTSVNQCSGDGRCALPF (SEQ ID NO: 5).

[0090] The UNIPROT number for LA_3271 is Q72TZ4_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_10870 PE=4 SV=1. f. LIC_12715 / LA_0934

[0091] The amino acid sequence for LA_0934 isMCNWKKIFIVVLLVLIGFGFEYGINPTPVDASSKIEYLVIQKPTDPPKDQPIKVIVS GEGKFCYGPDFSGGESYIIIEQCWQMHVMNARYDVFQRISYNINNTWLCITAPEK VIKAEKNWDYVHLRPCTINDPLQRWTIKDNSFWTADGVYRLKNYNWYGYISRN SGDRYNHTLDPSMNDWIKTVATPGNISIQTSIAWNLRTTEGQERYFIRWGGSDKN TTPLYYNPESGHLAQYDPISGSLYCMYSQVGGNQWNWVTWALCSDAAISKNNP AFWNVSLETDEGGIITDYKGNLLRVTRYGSNWGVAYAAKPDFVKKDTKNSPTSL FVVDKSLLDWTRYTYSNLGKTDQYCPAGNKENILHRRVKRTLPPDFQLTEEWIQ RLYAIATSASPTAQRSGVCGVCLLQTFQMLAELQEYHSQGPLSAGGYFFDTAPNT DPFISFRQRYPDLNLIIENLPIMFNVDLIGLRLLVLATAAYMLPQYEWIVSPEIETRP EMITHINSLINSPPGSFWLAMMDRQRPDGTIVGHAVPILRTSQGLVVMSTNWRSV SLDQYRQTLTPTMDPNQVIANLERPDRTLLLLITMQLGQVYHNPLDAMISNRNCT GEGEDRRGTGEYPSSASVNQCLGGDGRCVLSPIH (SEQ ID NO: 6).

[0092] The UNIPROT number for LA_0934 is Q72NW3_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_12715 PE=4 SV=1. g. LIC_11358 / LA_2628

[0093] The amino acid sequence for LA_2628 is MRKWKVSLLVLFVFIRADYSYGVNPAFTPIPSSVTGSIVQKSTDSPKDKAIKVVIH DGRKFCYGPIFSGGESYIIVEQCSEQHVMNARYDVFQRISYNINNTWLCITAPEKV IKAEQIWDYVHLRPCTINDSLQRWVLQGNAFWTADGNYRLKDINWYGYISRNSG DNYNHTLDSSMNDWVKTVATPGNISLQTSIAWDLNNSWGNERYFIRGGGSGKN TTLLYYNPESGHLAQYNSISGSLYCMYSQMGRSNWNWVKWALCNDAVISKDNP AFWNVSLGTDEGGIITDYQGNILRVTRYGSNWGVVYTAKPDFVKTDIRNSPTSLF VVTKDLLNWTRYVSSNLGKTEQYCPAGNHVSLGHKRIVRTLPPDFQLSEAWIRR LYQIAISTVDTTQGSGICGICTLQAFQMVAELQEYHSQEPLQSGGYFFDTAPNTDP FISFRQRYPFLDRLLTDIPRAYSSINVVPADNRNRLVTFASARTILPQYNWAISAEF TTRSEILSHVSSLIDSSHGSLWIAILGRRRPDGTLGWHAAPILRTSQGLVVMRTSLI SASLSFYRQSLTPSTDPIQVVDNQLGRSDRALIRLITIQLVGSYQNSFDFMISNRNC TGEGEDRRGTGEYPTSSLVNQCLEGRCTLQ (SEQ ID NO:7).

[0094] The UNIPROT number for LA_2628 is Q72SM1_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_11358 PE=4 SV=1. h. LIC_12340 / LA_1400

[0095] The amino acid sequence for LA_1400 is MGRWIVLRVSLLVLIGIGFEYGINHTSINASSKSDYSIAQKPADQPKDKSIQVVMH GGSNYCYSPVFTKGEGYIWIDYCSDNTAKARYDVFQRISYNINNTWLCITAPETVVKGEETWNYVNLRPCTINDPLQRWIVKDNSFWTANGRYRLKDTNWYGYISRNS GDRYDHTLNSSMDDWIKTVAIPGNISIQTSIAWDLQTTEGNERYFIRWGSSNKNT TPLYYNPESGHIAQYDPSSGLLNCMYSKMTDKYDWNWVKWGKCSDAPIKKDNP AFWNVFFETDKEGAITDYKGNVLRVTRYGLNWGVAYTVKPSYLEKDTTHSPTSL FVIDKDLLDWTRYTYSNLGKTDQYCPAGNKESLVRKRVKRNLNLPSDFQLTREW IQRLYEIARSSISRAIPCRGVCGVCMLHSYQMIAELLEYHSRGPLTGGGYFFDTAP NRDPFISFNQRYPQLNALLTNVPSYANRPGFGSTLVMLPQYEWTSSDTITTRSGRL LHARSLINSPPGSIWLGLLRGRDADGSTWGHAVPILRTSQGIVVIPTNSPTMSLNT YIRSLAPTMDPNEVINRLENGSTLTELTTIQPVRIYDIPFSLTVSTRDCTGDGDGRR GSGRYPTSSLINQCSGGRCILQ (SEQ ID NO:8).

[0096] The UNIPROT number for LA_1400 is Q72PX7_LEPIC an Uncharacterized protein OS=Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni (strain Fiocruz L1-130) OX=267671 GN=LIC_12340 PE=4 SV=1. i. LIC_12339 (L. borg has 1) / LA_1402

[0097] The amino acid sequence for LA_1402 is MFVLYVLWLCDDVLIFERMETKMGNWKNLVVVLLVSIGVGFGYHTLIHASSSK ANYSIAQKPTDPPKDKPINIVTHDGKTYCYSPVFSKGEGYVWIEKCGDNTAKARY DVFQRISYNINNTWLCITAPEPVVKGNARWGYVNLRPCTINDPLQRWIVKENSFW TADGKYRLKDTNWYGYISKTSGDNYNHTLNSSMDNWVKTVATPGNISIRTSISW NSGWGDGIWDINMAPSAYFIHSKGSSKEDIIPLYYNPESGHIAQYDPSSGLLSCMY SKMTDKYDWNWVQWGKCSDAPIKKENPAFWNVYFVANAGGMITDYKGNILRV TKEGPNWGVAYTAKPSYLEKDTTHSPTSVFTVDVDLLKWIRYTTSNLGKTDQYC PAGKKESRIYQRVKRNLPSDFQLSVAWVQRLYDIARSATFESANPGAIPQRHGAC GVCLLHSFQMIAELMEYHSREPLTSGGYFFNTASNRDPFLSFSQRYPELDRLVTN VPVDYANRGRVLAFASAMIMLPQYEWESSSPLTTRSDIQSHIRSLINSPPGSIWLG LLRRQRANGSISGHAVPILRTSEGLVVIPTNMPTASLNTYIQSLAPTMDPNEVINRL ENGRTLTTLTTIRPVGTYETPFSLTVSSRDCTGDGDDRRGSGRYPISSLINQCSGGR CILQ (SEQ ID NO:9).

[0098] The UNIPROT number for LA_1402 is Q72PX8_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_12339 PE=4 SV=1. j. LIC_12986 / LA_0589

[0099] The amino acid sequence for LA_0589 is MGRWIVLLLVLLISFGVGYSYGVNPDVVPISSSDIPGSIIQRPTDKPKDKPIKVNVS SGGTFCYGPNFSGGESYIVIEQCWQMHVMNARYDVFQRISYNINDTWLCITVPESVVKGETNWDYVHLRPCTINDPLQRWIVKENSFWTADERYRLKDTNWYAYISRN SGDRYNHTLDSSMSDWINTVATPGNISILTSIAWDLGSDRYFIRSGGSDKNTTPIY YNPESGHLAQYNPVSGLLSCMYSRVGSYDWNWVTWALCSDAPISKDNPAYWN VYLATEEGGMIMDYQGNALRVTRYGPNWGVAYAAKLSYLKKDTTYNPTSLFIV DRNLLNWVRYTVSNLGKTDQYCPAGKKENHNIRIKRTLPPDFQLTEEWLRRLYD IAISTSFTSEGQVHGICGTCLLQTFQMLAELQEYHSHGPIQGGGYFFNTAHDRDPF DSFRQRYPELDAMLVNIPIVYSQDGNVPRMGLASARTMLPQYDWTLSREFTTRS EMLSHITSLIASPPGSMWLSMFRVRRPDGTTGGHAVPILRTSQGLVVIPTNSASLSF FTYRRFATPTTDPVQVMNNLEMSSWTLEILVTAQLEGLYYNTFDFTISNRNCTGE GRDRRGSGGYPTRTTVNQCSGRGGGRCVLL (SEQ ID NO:10).

[0100] The UNIPROT number for LA_0589 is Q72N52_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_12986 PE=4 SV=1. k. LIC_10778 / LA_3388

[0101] The amino acid sequence for LA_3388 is MSRWIVLLLVLLISLGVGYSYGVNPDVVPISSNDIPSSIIQRPTDKPKDKPIKIVIHD GGTFCYGPTFSGGESYIVIEQCWQMNVMNARYDVFQRISYNINNTWLCITVPESV VKGETNWDYVHLRPCTINDPLQRWIVKDNSFWTADKRYRLKDYNWYAYISKNS GDRYNHTLDSSMSDWINTVATPGNISILTSIAWNLGSDRYFIRSGGSDKNTTPIYY NPESGHLAQYNPVSGSLYCMYSRVGSYNWNWVTWALCSDAPISKDNPAFWNVS YETEEGGMIMDYQGNALRVARYGPNWGVAYAAKLSYLKKDTTYNPTSLFIVDR NLLNWVRYTASNLGKTDQYCPAGKKENHNITIKRNLPPGFQLTEEWIRRLYDITN STLSAGEVQIHGICGVCLLHTFQMLAELQEYHSHGPIQGGGYFFDTAPNRDPFDSF RQRYPELDALLVNVPNVYGSAGSTTRLLTLASARIMLPQYNWILSREFTTRSEILS HIRSLIGSPAGSVWLALMLRYSPYARTLIWHAVPILRTSQGLVVIPTNWSGLPLNV YRLYLTPTTDPFQVISNLEIPSRPLLRLITIRLDRIYNNIFDFMISNRDCSGEGEDRR GTGRYPISTLVNQCSGGRCTLM (SEQ ID NO:11).

[0102] The UNIPROT number for LA_3388 is Q72U83_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_10778 PE=4 SV=1. l. LIC_12791 / LA_0835

[0103] The amino acid sequence for LA_0835 is MSRWIVLLLVLLISLGVGYSYGVNPDVVPISSNDIPSSIIQRPTDKPKDKPIKIVIHD GGTFCYGPTFSGGESYIVIEQCWQMNVMNARYDVFQRISYNINNTWLCITVPESV VKGETNWDYVHLRPCTINDPLQRWIVKDNSFWTADKRYRLKDYNWYAYISKNS GDRYNHTLDSSMSDWINTVATPGNISILTSIAWNLGSDRYFIRSGGSDKNTTPIYYNPESGHLAQYNPVSGSLYCMYSRVGSYNWNWVTWALCSDAPISKDNPAFWNVS YETEEGGMIMDYQGNALRVARYGPNWGVAYAVKLSYLKKDTTYNPTSLFIVDR NLLNWVRYTASNLGKTDQYCPAGKKESRAKRVKRTLPSDFQLTEEWIQRLYDIA ISTVTNPQVSGICGVCLLQTFQILAELQEYHSREPLQSGGYFFDTAPNRDPFISFRQ RYPELDSLLSHIPRVYNSDSTTLRLLTLVSAMNMMPLYDWTPSREFTTRSEILSHI TSLIDSPAGSIWLALMRRQRPDGTIAGHSVPILRTSEGLVVIPTRVPSSISLELYREY LTPTMDPIQAINNLEQPDRTLTYFVTIQLGEFYDNFTDLVISNRNCTGEGEGRRGT GEYPASATVNQCSESRCALPSQ (SEQ ID NO:12).

[0104] The UNIPROT number for LA_0835 is Q72NP1_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_12791 PE=4 SV=1. m. LIC_12985 / LA_0591

[0105] The amino acid sequence for LA_0591 is MGRWIVLLLVLLISLGVGYSYGVNPDQYCPASKKENHNIRIKRTLPPDFQLTEEW LRRLYDIATSASLTEGQIHGICGVCLLQTFQMLAELQEYHSHGPLQGGGYFFNTA PDTDPFDSFRQRYPELDTMLTDAATAYGPAYNTTRLLTLVSAMTMMPQYEWTPS REFTTRSDMHSHIRSLIDSPPGSIWLGLMQRRESDETLRWHALPILRTSQGLIVIQT RVSTMSFELYRLYLTPSTSIVQIINDYLEEADRTLTVLVTIQLEQAYQNLFDFMVS NMNCTGEGENRRGSGGYPTSATVNQCSGGRCALPNW (SEQ ID NO:13).

[0106] The UNIPROT number for LA_0591 is Q72N53_LEPIC DUF1561 (strain Fiocruz L1-130) OX=267671 GN=LIC_12985 PE=4 SV=1. 3. Vaccine candidates

[0107] The VM protein-based leptospirosis vaccine candidates provided herein are based on the novel identification of the leptospiral VM proteins as antigen(s) with pan-L. interrogans vaccine potential based on newly published in vivo proof-of-principle data.30The studies presented herein with show two VM proteins - LA1400 and LA0591 - as recombinant protein immunogens in one or more candidate vaccine formulation(s). Data generated in this study may well lead to several alternative future vaccine delivery approaches beyond traditional subunit vaccine approaches, including mRNA or other recombinant nucleic acid approaches, and new technological ways of vaccine delivery, e.g, through transcutaneously administered nanoparticles83or orally adminstered recombinant vaccinia (orthopox viral vector) such as that currently done with rabies vaccine (Raboral®) in animals / wildlife.84-87

[0108] The laboratory group led by the inventor spearheaded the global Leptospira genome project,88and used computational analysis to identify the VM protein family89, 90and its potential functions,78and experimentally demonstrated in vivo proof-of-principle of the potential of VM proteins as leptospirosis vaccine candidates.30The laboratory’s leptospirosis basic and translational science research program, enabled identification of a family of pathogenic Leptospira-encoded Virulence Modifying (VM) proteins.88-90As they recently published, VM proteins are multidomain secreted cytotoxins with an N- terminal bona fide ricin B chain-like lectins and C-terminal DNase domains.78Using a mouse model that recapitulates severe human disease,40the group very recently published a key paper showing that VM protein vaccination of mice prevented severe disease and death.30The VM protein family is expanded and highly conserved at the amino acid level (>85-99%) in the most highly pathogenic Leptospira species, particularly L. interrogans, serovars of which are almost exclusively associated with severe disease, including Copenhageni, Lai, Autumnalis, Canicola and others.3, 18, 88, 91-93The laboratory recently validated their originally proposed bioinformatics approach by making genetically inactivated mutants using the predicted active sites as previously described. These mutations eliminated DNase activity and cytopathic effect on HeLa cells (Figures 1A- 1D), showing that we can produce genetically inactivated proteins for testing as vaccine candidates. II. Methods for Making

[0109] The disclosure also provides methods for producing VM protein-based leptospirosis compositions. Examples of host cells which can be utilized to produce VM protein-based leptospirosis compositions, include, any mammalian or human cell line or primary cell. Non-limiting examples include, e.g., 293, HT1080, Jurkat, and SupT1 cells. Other examples are CHO, 293, Hela, Vero, L929, BHK, NIH 3T3, MRC-5, BAE-1, HEP- G2, NSO, U937, Namalwa, HL60, WEHI 231, YAC 1, U 266B1, SH-SY5Y, CHO, e.g., CHO-K1 (CCL-61), 293 (e.g., CRL-1573). Cells are cultured under conditions effective to produce transfection and expression. Such conditions include, e.g., the particular milieu needed to achieve protein production. Such a milieu, includes, e.g., appropriate buffers, oxidizing agents, reducing agents, pH, co-factors, temperature, ion concentrations, suitable age and / or stage of cell (such as, in particular part of the cell cycle, or at a particular stage where particular genes are being expressed) where cells are being used, culture conditions (including cell media, substrates, oxygen, carbon dioxide, glucose and other sugar substrates, serum, growth factors, etc.).III. Methods for Treatment

[0110] The present disclosure can be used in gene therapy and / or therapeutic approaches for the treatment of disease which involve the increase or decrease of a nucleotide sequence of interest in a host-cell. In these embodiments, the expressible heterologous nucleotide sequence may be derived from a mammalian genome. It may be particularly useful in some embodiments to have the expressible heterologous nucleotide sequence derived from a human genome, wherein expression of the wild-type RNA and / or protein can produce therapeutic effects in a patient. For example, the expressible heterologous nucleotide sequence can encode CFTR, NeuroD1, Cas9 and Guide RNAs, or any other such sequence. In other embodiments, the heterologous nucleotide sequence encodes a secreted protein.

[0111] In other embodiments, the expressible heterologous nucleotide sequence responds to positive selection stimuli. In other embodiments, the expressible heterologous nucleotide sequence also responds to negative selection stimuli. In further embodiments, it may be useful for the polynucleotide sequences to further comprise a reporter gene. For example, the report gene can be a luciferase or green fluorescent protein.

[0112] In some embodiments, vectors used herein expresses one or more nucleotide sequences (e.g., siRNAs) that modify the translation and / or transcription of a host-cell nucleotide sequence of interest within a host cell. In some embodiments, transcription and / or translation of the expressible heterologous nucleotide sequence is modified so that its nucleotide sequence is codon degenerated with respect to the endogenous gene in a cell. Additionally, the expressible heterologous nucleotide sequence can be modified so that it co-expresses inhibitory or silencing sequences capable of inhibiting or silencing a host-cell nucleotide sequence of interest within a host cell.

