Attenuated heterologous live vaccine for leptospires

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application 62 / 239,136 filed on October 8, 2015.FIELD OF THE INVENTION

[0002] The present disclosure relates to live attenuated vaccines that provide heterologous protection from infection by Leptospira spirochetes. The present invention also relates to attenuated Leptospirra interrogans or compositions or vaccines comprising attenuated Leptospirra interrogans, for use in a method of vaccinating an animal or for inducing an immunogenic or protective response against Leptospira infection.BACKGROUND

[0003] Leptospirosis is a life-threatening disease representing the most spread zoonosis worldwide, affecting every mammalian species to include humans (B. Adler, 2015, Leptospira and Leptospirosis, vol. 387, B. Adler, Ed. Berlin, Heidelberg: Springer Berlin Heidelberg, pp. 1-9). This neglected and emerging infection is caused by the spirochete Leptospira, a helically-shaped bacteria with characteristic hook and spiral-shaped ends (Charon NW et al., 2002, Annu. Rev. Genet., vol. 36, no. 1, pp. 47-73). Infection with one of the 10 pathogenic species, including Leptospira interrogans, can result in a broad spectrum of symptoms from subclinical infection to multiple organ failure with a mortality rate of 10 to 50%. However, there is a considerable deficit in the understanding of basic aspects of the biology and virulence mechanisms of pathogenic Leptospira. This is mainly due to the lack of adequate and efficient genetic tools for these slow-growing bacteria. Therefore, only a limited number of virulence factors have been identified so far. Virulence mechanisms that have been described include pathogen adhesion and persistence in the host, host tissue damage, renal colonization, and pathogen entry and dissemination in the host (Murray GL, 2015, Curr. Top. Microbiol. Immunol., vol. 387, pp. 139-185).

[0004] The motility of spirochetes is unique in that they can swim in highly viscous gel-like media that slow or stop the motility of other peritrichously flagellated bacteria (C. Li et al., 2000, J. Mol. Microbiol. Biotechnol., vol. 2, no. 4, pp. 345-354). This motility is conferred by two endoflagella inserted subterminally in the periplasm at each pole, extending toward the middle of the cell without overlapping. Whether the poles are hook or spiral-shaped is defined by flagella rotation direction. Rotation in counterclockwise direction (CCW, as seen from the center of the cell) forms the spiral-shaped ends whereas rotation in clockwise direction (CW) results in hook-shaped ends. Symmetric rotation of flagella leads to non-translational motility. Translational motility only occurs when flagella are rotating in opposite direction, with the anterior end in hook-shape and the posterior end in corkscrew shape (Charon NW et al., 2002, supra).

[0005] Flagella of model systems Escherichia coli and Salmonella enterica have been widely studied (Berg HC, 2003, Annu. Rev. Biochem., vol. 72, pp. 19-54; Macnab RM, 2003, Annu. Rev. Microbiol., vol. 57, pp. 77-100; Terashima H et al., 2008, Int. Rev. Cell Mol. Biol., vol. 270, pp. 39-85; S. Kojima S et al., 2004, Int. Rev. Cytol., vol. 233, pp. 93-134; Sowa Y et al., 2008, Q. Rev. Biophys., vol. 41, no. 2, pp. 103-132). They are classically composed of a filament, a hook and a motor constituting the basal body, and their structure seems to be highly conserved within the bacterial kingdom (Zhao X et al., 2014, Biochemistry (Mosc.), vol. 53, no. 27, pp. 4323-4333). The flagellar filaments of spirochetes, however, are not composed of a single flagellin polymer like in enterobacteria, but comprise a complex structure of several subunits (Slamti L et al., 2011, J. Bacteriol., vol. 193, no. 22, pp. 6266-6275). In Leptospira, site-directed or random mutagenesis allowed the study of some endoflagellum components, including the flagellar proteins FlaA, FlaB (Picardeau M et al., 2001, Mol. Microbiol., vol. 40, no. 1, pp. 189-199; Lambert A et al., 2012, Infect. Immun., vol. 80, no. 6, pp. 2019-2025), as well as FliY, a protein involved in flagellar motor and likely a part of the C-ring (Zhao X et al., 2014, supra; Liao S et al., 2009, BMC Microbiol., vol. 9, no. 1, p. 253). Cryo-electron tomography of Leptospira spp. also provides insights of the structure of the endoflagellum (Zhao X et al., 2014, supra; Zhao X et al., , 2013, PNAS, vol. 110, no. 35, pp. 14390-14395; Raddi G et al., 2012, J. Bacteriol., vol. 194, no. 6, pp. 1299-1306). Zhao and coworkers showed that leptospiral flagellar motor is composed of the MS ring (the base of the motor), the C ring, responsible for switch of motor rotation, the rod with L and P rings connecting the MS ring to the hook, a spirochete-unique structure called the collar surrounding the MS-ring, a complex export apparatus responsible for flagellar type III proteins including subunits of the rod, the hook and the filament, and finally the stator, which is the motor force generator. It is thought that C-ring is composed in its center of FliM in complex with FliN. Chemotaxis signaling protein CheY-P (CheY phosphorylated) binds to FliM and possibly FliN, leading by direct interaction to conformational change of FliG, rearranging the interaction between FliG and the stator, allowing switch rotation direction of the motor either in CW or CCW direction (Ahn DR et al., 2013, Int. J. Biol. Macromol., vol. 54, pp. 76-83; Y. Morimoto Y et al., 2014, Biomolecules, vol. 4, no. 1, pp. 217-234).

[0006] Srikram et al. discloses cross-protective immunity against Leptospirosis elicited by a live, attenuated lipopolysaccharide mutant (Srikram et al., 2011, J. Infectious Diseases, vol. 203, no. 6, pp 870-879).

[0007] The first bacterins against Leptospirosis were developed more than one hundred years ago. As of today inactivated whole cell preparations remain the only licensed products for use in animal and human health. They elicit antibody responses against the major immunodominant bacterial antigen, the lipopolysaccharide (LPS). Its composition varies between serovars, hence a narrow-spectrum serogroup specific protective response. Humans, however, as well as animals - dogs in particular - are exposed to multiple serovars, with strong temporal and regional variations in the epidemiology. This is an important problem for the vaccine industry because there is a long and costly regional development by species and by geographic location.

[0008] Accordingly, there is an urgent need for an effective vaccine against broad range Leptospira spirochetes infection in canines and felines.SUMMARY OF THE INVENTION

[0009] The invention is defined by the appended claims. Any part of the description which does not fall under the scope of the appended claims is for illustrative purposes only. In particular, any subject-matter referred to as "disclosure" which is not falling under the scope of the appended claims does not form part of the invention.

[0010] Any references in the description to methods of treatment refer to the isolated attenuated Leptospira interrogans or compositions or vaccinations of the present invention for use in a method of treatment of the human (or animal) body by therapy.

[0011] The invention provides a composition or vaccine that contains a live attenuated Leptospira interrogans. In particular, the present invention provides a recombinant or attenuated Leptospira interrogans that comprises a mutated fliM gene comprising SEQ ID NO: 14. The fliM gene may encode a mutated fliM protein wherein the FliM protein is not translated or the C-terminal region of the fliM protein is deleted.

[0012] The present invention relates to an attenuated Leptospira interrogans strain. The disclosure provides the Leptospira interrogans strains under the CNCM deposit Nos. CNCM I-5132 and CNCM I-5133, or any descendant or progeny of the strains. The descendants or progenies of the deposited strains may have the entire fliM gene deleted.