[0113] In certain embodiments of the disclosure, VM protein-based leptospirosis compositions can be utilized to prepare antigenic preparations that be used as vaccines. Any suitable antigen(s) can be prepared in accordance with the disclosure, including antigens obtained from prions, viruses, mycobacterium, protozoa (e.g., Plasmodium falciparum (malaria)), trypanosomes, bacteria (e.g., Streptococcus, Neisseria, etc.), etc.

[0114] Host cells can be transfected with single VM protein-based leptospirosis particles containing one or more heterologous polynucleotide sequences, or with a plurality of VM protein-based leptospirosis particles, where each comprises the same or different heterologous polynucleotide sequence(s). For example, a multi-subunit antigen (including intracellular and cell-surface multi-subunit components) can be prepared byexpressing the individual subunits on separate vectors, but infecting the same host cell with all the vectors, such that assembly occurs within the host cell.

[0115] Vaccines often contain a plurality of antigen components, e.g., derived from different proteins, and / or from different epitopic regions of the same protein. For example, a vaccine against a bacterial disease can comprise one or more polypeptide sequences obtained from the bacteria which, when administered to a host, elicit an immunogenic or protective response to bacterial challenge.

[0116] The disclosure can also be utilized to prepare polypeptide multimers, e.g., where an antigenic preparation is produced which is comprised of more than one polypeptide. For instance, virus capsids can be made up of more than one polypeptide subunit. By transducing a host cell with vectors carrying different viral envelope sequences, the proteins, when expressed in the cell, can self-assemble into three- dimensional structures containing more than one protein subunit (e.g., in their native configuration). IV. Methods for Administration A. Administration by Vaccination

[0117] The present invention includes methods of vaccinating a subject by administering compositions as described herein to a subject in need thereof. In particular, the disclosure provides vaccines against leptospirosis.

[0118] The disclosure also provides various treatment methods involving delivering VM protein-based leptospirosis compositions to host cells in vivo. In some embodiments, VM protein-based leptospirosis compositions are delivered into a subject for treating or preventing leptospirosis.

[0119] It is contemplated that when used to treat leptospirosis, the compositions and methods of the disclosure can be combined with other therapeutic agents suitable for the same or similar diseases. Also, two or more embodiments of the disclosure may be also co-administered to generate additive or synergistic effects. When co-administered with a second therapeutic agent, the embodiment of the disclosure and the second therapeutic agent may be simultaneously or sequentially (in any order). Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0120] As a non-limiting example, the disclosure can be combined with other therapies that block inflammation through (e.g., via inhibition, reduction and / or blockage of IL1, INFα / β, IL6, TNF, L13, IL23, etc.). In some embodiments, compositions andmethods disclosed herein are useful to enhance the efficacy of vaccines directed to leptospirosis infections. The compositions and methods of the disclosure can be administered to a subject either simultaneously with or before (e.g., 1-30 days before) a reagent (including but not limited to small molecules, antibodies, or cellular reagents) that acts to elicit an immune response (e.g., to treat cancer or an infection). The compositions and methods of the disclosure can be also administered in combination with an anti- bacterial therapeutic. The compositions and methods of the disclosure can be also administered in combination with an anti-tumor antibody or an antibody directed at a pathogenic antigen or allergen.

[0121] The pharmaceutical compositions of the invention can be readily employed in a variety of therapeutic or prophylactic applications, e.g., for treating leptospirosis infection or eliciting an immune response to leptospirosis in a subject. Thus, the vaccine compositions of the invention can be used in diverse clinical settings for treating or preventing infections caused by various bacteria. As exemplification, a VM protein-based leptospirosis vaccine composition can be administered to a subject to induce an immune response to leptospirosis, e.g., to induce production of broadly neutralizing antibodies to the bacteria. For subjects at risk of developing a leptospirosis infection, a vaccine composition of the invention can be administered to provide prophylactic protection against bacterial infection. Therapeutic and prophylactic applications of vaccines derived from the other immunogens described herein can be similarly performed. Depending on the specific subject and conditions, pharmaceutical compositions of the invention can be administered to subjects by a variety of administration modes known to the person of ordinary skill in the art, for example, topical, oral, intranasal, intramuscular, subcutaneous, intravenous, intra-arterial, intra- articular, intraperitoneal, or parenteral routes. In some aspects, administration is to a mucosal surface. A vaccine may be administered by mass administration techniques such as by placing the vaccine in drinking water or by spraying the animals' environment. When administered by injection, the immunogenic composition or vaccine may be administered parenterally. Parenteral administration includes, for example, administration by intravenous, subcutaneous, intramuscular, or intraperitoneal injection. B. Booster Vaccines

[0122] The present disclosure provides for the administration of a booster VM protein-based vaccine against leptospirosis infection for use in such a method forinducing in a human subject an immune response, wherein said subject has previously received a primary vaccination against leptospirosis.

[0123] The method of booster vaccination according to the disclosure comprises the step of administering the vaccine composition to the subject.

[0124] The immune response induced by the vaccine composition of the disclosure or by the method of the disclosure is preferably a humoral response, especially a response comprising the production of neutralizing antibodies against leptospirosis infection, i.e. a neutralizing antibody response. V. Diagnostics

[0125] The present disclosure further provides methods of identification and diagnosis based on the leptospiral VM proteins of the present invention. Novel assays to identify patients at particular risk for severe leptospirosis are contemplated to be within scope of the present invention. Further, therapeutics targeting VM proteins may be useful in reversing severe leptospirosis pathophysiology with to-be-developed therapeutics such as monoclonal antibodies or small molecule drugs.

[0126] In particular, the present disclosure provides rapid, point-of-care, specific leptospirosis antigen-detection diagnostic tests useful with body fluids, particularly blood, including serum and plasma, urine, and saliva. A. Leptospirosis diagnostics

[0127] There is a vast global need for improved leptospirosis diagnostics. Reliable, timely, and clinically actionable diagnosis is not available in both high-income or low- and middle-income (LMIC) countries. The comprehensive current toolbox to diagnose human leptospirosis includes dark field microscopy examination of blood (neither sensitive nor specific), nucleic acid amplification (which requires either central reference laboratories or sophisticated research laboratory facilities generally not found in leptospirosis-affected settings), rapid antibody-detection-based tests, including ELISA, and L. biflexa-based dipstick and indirect hemagglutination tests that do not detect diagnostic antibodies in many regions and / or do not necessarily distinguish current illness from past infection in other endemic / epidemic regions.110The most common commercially available tests to diagnose acute leptospirosis are based on detection of IgM antibodies. These antibody-detecting serological tests are typically based on genus-level antigens using lysates of the non-pathogenic L. biflexa serovar Patoc as antigen and are the basis for two FDA-approved tests based on indirect hemagglutination or ELISA110.These tests are inaccurate because they are neither sensitive nor specific in most clinical and epidemiological contexts.6, 18, 80, 111-115Culture, while definitive,6, 18, 110is difficult and usually not available and is insensitive and slow (taking many weeks) so that clinically actionable results are rarely available. In-house custom solid phase assays have been used on a local basis but have not been developed nor deployed for general clinical use.116, 117

[0128] Academic laboratories have developed antigen-detection test prototypes, but they are of suboptimal sensitivity to diagnose leptospirosis. While other research groups have described various nucleic acid amplification tests to diagnose leptospirosis, such tests have not found significant impact because they are relatively expensive, require technology to carry out and do not have protected IP.30, 78, 122-125Additionally, their reagents are generally expensive and not available, and no diagnostic kits have been commercially produced based on such technology over more than 20 years of publications.88, 1261. VM protein-based leptospirosis diagnostic

[0129] Provided herein is a rapid, point-of-care, specific leptospirosis diagnostic test based on antigen detection in body fluids, particularly blood and urine. The diagnostic will first be based on VM proteins (VMP) to rapidly diagnose severe disease. Subsequent iterations will advance multiplex lateral flow assays (LFAs) based on additional antigen detection to enhance test performance and supplement diagnosis with epidemiological risk / exposure information.

[0130] Described herein are antigen detection solid phase assays with novel components and standard operating procedures (SOPs) for manufacturing and validation in both animal models and human samples from leptospirosis field studies in endemic regions. The assays will be equally applicable to diverse aspects of the animal health industries, including dogs, cattle, pigs, and horses, in which leptospirosis is caused by the same Leptospira and hence detectable using the same technologies as developed for humans. An animal health application is important for identifying potential sources of human infection and for expanding our market.

[0131] The acute leptospirosis diagnostic described herein will fill two major gaps in the leptospirosis field: 1) enable actionable diagnosis for clinical management; and 2) provide an essential, still-missing tool for burden of disease quantification, which drives the global business proposition for vaccine development and new approaches to public health interventions on a global basis.

[0132] The disclosed leptospirosis diagnostic is based on two key innovations: 1) The inventor’s laboratory has shown the ability to use a dual monoclonal antibody (mAb)- based capture ELISA that detects and quantifies leptospiral VM proteins in the blood of infected animals, and 2) the use of glycoconjugate technology in developing high affinity mAbs against poorly immunogenic antigens, such as leptospiral lipopolysaccharide (LPS).118, 120As shown below, Luna Biosciences’ preliminary capture ELISA and Western blot data indicate that our technology is sensitive and can detect the highly conserved VM protein antigen family119-121at low nanogram / high picogram levels per ml in the blood of infected hamsters. Not only do these data directly support VM protein detection as the basis for a novel diagnostic test, but they are also the first to demonstrate and quantify the presence of a secreted, pathogenic bacterial toxin that mediates disease pathogenesis in the blood of infected animals. This has important implications for vaccine and therapeutics development, and is recognized by major clinical laboratories and academic diagnostic developers as addressing a major gap and being innovative. 2. Leptospiral LPS and LipL32 as complementary targets for antigen detection diagnostic testing

[0133] Leptospiral LPS and LipL32 are the most abundant macromolecules in Leptospira.127LPS forms a dense, confluent surface on the bacterial cell, and LipL32 is the most abundant (subsurface5, 88, 127) leptospiral protein present, measured at 40,000 copies per cell.128LipL32 is highly conserved (~99% identical at amino acid level) in all of the pathogenic Group 1 Leptospira.5Tests have been developed for LipL32 antigen detection but are not generally available, commercially developed, or particularly sensitive.88Together with VM protein detection in a multiplex assay, leptospiral LPS and LipL32 antigen detection described herein will enable the most sensitive laboratory diagnosis with a determination of serogroup-specific and non-L. interrogans-specific infection, the key to both clinical management of the full spectrum of leptospirosis and enabling epidemiology / exposure risk-driven public health interventions, including animal reservoir identification.

[0134] Solid phase antibody-based antigen detection has major advantages over current molecular techniques in being inexpensive and available as Rapid Diagnostic Tests (RDTs) at point of care in diverse and relevant epidemiological contexts (similar to the use of malaria RDTs around the world, which use whole blood as test matrix).129This disclosure provides both a single antigen detection assay (VMP-based) and multiplex(VMP, LPS and LipL32) assay that would have clinical and public health impact. No such assay currently exists.

[0135] Leptospira genomics have been used to identify candidates for a pan- leptospirosis vaccine,50-52, 60but we are the only group to date to have succeeded in demonstrating in vivo proof-of-principle of such a vaccine, which is based on the same antigens as our proposed antigen detection test.

[0136] The inventor’s laboratory has led the global Leptospira genome project,121and his research group used computational analysis to identify the VM protein family89,90and its potential functions.119His laboratory experimentally demonstrated in vivo proof- of-principle of the potential of VM proteins as candidates for antigen detection.1203. Leptospirosis diagnostic markers

[0137] Severe clinical manifestations of human leptospirosis include jaundice, acute kidney injury (sometimes requiring dialysis), refractory shock, and pulmonary hemorrhage (Weil’s Syndrome).28, 29L. interrogans infections were associated with acute kidney injury and cardiovascular involvement, while both L. interrogans and L. borgpetersenii infections were linked to thrombocytopenia and liver impairment19. Three L. borgpetersenii isolates were found in male patients from the dry zone, indicating exposure during outdoor activities common in rice paddy farming areas with cattle and buffalo. These findings provide insights into strain-level factors, environmental responsiveness, and species-specific host interactions.

[0138] Despite identifying potential virulence factors, the clinical pathogenesis of leptospirosis remains poorly understood. For over a century, there has been speculation about the presence of a leptospiral toxin responsible for the clinical progression of the disease1, 53, 54, 155-159. Our recent discovery of the PF07598 gene family, unique to pathogenic Leptospira group I, encodes Virulence Modifying Proteins (VMPs) that likely play a crucial role in the disease's clinical progression, addressing a long-standing knowledge gap18, 88. These VMPs, comprising 12 / 13 conserved paralogs in L. interrogans serovars, are massively transcriptionally upregulated in vivo and are secreted exotoxins18. They are novel R-type lectins containing N-terminal ricin B chain-like lectin domains (RBL1 and RBL2) and C-terminal toxin domain78, 160. LA0591, a variant lacking RBLs, may function differently in pathogenesis, possibly originating intracellularly160. Furthermore, vaccination with two VMPs—LA1400 (LIC12340 orthologs in Copenhageni) and LA0591 (LIC12985)—has shown efficacy in protecting mice from death and reducing bacterial load in key target organs, liver, and kidney103.

[0139] Given our observations that VMPs mediate the pathogenesis of leptospirosis, leptospiral anti-VMPs antibodies diagnostic makers for early screening of leptospirosisare provided herein. The dislosure discloses a study evaluating the potential of recombinant VMPs to detect anti-VMP leptospiral antibodies in patients’sera collected in Sri Lanka19. The efficacy of VMPs was assessed by comparing the diagnostic potential of full-length LA3490 (one of the highly upregulated in vivo and cytotoxic to HeLa cells18, 160), N- terminal encoding ricin-like lectin domain (RBLs), and C-terminal encoding natural variant LA0591. The disclosure provides VMPs-based leptospiral prognostic and diagnosis markers to address the under-reporting of this neglected disease and to enable early intervention to prevent disease progression.

[0140] In the studies presented herein, we tested and assessed the hypothesis that VMPs antigens could predict and diagnose early anti-VMPs antibodies in serologically confirmed acute leptospirosis / febrile patient serum in an endemic region in Sri Lanka. The serum samples were previously evaluated by qPCR and MAT systematically using paired serum samples without selection bias. Out of 222 patient samples, 26.6% and 21.7% of cases were definite positive by qPCR and MAT respectively. However, 16% and 19% of cases were suspected / probable cases by qPCR and MAT. Our current study, for the first time, evaluated the potential of both recombinant full-length VMPs (LA3490) and their domains (RBLs and C-terminals; LA0591) for their ability to detect leptospiral IgG-specific anti-VMPs antibodies in definite and suspected patient serum and eventually differentiate healthy controls. LA3490 (full-length) and LA0591 (C-terminal domain) showed remarkable distinguished and statistically significant potential to detect anti- VMPs antibodies in confirmed and probable cases versus healthy controls. LA0591 is a superior antigen because it was able to well–differentiate the groups into distinct subsets, and substantially distinguishes positive sera from non-reactive / reactive serum; however, LA3490 and RBLs separated healthy controls from confirmed / probable cases. VMPs were able to detect antibodies in serum samples that tested negative in the MAT, indicating the potential omission of certain serovars necessary for accurately detecting agglutinating antibodies. Nonetheless, the use of VMPs has significantly improved the detection of antibodies and has helped reduce false negative results.

[0141] Earlier studies show that ELISA-based diagnosis of leptospirosis offers several advantages over the conventional methods such as MAT, culture, and serological detection of antibodies. MAT, considered the gold standard, detects agglutinating (IgM and IgG) antibodies and is serovar-specific. However, it can produce false negatives if aserovar is omitted and cannot distinguish early / late infections, limiting its utility in acute illness settings. MAT also requires technical expertise, a broad range of live pathogenic Leptospira cultures, and paired sera collected at specific intervals for accurate interpretation of results. On the other hand qPCR detects bacterial load but does not provide information on disease severity. ELISA, in contrast, is rapid, simpler than MAT, and offers high sensitivity and specificity161, 164.

[0142] Leptospirosis, an acute undifferentiated illness, can be life-threatening, if severe and undiagnosed, leading to Weil's disease with multi-organ involvement, including jaundice (liver injury), renal failure (kidney injury), and potentially fatal pulmonary hemorrhage2, 4, 29, 165. The absence of reliable diagnostic markers increases morbidity and mortality in humans, livestock, and companion animals, impacting veterinary health and economics significantly. Our recently discovered PF07598 gene family encoding VMPs belong to the classical secreted toxin paradigm but have a unique ABC domain architecture, unlike classical AB toxins such as diphtheria toxin, pertussis toxin, shiga toxin, or ricin toxin which are typically encoded by two or more genes and self-assembled into a multi-domain holotoxin160. VMPs possess secretory signal peptides and are secreted exotoxins with discrete N-terminal ricin B-like domains involved in host cell surface binding and a C-terminal DNase / toxin domain, mediating cell death78, 160. The current study underscores the potential VMPs antigens (LA3490 and LA0591) to detect antibody onset on day 2 since the patient showed the first symptom. LA0591 is superior antigen in detecting early anti-VMPs antibodies. LA0591 is exclusively a natural variant that encodes only the C-terminal domain and lacks N-terminal RBLs (RBL1 and RBL2), suggesting that this natural variant does not require binding and internalization into the host cell, rather it may produce intracellularly and imply a unique role in pathogenesis.