[0013] The present invention showed surprising benefit of the vaccination using the live attenuated Leptospira interrogans vaccine to protect animals against homologous and heterologous Leptospira challenges. The present invention also demonstrated surprisingly that cross protection can only be achieved by using a virulence attenuated strain directly issued from a fully virulent isolate but not with an avirulent saprophytic strain.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1A is L. interrrogans serogroup autralis mutant strain 702 fliM-< . FIG. 1B is L. interrrogans serogroup autralis mutant strain 702 fliM- / +< restored with a functional fliM gene. FIG. 1C is wild type L. interrrogans serogroup autralis strain. FIG. 1D is L. interrrogans serogroup autralis mutant strain 702 fliM-< ; time lapse at 0s. FIG. 1E is L. interrrogans serogroup autralis mutant strain 702 fliM-< ; time lapse at 0.1s. FIG. 1F is L. interrrogans serogroup autralis mutant strain 702 fliM-< ; time lapse at 0.2s. FIG. 1G is L. interrrogans serogroup autralis mutant strain 702 fliM-< ; time lapse at 0.3s. FIG. 1H is L. interrrogans serogroup autralis mutant strain 702 fliM-< ; time lapse at 0.4s. FIG. 1I is L. interrrogans serogroup autralis mutant strain 702 fliM-< ; time lapse at 0.5s. FIG. 1J is L. interrrogans serogroup autralis mutant strain 702 fliM-< ; time lapse at 0.6s. FIG. 1K is L. interrrogans serogroup autralis mutant 702 fliM- / +< restored with a functional fliM gene; time lapse at 0s. FIG. 1L is L. interrrogans serogroup autralis mutant 702 fliM- / +< restored with a functional fliM gene; time lapse at 0.1s. FIG. 1M is L. interrrogans serogroup autralis mutant 702 fliM- / +< restored with a functional fliM gene; time lapse at 0.2s. FIG. 1N is L. interrrogans serogroup autralis mutant 702 fliM- / +< restored with a functional fliM gene; time lapse at 0.3s. FIG. 1O is L. interrrogans serogroup autralis mutant 702 fliM- / +< restored with a functional fliM gene; time lapse at 0.4s. FIG. 1P is L. interrrogans serogroup autralis mutant 702 fliM- / +< restored with a functional fliM gene; time lapse at 0.5s. FIG. 1Q is L. interrrogans serogroup autralis mutant 702 fliM- / +< restored with a functional fliM gene; time lapse at 0.6s. FIG. 1R is wild type L. interrrogans serogroup autralis strain; time lapse at 0s. FIG. 1S is wild type L. interrrogans serogroup autralis strain; time lapse at 0.1s. FIG. 1T is wild type L. interrrogans serogroup autralis strain; time lapse at 0.2s. FIG. 1U is wild type L. interrrogans serogroup autralis strain; time lapse at 0.3s. FIG. 1V is wild type L. interrrogans serogroup autralis strain; time lapse at 0.4s. FIG. 1W is wild type L. interrrogans serogroup autralis strain; time lapse at 0.5s. FIG. 1X is wild type L. interrrogans serogroup autralis strain; time lapse at 0.6s. FIG. 2A is L. interrrogans serogroup autralis mutant strain 702 fliM-< motility after 10 days of incubation. FIG. 2B is L. interrrogans serogroup autralis mutant strain 702 fliM- / +< restored with a functional fliM gene motility after 10 days of incubation. FIG. 2C is wild type L. interrrogans serogroup autralis strain motility after 10 days of incubation. FIG. 3A is a diagram showing the location of the SNP of the L. interrrogans serogroup autralis mutant strain 702 fliM-< . FIG. 3B is a diagram comparing the nucleotide sequences of wild type L. interrrogans serogroup autralis strain with the L. interrrogans serogroup autralis mutant strain 702 fliM-< . FIG. 4A is a FlaB immunofluorescence assay of L. interrrogans serogroup autralis mutant strain 702 fliM-< . FIG. 4B is a FlaB immunofluorescence assay of