[0143] L. interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. weilli, L. kirschneri, and L. alexanderi pathogens are the leading causes of the disease5, 29. Interestingly, both L. interrogans and L. borgpetersenii were cultured from two patient serum samples each. These four patients also exhibited detectable in vivo expression of anti-VMPs antibodies (LA3490 and LA0591) early in the course of infection. The expression of anti-LA0591 antibodies was relatively higher in L. interrogans, a species that infects humans, compared to the animal-infecting L. borgpetersenii, while the opposite was observed for anti-LA3490 antibodies. Recently, Putz et al. conducted a study that found the serologically identical L. borgpetersenii serovar Hardjo strains JB197 andHB203 exhibit varying levels of disease severity in the hamster model88. Despite their genomic and nucleotide level similarities, JB197 causes a severe acute infection, while HB203 results in a persistent chronic infection. Proteome analysis of these two strains,isolated from experimentally challenged hamsters at 29◦C and 37◦C, revealed that theVMP–Q04V07 (LIC12339 orthologs) a member of the PF07598 gene family, wassignificantly upregulated at 29◦C and 37◦C (3.26 and 6.43 log2-fold, respectively) in acutedisease-causing strain JB197 compared to chronic strain HB203. This finding enhances our understanding of VMP-mediated host-pathogen interactions and contributes to potential improvements in vaccine and diagnostic strategies88.

[0144] Previously, in the field, various recombinant leptospiral proteins, including rLipL21166, rLipL32144, 167, 168, rLipL41168, 169, the D5-CBD fragment of LigA170, and rGroEL168, 171, were evaluated for detecting anti-leptospiral antibodies but lacked adequate sensitivity for identifying acute-phase leptospirosis. Lig proteins are sensitive markers for acute infection, although their usefulness is mainly evaluated in urban Brazilian outbreaks167, 172. Additionally, rLipL21, rLoa22, rLipL32, and rLigACon4-8 were evaluated in equine and canine samples173, 174. The LIC13341 antigen, capable of binding to various host matrices, showed diagnostic potential in human and bovine leptospirosis serum175. A recent study highlighted rChi2, a multiepitope chimeric protein that recognizes antibodies in both onset (MAT-, 75%) and convalescent (MAT+, 82%) phases176, suggesting as a promising candidate for an early, broad, and cross-reactive diagnostic test. Eight recombinant fragments of LigA, LigB, and LipL32, ranging from 31-70 kDa and encoded by various serovars were assessed for canine leptospirosis diagnosis, using the Multi-antigen Print Immunoassay (MAPIA) platform, demonstrating promising accuracy (AUC = 0.826 to 0.869) with specificity at 70% and sensitivity ranging from 89% to 95%177. Additionally, studies have explored urine screening for anti-leptospiral antibodies178and specific antigens such as LipL32, LipL41, HbpA, and sphingomyelinase164, aiding in the differentiation of patients with dengue who tested negative for these leptospiral antigens164.

[0145] The compositions and methods provided herein offer advantages in detecting IgG antibodies in early infection even on day 2, compared to earlier studies. The current study also highlights the diagnostic potential VMPs, which can even detect the circulatory antibodies generated from previous exposure and reaffirm the diagnostic accuracy. The AUC for LA0591 was 0.947 (SE = 0.017, p< 0.001), relatively superior to LA34900.930(SE = 0.020, p < 0.001) in distinguishing the potential positive and negative cases and suggesting they are a reliable diagnostic marker and further superior to qPCR or MAT tests for detecting leptospirosis illness. EXAMPLES

[0146] Presented herein are leptospiral VM proteins as vaccine candidates for human and animal leptospirosis, for which there is a worldwide market and major unmet need. The inventor’s laboratory recently demonstrated that the newly discovered leptospiral VM proteins are viable antigens on which to base a leptospirosis vaccine. They also demonstrated the feasibility of genetically detoxifying VM proteins by LA0591, which will be extended to the closely related LA1400 antigen. Presented herein are studies of leptospiral VM proteins as candidate immunogens for a human leptospirosis vaccine. Materials and Methods

[0147] Suitability of animal models for leptospirosis vaccine development: Rodent models—mice, hamsters—recapitulate the clinical manifestations of human leptospirosis.34, 40The hamster model remains the standard for assessing leptospirosis vaccine efficacy for the animal health industry;41, 42this model is required by USDA30(and US Federal law43) for approval of veterinary leptospirosis vaccines.44-49The hamster model recapitulates the clinical manifestations of human leptospirosis. The experiments proposed involve the use of commercially available outbred Syrian Golden hamsters for 1) vaccination experiments, 2) and maintaining leptospiral virulence.

[0148] In initial experiments, male hamsters will be used for various vaccination and passive immunization protocols, receiving intramuscular injections of graded doses of recombinant proteins in combination of adjuvant or ip injection of immune sera as indicated. The recombinant proteins will be ensured to be endotoxin-free as assessed by the limulus lysate assay. The adjuvants used (GLA- or MPL-squalene oil-in-water) are safe for use in animals and approved by FDA for human use; no Freund’s type or other potential harmful adjuvants will be used. Polyclonal sera for passive transfer experiments will be obtained from various groups of hamsters retro-orbitally under injection (L / X) anesthesia because obtaining sufficient quantity of blood from hamsters by saphenous vein or tail bleeding is otherwise not practical.

[0149] To take into account sex as a biological variable, female hamsters will be used to replicate male hamster vaccination results when experimental results indicate vaccine-induced protective immunity in male hamsters.

[0150] Protective immune responses will be assessed by correlates with protection from death, clinical disease, and determination of bacterial load and viability in liver and kidney at various time points by qPCR and culture, respectively. To enhance rigor of the proposed experiments, we will confirm that protective immune responses are due to anti- VM protein antibodies induced by vaccination, and passive transfer experiments will be carried out in which vaccine-generated sera (polyclonal sera) will be injected into naïve hamsters followed by challenge infection. Characterization of VM-protein-specific antibody responses will assess the anti-VM protein cross-reactivity within different L. interrogans serovars and quantify the affinity of vaccine-induced IgG responses in relation to protection against death and bacterial load.

[0151] Statistical Analysis: This analysis was conducted in collaboration with Dr. Armin Schwartzman (Biosketch, Letter of Support). Experiments will test the hypothesis that vaccination with recombinant VM proteins will be non-inferior to a tetravalent bacterin vaccine in terms of protecting from mortality, which has been reported to be zero for one canine vaccine (Nobivac, Merck).94, 95Sample size calculation for the non- inferiority test: The efficacy rate in terms of preventing mortality in vaccinating hamsters treated with the Nobivac tetravalent leptospirosis bacterin vaccine is unknown. In a trial with the same four challenges as ours, comparing 20 vaccinated dogs to 10 animals in the placebo group in each challenge, Merck reported zero mortality cases in vaccinees group with mortality confirmed in the placebo groups.94, 95We estimated the mortality rate as the highest value for which the reported result would be most likely, i.e. the probability of such an outcome is greater than 50%. Our analysis shows that the Merck results are most likely if the mortality rate is no higher than 0.0065 (data not shown because of space constraints). Taking this as the reference mortality rate, we used the two-sample non- inferiority test of ratio of Poisson rates to calculate the sample size required to obtain 80% statistical power to claim non-inferiority, including the Bonferroni correction. A sample size of 20 animals corresponds to a margin of 3.5 fold, equal to a mortality rate of 2.2%. Sample size calculation for comparison to placebo: Assuming a two-sample test of proportions as described above, a straightforward power calculation shows that with 20 animals in the vaccine group and 10 animals in the placebo group, a difference of mortality rates between 2% and 50% respectively can be detected with 87% power. Statistical analysis of outcomes: The primary endpoint is mortality rate 14 days after exposure. A two-sided two-sample test of proportions will be conducted comparing the VM protein groups to the placebo group for each infectious challenge. Since we expectthe mortality rate to be low in the comparison canine bacterin vaccine group and high in the placebo group,94a z-test may be inaccurate and we will use Fisher’s Exact test to assess significance.45P-values will be adjusted for multiple testing using a Bonferroni correction. For a more precise description of the vaccine effect, log rank analysis of Kaplan-Meier curves will be used. Non-inferiority test: A non-inferiority test of proportions will be to see if the mortality rate of VM protein groups is non-inferior to that of the Merck vaccine. The binomial distribution analysis will test the mortality rate ratios as Poisson rates.96, 97

[0152] Provided herein are compositions and methods of using VMPs as diagnostic antigens for ELISA. We produced E. coli-expressed and AKTA Pure FLPC system- purified recombinant VMPs as diagnostic antigens for ELISA. As we have already published162, 166, 57, 58and according to our unpublished data cited here, we have used Cytiva HisTrap columns to obtain >95% pure His6-tagged recombinant VMPS: LA0591, LA3490, LA1402, LA1400, and RBL1+2.

[0153] ELISA assay protocol: We have created a preliminary procedure for VMP antibody-detecting ELISA based on adsorption of His6-tagged recombinant VMPS to BSA-blocked, nickel-coated 96-well plates (Pierce™ Nickel-Coated Plates, ThermoFisher, catalog #15442). VMP antigen is added to wells (100 ng in 100 µL / well), followed by 100 µL of 1 / 500 diluted serum, followed by species-specific horseradish peroxidase (HRP) labeled secondary antibody Substrate (tetramethylbenzidine (TMB)),the reaction stopped with 2M H2SO4, amd absorbance quantified colorimetrically at 450 nm using a Molecular Devices SpectraMax M5 with up-to-date software that includes a system audit trail. Controls include, negative controls, antigen blank, and primary and secondary antibody blanks. Positive controls are sera from vaccinated mice and hamsters (already available in the laboratory). ELISA results are generally interpreted using a positive result considered at 3 standard deviations above the mean of the negative controls.

[0154] Kinetics of ELISA-determined anti-VMP antibodies in experimentally infected rhesus macaques: Three adult female retired breeders (Xena, Skeeter, Melody) in the Yale University Animal Resource Center, under Yale IACUC-approved protocols, were inoculated with 250 x 106freshly passaged L. interrogans serovar Copenhageni (split among subcutaneous injection and conjunctival and nasal instillation) and serially bled at days 0, 4, 8, 14, 21 (Xena at day 24). At a 1 / 500 dilution of serum, there was arobust IgM antibody response detectable in all monkeys at day 4 and a robust IgG response at day 8. These results indicate that diagnostic anti-VMP protein antibodies are detectable early in leptospiral infection, earlier and more robustly than reported for MAT or L. biflexa-based solid phase assays. In this small number (N=3) of monkeys, it appears that LA0591 plus one other VMP (i.e. LA3490) may be sufficient antigens on which to base the solid phase assay. This will be assessed in this project with mice, rats and dogs, and with sera from known leptospirosis-infected humans.

[0155] Description of human sample collections and preliminary anti-VMP antibody ELISA data from a Sri Lankan human longitudinal study: The Yale collaborator already has obtained well-characterized samples47(from the leptospirosis diagnostics point of view: MAT, PCR and sometimes culture isolates, from a longitudinal Sri Lankan cohort, N=1,192 total subjects, including positives and negatives, and negative controls), and has these samples as deidentified, biobanked, human samples from protocols in which additional studies on samples are formally consented to by the human subjects involved in the studies. In addition, the Yale collaborator’s laboratory has N=589) population- based serum sample controls to assess the prevalence and level of anti-VMP antibodies in co-endemic controls and as a contrast to the Sri Lankan cohort. We have described the cohort and diagnostic characterization from whom the serum samples were obtained.47The study sample reported here included 224 serum samples from suspected cases of leptospirosis / febrile patients and 45 healthy controls. 1,192 subjects were enrolled but only 224 patient randomly-sampled samples, about a 1 / 6thsubsets of the sample collection, have yet been studied.70 (31.3%) were confirmed as positive, and 42 (18.8%) were categorized as probable cases of leptospirosis, with a single well positive out of two or more replicates on the qPCR plate. Another 93 (41.5%) were categorized as Leptospira ‘not detected’ in qPCR. For 19 cases (8.5%), qPCR results were not available. As for the MAT results, 57 (25.4%) tested positive, 50 (22.3%) exhibited a reactive response (titer < 400), and 105 cases (46.9%) were not reactive. MAT results were not available for 12 samples. We conducted Receiving Operating Curve (ROC) analysis to evaluate the diagnostic accuracy of using two recombinant proteins to detect anti-VMP IgG antibodies, LA0591 and LA3490, in acute serum samples, that is, specimens obtained at the time of clinical presentation. Only confirmed leptospirosis cases (n=103) and healthy non-febrile, co-endemic region adults were included in this analysis. The AUC for LA0591 was 0.957 (SE = 0.015, p < 0.001, 95% CI: 0.928-0.987), indicating a high discriminatory power in distinguishing between positive and negative cases. Similarly, the AUC for LA3490 was0.927 (SE = 0.021, p < 0.001, 95% CI: 0.886-0.967), further suggesting its potential as a reliable diagnostic marker for Leptospira infection. The Gini Index values were 0.915 (LA0591) and 0.853 (LA3490), confirming their strong discriminatory abilities. Optimal cut-off values for classification, determined by maximum K-S metrics, were 0.505333 (LA0591) and 0.42317 (LA3490). We conclude that at the time of clinical presentation in a real world setting, with symptom duration as short as 2 days, that ELISA with two VMPs LA0591 and LA3490) is provisionally as good as or superior to PCR and other “gold standard”25, 26diagnostics. Example 1: Optimize dose and dosing regimen of VM protein-based vaccine against leptospirosis infection.

[0156] To further develop prioritized wildtype VM proteins as leptospirosis vaccine candidates. Using a lethal hamster challenge model of leptospirosis, we will optimize dose, delivery, form and composition of a tagless, recombinant E. coli-produced VM protein-based experimental vaccine in combination with human- compatible, approved adjuvant(s) to determine protective efficacy. We will compare commercially-obtained animal bacterin vaccines to our candidate recombinant VM proteins. Duration (within project timeline) and quality (i.e. VM protein toxin neutralization, affinity) of IgG- mediated protective immunity will be measured.

[0157] The dosing frequency and composition of recombinant E. coli-produced wildtype VM proteins and human-compatible adjuvant must be optimized to induce protective efficacy. Using the lethal hamster challenge model of leptospirosis, commercially available bacterin vaccines will be compared to varying doses and dose regimens of candidate recombinant VM proteins. Duration and quality of IgG-mediated protective immunity mediated by VM protein vaccination will be measured after cross- serovar challenge of hamsters with

[0158] L. interrogans serovar Copenhageni and L. interrogans serovar Canicola. The primary outcome will be comparison of mortality in VM protein vaccine groups vs. negative control and commercially available vaccine. In addition to mortality and severity of clinical disease, determination of bacterial load and viability in liver and kidney at various time points will be measured by qPCR and culture. To confirm that protective immune responses are due to anti-VM protein antibodies induced by vaccination, passive transfer experiments will be carried out in which vaccine-generated sera are injected into naïve hamsters followed by challenge infection.

[0159] Milestones: Demonstration of non-inferiority of VM protein-based vaccine candidate vs. bacterin; identification of antibody correlates of protective immunity.

[0160] Approach

[0161] 1) Antigen production and characterization. Recombinant LA1400 and LA0591 (sequences derived from serovar Lai) will be produced in E. coli as endotoxin- free, purified recombinant proteins as published.11, 30, 78For tagless recombinant proteins, we will use the pFINA vector and glutathione reductase-deficient E. coli from Fina BioSolutions, as published98-102. Protocols for laboratory scale column chromatography purification, suitable for later scale- up, will be established including, for example, Ni-NTA affinity, etc. for His6tagged proteins; ion exchange,hydrophobic interaction andsize exclusion chromatography for tagless proteins. Tagless VM proteins (i.e., lacking fusion partners; comprised only of the antigen of interest) will be produced using kanamycin resistance markers, and polymyxin columns will be used to eliminate endotoxin to acceptable levels (as determined by the standard limulus amebocyte lysate assay106) for downstream use. The identity and purity of recombinant VM proteins will be verified by SDS-PAGE, Western immunoblot and mass spectrometry as shown above.

[0162] Downstream process development for purification of tagless, human vaccine- compatible, recombinant E. coli- produced VM protein antigens based on single-step, custom resin affinity purification will be considered to maximize manufacturing efficiency and reduce the cost of goods (with Avitide, Chavarria, Letter of Support).

[0163] 2) Vaccination of Syrian golden hamsters will be carried out as outlined in Table 2, followed by lethal L. interrogans serovar Copenhageni strain FIOCRUZ challenge. Vaccination regimens will be dose-finding, ranging from 0.5 to 25 µg per injection (in combination with a range of adjuvants including squalene / oil-in-water along with TLR agonists such as GLA-SE, synthetic monophosphoryl lipid A, with 2-3 vaccinations performed 4 weeks apart. Initial experiments will compare bacterin canine vaccines containing various L. interrogans serovars (obtained commercially or produced in the Yale laboratory according to standard procedures31, 107, 108) to the VM protein antigen groups. Vaccine groups are summarized as follows: For statistical and experimental robustness, each vaccination test group will have 20 outbred Syrian golden hamsters, 15 of which will have death as an endpoint, and 5 of which will be serially sacrificed for sampling blood and tissues for histopathology, culture and qRT-PCR assessment of bacterial load. Groups 2-7 will have graded amounts of VM protein rangingfrom a total of 0.5 to 25 μg and will be done with either 2 or 3 injections 3 weeks apart. Initial experiments will use the human compatible GLA-SE adjuvant as we have observed. This adjuvant is available to us (see Letter of Support, Dr. Sanjay Singh, Gennova), and other experimental adjuvants will be tested as well (see Letter of Support, Dr. Darrick Carter, PAI Life Science) given the importance of cost of goods in developing leptospirosis vaccines. We recognize that all such experiments cannot be done simultaneously given the volume of work required; go / no-go decisions will be made after each vaccination / challenge experiment. For example, if we find that both LA1400 and LA0591 tagless proteins together generate protective immunity better than bacterins and better than either alone, VM protein dose finding and adjuvant testing experiments will follow up on the most promising results. We will also follow up these experiments with other forms of recombinant immunogens (chemically inactivated, inclusion bodies) if tagless proteins are too difficult to produce at scale. If, as we expect, VM protein vaccination is statistically non-inferior to bacterins, or, indeed superior (i.e. in terms of preventing death with cross-serovar challenge, lower bacterial loads or non-viable bacteria in liver and kidney), duration of protective immunity and antibody responses will be assessed within the project period. Challenge infection will be done using low passage (<5) L. interrogans serovar Copenhageni; later, depending on results, additional L. interrogans serovars will be used to validate cross-serovar / species challenge infections will include Canicola, Autumnalis, Australis, Lai and L. kirschneri serovar Grippotyphosa, which we have in hand. Progression in cross-serovar challenge infections will be rationally pursued; such cross-strain challenge experiments will be pursued with the goal of testing whether the VM protein vaccine candidates are pan- leptospirosis vaccine candidates (for animals and humans). Assessments will include overall clinical assessment (eating, drinking, grooming, energy level, daily weights) and death as an endpoint. Animals surviving to 14 days after challenge infection will be sacrificed to determine bacterial load in kidney and liver.

[0164] Table 2

[0165] Passive immunization experiments: To test the hypothesis that antibodies mediate protective immunity, post- vaccination sera will be obtained at the time of sacrifice and from negative control (PBS alone vaccine) groups, pooled and used to inject (0.5ml) ip prior to L. interrogans serovar Copenhageni challenge infection. Similarly, we have ongoing monoclonal antibody production against VM proteins (outside the scope of the present proposal); to date we have obtained 6 mAbs with high affinity, 3 of which cross-react against LA0591, LA1402 and LA3490. We will be testing these mAbs for clinical and microbiological outcomes of challenge infection, which will be assessed as above.

[0166] Data analysis: Kaplan-Meier curves will be used to analyze survival times. The Kruskal-Wallis test will be used to determine significant differences in leptospiremia and leptospiruria, and compare bacterial load in kidney or liver among the survivors from different immunization groups. The results will also be analyzed by the non- parametric Mann–Whitney test to determine organism load differences between groups at p<0.05. Example 2: Demonstrate safety of VM protein-based vaccine.

[0167] To compare wild type and genetically mutated toxoid-forms of either fusion or tagless, recombinant E. coli-produced VM proteins in a lethal hamster model.

[0168] We will compare protective immunity induced by wild type and genetically mutated toxoid-forms of tagless, recombinant E. coli-produced VM proteins in a lethal hamster model. Because leptospiral VM proteins are toxins, we must eliminate potential adverse effects from a candidate vaccine. We will compare the immunogenicity of wild type VM proteins and genetically inactivated VM proteins (toxoids) vs. commercially available bacterin vaccines as in Experiment 1.

[0169] Laboratory correlates of protective immune responses will be assessed as in Aim 1. Milestones: SOP development for producing tagless VM protein vaccine candidates at-scale; demonstrate protective immunity induced by tagless, genetically inactivated VM protein toxoids; identify correlates of protective immunity.

[0170] Expected Outcomes: This Phase I project will yield one or more VM-protein- based vaccine candidates to advance to further human clinical development and determine in vitro correlates of protective immunity to guide clinical development.

[0171] Plans for Phase II. The inventors will focus on pilot lot production, preclinical toxicology, immunogenicity studies, process development (fermentation, purification, characterization, QA / QC, assays to predict efficacy), and clinical trials in dogs with input from experts in animal and human vaccine development regulatory pathways.

[0172] Commercial Application The inventors intend first to commercialize a VM protein-based leptospirosis vaccine for dogs; a pivotal dog vaccine trial will provide key data for an FDA IND submission.

[0173] Approach:

[0174] Recombinant, non-toxic mutant VM proteins (LA1400, LA0591) will be produced for vaccination experiments; the core experimental design (Table 3) includes 20 animals per vaccine group; 10 in placebo group.

[0175] Table 3: Core experimental design

[0176] We will use our previously established assays to demonstrate abrogation of toxicity (loss of cytotoxicity and DNase activity), which will be incorporated into QA / QC in future process development. We focus here on making genetically-inactivated mutant VM proteins following what has been previously shown for diphtheria toxin (CRM197)99,104and tetanus toxin.105We combined bioinformatic approaches (FTMAP,12, 13PrankWeb / P2Rank,14Deepsite27) to identify likely active site residues of the VM protein toxin activity (data not shown due to space limitation); these Glu, His and Asp residueswill be mutated to Ala and synthetic mutant genes made (as done previously78) in pET32b and in pFINA (tagless). We do not propose to mutate the ricin B- like domain-1 of LA1400 (LA0591 does not have such a domain) because it does not exert cytopathic effect.78Data analysis, interpretation, Potential Limitations, Pitfalls, Alternative Approaches: Experiments in Specific Aims 1 and 2 are focused on determining the basic feasibility of LA1400 and LA0591 VM protein antigens in inducing protective immunity against lethal challenge infection in a hamster model, both with wild type and mutant proteins. For recombinant vaccinogen production, we will produce purified tagless recombinant VM proteins by scaling up expression conditions and purification protocols, with which our collaborator Dr. Andrew Lees, Fina Biosolutions has extensive experience (Dr. Andrew Lees Letter of Support).98-102Alternative forms of recombinant protein immunogen production may include chemical inactivation (as is standard in producing tetanus toxoid105, 109) or proteins purified from urea-solubilized and affinity purification proteins from inclusion bodies, which, if necessary, will be assessed for immunogenicity and induction of protective immunity.

[0177] Statistical analysis will be carried out as described above (Armin Schwartzman, Letter of Support). Given the constraints of time and budget, for challenge infections, we will start with using one L. interrogans heterologous challenge isolate, but will have the opportunity to carry out further challenges with additional heterologous L. interrogans serovars and other species which we already have in hand (L. kirschneri, which is very closely related to L. interrogans and is known to cause human disease;39and L. borgpetersenii, which infects both livestock and humans19). Example 3: Optimize and validate solid phase virulence modifying protein (VMP)-based antigen- detection leptospirosis diagnostic tests

[0178] Preliminary Data

[0179] Figures 2A-2F show that we have specific anti-VMP mAbs and that VMP antigen is readily detected in the serum of infected hamsters (Figures 3A-3C), in direct support of the proposed diagnostic test development. We have characterized three murine mAbs raised against LA0591, which has >95% amino identity for the C- terminal toxin domain across all L. interrogans VM proteins.1) 5F8 and 5G10 have affinity constants of Kd~10- 9, 6A5 Kd~10-12; 2) mAbs 5F8 and 5G10 (IgG1) bind to overlapping linear peptide epitopes that are spatially distinct from another linear epitope recognized by mAb 6A5 (IgG2b) (not shown); 3) the 5F8 / 5G10 and 6A5 epitopes are conserved >90-85% among all serovars of L. interrogans and between L. interrogans and all serovars of L.borgpetersenii, a major cattle- and human-infecting leptospire; and 4) all three mAbs recognize native L. interrogans serovar Lai, Copenhageni and Canicola LA0591 VM proteins (FIGs. 2A-2F), in addition to recognizing multiple recombinant VM proteins conserved across Group 1 pathogenic members of the genus (LA3490, LA1400, LA1402, LA0591; data not shown); 5) Notably, VMP antigen expression is massively upregulated by L. interrogans serovar Copenhageni under high salt conditions (120 mM NaCl added to EMJH culture medium) reported to mimic in vivo conditions and pathogenesis-related gene expression, potentially providing an explanation of why Copenhageni is so virulent (and hence useful in a diagnostic test).1-4, 130Further, mAb 5F8 cross-reacts with SL-23, a L. borgpetersenii we isolated from humans in Sri Lanka,24and which causes lethal pulmonary hemorrhagic leptospirosis in hamsters (data not shown). IP for this is submitted as patents PCT / US22 / 74601 and PCT / US2021 / 016564. As essential proof-of- principle for this proposal, VMPs are readily detectable in the serum of L. interrogans Copenhageni-infected hamsters during infection (FIGs. 3A-3D). At the time of serum collection in this experiment (day 4), animals did not manifest clinical illness.23

[0180] Experimental design

[0181] Lateral flow assay (LFA) prototype development: ELISA-validated mAb pairs will be advanced to LFA prototype development. The development of a prototype LFA device for a rapid, point-of-care test will be carried out jointly by personnel from Luna Bioscience and nanoComposix / Fortis Life Sciences. We will focus on the development of LFA prototypes for the detection of leptospirosis in body fluids, particularly whole blood, serum / plasma and urine.

[0182] VM proteins are present in infected blood at 500pg-5ng / mL and we expect that leptospiral antigens LPS and LipL32 are secreted / released into the blood as well. Despite the presence of Leptospira organisms in blood at 102to 106 / ml, as noted above, we aim to detect soluble circulating antigen. Detecting such secreted / released antigens in the blood as the basis of infectious disease diagnosis has robust antecedents as exemplified with malaria antigen detection by LFA; soluble antigen is detected independent of cell-associated antigen.131-133We propose to optimize and quantify prototype performance using blood-based matrices spiked with leptospiral antigens, with subsequent testing of samples from infected hamsters and well-characterized human specimens in the Yale collaborator’s biobank. Specifically, nanoComposix will be responsible for the following in association with Dr. Chaurasia: 1) Lateral Flow Material Preparation: Test strips and conjugates will be prepared using each antibody as it isimportant to evaluate both orientations of the antibodies on the strip. The presentation of the antigen to the antibody can be quite different depending on its position in the assay. Antibodies with lower binding affinities perform better conjugated to a reporter particle (i.e. colloidal gold), whereas high affinity antibodies are best used on the test line due to the short antigen exposure time; 2) Reagent Pair Down Selection: After material preparation, Dr. Chaurasia and nanoComposix will functionally evaluate the antibody pairs in both possible orientations using a panel of contrived samples prepared in a control solution (e.g., PBS followed by blood, serum / plasma and urine matrices). Contrived samples will be prepared at a wide range of concentrations to assess assay performance. Combinations of material will be evaluated based on analytical sensitivity and specificity. Additional considerations, such as stability and precision, will be examined during optimization; 3) Volumes of sample: Spiked matrices and animal- derived samples will be added to prototype devices from 5-100 uL, taking into account proper sample / buffer flow.4) Buffer Selection and Optimization: Samples will be treated with running buffers that contain pH stabilizers (optimum pH 7.5-8.0), non-ionic detergents at graded concentrations (Tween-200.05-1%, 0.05 to 2% Triton X100, NP40, sarcosyl, in Tris / potassium phosphate / NaCl / imidazole buffers) to optimize cell lysis and exposure of epitopes; one such example includes 100 mM potassium phosphate pH = 8.0, 600 mM NaCl, 250 mM imidazole, 2% Triton X-100, but the lysis / running buffer composition will be systematically tested for optimal performance. We will also test buffers from proprietary malaria LFA kits (such as SD BIOLINE Malaria Ag Pf / Pan) for malaria HRP and LDH detection of whole blood (buffer composition not publicly available). Selected top assay(s) will then undergo rapid optimization. Optimization will focus on matrix (e.g., serum / plasma / whole blood / urine), running buffer components and conjugate dry down, cartridge integration, and analytical performance. Correlation testing against the ELISA developed by Luna will be included in further optimization efforts with available animal and human clinical samples. At the conclusion of the project, Luna and nanoComposix will have collected and provided objective evidence to demonstrate feasibility of employing a lateral flow immunoassay prototype (FIG. 4) for detection of leptospiral antigens in human serum / plasma / whole blood. Luna will then use the prototype performance data to determine whether to move forward with further product development.

[0183] Determine the performance of new ELISA and lateral flow assay prototypes:

[0184] In vitro testing. Determine sensitivity of ELISA and LFA prototypes for detecting VMP antigen in spiked L. human plasma, serum and urine matrices. Serum and urine from normal humans (locally obtained) will be spiked with known quantities of purified recombinant VMP and analytical sensitivity / standard curves determined by ELISA. Optimal buffers (as noted above) will be determined.

[0185] Animal model testing. ELISA and LFA prototypes will be used to detect and measure VMP in L. interrogans serovar Copenhageni infected hamsters, an animal model of acute leptospirosis that recapitulates human disease.134, 135This model will be used to determine analytical sensitivity of the prototype ELISAs and LFAs. Hamsters were chosen because sufficient quantity of blood (200 μL) can be obtained at sequential time points over the first 7 days of infection, up to 14 d after infection. We propose to compare the kinetics of leptospiremia and leptospiruria over 2 weeks in infected hamsters models by qPCR24, 30, 124, 136with ELISA-based antigen detection using new mAbs. Groups of 4 hamsters per cage (with 4 replicate cages totaling 32 hamsters, respectively, for the first experiment) will be used, with inoculation of 103low passage organisms, to provide a first estimate of antigen-detection limits. Animals will be serially sampled for blood every 3 days (200 μL per sample), and daily for 200-500 μL of urine (using standard noninvasive methods) as we have previously performed.137, 138Appropriate negative controls (i.e. water or body fluid matrix extracted at time of sample extraction) will be performed. These hamster experiments will yield qPCR quantification of leptospiremia and leptospiruria in acute infection, against which soluble VMPs, LPS and LipL32 antigen detection will be benchmarked to determine sensitivity / specificity, with replicates done to ensure rigor.

[0186] Pilot human specimen testing. Using de-identified human blood and urine samples originally from our NIH-funded leptospirosis-endemic sites in Peru and Sri Lanka (>1000 well-characterized samples from subjects are available in our specimen collections (eg.30, 125, 92, 139, 140)), we will test ELISA and LFA prototypes in pilot studies. We propose to test serum and urine samples by ELISA and LFA from 100 well- characterized leptospirosis patients (diagnostic serology, qPCR, infecting serovar identification focusing on infections by serogroup Icterohaemorrhagiae which includes Copenhageni) plus an equal number of co-endemic human controls found not to be infected by Leptospira. These samples are available and will be processed using methodsdescribed above. Data analysis using negative and positive controls will be carried out as described above.

[0187] We will develop a capture ELISA that will determine and quantify the natural history of VMP antigenemia in the acute leptospirosis hamster model. As milestones, in comparing the VMP antigen detection ELISA and LFA to PCR we expect to see 100% sensitivity and 100% specificity in spiked matrices, and 90% sensitivity and 100% specificity in animal and human specimens. We expect to extend the animal models to assess capture ELISA performance for VMP detection in recombinant VMP-spiked human body fluid matrices, and during natural human infection applying the assays to human samples from leptospirosis-confirmed cases from endemic sites. We will compare new LFA devices to ELISA on the same sample sets (animal, spiked matrices, confirmed human cases) and benchmark to qPCR assays as gold standard. The new LFAs may not be as sensitive as ELISA, but this approach is important to pursue. Example 4: Develop multiplex antigen-detection solid phase assays based on additional antigens, leptospiral LPS and the pan-pathogenic leptospiral antigen, LipL32

[0188] Preliminary data

[0189] We modified a standard method of LPS extraction (FIG. 5A-5D) to yield a novel, high MW form of Copenhageni LPS with large immune-reactive repeating subunits (FIG.5A); rabbit polyclonal antiserum to serovar Copenhageni cross-reacts with co-serogroup member, serovar Lai. This material will be conjugated to a immunogenic protein partner, CRM197 (inactive diphtheria toxin), and used to immunize mice to create high affinity mAbs, as published.125Leptospiral LipL32 (rLipL32) is the most highly abundant leptospiral protein128and is highly conserved among pathogenic Leptospira serovars.5It is reasonable to expect that a diagnostic test for human leptospirosis based on LipL32 antigen detection itself will be broadly useful.5, 121, 141We have the expression plasmids and have already produced and purified LipL32, as published.142We have confirmed that rabbits immunized with our LipL32, generate polyclonal antisera that detects LipL32 across pathogenic Leptospira (data not shown). With the goal of enhancing the yield of high affinity anti-LipL32 mAbs,143, 144anti-rLipL32 mouse mAbs will be made by vaccinating mice with rLipL32 with glucopyranosyl lipid A [GLA] / SE emulsion, which produces high affinity antibodies.145, 146

[0190] Experimental design1. Produce, characterize and validate high affinity monoclonal antibodies for serogroup (LPS) and genus levels (LipL32) leptospiral antigen detection.

[0191] Prepare leptospiral LPS- CRM197 immunoconjugates as immunogen. The novel, high molecular weight form of L. interrogans serovar, Copenhageni LPS O-antigen polysaccharide, described above will be covalently conjugated to CRM197 carrier protein to generate high-affinity mouse mAbs (Letter of Support, Andy Lees, PhD, Fina Biosolutions). CRM197 was chosen as the immunoconjugate partner because 1) CRM197 is highly immunogenic when conjugated to polysaccharides,147and 2) CRM197 has been successfully used as an immunoconjugate partner for human vaccines (e.g. Hemophilus influenzae type b, pneumococcus and meningococcus148-151). We have already obtained more than 60 mg of purified L. interrogans serovar Copenhageni O-antigen using the above-described methods and will prepare more as necessary. Fina BioSolutions will use sodium metaperiodate152to oxidize the LPS for CRP197 immunoconjugation, in which the oxidized galactoses are transformed to aldehydes that will react with amine groups on the CRM197. We will confirm that this material retains immunogenicity by Western immunoblot using a reference anti-Copenhageni antiserum (obtained from National Veterinary Services Laboratory, USDA, Ames, IA), as in Preliminary Data above. mAbs / hybridomas will be produced at a contractor (Precision Antibody, Inc.) will be screened against the primary antigens—Copenhageni LPS and LipL32—by ELISA, and cross-screened by ELISA against the conjugate partner CRM197 to ensure specificity. Recombinant leptospiral LipL32 / GLA / SE will be used to generate high-affinity mouse mAbs (contractor; quotation in budget justification). 2. Develop solid phase assays (ELISA, lateral flow assay) for LPS and LipL32 antigen detection.