L. interrrogans serogroup autralis mutant strain 702 fliM- / +< . FIG. 4C is a FlaB immunofluorescence assay of wild type L. interrrogans serogroup autralis strain. FIG. 4D is a flagella preparation after uranyl acetate staining of L. interrrogans serogroup autralis mutant 702 fliM-< . FIG. 4E is a flagella preparation after uranyl acetate staining of L. interrrogans serogroup autralis mutant 702 fliM- / +< . FIG. 4F is a flagella preparation after uranyl acetate staining of wild type L. interrrogans serogroup autralis strain. FIG. 4G is a flagella preparation after uranyl acetate staining of L. interrrogans serogroup autralis mutant strain 702 fliM-< , higher magnification than 5D. FIG. 4H is a flagella preparation after uranyl acetate staining of L. interrrogans serogroup autralis mutant strain 702 fliM- / +< , higher magnification than 5E. FIG. 4I is a flagella preparation after uranyl acetate staining of wild type L. interrrogans serogroup autralis strain, higher magnification than 5F. FIG. 5A is a coomassie stain of purified flagellar proteins from L. interrrogans serogroup autralis mutant strain 702 fliM-< , L. interrrogans serogroup autralis mutant strain 702 fliM- / +< and wild type L. interrrogans serogroup autralis strain. FIG. 5B is a western blot using FlaA2 antibody of purified flagellar proteins from L. interrrogans serogroup autralis mutant strain 702 fliM-< , L. interrrogans serogroup autralis mutant strain 702 fliM- / +< and wild type L. interrrogans serogroup autralis strain. FIG. 5C is a western blot using FlaB antibody of purified flagellar proteins from L. interrrogans serogroup autralis mutant strain 702 fliM-< , L. interrrogans serogroup autralis mutant strain 702 fliM- / +< and wild type L. interrrogans serogroup autralis strain. FIG. 5D is a western blot using FliM antibody of whole cell lysate from L. interrrogans serogroup autralis mutant strain 702 fliM-< , L. interrrogans serogroup autralis mutant strain 702 fliM- / +< and wild type L. interrrogans serogroup autralis strain. FIG. 5E is a western blot using LipL41 antibody of whole cell lysate from L. interrrogans serogroup autralis mutant strain 702 fliM-< , L. interrrogans serogroup autralis mutant strain 702 fliM- / +< and wild type L. interrrogans serogroup autralis strain. This blot was performed for protein expression normalization. FIG. 6 is a graph representing hamster survival following injection with 10 8< of either L. interrrogans serogroup autralis mutant strain 702 fliM-< , L. interrrogans serogroup autralis mutant strain 702 fliM- / +< or wild type L. interrrogans serogroup autralis strain. FIG. 7 is a graph showing the mortality of hamsters immunized with live 702 fliM-< or Patoc or with culture medium alone and challenged with virulent strains of L. interrrogans serogroup Icterohaemorrhagiae (open bars) or Australis (hatched bars). FIGs.8A-8D depict the DNA and protein sequences and sequence alignments. FIG. 9 is a table showing the SEQ ID NOs assigned to the DNA and protein sequences. FIG. 10 depicts the Il17 response in vaccinated and unvaccinated dogs. FIG. 11 depicts the platelets result in vaccinated, unvaccinated and shedding control dogs. FIG. 12 depicts the ASAT result in vaccinated, unvaccinated and shedding control dogs. FIG. 13 depicts the creatinemia result in vaccinated, unvaccinated and shedding control dogs. FIG. 14 depicts the uremia result in vaccinated, unvaccinated and shedding control dogs. FIG. 15 depicts the ALAT result in vaccinated, unvaccinated and shedding control dogs. FIG. 16 depicts the PAL result in vaccinated, unvaccinated and shedding control dogs. DETAILED DESCRIPTION OF THE INVENTION

[0015] It is noted that in this disclosure and particularly in the claims, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0016] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise. The word "or" means any one member of a particular list and also includes any combination of members of that list.

[0017] The term "animal" is used herein to include all mammals, birds and fish. The animal as used herein may be selected from the group consisting of equine (e.g., horse), canine (e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domestic cats, wild cats, other big cats, and other felines including cheetahs and lynx), bovine (e.g., cattle), swine (pig), ovine (e.g., sheep, goats, lamas, bisons), avian (e.g., chicken, duck, goose, turkey, quail, pheasant, parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian, tarsier, monkey, gibbon, ape), humans, and fish. The term "animal" also includes an individual animal in all stages of development, including embryonic and fetal stages.

[0018] The term "nucleic acid", "nucleotide", and "polynucleotide" are used interchangeably and refer to RNA, DNA, cDNA, or cRNA and derivatives thereof, such as those containing modified backbones. It should be appreciated that the invention provides polynucleotides comprising sequences complementary to those described herein. The "polynucleotide" contemplated in the present invention includes both the forward strand (5' to 3') and reverse complementary strand (3' to 5'). Polynucleotides according to the invention can be prepared in different ways (e.g. by chemical synthesis, by gene cloning etc.) and can take various forms (e.g. linear or branched, single or double stranded, or a hybrid thereof, primers, probes etc.).

[0019] The term "genomic DNA", or "genome" is used interchangeably and refers to the heritable genetic information of a host organism. The genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also the DNA of the plastids (e.g., chloroplasts) and other cellular organelles (e.g., mitochondria). The genomic DNA or genome contemplated in the present invention also refers to the RNA of a virus. The RNA may be a positive strand or a negative strand RNA. The term "genomic DNA" contemplated in the present invention includes the genomic DNA containing sequences complementary to those described herein. The term "genomic DNA" also refers to messenger RNA (mRNA), complementary DNA (cDNA), and complementary RNA (cRNA).

[0020] The term "gene" is used broadly to refer to any segment of polynucleotide associated with a biological function. Thus, genes or polynucleotides include introns and exons as in genomic sequence, or just the coding sequences as in cDNAs , such as an open reading frame (ORF), starting from the start codon (methionine codon) and ending with a termination signal (stop codon). Genes and polynucleotides can also include regions that regulate their expression, such as transcription initiation, translation and transcription termination. Thus, also included are promoters and ribosome binding regions (in general these regulatory elements lie approximately between 60 and 250 nucleotides upstream of the start codon of the coding sequence or gene; Doree S M et al.; Pandher K et al.; Chung J Y et al.), transcription terminators (in general the terminator is located within approximately 50 nucleotides downstream of the stop codon of the coding sequence or gene; Ward C K et al.). Gene or polynucleotide also refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences.

[0021] The term "heterologous DNA" as used herein refers to the DNA derived from a different organism, such as a different cell type or a different species from the recipient. The term also refers to a DNA or fragment thereof on the same genome of the host DNA wherein the heterologous DNA is inserted into a region of the genome which is different from its original location.

[0022] As used herein, the term "antigen" or "immunogen" means a substance that induces a specific immune response in a host animal. The antigen may comprise a whole organism, killed, attenuated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a polypeptide, an epitope, a hapten, or any combination thereof. Alternately, the immunogen or antigen may comprise a toxin or antitoxin.

[0023] 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.

[0024] The terms "recombinant" and "genetically modified" are used interchangeably and refer to any modification, alteration or engineering of a polynucleotide or protein in its native form or structure, or any modification, alteration or engineering of a polynucleotide or protein in its native environment or surrounding. The modification, alteration or engineering of a polynucleotide or protein may include, but is not limited to, deletion of one or more nucleotides or amino acids, deletion of an entire gene, codon-optimization of a gene, conservative substitution of amino acids, insertion of one or more heterologous polynucleotides.

[0025] 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.

[0026] In one embodiment, the present invention provides a L. interrogans composition or vaccine comprising an attenuated or recombinant L. interrogans (LI) strain that contains a mutated fliM (LIC11836) gene comprising SEQ ID NO: 14. The term "mutated fliM gene" refers to the fliM gene of L. interrogans that is altered or engineered which results in a non-functional fliM protein upon expression. The alteration or engineering of the fliM gene includes mutation or deletion of a segment of the fliM gene which is essential for the expression of a functional fliM protein. The alteration or engineering of the fliM gene also includes mutation or deletion of one or more nucleotides which are essential for the expression of a functional fliM protein.