[0192] ELISA development. In the Luna and Yale laboratories, mAbs against LPS and LipL32 will be tested by Western immunoblot and ELISA on purified unconjugated Copenhageni LPS and lysates of standard MAT panel of Leptospira serovars to assess reactivity and cross-serovar specificity, as published and tested for non- specific binding to unrelated bacteria.125, 153, 154Criteria for advancing the development of mAb pairs to final ELISA and LFA development include: 1) mAb pair capturing and detecting purified LPS and LipL32, respectively, as expected; 2) specificity of binding to Copenhageni LPS; 3) possible broad, genus-level LPS binding as determined by reactivity of mAbs to the leptospiral MAT panel, and lack of binding to unrelated bacteria, such as a panel of Gram negative and Gram positive bacteria, obtained from ATCC and Sigma. We havesucceeded in this approach as we published for mAb-mediated Brucella melitensis LPS antigen detection.125

[0193] Lateral flow assay prototype development. ELISA-validated mAb pairs will be advanced to lateral flow assay prototype development as described above.

[0194] We will compare the multiplex antigen detection ELISA and LFA to PCR. We expect to see 100% sensitivity and 100% specificity in spiked matrices, and 90% sensitivity and 100% specificity in the animal and human specimens.

[0195] This disclosure provides compositions and methods for the development of solid phase-based antigen detection assays for the diagnosis of leptospirosis, first with a single antigen test based on VMP detection, followed by development of additional complementary detection of clinically relevant leptospiral LPS and LipL32 antigens for multiplex test development. ELISAs will be analyzed by rigorous statistical testing, including Student’s t test with cutoffs 3 SD above negative controls. Optimized and validated capture antibodies (polyclonal capture plus mAb detection and mAb pairs) will both be tested; mAb pairs are preferred for manufacture, QA / QC, cost of goods and reproducibility. Risks and Pitfalls: Rabbit polyclonal antibody to capture antigen may perform better than mAb pairs. Although high affinity mAbs to serovar Copenhageni LPS will not detect other clinically important serogroups, similar mAb development for other clinically relevant serovar LPSs will follow on the present work. We do not yet know whether the LPS and / or LipL32 antigen detection will improve upon VMP antigen detection alone; this proof-of-principle project will determine whether this is so using an appropriate animal model and well-characterized human samples. Example 5: Clinical assessment of leptospirosis illness by early detection of Virulence– Modifying biomarker

[0196] Materials and Methods

[0197] Patient samples: We used a subset of samples that were systematically collected as part of a comprehensive investigation into the leptospirosis disease burden in Sri Lanka19. These samples were derived from a prospective fever surveillance study that implemented consecutive sampling to minimize selection bias. The study enrolled patients presenting with acute febrile illness lasting less than 14 days, who lacked a definitive diagnosis at the time of hospital admission. For the analysis, we employed only the acute- stage samples. The diagnosis of leptospirosis was determined using validated quantitative PCR (qPCR) techniques, employing using lipl32 gene and, MAT employing both lipl32and 16s rrs primer pairs, and a Microscopic Agglutination Test (MAT) with a wide- ranging panel of serovars161using acute as well as convalescent sera.

[0198] Cloning: VMPs were produced using the protocol we had previously protocol78. Briefly, E. coli codon-optimized VM genes, specifically la3490 (Uniprot ID: Q8F0K3), la0591 (Q8F8G6), and truncated N-terminal ricin binding domain, RBLs were synthetically synthesized, and linked to mCherry (X5DSL3) via a glycine-serine hinge (G4S)3 then cloned into pET32b (+) (Gene Universal Inc., USA) between enterokinase cleavage sites, facilitating the easy removal of the mCherry fluorescent tag. Dual histidine (His6) tags were introduced at the N and C-terminals to enable nickel-NTA purification. To ensure accuracy, the gene sequences and their orientations within the constructs were confirmed through restriction digestion and sequencing before utilization162. It is worth mentioning that la0591 was produced without mCherry tag. Earlier studies showed that mCherry tag does not interfere with the functional studies78, 162.

[0199] Expression and purification of soluble VMPs: Cysteine-rich VM proteins LA3490, LA0591, or RBLs were expressed in SHuffle®T7 competent E. coli cells (New England Biolabs, USA), leveraging their cytoplasmic disulfide bond-forming capabilities for proper protein folding78, 162. Expression and purification were performed by following our previous published protocol78, 162. Transformants were cultured in Luria- Bertani (LB) medium with 100 µg / mL ampicillin. Upon reaching an OD of 0.6, expression was induced at 16°C and 250 rpm for 24 h with 1 mM isopropyl-^-D- thiogalactoside (IPTG; Sigma-Aldrich, USA). Post-induction, cells were pelleted, lysed in CelLytic™ B (Sigma-Aldrich, USA) with benzonase nuclease (Sigma-Aldrich, USA), lysozyme, protease inhibitor cocktail (Roche, USA), and 1 mM PMSF, and centrifuged 4°C and 18,514 g for 10 min.Supernatants and pellets were analyzed on 4-12% bis-tris SDS-PAGE, and protein concentrations were determined using a BCA assay (Thermo Fisher Scientific, USA). Recombinant proteins were purified using a 1 mL pre-packed Ni- Sepharose AKTA Hi-TRAP column (GE Healthcare, USA) equilibrated with a buffer containing 100 mM NaH2PO4, 10 mM Tris-HCl, and 25 mM imidazole (pH 8.0). The bound fusion protein was eluted with 500 mM imidazole (pH 8.0), pooled, concentrated using a10 kDa Amicon® Ultra centrifugal filter, and subjected to a high-capacity endotoxin-removal spin column (Thermo Fisher Scientific, USA) for lipopolysaccharide elimination. The recombinant purified protein was dialyzed overnight in 1X PBS (pH 7.4) with gentle stirring (350 rpm) at 4°C using a 10 kDa cutoff Slide- A-Lyzer (ThermoScientific™, USA) and further desalted using 7 kDa Zeba™ desalting spin column (Thermo Fisher Scientific, USA).

[0200] Protein Analysis and Western blot: SDS-PAGE analyzed purified proteins according to the method of Laemmli163, then proteins were transferred to nitrocellulose membrane and blocked for 2 h with 5% non-fat dry milk dissolved in 1X TBST buffer (AmericanBio, USA). The membrane with LA3490 and LA0591 was probed with mouse anti-LA0591 monoclonal antibodies (1:2,000 dilution) at 4ºC overnight on the rocker. It is noteworthy that monoclonal antibodies (5G10) targeting the C-terminal domain of VMPs are generated against the natural variant LA0591, which lacks RBLs. Therefore, 5G10 mAbs exhibit cross-reactivity with the entire protein but not with RBLs alone. The membrane with RBLs was probed with a laboratory source, rabbit polyclonal antibodies (1:2,000 dilution) generated against full-length VMP (LA1402). After three washes, membranes were incubated for 2½ h with alkaline phosphatase-conjugated goat anti- mouse (for monoclonal) and anti-rabbit (for polyclonal) IgG (H+L) (dilution 1:5000, KPL, USA). Blots were developedin 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium solution (BCIP / NBT; KPL, USA).

[0201] Demonstration of anti-VMPs antibodies in patient’s serum by Indirect- ELISA: Serologically defined patient serums were screened for anti-VMPs antibodies. ELISA was performed following the previously published protocol103. Briefly, a 96-well microtiter ELISA plate (Corning, USA) was coated with 100 ng / wells of each recombinant VMPs (LA3490, LA0591, and RBL1+RBL2; RBLs, in equimolar ratio), in 100 μL of bicarbonate / carbonate coating buffer. Subsequently, 100 μL of the patient's serum sample was diluted at 1:50 and added into the wells followed by the addition of 1:5000 dilution of goat anti-human IgG (Fc specific) HRP-conjugate (KPL, USA), andfurther procedures were carried out as mentioned earlier. The reaction was stopped with2 M H2SO4and absorbance was read at 450 nm using aSpectraMax® M2e MicroplateReader (Molecular Devices, USA). Antigen and antibody blanks were served as a control.

[0202] Results

[0203] The study sample comprised 222 serum samples from individuals suspected of having leptospirosis or presenting with fever, along with 41 healthy controls. Among the 222 patient samples, 70 (26.6%) confirmed positive, and 42 (16.0%) were categorized as probable cases of leptospirosis, showing a positive result in at least one of two or more replicates on the qPCR plate. An additional 93 (35.4%) were categorized as Leptospiraantigen ‘not detected’ in qPCR. For 17 cases, qPCR results were not available. As for the MAT results, 57 (21.7%) were positive, 50 (19.0%) showed a reactive response (titer < 400), and 109 cases (41.4%) were non-reactive. MAT results were not available for 6 samples.

[0204] These samples underwent screening using AKTA-purified soluble VMPs encoding full-length LA3490, the N-terminal ricin binding domain (RBLs), and the C- terminal toxin domain individually FIG. 6A-6B) aiming to assess their ability to detect anti-VMP antibodies in acute leptospirosis. Previously, we evaluated the secondary structure of VMPs (helix and ß-sheet) using CD-spectroscopy and assessed their stability through molecular dynamics simulation160, 162. VMPs–based IgG–specific ELISA suggested that anti–LA0591 and anti–LA3490 antibodies were significantly higher in MAT–confirmed / reactive and qPCR–confirmed / probable cases compared to healthy controls. Remarkably, this pattern was seen in over 97.5% of cases, compared to healthy control. The results for the N-terminal ricin binding domain (RBLs) followed a similar pattern, albeit with some overlap and notably higher OD values for suspected cases compared to confirmed or probable cases. When comparing the collective final diagnosis of confirmed and probable cases versus healthy controls, it is evident that the OD values of LA0591 and LA3490 are distinctly different between confirmed and suspected cases (FIG.7A-7I)

[0205] ANOVA, after excluding test-negative cases, showed significant differences in OD values between healthy subjects and both confirmed and probable / reactive cases (Table 4). Follow-up post hoc analysis using Tukey’s HSD test identified distinct groupings within the data. Specifically, LA0591 levels were distinct enough to categorize each group into separate homogeneous subsets. In contrast, the other two markers distinguished healthy controls from confirmed / probable cases, forming two homogeneous subsets.

[0206] Table 4: Descriptive and ANOVA table for OD values by leptospirosis confirmatory test results.

[0207] Antibodies to the VM protein were tested only in acute-phase samples. Of the 222 samples tested, 118 were confirmed to have leptospirosis using the MAT with paired serum samples. Among these 118 patients, 33 had a positive MAT titer (≥400) in the acute sample, while an additional 24 were confirmed retrospectively using convalescent serum samples (≥400, seroconversion, or a fourfold rise). None of these patients were confirmed as laboratory-confirmed leptospirosis cases during their hospital stay. Figures 8A-8C show the OD values of anti-VM antibodies during the acute stage, grouped by paired sample MAT results. However, RBLs detect anti-RBLs antibodies inreactive and non–reactive cases but are unable to distinguish confirmed / reactive and confirmed / probable cases compared to healthy controls (FIG.8A-8C, Table 4).

[0208] These findings indicate that individuals with diagnostic MAT titers during the acute stage had notably higher mean OD values compared to healthy subjects. Moreover, individuals who tested negative during the acute stage but had diagnostic titers in convalescent samples (indicating they were diagnosed later) also exhibited significantly high levels of VM protein antibodies in the early stages of the illness (Table 5). The VM protein LA0591 differentiated positive from non-reactive / reactive results, while LA3490 differentiated samples with non-reactive results from the rest, but not RBLs.

[0209] Table 5: ANOVA and Tukey HSD results for OD values by paired sera MAT results.

[0210] To determine the optimal day for the sample collection, we analyzed the mean OD values distribution based on symptom duration at the sample collection (FIG. 8A-8C).The results show that VM antibodies were detectable as early as day 2 since the first patient- reported symptom.

[0211] Among the analyzed serum samples, four samples yielded isolation of Leptospira, these patients comprised two cases of L. interrogans and two of L. borgpetersenii. For L. interrogans infections, the OD values for the LA0591 antibodies ranged from 0.7183 to 1.1937, while those for the LA03490 antibodies ranged from 0.759 to 0.855. Regarding L. borgpetersenii infections, the OD values for LA0591 were recorded as 0.2243 to 0.887, while those for LA03490 were recorded as 0.775 and 1.115.

[0212] We performed a ROC analysis to assess the diagnostic accuracy of LA0591, LA3490, and RBLs (FIG.9), using only confirmed cases and healthy adults. The AUC for LA0591 was 0.947 (SE = 0.017, p < 0.001), indicating excellent discriminatory power in distinguishing positive and negative cases. Similarly, the AUC for LA3490 was 0.930 (SE = 0.020, p < 0.001), suggesting its potential as a reliable diagnostic marker for leptospirosis illness. The Gini Index values were 0.893 for LA0591 and 0.860 for LA3490, confirming their strong discriminatory abilities. The optimal cut-off values for classification, determined by maximum K-S metrics, were 0.505333 for LA0591 and 0.42317 for LA3490.

[0213] The diagnostic accuracy of the VMPs to detect anti–VMPs antibodies presented here surpasses that of reported qPCR or MAT tests. Furthermore, among confirmed cases, there was no discernible difference in the distribution of anti–VMPs antibodies between complicated and uncomplicated cases, including those with mild, moderate, or severe renal / liver impairment. Example 6: Create prototype solid phase Virulence Modifying Protein-based antibody- detection leptospirosis diagnostic tests.

[0214] First, we will be optimizing solid phase VMP antibody-detecting assays, starting with ELISA followed by LFA development based on positive control sera obtained from experimentally- infected animals. Second, we will examine the kinetics of anti-VMP antibody development in blood samples from outbred mice, hamsters, and dogs experimentally infected with different leptospiral species and serovars, which also enables obtaining the positive control samples for ELISA assay development. Using these samples, a subset of recombinant VMPs will be chosen for use in developing a Lateral Flow Assay (LFA)-based diagnostic test. Milestones: Sensitivity and specificity >90% using Leptospira-infected mouse hamster blood samples for infections caused by five different serovars (Copenhageni / Icterohaemorrrhagie, Canicola, Grippotyphosa,Australis, Pomona), within three different leptospiral species (L. interrogans, L. kirchneri and L. borgpetersenii) resulting from experimental infection of outbred mice, hamsters and dogs.

[0215] Experimental animal infections. Five serovars (Copenhageni / Icterohaemorrrhagie), Canicola, Grippotyphosa, Australis, Pomona from three leptospiral species (L. interrogans, L. kirchneri and L. borgpetersenii) will be inoculated ip (~104to 107; dose determined per strain as previously done in the laboratory for each isolate) into outbred mice and hamsters. Dogs will be inoculated conjunctivally on three successive days with 5 x 107organisms, as reported.172Blood will be obtained from experimentally infected animals on days 0, 4, 7 and 14 for determination of kinetics of anti-VMP IgM and IgG antibodies by ELISA, using commercially available species- specific HRP-labelled secondary antibodies. Terminal bleeds of the mice and hamsters will provide sufficient blood for positive controls; 20-30 ml of blood will be obtained from dogs at days 14 and 30 for later use as positive controls.

[0216] ELISA and Lateral flow assay (LFA) prototype development: ELISAs will be developed first confirming each individual antigen (LA0591, LA3490, LA1400, LA1402, RBL1 / 2), then mixing the ELISA VMP antigens to determine the optimal, minimal number of antigens needed for optimal, best performance, IgM and IgG antibody detection. VMP antigens determined to be optimal in ELISA will be advanced to LFA prototype development. The development of a prototype LFA device for a rapid, point-of- care test will be carried out jointly by personnel from Luna Bioscience and nanoComposix / Fortis Life Sciences (Andre Alfaro, nanoComposix, Letter of Support). Dr. Chaurasia, PI, will travel to nanoComposix to work on assay development on site. nanoComposix has experience in all aspects of LFA fabrication and scaled-up manufacture including buffer selection, sample preparation, and optimizing device configuration based on the physics of sample flow and design of pads within the device for one or multiple analyte detection capabilities. In this Phase I scope of work, Luna will provide nanoComposix with mAbs, polyclonal antibody and control antigen for joint work to create an LFA prototype. We propose to optimize and quantify prototype performance using fresh blood-based matrices spiked with anti-VMP antibodies, with subsequent testing of samples from infected mice, hamsters (see Preliminary Data above) and well-characterized human specimens in the Yale collaborator’s biobank. Specifically, nanoComposix will be responsible for the following in association with Dr. Chaurasia: 1) Lateral Flow Material Preparation: Test strips and conjugates will be prepared using therecombinant VMP combination. The presentation of antigen to the blood samples can be quite different depending on position in the assay. Secondary antibodies with lower binding affinities perform better conjugated to a reporter particle (i.e. colloidal gold), whereas high affinity antibodies are best used on the test line due to the short antigen exposure time; 2) Reagent Pair Down Selection: After material preparation, Dr. Chaurasia and nanoComposix will functionally evaluate the antigens using a panel of contrived samples prepared in a control matrix (e.g., PBS, followed by whole blood, serum / plasma matrices). Contrived samples will be prepared at a wide range of concentrations to assess assay performance.Combinations of material will be evaluated based on analytical sensitivity and specificity. Additional considerations, such as stability and precision, will be examined during optimization; 3) Volumes of sample: Spiked matrices and animal-derived samples will be added to prototype devices from 5-100 uL, taking into account proper sample / buffer flow. 4) Buffer Selection and Optimization: Samples will be treated with running buffers that contain pH stabilizers (optimum pH 7.5- 8.0), non-ionic detergents at graded concentrations (Tween-20 0.05-1%, 0.05 to 2% Triton X100, NP40, sarcosyl, in Tris / potassium phosphate / NaCl / imidazole buffers) to optimize cell lysis and exposure of epitopes; one such example includes 100 mM potassium phosphate pH = 8.0, 600 mM NaCl, 250 mM imidazole, 2% Triton X-100, but the lysis / running buffer composition will be systematically tested for optimal performance. Optimization will focus on matrix (e.g., serum / plasma / whole blood), running buffer components and conjugate dry down, cartridge integration, and analytical performance. Correlation testing against the ELISA developed by Luna will be included in further optimization efforts with animal and human clinical samples. Luna and nanoComposix will have collected and provided objective evidence to demonstrate feasibility of employing a lateral flow immunoassay prototype (Figure 4) for detection of leptospiral anti-VMP antibodies in human serum / plasma / whole blood. The performance data will determine go / no- go decisions regarding advancing product development.

[0217] Determination of the performance of new ELISA and lateral flow assay prototypes:

[0218] In vitro testing. Serum from normal humans (locally obtained) will be spiked with known quantities of positive control antibodies from different animal species to develop the prototype and analytical sensitivity / standard curves determined by ELISA. Optimal buffers (as noted above) will be determined. Analysis of human samples, very precious, will follow on, as described below.

[0219] Animal model testing. ELISA and LFA prototypes will be used to detect and measure the kinetics anti-VMP antibodies over the course of 14 days, starting with a diverse set of Leptospira all of which we have succeeded with in infecting rodents (L. interrogans serovars Copenhageni, Canicola and Pomona; L. kirschneri serovar Grippotyphosa; L. borgpetersenii serovar SL23 isolated from humans in Sri Lanka) used to experimentally infect outbred mice and hamsters, and then experiments will progress to dogs, an important target animal species. Each is a relevant animal model of leptospirosis that both recapitulate human disease and the carrier / transmission state.171, 134, 135This model will be used to determine analytical sensitivity of the prototype ELISAs and LFAs. Mice and hamsters were chosen because a sufficient quantity of blood (50 μL) from multiple animals can be obtained at sequential time points over the first 7 days of infection, up to 14 d after infection.

[0220] We propose to measure the kinetics of IgM and IgG anti-VMP antibodies. Groups of 4 mice and hamsters per cage (with 4 replicate cages totaling 32 of each species, respectively, for the different leptospiral infections) will be used, with inoculation of 104-7low passage isolated organisms, to ensure infection. Animals will be serially sampled for blood on days 0, 4, 7 and 14 days (50 μL per sample), as we have previously performed.137, 138These outbred rodent experiments will yield serum samples both to determine kinetics and to serve as positive controls, with replicates done to ensure rigor. Dogs will be experimentally infected with a fewer Leptospira isolates and are selected to be those of clinical relevance to dogs and humans. Smaller numbers of dogs will be used (3 per Leptospira challenge) but larger amounts of serum can be obtained from these priority animals as positive controls for dog diagnostics (an additional market). ELISA and LFA prototypes will be used to quantify the kinetics of anti-VMP antibody development using serially-obtained blood samples.

[0221] Expected outcomes, data analysis and limitations: We will develop solid phase tests (ELISA, LFA prototypes) that will determine and quantify the natural history of VMP antibody development in rigorous testing in multiple relevant animal species. As milestones, in comparing VMP antibody detection in ELISA and LFA to PCR we expect to see 100% sensitivity and 100% specificity in blood samples, and 90% sensitivity and 100% specificity in animal and human specimens. We expect to extend the animal models to assess ELISA performance for VMP detection in natural human infection in SpecificAim 2. We will compare new LFA devices to ELISA on the same sample sets and benchmark to commercial diagnostic tests. Example 7: Test well-characterized biobanked, deidentified sera from humans with known leptospirosis anti-VMP antibodies.

[0222] ELISA and LFA will be tested with deidentified, biobanked human leptospirosis patient sera using prototype assays / devices. Optimal recombinant VMP antigens for pan- leptospirosis diagnostic testing will be identified for subsequent scaled- up manufacture of assay prototypes.

[0223] SA2 experimental design: Human specimen testing: We will use ELISA and LFA to test de-identified human blood and urine samples originally from our NIH-funded leptospirosis-endemic sites in Peru and Sri Lanka (1,192 well-characterized samples from Sri Lanka subjects; >500 serum samples from Peru including 160 from leptospirosis patients and controls are available in our specimen collections92,117, 139, 140, 167. We will test ELISA and LFA prototypes on these sample collections and generate sensitivity / specificity data which will be displayed in ROC curves. Data analysis using negative and positive controls will be carried out as described above. We will compare the performance of our VMP-based ELISA and LFA with the only FDA- approved commercially available test, the Leptodot IgM test (GenBio, San Diego), and a research use-only test from Serion. Both of these are based on genus-level antigens derived from lysates of L. biflexa serovar Patoc 1, which, as noted above, are insensitive for early diagnosis of leptospirosis.

[0224] Described herein is the development of solid phase-based antibody detection assays for the diagnosis of leptospirosis, first with an ELISA based on anti-VMP antibody detection in experimentally infected mice, hamsters and dogs, followed by testing biobanked human samples from epidemiologically distinct sites (Sri Lanka, Peru) using prototype ELISA then LFAs developed using sera from the animal work. ELISAs will be analyzed by rigorous statistical testing, including Student’s t test with cutoffs 3 SD above negative controls; in humans Receiver Operator Curves (ROC) will be used to analyze assay performance compared to “gold standard” diagnostics in well- characterized specimen banks. Optimized and validated single and combinations of VMP antigens will be tested to have the minimum number of antigens in the solid phase assays that detect the maximum number of antibody positives. Risks, Pitfalls and Alternatives: The main risk and pitfall are the translation of the ELISA to LFA format with potential loss of sensitivity. To address this issue, in addition to optimizing the quantity and identity ofantibody-detecting VMP antigen in LFA devices and buffers as an alternative, we will consider using additional immunodominant leptospiral protein antigens simultaneously with the VMPs based on published data,168, 169as well as purified serovar-specific LPS as antigen, with which we have experience.170

[0225] The methods described herein are expected to l yield prototype VMP-based pan-leptospirosis antibody detection diagnostic ELISA and LFA tests for advancement to a Phase 2 STTR project, in which scaled-up manufacturing and prospective comparative testing in endemic leptospirosis settings will be carried out. The use of serially obtained serum specimens from multiple animal species, especially the important target species and model for human leptospirosis, dogs, adds rigor to understanding the kinetics of VMP antibody development and provides positive controls for tests in leptospirosis-endemic areas with diverse Leptospira.

[0226]