[0027] The term "composition" comprises any vaccine or immunological composition, once it has been injected to a host, including canines, felines, equine and humans, that induces an immune response in the host, and / or protects the host from leukemia, and / or which may prevent implantation of the parasite, and / or which may prevent disease progression in infected subjects, and / or which may limit the diffusion of runaway parasites to internal organs. This may be accomplished upon vaccination according to the present invention through the induction of cytokine secretion, notably IFN-gamma secretion (as example of a method of measurement of IFN-gamma secretion, the Quantikine ®< immunoassay from R&D Systems Inc. (catalog number# CAIF00) could be used (Djoba Siawaya JF et al.)).

[0028] In another embodiment, the present invention provides an attenuated or recombinant L. interrogans wherein the fliM gene is mutated to produce one or more pre-mature stop codons within the gene. The term "fliM gene" includes any gene or polynucleotide that encodes the fliM of L. interrogans, and homologs, fragments or variants thereof. The fliM gene may encode a fliM protein having at least 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO: 17, 18, 19 or 20, or a variant thereof. The disclosure also provides the fliM gene having at least 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to SEQ ID NO:17, 18, 19 or 20. In another aspect, the present invention provides a recombinant L. interrogans wherein the fliM gene in the native (wild-type) L. interrogans genome encoding thefliM protein is altered or engineered resulting in a mutated flim protein.

[0029] The disclosure provides L. interrogans strains under the CNCM deposit numbers CNCM I-5132 and CNCM I-5133, or any parent, descendant or progeny of the deposited strains.

[0030] The disclosure also provides a live attenuated L. interrogans strain. The strains were deposited at CNCM (Collection Nationale de Cultures de Micro-organismes) on September 1, 2016 in accordance with Budapest Treaty and were accorded Accession Nos. CNCM I-5132 and CNCM I-5133. The disclosure also contemplates preparation and isolation of a progeny or descendant of a L. interrogans strain that has been deposited at CNCM under the Accession Numbers CNCM I-5132 and CNCM I-5133. The disclosure therefore also extends to L. interrogans strains which are derived from the deposited strains through propagation or alteration in an identical or divergent form. The progenies or descendants of CNCM I-5132 and CNCM 1-5133 strains include the L. interrogans strain wherein the fliM gene is deleted. The progenies or descendants may comprise a mutated fliM gene encoding a FliM protein having at least 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the sequence as set forth in SEQ ID NO: 17, 18, 19 or 20. The progeny or descendant may comprise a polynucleotide having at least 75%, 80%, 85%, 90%, 95%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to the sequence as set forth in SEQ ID NO: 13, 14, 15, or 16.

[0031] In one embodiment, the invention provides for the administration of a therapeutically effective amount of a vaccine or composition for the delivery an L. interrogans antigen in a target cell. Determination of the therapeutically effective amount is routine experimentation for one of ordinary skill in the art. In another embodiment, the vaccine or composition comprises an attenuated L. interrogans strain wherein the Leptospira interrogans comprises a mutated fliM gene comprising SEQ ID NO: 14, and a pharmaceutically or veterinarily acceptable carrier, adjuvant, vehicle or excipient. In an embodiment, the pharmaceutically or veterinarily acceptable carrier, vehicle, adjuvant, or excipient facilitates transfection and / or improves preservation of the virus, bacterium or protein.

[0032] The pharmaceutically or veterinarily acceptable carriers, adjuvants, vehicles, or excipients are well known to the one skilled in the art. For example, a pharmaceutically or veterinarily acceptable carrier or vehicle or excipient can be a 0.9% NaCl (e.g., saline) solution or a phosphate buffer. Other pharmaceutically or veterinarily acceptable carrier, adjuvant, vehicle, or excipients that can be used for methods of this invention include, but are not limited to, poly-(L-glutamate) or polyvinylpyrrolidone. The pharmaceutically or veterinarily acceptable carrier, adjuvant, vehicle, or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro); the carrier, vehicle, adjuvant, or excipient may facilitate transfection and / or improve preservation of the vector (or protein). Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure read in conjunction with the knowledge in the art, without any undue experimentation.