[0227] The complete disclosure of all patents, patent applications, and publications,andelectronically available material (including, for instance, nucleotide sequencesubmissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.Bibliography / Citations 1. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM, 2003. Leptospirosis: A zoonotic disease of global importance. Lancet Infect Dis 3: 757-771. 2. Levett PN, 2001. Leptospirosis. Clin Microbiol Rev 14: 296-326. 3. Ko AI, Galvao Reis M, Ribeiro Dourado CM, Johnson WD, Jr., Riley LW, 1999. Urban epidemic of severe leptospirosis in Brazil. Salvador Leptospirosis Study Group. Lancet 354: 820-5. 4. Ko AI, Goarant C, Picardeau M, 2009. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7: 736-47. 5. Evangelista KV, Coburn J, 2010. Leptospira as an emerging pathogen: a review of its biology, pathogenesis and host immune responses. Future Microbiology 5: 1413-25. 6. NIAID, 2022. https: / / www.niaid.nih.gov / research / emerging-infectious-diseases- pathogens. Accessed. 7. Costa F, Hagan JE, Calcagno J, Kane M, Torgerson P, Martinez-Silveira MS, Stein C, Abela-Ridder B, Ko AI, 2015. Global Morbidity and Mortality of Leptospirosis: A Systematic Review. PLoS Negl Trop Dis 9: e0003898. 8. Torgerson PR, Hagan JE, Costa F, Calcagno J, Kane M, Martinez-Silveira MS, Goris MG, Stein C, Ko AI, Abela-Ridder B, 2015. Global Burden of Leptospirosis: Estimated in Terms of Disability Adjusted Life Years. PLoS Negl Trop Dis 9: e0004122. 9. Masunga DS, Rai A, Abbass M, Uwishema O, Wellington J, Uweis L, El Saleh R, Arab S, Onyeaka CVP, Onyeaka H, 2022. Leptospirosis outbreak in Tanzania: An alarming situation. Ann Med Surg (Lond) 80: 104347. 10. Bourque DL, Vinetz JM, 2018. Illnesses Associated with Freshwater Recreation During International Travel. Curr Infect Dis Rep 20: 19.Chaurasia R, Marroquin AS, Vinetz JM, Matthias MA, 2022. Pathogenic Leptospira Evolved a Unique Gene Family Comprised of Ricin B-Like Lectin Domain-Containing Cytotoxins. Front Microbiol 13: 859680. Brenke R, Kozakov D, Chuang GY, Beglov D, Hall D, Landon MR, Mattos C, Vajda S, 2009. Fragment- based identification of druggable 'hot spots' of proteins using Fourier domain correlation techniques. Bioinformatics 25: 621-7. Kozakov D, Hall DR, Chuang GY, Cencic R, Brenke R, Grove LE, Beglov D, Pelletier J, Whitty A, Vajda S, 2011. Structural conservation of druggable hot spots in protein-protein interfaces. Proc Natl Acad Sci U S A 108: 13528-33. Jendele L, Krivak R, Skoda P, Novotny M, Hoksza D, 2019. PrankWeb: a web server for ligand binding site prediction and visualization. Nucleic Acids Res 47: W345-W349. WHO, 2022. https: / / www.who.int / emergencies / disease-outbreak-news / item / 2022- DON403. Accessed. WHO, 2000. Weekly epidemiological record.75: 217-224. WHO, 2001. Leptospirosis worldwide, 1999. Weekly Epidemiological Record 76: 109-116. Lehmann JS, Fouts DE, Haft DH, Cannella AP, Ricaldi JN, Brinkac L, Harkins D, Durkin S, Sanka R, Sutton G, Moreno A, Vinetz JM, Matthias MA, 2013. Pathogenomic inference of virulence-associated genes in Leptospira interrogans. PLoS Negl Trop Dis 7: e2468. Jayasundara D, Senavirathna I, Warnasekara J, Gamage C, Siribaddana S, Kularatne SAM, Matthias M, Mariet JF, Picardeau M, Agampodi S, J MV, 2021. 12 Novel clonal groups of Leptospira infecting humans in multiple contrasting epidemiological contexts in Sri Lanka. PLoS Negl Trop Dis 15: e0009272. Picardeau M, 2020. Leptospira and Leptospirosis. Methods Mol Biol 2134: 271- 275.Grillova L, Picardeau M, 2020. Core Genome Multi-locus Sequence Typing Analyses of Leptospira spp. Using the Bacterial Isolate Genome Sequence Database. Methods Mol Biol 2134: 11-21. Vincent AT, Schiettekatte O, Goarant C, Neela VK, Bernet E, Thibeaux R, Ismail N, Mohd Khalid MKN, Amran F, Masuzawa T, Nakao R, Amara Korba A, Bourhy P, Veyrier FJ, Picardeau M, 2019. Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics. PLoS Negl Trop Dis 13: e0007270. Thibeaux R, Iraola G, Ferres I, Bierque E, Girault D, Soupe-Gilbert ME, Picardeau M, Goarant C, 2018. Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence. Microb Genom 4. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Willig MR, Gotuzzo E, Vinetz JM, Peru-United States Leptospirosis C, 2003. Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3: 757-71. Haake DA, Mazel MK, McCoy AM, Milward F, Chao G, Matsunaga J, Wagar EA, 1999. Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection. Infect Immun 67: 6572-82. Truong KN, Coburn J, 2011. The emergence of severe pulmonary hemorrhagic leptospirosis: questions to consider. Front Cell Infect Microbiol 1: 24. Jimenez J, Doerr S, Martinez-Rosell G, Rose AS, De Fabritiis G, 2017. DeepSite: protein-binding site predictor using 3D-convolutional neural networks. Bioinformatics 33: 3036-3042. Adler B, de la Pena Moctezuma A, 2010. Leptospira and leptospirosis. Vet Microbiol 140: 287-96. Haake DA, Levett PN, 2015. Leptospirosis in humans. Curr Top Microbiol Immunol 387: 65-97.Chaurasia R, Salovey A, Guo X, Desir G, Vinetz J, M., 2022. Vaccination with Leptospira interrogans PF07598 gene family-encoded Virulence Modifying Proteins protects mice from severe leptospirosis and reduces bacterial load in liver and kidney. Frontiers in Cellular and Infection Microbiology doi: 10.3389 / fcimb.2022.926994. Adler B, 2015. Vaccines against leptospirosis. Curr Top Microbiol Immunol 387: 251-72. Rodriguez-Gonzalez I, Fillonneau C, Blanchet B, Suard I, Catilina P, Andre- Fontaine G, 2004. [Efficacy of Spirolept vaccine against human leptospirosis as estimated by passive protection of laboratory rodents]. Med Mal Infect 34: 196- 200. Teixeira AF, Fernandes LGV, Cavenague MF, Takahashi MB, Santos JC, Passalia FJ, Daroz BB, Kochi LT, Vieira ML, Nascimento A, 2019. Adjuvanted leptospiral vaccines: Challenges and future development of new leptospirosis vaccines. Vaccine 37: 3961-3973. Gomes-Solecki M, Santecchia I, Werts C, 2017. Animal Models of Leptospirosis: Of Mice and Hamsters. Front Immunol 8: 58. Benoist F, Lataste-Dorolle C, 1970. [Preliminary research on the vaccination of sewage workers against occupational leptospirosis]. Ann Med Interne (Paris) 121: 489-96. Martinez Sanchez R, Obregon Fuentes AM, Perez Sierra A, Baly Gil A, Diaz Gonzalez M, Baro Suarez M, Menendez Capote R, Ruiz Perez A, Sierra Gonzalez G, Lopez Chavez AU, 1998. The reactogenicity and immunogenicity of the first Cuban vaccine against human leptospirosis. Rev Cubana Med Trop 50: 159-66. Martinez Sanchez R, Perez Sierra A, Baro Suarez M, Alvarez AM, Menendez Hernandez J, Diaz Gonzalez M, Cruz de la Paz R, de los Reyes G, Montoya Batista B, Sierra Gonzalez G, Armesto del Rio M, Saltaren Cobas A, Sabournin Ramos O, 2000. Evaluation of the effectiveness of a new vaccine against human leptospirosis in groups at risk. Rev Panam Salud Publica 8: 385-92.Wunder EA, Adhikarla H, Hamond C, Owers Bonner KA, Liang L, Rodrigues CB, Bisht V, Nally JE, Alt DP, Reis MG, Diggle PJ, Felgner PL, Ko A, 2021. A live attenuated-vaccine model confers cross- protective immunity against different species of the Leptospira genus. Elife 10. Philip N, Bahtiar Affendy N, Ramli SNA, Arif M, Raja P, Nagandran E, Renganathan P, Taib NM, Masri SN, Yuhana MY, Than LTL, Seganathirajah M, Goarant C, Goris MGA, Sekawi Z, Neela VK, 2020. Leptospira interrogans and Leptospira kirschneri are the dominant Leptospira species causing human leptospirosis in Central Malaysia. PLoS Negl Trop Dis 14: e0008197. Viriyakosol S, Matthias MA, Swancutt MA, Kirkland TN, Vinetz JM, 2006. Toll- like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden. Infect Immun 74: 887-95. Putz EJ, Andreasen CB, Stasko JA, Fernandes LGV, Palmer MV, Rauh MJ, Nally JE, 2021. Circulating Foamy Macrophages in the Golden Syrian Hamster (Mesocricetus auratus) Model of Leptospirosis. J Comp Pathol 189: 98-109. Zuerner RL, Alt DP, Palmer MV, 2012. Development of chronic and acute golden Syrian hamster infection models with Leptospira borgpetersenii serovar Hardjo. Vet Pathol 49: 403-11. USCFR, 2022. United States Code of Federal Regulations https: / / www.ecfr.gov / current / title-9 / chapter- I / subchapter-E / part-113. Accessed. Ruby KW, Srinivas GB, 2013. Development of in vitro assays for measuring the relative potency of leptospiral bacterins containing serogroups canicola, grippotyphosa, icterohaemorrhagiae, and pomona. Biologicals 41: 308-14. Klaasen HL, van der Veen M, Sutton D, Molkenboer MJ, 2014. A new tetravalent canine leptospirosis vaccine provides at least 12 months immunity against infection. Vet Immunol Immunopathol 158: 26-9. Goldstein RE, 2010. Canine leptospirosis. Vet Clin North Am Small Anim Pract 40: 1091-101.Eric Klaasen HL, Adler B, 2015. Recent advances in canine leptospirosis: focus on vaccine development. Vet Med (Auckl) 6: 245-260. Andre-Fontaine G, Branger C, Gray AW, Klaasen HL, 2003. Comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis. Vet Rec 153: 165-9. USDA, 2022. https: / / www.aphis.usda.gov / aphis / ourfocus / animalhealth / veterinary-biologics. Grassmann AA, Kremer FS, Dos Santos JC, Souza JD, Pinto LDS, McBride AJA, 2017. Discovery of Novel Leptospirosis Vaccine Candidates Using Reverse and Structural Vaccinology. Front Immunol 8: 463. Grassmann AA, Souza JD, McBride AJ, 2017. A Universal Vaccine against Leptospirosis: Are We Going in the Right Direction? Front Immunol 8: 256. Barazzone GC, Teixeira AF, Azevedo BOP, Damiano DK, Oliveira MP, Nascimento A, Lopes APY, 2021. Revisiting the Development of Vaccines Against Pathogenic Leptospira: Innovative Approaches, Present Challenges, and Future Perspectives. Front Immunol 12: 760291. Noguchi H, 1917. Spirochaeta Icterohaemorrhagiae in American Wild Rats and Its Relation to the Japanese and European Strains : First Paper. J Exp Med 25: 755- 63. Inada R, Ido Y, Hoki R, al. e, 1915. The etiology, mode of infection, and specific therapy of Weil's Disease (Spirochaetosis Icterohaemorrhagica). The Journal of Experimental Medicine XXIII: 377-402. Martin LE, Wiggans KT, Wennogle SA, Curtis K, Chandrashekar R, Lappin MR, 2014. Vaccine- associated Leptospira antibodies in client-owned dogs. J Vet Intern Med 28: 789-92. Balakrishnan G, Roy P, 2014. Comparision of efficacy of two experimental bovine leptospira vaccines under laboratory and field. Vet Immunol Immunopathol 159: 11-5.Bouvet J, Lemaitre L, Cariou C, Scotto M, Blain C, Oberli F, Cupillard L, Guigal PM, 2020. A canine vaccine against Leptospira serovars Icterohaemorrhagiae, Canicola and Grippotyphosa provides cross protection against Leptospira serovar Copenhageni. Vet Immunol Immunopathol 219: 109985. Brown RA, Blumerman S, Gay C, Bolin C, Duby R, Baldwin CL, 2003. Comparison of three different leptospiral vaccines for induction of a type 1 immune response to Leptospira borgpetersenii serovar Hardjo. Vaccine 21: 4448- 58. Felix CR, Siedler BS, Barbosa LN, Timm GR, McFadden J, McBride AJA, 2020. An overview of human leptospirosis vaccine design and future perspectives. Expert Opin Drug Discov 15: 179-188. Dellagostin OA, Grassmann AA, Rizzi C, Schuch RA, Jorge S, Oliveira TL, McBride AJ, Hartwig DD, 2017. Reverse Vaccinology: An Approach for Identifying Leptospiral Vaccine Candidates. Int J Mol Sci 18. Palaniappan RU, Ramanujam S, Chang YF, 2007. Leptospirosis: pathogenesis, immunity, and diagnosis. Curr Opin Infect Dis 20: 284-92. Bashiru G, Bahaman AR, 2018. Advances & challenges in leptospiral vaccine development. Indian J Med Res 147: 15-22. Fish NA, Kingscote B, 1973. Protection of gilts against leptospirosis by use of a live vaccine. Can Vet J 14: 12-5. Stringfellow DA, Brown RR, Hanson LE, Schnurrenberger PR, Johnson J, 1983. Can antibody responses in cattle vaccinated with a multivalent leptospiral bacterin interfere with serologic diagnosis of disease? J Am Vet Med Assoc 182: 165-7. Bolin CA, Cassells JA, Zuerner RL, Trueba G, 1991. Effect of vaccination with a monovalent Leptospira interrogans serovar hardjo type hardjo-bovis vaccine on type hardjo-bovis infection of cattle. Am J Vet Res 52: 1639-43.Jost BH, Adler B, Faine S, 1989. Experimental immunisation of hamsters with lipopolysaccharide antigens of Leptospira interrogans. J Med Microbiol 29: 115- 20. Jost BH, Adler B, Vinh T, Faine S, 1986. A monoclonal antibody reacting with a determinant on leptospiral lipopolysaccharide protects guinea pigs against leptospirosis. J Med Microbiol 22: 269-75. Wang Z, Jin L, Wegrzyn A, 2007. Leptospirosis vaccines. Microb Cell Fact 6: 39. Midwinter A, Faine S, Adler B, 1990. Vaccination of mice with lipopolysaccharide (LPS) and LPS- derived immuno-conjugates from Leptospira interrogans. J Med Microbiol 33: 199-204. Branger C, Sonrier C, Chatrenet B, Klonjkowski B, Ruvoen-Clouet N, Aubert A, Andre-Fontaine G, Eloit M, 2001. Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination. Infect Immun 69: 6831-8. Grassmann AA, Felix SR, dos Santos CX, Amaral MG, Seixas Neto AC, Fagundes MQ, Seixas FK, da Silva EF, Conceicao FR, Dellagostin OA, 2012. Protection against lethal leptospirosis after vaccination with LipL32 coupled or coadministered with the B subunit of Escherichia coli heat-labile enterotoxin. Clin Vaccine Immunol 19: 740-5. Faisal SM, Yan W, McDonough SP, Chang YF, 2009. Leptospira immunoglobulin-like protein A variable region (LigAvar) incorporated in liposomes and PLGA microspheres produces a robust immune response correlating to protective immunity. Vaccine 27: 378-87. Llanos Salinas SP, Castillo Sanchez LO, Castaneda Miranda G, Rodriguez Reyes EA, Ordonez Lopez L, Mena Banuelos R, Alcaraz Sosa LE, Nunez Carrera MG, Jose Manuel RO, Carmona Gasca CA, Matsunaga J, Haake DA, Candanosa Aranda IE, de la Pena-Moctezuma A, 2020. GspD, The Type II Secretion System Secretin of Leptospira, Protects Hamsters against Lethal Infection with a Virulent L. interrogans Isolate. Vaccines (Basel) 8.Evangelista KV, Lourdault K, Matsunaga J, Haake DA, 2017. Immunoprotective properties of recombinant LigA and LigB in a hamster model of acute leptospirosis. PLoS One 12: e0180004. Conrad NL, Cruz McBride FW, Souza JD, Silveira MM, Felix S, Mendonca KS, Santos CS, Athanazio DA, Medeiros MA, Reis MG, Dellagostin OA, McBride AJ, 2017. LigB subunit vaccine confers sterile immunity against challenge in the hamster model of leptospirosis. PLoS Negl Trop Dis 11: e0005441. Faisal SM, Yan W, Chen CS, Palaniappan RU, McDonough SP, Chang YF, 2008. Evaluation of protective immunity of Leptospira immunoglobulin like protein A (LigA) DNA vaccine against challenge in hamsters. Vaccine 26: 277-87. Murray GL, Simawaranon T, Kaewraemruaen C, Adler B, Sermswan RW, 2018. Heterologous protection elicited by a live, attenuated, Leptospira vaccine. Vet Microbiol 223: 47-50. Chaurasia R, Marroquin AS, Vinetz JM, Matthias MA, 2022. Pathogenic Leptospira evolved a unique gene family comprised of ricin B-like lectin domain- containing cytotoxins. Frontiers in Microbiology 12: 859680. ExpertMarketResearch, 2022. Global Cholera Vaccines Market. Accessed 2022. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, Ahn SY, Alvarado M, Anderson HR, Anderson LM, Andrews KG, Atkinson C, Baddour LM, Barker-Collo S, Bartels DH, Bell ML, Benjamin EJ, Bennett D, Bhalla K, Bikbov B, Bin Abdulhak A, Birbeck G, Blyth F, Bolliger I, Boufous S, Bucello C, Burch M, Burney P, Carapetis J, Chen H, Chou D, Chugh SS, Coffeng LE, Colan SD, Colquhoun S, Colson KE, Condon J, Connor MD, Cooper LT, Corriere M, Cortinovis M, de Vaccaro KC, Couser W, Cowie BC, Criqui MH, Cross M, Dabhadkar KC, Dahodwala N, De Leo D, Degenhardt L, Delossantos A, Denenberg J, Des Jarlais DC, Dharmaratne SD, Dorsey ER, Driscoll T, Duber H, Ebel B, Erwin PJ, Espindola P, Ezzati M, Feigin V, Flaxman AD, Forouzanfar MH, Fowkes FG, Franklin R, Fransen M, Freeman MK, Gabriel SE, Gakidou E, Gaspari F, Gillum RF, Gonzalez-Medina D, Halasa YA, Haring D, Harrison JE, Havmoeller R, Hay RJ, Hoen B, Hotez PJ, Hoy D,Jacobsen KH, James SL, Jasrasaria R, Jayaraman S, Johns N, Karthikeyan G, Kassebaum N, Keren A, Khoo JP, Knowlton LM, Kobusingye O, Koranteng A, Krishnamurthi R, Lipnick M, Lipshultz SE, Ohno SL, et al., 2012. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380: 2095- 128. Xu Y, Ye Q, 2018. Human leptospirosis vaccines in China. Hum Vaccin Immunother 14: 984-993. BCCMarketReport, 2022. https: / / www.bccresearch.com / market- research / healthcare / animal- therapeutics-diagnostics-markets-report.html. Pielenhofer J, Sohl J, Windbergs M, Langguth P, Radsak MP, 2020. Current Progress in Particle-Based Systems for Transdermal Vaccine Delivery. Front Immunol 11: 266. Centers for Disease C, Prevention, 2013. Human contacts with oral rabies vaccine baits distributed for wildlife rabies management--Ohio, 2012. MMWR Morb Mortal Wkly Rep 62: 267-9. Artois M, Masson E, Barrat J, Aubert MF, 1993. Efficacy of three oral rabies vaccine-baits in the red fox: a comparison. Vet Microbiol 38: 167-72. Rupprecht CE, Wiktor TJ, Johnston DH, Hamir AN, Dietzschold B, Wunner WH, Glickman LT, Koprowski H, 1986. Oral immunization and protection of raccoons (Procyon lotor) with a vaccinia-rabies glycoprotein recombinant virus vaccine. Proc Natl Acad Sci U S A 83: 7947-50. Koprowski H, 1989. Rabies oral immunization. Curr Top Microbiol Immunol 146: 137-51. Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL, Haake DA, Haft DH, Hartskeerl R, Ko AI, Levett PN, Matsunaga J, Mechaly AE, Monk JM, Nascimento AL, Nelson KE, Palsson B, Peacock SJ, Picardeau M, Ricaldi JN, Thaipandungpanit J, Wunder EA, Jr., Yang XF, Zhang JJ, Vinetz JM, 2016. WhatMakes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira. PLoS Negl Trop Dis 10: e0004403. Lehmann JS, Fouts DE, Haft DH, Cannella AP, Ricaldi JN, Brinkac L, Harkins D, Durkin S, Sanka R, Sutton G, Moreno A, Vinetz JM, Matthias MA, , 2013. Pathogenomic Inference of Virulence-Associated Genes in Leptospira interrogans. PLoS Negl Trop Dis 7(10): e2468. doi:10.1371 / journal.pntd.0002468. Lehmann JS, Matthias MA, Vinetz JM, Fouts DE, 2014. Leptospiral pathogenomics. Pathogens 3: 280- 308. Agampodi SB, Dahanayaka NJ, Bandaranayaka AK, Perera M, Priyankara S, Weerawansa P, Matthias MA, Vinetz JM, 2014. Regional differences of leptospirosis in Sri Lanka: observations from a flood- associated outbreak in 2011. PLoS Negl Trop Dis 8: e2626. Ganoza CA, Matthias MA, Collins-Richards D, Brouwer KC, Cunningham CB, Segura ER, Gilman RH, Gotuzzo E, Vinetz JM, 2006. Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira. PLoS Med 3: e308. Spichler AS, Vilaca PJ, Athanazio DA, Albuquerque JO, Buzzar M, Castro B, Seguro A, Vinetz JM, 2008. Predictors of lethality in severe leptospirosis in urban Brazil. Am J Trop Med Hyg 79: 911-4. Merck, 2022. https: / / www.merck-animal-health-usa.com / nobivac / stop- lepto / lepto4-vaccine. Accessed. LaFleur R, Dant J, Wasmoen T, 2011. Prevention of disease and mortality in vaccinated dogs following experimental challenge with virulent leptospira. J Vet Int Med 25: 747. Ng HK, Tang ML, 2005. Testing the equality of two Poisson means using the rate ratio. Stat Med 24: 955-65. Zhu H, Lakkis H, 2014. Sample size calculation for comparing two negative binomial rates. Stat Med 33: 376-87.Chang MJ, Ollivault-Shiflett M, Schuman R, Ngoc Nguyen S, Kaltashov IA, Bobst C, Rajagopal SP, Przedpelski A, Barbieri JT, Lees A, 2022. Genetically detoxified tetanus toxin as a vaccine and conjugate carrier protein. Vaccine 40: 5103-5113. Scaria PV, Chen BB, Rowe CG, Alani N, Muratova OV, Barnafo EK, Lambert LE, Zaidi IU, Lees A, Rausch KM, Narum DL, Duffy PE, 2020. Comparison of carrier proteins to conjugate malaria transmission blocking vaccine antigens, Pfs25 and Pfs230. Vaccine 38: 5480-5489. Baruffaldi F, Kelcher AH, Laudenbach M, Gradinati V, Limkar A, Roslawski M, Birnbaum A, Lees A, Hassler C, Runyon S, Pravetoni M, 2018. Preclinical Efficacy and Characterization of Candidate Vaccines for Treatment of Opioid Use Disorders Using Clinically Viable Carrier Proteins. Mol Pharm 15: 4947-4962. Hegerle N, Choi M, Sinclair J, Amin MN, Ollivault-Shiflett M, Curtis B, Laufer RS, Shridhar S, Brammer J, Toapanta FR, Holder IA, Pasetti MF, Lees A, Tennant SM, Cross AS, Simon R, 2018. Development of a broad spectrum glycoconjugate vaccine to prevent wound and disseminated infections with Klebsiella pneumoniae and Pseudomonas aeruginosa. PLoS One 13: e0203143. Baliban SM, Yang M, Ramachandran G, Curtis B, Shridhar S, Laufer RS, Wang JY, Van Druff J, Higginson EE, Hegerle N, Varney KM, Galen JE, Tennant SM, Lees A, MacKerell AD, Jr., Levine MM, Simon R, 2017. Development of a glycoconjugate vaccine to prevent invasive Salmonella Typhimurium infections in sub-Saharan Africa. PLoS Negl Trop Dis 11: e0005493. Chaurasia R, Salovey A, Guo X, Desir G, Vinetz JM, 2022. Vaccination With Leptospira interrogans PF07598 Gene Family-Encoded Virulence Modifying Proteins Protects Mice From Severe Leptospirosis and Reduces Bacterial Load in the Liver and Kidney. Front Cell Infect Microbiol 12: 926994. Anderson P, Pichichero ME, Insel RA, 1985. Immunogens consisting of oligosaccharides from the capsule of Haemophilus influenzae type b coupled to diphtheria toxoid or the toxin protein CRM197. J Clin Invest 76: 52-9.Przedpelski A, Tepp WH, Pellett S, Johnson EA, Barbieri JT, 2020. A Novel High-Potency Tetanus Vaccine. mBio 11. Bryans TD, Braithwaite C, Broad J, Cooper JF, Darnell KR, Hitchins VM, Karren AJ, Lee PS, 2004. Bacterial endotoxin testing: a report on the methods, background, data, and regulatory history of extraction recovery efficiency. Biomed Instrum Technol 38: 73-8. Sonrier C, Branger C, Michel V, Ruvoen-Clouet N, Ganiere JP, Andre-Fontaine G, 2000. Evidence of cross-protection within Leptospira interrogans in an experimental model. Vaccine 19: 86-94. Wilson-Welder JH, Alt DP, Nally JE, Olsen SC, 2021. Bovine Immune Response to Vaccination and Infection with Leptospira borgpetersenii Serovar Hardjo. mSphere 6. WHO, 1973. McBride AJ, Athanazio DA, Reis MG, Ko AI, 2005. Leptospirosis. Curr Opin Infect Dis 18: 376-86. Pinto GV, Senthilkumar K, Rai P, Kabekkodu SP, Karunasagar I, Kumar BK, 2022. Current methods for the diagnosis of leptospirosis: Issues and challenges. J Microbiol Methods 195: 106438. Cespedes M, Glenny M, Felices V, Balfa L, Suarez V, 2002. Prueba de ELISA indirecta para la deteccion de anticuerpos IgM para el diagnostico de leptospirosis humana. Rev Peru Med Exp Salud Publica 19: 24-27. Goris MG, Leeflang MM, Loden M, Wagenaar JF, Klatser PR, Hartskeerl RA, Boer KR, 2013. Prospective evaluation of three rapid diagnostic tests for diagnosis of human leptospirosis. PLoS Negl Trop Dis 7: e2290. Waggoner JJ, Balassiano I, Mohamed-Hadley A, Vital-Brazil JM, Sahoo MK, Pinsky BA, 2015. Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing. PLoS One 10: e0132988.Waggoner JJ, Pinsky BA, 2016. Molecular diagnostics for human leptospirosis. Curr Opin Infect Dis 29: 440-5. Agampodi SB, Dahanayaka NJ, Nockler K, Mayer-Scholl A, Vinetz JM, 2016. Redefining Gold Standard Testing for Diagnosing Leptospirosis: Further Evidence from a Well-Characterized, Flood-Related Outbreak in Sri Lanka. Am J Trop Med Hyg 95: 531-536. Agampodi SB, Matthias MA, Moreno AC, Vinetz JM, 2012. Utility of quantitative polymerase chain reaction in leptospirosis diagnosis: association of level of leptospiremia and clinical manifestations in Sri Lanka. Clin Infect Dis 54: 1249-55. Matsunaga J, Sanchez Y, Xu X, Haake DA, 2005. Osmolarity, a key environmental signal controlling expression of leptospiral proteins LigA and LigB and the extracellular release of LigA. Infect Immun 73: 70-8. Matsunaga J, Haake DA, 2018. cis-Acting Determinant Limiting Expression of Sphingomyelinase Gene sph2 in Leptospira interrogans, Identified with a gfp Reporter Plasmid. Appl Environ Microbiol 84. Dhandapani G, Sikha T, Pinto SM, Kiran Kumar M, Patel K, Kumar M, Kumar V, Tennyson J, Satheeshkumar PK, Gowda H, Keshava Prasad TS, Madanan MG, 2018. Proteomic approach and expression analysis revealed the differential expression of predicted leptospiral proteases capable of ECM degradation. Biochim Biophys Acta Proteins Proteom 1866: 712-721. Narayanavari SA, Lourdault K, Sritharan M, Haake DA, Matsunaga J, 2015. Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans. PLoS Negl Trop Dis 9: e0003952. Agampodi SB, Vinetz JM, 2021. Next-Generation Sequencing Analysis of Pathogenic Leptospira: A Way Forward for Understanding Infectious Disease Dynamics in Low / Middle-Income, Disease-Endemic Settings. Am J Trop Med Hyg 104: 1625-1627.Jampasa S, Lae-Ngee P, Patarakul K, Ngamrojanavanich N, Chailapakul O, Rodthongkum N, 2019. Electrochemical immunosensor based on gold-labeled monoclonal anti-LipL32 for leptospirosis diagnosis. Biosens Bioelectron 142: 111539. Saengjaruk P, Chaicumpa W, Watt G, Bunyaraksyotin G, Wuthiekanun V, Tapchaisri P, Sittinont C, Panaphut T, Tomanakan K, Sakolvaree Y, Chongsa- Nguan M, Mahakunkijcharoen Y, Kalambaheti T, Naigowit P, Wambangco MA, Kurazono H, Hayashi H, 2002. Diagnosis of human leptospirosis by monoclonal antibody-based antigen detection in urine. J Clin Microbiol 40: 480-9. Patra KP, Saito M, Atluri VL, Rolan HG, Tsolis RM, Smits H, Leng D, Gotuzzo E, Gilman RH, Vinetz JM, 2014. A Protein-Conjugate Approach to Develop a Rapid Antigen Detection Test for the Diagnosis of Human Brucellosis. PLoS Negl. Trop. Dis. In revision. Haake DA, Zuckert WR, 2015. The leptospiral outer membrane. Curr Top Microbiol Immunol 387: 187- 221. Pinne M, Haake DA, 2013. LipL32 Is a Subsurface Lipoprotein of Leptospira interrogans: presentation of new data and reevaluation of previous studies. PLoS One 8: e51025. Malmstrom J, Beck M, Schmidt A, Lange V, Deutsch EW, Aebersold R, 2009. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans. Nature 460: 762-5. Boyce MR, O’Meara WP, 2017. Use of malaria RDTs in various health contexts across sub-Saharan Africa: a systematic review. BMC Public Health 17: 470. Matsunaga J, Lo M, Bulach DM, Zuerner RL, Adler B, Haake DA, 2007. Response of Leptospira interrogans to physiologic osmolarity: relevance in signaling the environment-to-host transition. Infect Immun 75: 2864-74. Parra ME, Evans CB, Taylor DW, 1991. Identification of Plasmodium falciparum histidine-rich protein 2 in the plasma of humans with malaria. J Clin Microbiol 29: 1629-34.Watson JA, Uyoga S, Wanjiku P, Makale J, Nyutu GM, Mturi N, George EC, Woodrow CJ, Day NPJ, Bejon P, Opoka RO, Dondorp AM, John CC, Maitland K, Williams TN, White NJ, 2022. Improving the diagnosis of severe malaria in African children using platelet counts and plasma PfHRP2 concentrations. Sci Transl Med 14: eabn5040. Howard RJ, Uni S, Aikawa M, Aley SB, Leech JH, Lew AM, Wellems TE, Rener J, Taylor DW, 1986. Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes. J Cell Biol 103: 1269-77. Wunder EA, Jr., Figueira CP, Santos GR, Lourdault K, Matthias MA, Vinetz JM, Ramos E, Haake DA, Picardeau M, Dos Reis MG, Ko AI, 2016. Real-Time PCR Reveals Rapid Dissemination of Leptospira interrogans after Intraperitoneal and Conjunctival Inoculation of Hamsters. Infect Immun 84: 2105-15. Lourdault K, Matsunaga J, Evangelista KV, Haake DA, 2017. High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection. J Vis Exp. Agampodi SB, Nugegoda DB, Thevanesam V, Vinetz JM, 2015. Characteristics of rural leptospirosis patients admitted to referral hospitals during the 2008 leptospirosis outbreak in Sri Lanka: implications for developing public health control measures. Am J Trop Med Hyg 92: 139-44. Kurien BT, Everds NE, Scofield RH, 2004. Experimental animal urine collection: a review. Lab Anim 38: 333-61. RamachandraRao SP, Matthias MA, Kokoy-Mondragon C, Aghania E, Park C, Kong C, Ishaya M, Madrigal A, Horng J, Khoshaba R, Bounkhoun A, Basilico F, De Palma A, Agresta AM, Awdishu L, Naviaux RK, Vinetz JM, Mauri P, 2015. Proteomic analysis of urine exosomes reveals renal tubule response to leptospiral colonization in experimentally infected rats. PLoS Negl Trop Dis 9: e0003640. Segura ER, Ganoza CA, Campos K, Ricaldi JN, Torres S, Silva H, Cespedes MJ, Matthias MA, Swancutt MA, Lopez Linan R, Gotuzzo E, Guerra H, Gilman RH, Vinetz JM, Peru-United States Leptospirosis C, 2005. Clinical spectrum ofpulmonary involvement in leptospirosis in a region of endemicity, with quantification of leptospiral burden. Clin Infect Dis 40: 343-51. Ganoza CA, Matthias MA, Saito M, Cespedes M, Gotuzzo E, Vinetz JM, 2010. Asymptomatic renal colonization of humans in the peruvian Amazon by Leptospira. PLoS Negl Trop Dis 4: e612. Haake DA, Suchard MA, Kelley MM, Dundoo M, Alt DP, Zuerner RL, 2004. Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer. J Bacteriol 186: 2818-28. Haake DA, Chao G, Zuerner RL, Barnett JK, Barnett D, Mazel M, Matsunaga J, Levett PN, Bolin CA, 2000. The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun 68: 2276-85. Chirathaworn C, Janwitthayanan W, Sereemaspun A, Lertpocasombat K, Rungpanich U, Ekpo P, Suwancharoen D, 2014. Development of an immunochromatographic test with anti-LipL32-coupled gold nanoparticles for Leptospira detection. New Microbiol 37: 201-7. Boonyod D, Poovorawan Y, Bhattarakosol P, Chirathaworn C, 2005. LipL32, an outer membrane protein of Leptospira, as an antigen in a dipstick assay for diagnosis of leptospirosis. Asian Pac J Allergy Immunol 23: 133-41. Patra KP, Li F, Carter D, Gregory JA, Baga S, Reed SG, Mayfield SP, Vinetz JM, 2015. Alga-produced malaria transmission-blocking vaccine candidate Pfs25 formulated with a human use-compatible potent adjuvant induces high-affinity antibodies that block Plasmodium falciparum infection of mosquitoes. Infect Immun 83: 1799-808. Desbien AL, Reed SJ, Bailor HR, Dubois Cauwelaert N, Laurance JD, Orr MT, Fox CB, Carter D, Reed SG, Duthie MS, 2015. Squalene emulsion potentiates the adjuvant activity of the TLR4 agonist, GLA, via inflammatory caspases, IL-18, and IFN-gamma. Eur J Immunol 45: 407-17.Tamigney Kenfack M, Mazur M, Nualnoi T, Shaffer TL, Ngassimou A, Bleriot Y, Marrot J, Marchetti R, Sintiprungrat K, Chantratita N, Silipo A, Molinaro A, AuCoin DP, Burtnick MN, Brett PJ, Gauthier C, 2017. Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens. Nat Commun 8: 115. Pichichero ME, 2013. Protein carriers of conjugate vaccines: characteristics, development, and clinical trials. Hum Vaccin Immunother 9: 2505-23. Schumann B, Hahm HS, Parameswarappa SG, Reppe K, Wahlbrink A, Govindan S, Kaplonek P, Pirofski LA, Witzenrath M, Anish C, Pereira CL, Seeberger PH, 2017. A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine. Sci Transl Med 9. Tontini M, Romano MR, Proietti D, Balducci E, Micoli F, Balocchi C, Santini L, Masignani V, Berti F, Costantino P, 2016. Preclinical studies on new proteins as carrier for glycoconjugate vaccines. Vaccine 34: 4235-42. Badahdah AM, Rashid H, Khatami A, 2016. Update on the use of meningococcal serogroup C CRM(1)(9)(7)-conjugate vaccine (Meningitec) against meningitis. Expert Rev Vaccines 15: 9-29. Meikle PJ, Young NM, Bundle DR, 1990. O-antigen biotin conjugates. Preparation and use in direct competitive enzyme immunoassays. J Immunol Methods 132: 255-61. Murray CK, Gray MR, Mende K, Parker TM, Samir A, Rahman BA, Habashy EE, Hospenthal DR, Pimentel G, 2011. Use of patient-specific Leptospira isolates in the diagnosis of leptospirosis employing microscopic agglutination testing (MAT). Trans R Soc Trop Med Hyg 105: 209-13. Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, Plikaytis BD, Perkins BA, Phelan M, Levett PN, Weyant RS, 2003. Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis. J Clin Microbiol 41: 803-9.Sun AH, Liu XX, Yan J. Leptospirosis is an invasive infectious and systemic inflammatory disease. Biomed J 2020; 43(1): 24-31. Guerra MA. Leptospirosis. J Am Vet Med Assoc 2009; 234(4): 472-8, 30. Picardeau M. Virulence of the zoonotic agent of leptospirosis: still terra incognita? Nat Rev Microbiol 2017; 15(5): 297-307 Noguchi H. Etiology of Yellow Fever : Vii. Demonstration of Leptospira Icteroides in the Blood, Tissues, and Urine of Yellow Fever Patients and of Animals Experimentally Infected with the Organism. J Exp Med 1919; 30(2): 87- 93. Noguchi H. Etiology of Yellow Fever : Vi. Cultivation, Morphology, Virulence, and Biological Properties of Leptospira Icteroides. J Exp Med 1919; 30(1): 13-29. Chaurasia R, Vinetz JM. In silico prediction of molecular mechanisms of toxicity mediated by the leptospiral PF07598 gene family-encoded virulence-modifying proteins. Front Mol Biosci 2022; 9: 1092197. Niloofa R, Fernando N, de Silva NL, et al. Diagnosis of Leptospirosis: Comparison between Microscopic Agglutination Test, IgM-ELISA and IgM Rapid Immunochromatography Test. PLoS One 2015; 10(6): e0129236. Chaurasia R, Liang C, How K, Vieira DS, Vinetz JM. Production and Purification of Cysteine-Rich Leptospiral Virulence-Modifying Proteins with or Without mCherry Fusion. Protein J 2023; 42(6): 792-801. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-5. Chaurasia R, Thresiamma KC, Eapen CK, Zachariah BJ, Paul R, Sritharan M. Pathogen- specific leptospiral proteins in urine of patients with febrile illness aids in differential diagnosis of leptospirosis from dengue. Eur J Clin Microbiol Infect Dis 2018; 37(3): 423-33. Sehgal SC, Murhekar MV, Sugunan AP. Outbreak of leptospirosis with pulmonary involvement in north Andaman. Indian J Med Res 1995; 102: 9-12.Chaurasia R, Liang C, How K, Vieira DS, Vinetz JM, 2023. mCherry Fusion Proteins Facilitate Production of Recombinant, Cysteine-Rich Leptospira interrogans Proteins in Escherichia coli. Protein J.42: 792-801. Agampodi SB, Moreno AC, Vinetz JM, Matthias MA, 2013. Utility and limitations of direct multi-locus sequence typing on qPCR-positive blood to determine infecting Leptospira strain. Am J Trop Med Hyg 88: 184-5. Lessa-Aquino C, Borges Rodrigues C, Pablo J, Sasaki R, Jasinskas A, Liang L, Wunder EA, Jr., Ribeiro GS, Vigil A, Galler R, Molina D, Liang X, Reis MG, Ko AI, Medeiros MA, Felgner PL, 2013. Identification of seroreactive proteins of Leptospira interrogans serovar copenhageni using a high- density protein microarray approach. PLoS Negl Trop Dis 7: e2499. Lessa-Aquino C, Wunder EA, Jr., Lindow JC, Rodrigues CB, Pablo J, Nakajima R, Jasinskas A, Liang L, Reis MG, Ko AI, Medeiros MA, Felgner PL, 2015. Proteomic features predict seroreactivity against leptospiral antigens in leptospirosis patients. J Proteome Res 14: 549-56. Patra KP, Choudhury B, Matthias MM, Baga S, Bandyopadhya K, Vinetz JM, 2015. Comparative analysis of lipopolysaccharides of pathogenic and intermediately pathogenic Leptospira species. BMC Microbiol 15: 244. Nair N, Gomes-Solecki M, 2020. A Mouse Model of Sublethal Leptospirosis: Protocols for Infection with Leptospira Through Natural Transmission Routes, for Monitoring Clinical and Molecular Scores of Disease, and for Evaluation of the Host Immune Response. Curr Protoc Microbiol 59: e127. Greenlee JJ, Alt DP, Bolin CA, Zuerner RL, Andreasen CB, 2005. Experimental canine leptospirosis caused by Leptospira interrogans serovars pomona and bratislava. American Journal of Veterinary Research 66: 1816-22.

Claims

What is claimed is:

1. A virulence modifying (VM) protein-based composition or vaccine for administration to a subject in need thereof comprising a recombinant VM protein antigen or a recombinant viral vector expressing a VM protein antigen and a pharmaceutically or veterinarily acceptable adjuvant.

2. The composition or vaccine of claim 1, wherein the VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13.

3. The composition or vaccine of claim 1, wherein the VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 12, 8, 2 or 9.

4. The composition or vaccine of claim 1, wherein the VM protein is tagless.

5. The composition or vaccine of claim 1, wherein the viral vector is an orthopox viral vector.

6. The composition or vaccine of claim 1, wherein the composition or vaccine further comprises a pharmaceutically or veterinarily acceptable carrier, vehicle or, excipient.

7. The composition or vaccine of claim 6, wherein the pharmaceutically or veterinarily acceptable carrier, excipient, adjuvant, or vehicle is selected from the group consisting of polyacrylic acid, LF2 emulsion, LR6 emulsion, TS6 emulsion, LR4 emulsion, carbomer, aluminum hydroxide, aluminum phosphate, saponin, CpG, water-in- oil emulsion, oil-in-water emulsion, carbomer-based adjuvant, and adjuvant composition comprising a lipophile, a polymer of acrylic or methacrylic acid, saline, cholesterol, a saponin, and sodium hydroxide.

8. The composition or vaccine of claim 1, wherein the subject is a human or an animal.

9. The composition or vaccine of claim 8, wherein the animal is selected from a group consisting of equine, canine, feline, ovine, bovine, porcine, caprine, avian, primate, and fish.

10. The composition or vaccine of claim 8, wherein the animal is susceptible to or has leptospirosis.

11. A method for vaccinating a subject susceptible to leptospirosis infection or eliciting an immune response in the subject against leptospirosis comprising administering to the subject a vaccine according to claim 1.

12. The method of claim 11 further comprising administering to the subject boost- vaccine, wherein at the boost-vaccine is the vaccine according to claim 1.

13. The method of claim 11, vaccine comprises a VM protein antigen, a recombinant viral vector that expresses, in vivo, a VM protein antigen, or both, and wherein the method protects the subject from leptospirosis infection, prevents leptospirosis disease progression in the subject, or the combination thereof.

14. The method of claim 11, wherein the vaccine comprises a VM protein antigen, a recombinant viral vector that expresses, in vivo, a VM protein antigen, or both, wherein the boost-vaccine is the vaccine according to claim 1, and wherein the method protects the animal from leptospirosis infection, prevents leptospirosis disease progression in the subject, or the combination thereof.

15. A method for diagnosing leptospirosis in a subject in need thereof comprising: a) obtaining a sample from the subject; b) detecting leptospiral VM proteins antigen the sample; and c) quantifying leptospiral VM proteins antigen; wherein the leptospiral VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13. wherein the method quantifies the presence of a secreted, leptospiral VM protein antigen that mediates disease pathogenesis in the subject.

16. The method of claim 15, wherein the leptospiral VM protein antigen is selected from antigens having at least 99% sequence identity to SEQ ID NO: 12, 8, 2 or 9.

17. The method of claim 15, wherein the subject is a human or an animal.

18. The method of claim 17, wherein the animal is selected from a group consisting of equine, canine, feline, ovine, bovine, porcine, caprine, avian, primate, and fish.

19. The method of claim 15, wherein the subject is susceptible to or has leptospirosis.

20. The method of claim 15, wherein the sample is selected from bodily fluids selected from blood, serum, plasma or urine.