[0033] The immunogenic compositions and vaccines according to the invention may comprise or consist essentially of one or more adjuvants. Suitable adjuvants for use in the practice of the present invention are (1) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (2) immunostimulating sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more non-methylated CpG units (Klinman D. M. et al., Proc. Natl. Acad. Sci., USA, 1996, 93, 2879-2883; WO98 / 16247), (3) an oil in water emulsion, such as the SPT emulsion described on p 147 of "Vaccine Design, The Subunit and Adjuvant Approach" published by M. Powell, M. Newman, Plenum Press 1995, and the emulsion MF59 described on p 183 of the same work, (4) cation lipids containing a quaternary ammonium salt, (5) cytokines, (6) aluminum hydroxide or aluminum phosphate or (7) saponin, (8) Dimethyldioctadecyl ammonium bromide (Vaccine Design p. 157), (9) Aridine (Vaccine Design p. 148) other adjuvants discussed in any document cited, or (8) any combinations or mixtures thereof.

[0034] The oil in water emulsion (3), can be based on: light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate / caprate), glycerol tri(caprylate / caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters.

[0035] The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g. anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, or polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121. Some of the emulsions, such as TS6, TS7, TS8 and TS9 emulsions, are described in US 7,608,279 and US 7,371,395.

[0036] The polymers of acrylic or methacrylic acid (1) are preferably cross-linked, in particular with polyalkenyl ethers of sugars or polyalcohols. These compounds are known under the term carbomer (Pharmeuropa vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 describing such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced with unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name CARBOPOL ™< (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allylpentaerythritol. Among them, there may be mentioned CARBOPOL ™< 974P, 934P and 971 P.

[0037] Among the copolymers of maleic anhydride and of alkenyl derivative, the EMA ™< copolymers (Monsanto) which are copolymers of maleic anhydride and of ethylene, which are linear or cross-linked, for example cross-linked with divinyl ether, are preferred.

[0038] The proportions of adjuvant which are useful are well known and readily available to the one skilled in the art. By way of example, the concentration of polymers of acrylic or methacrylic acid or of anhydride maleic and alkenyl copolymers in the final vaccine composition will be from 0.01% to 1.5% W / V, more particularly from 0.05 to 1% W / V, preferably from 0.1 to 0.4% W / V.

[0039] In one embodiment, the adjuvant may include TS6 (US7,371,395), LR2, LR3 and LR4 (US7,691,368), TSAP (US20110129494), TRIGEN ™< (Newport Labs), synthetic dsRNAs (e.g. poly-IC, poly-ICLC [HILTONOL ®< ]), and MONTANIDE ™< adjuvants (W / O, W / O / W, O / W, IMS and Gel; all produced by SEPPIC).

[0040] In yet another embodiment, the adjuvant may include interleukin-2 (IL-2), IL-12, interferon α (IFNα), polyinosinic and polycytidylic acid, and cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODN), which are known to significantly enhance CMI response to CIV vaccines (Vet. Immuno. and Immunopath. Vol. 129, Issues 1-2, 15 May 2009, Pages 1-13).

[0041] In a specific embodiment, the pharmaceutical composition is directly administered in vivo. Advantageously, the pharmaceutical and / or therapeutic compositions and / or formulations according to the invention comprise or consist essentially of or consist of an effective quantity to elicit a therapeutic response of one or more expression vectors and / or polypeptides as discussed herein; and, an effective quantity can be determined from this disclosure, and the knowledge in the art, without undue experimentation.

[0042] The composition or vaccine may contain a dose from about 10 2< to about 10 20< , about 10 3< to about 10 18< , about 10 4< to about 10 16< , about 10 5< to about 10 12< VLPs (virus like particles) produced in vitro or in vivo from a viral vector, a plasmid, or baculovirus. The viral vector may be titrated based on any virus titration methods including, but not limited to, FFA (Focus Forming Assay) or FFU (Focus Forming Unit), TCID 50 (50% Tissue Culture Infective Dose), PFU (Plaque Forming Units), and FAID 50 (50% Fluorescent Antibody Infectious Dose), and the VLPs produced in vitro can be titrated by hemagglutination assay, ELISA, and electron microscopy. Other methods may also be applicable depending on the type of VLP.

[0043] The composition or vaccine may contain from about 10 2.0< to about 10 10.0< bacterial counts (for example, number of leptospires) / dose. The composition or vaccine may contain equivalent bacterial counts in the case of inactivated / killed composition or vaccine. The dose volumes can be between about 0.1 and about 10 ml, between about 0.2 and about 5 ml. By definition, the volume of one dose means the total volume of vaccine administered at once to one animal.

[0044] The present invention contemplates at least one administration to an animal of an efficient amount of the therapeutic composition made according to the invention. The animal may be male, female, pregnant female and newborn. This administration may be via various routes including, but not limited to, intramuscular (IM), intradermal (ID) or subcutaneous (SC) injection or via intranasal or oral administration. The therapeutic composition according to the invention can also be administered by a needleless apparatus (as, for example with a Pigjet, Biojector or Vitajet apparatus (Bioject, Oreg., USA)).

[0045] The composition or vaccine is administered to a dog or a cat. A booster administration can be done if necessary around 2 to 11 weeks after the first administration.

[0046] Liquid jet needle-free injectors are devices performing injections of a certain amount of liquid under high pressure through a minute orifice. In an embodiment, the needle-free injection is a DERMA-VAC NF transdermal vaccinator system.

[0047] Optionally, the administration can be repeated, as booster administration, at suitable intervals if necessary or desirable, e.g. from about 2 to about 11 weeks after the first administration. A booster administration can also be repeated every 6 months or every year.

[0048] In one embodiment of the invention, a prime-boost regimen can be employed, which is comprised of at least one primary administration and at least one booster administration using at least one common polypeptide, antigen, epitope or immunogen. Typically the same composition or vaccine is used as the primary administration and the boost. This administration protocol is called "prime-boost". However, different compositions or vaccines may be used as the prime administration and the boost.

[0049] The disclosure also provides a vaccination kit or set, comprising at least one vaccine vial containing the vaccine of the present invention, and operatively assembled to perform the administration of the vaccine to an animal of the canine family. Such vaccination kit or set is able to elicit a safe and protective immune response against influenza infection.

[0050] The invention will now be further described by way of the following non-limiting examples.EXAMPLES

[0051] Construction of DNA inserts, plasmids and recombinant viral vectors was carried out using the standard molecular biology techniques described by J. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2014).Example 1 Isolation and Identification of L. interrogans Mutant and Vaccination Study in Hamsters against Homologous and Heterologous ChallengeIntroduction

[0052] Pathogenic Leptospira are highly invasive spirochetes responsible for leptospirosis, the most widespread zoonosis worldwide. Symptoms vary from chronical infection to multiple organ failure with a significant mortality rate. Virulence factors are not well known due to a lack of adequate and efficient genetic tools for these slow-growing bacteria. Among identified pathogenic determinants, motility is reported as critical as it may participate in pathogen entry and dissemination in the host. However, direct link between motility and virulence has not been demonstrated yet in Leptospira. In this study, we isolated a mutated L. interrogans strain lacking both motility and virulence. Whole genome sequencing allowed to identify a single deletion in fliM gene disrupting the whole protein. FliM is annotated as a flagellar motor switch protein and a component of the C-ring, a central unit of flagellar motor regulating its rotation direction. In this study, we confirmed the implication of fliM in flagellar rotation and assembly. Flagella were indeed either absent from the cell or present at one or both extremities in a truncated form, and rotation movements of cell extremities were lacking. Shorter and non-functional flagella induced loss of translational and non-translational motility as well as morphological defects: hook and spiral shaped-ends were lacking and long chains of bacteria were observed, indicating division defects. In addition, our strain failed to induce an infection on hamster model even at high infectious dose (10 8< bacteria). Genetic complementation in trans with a replicative plasmid restored wild type morphology, flagellar length and rotation, motility and infectivity. We therefore confirmed the function of FliM in Leptospira as fundamental for flagellar structure and rotation, both required for translational motility. Besides, we validate that absence of motility is directly correlated to lack of infectivity, proving that motility is essential for virulence.Strains

[0053] Leptospira interrogans serogroup Australis strains 702 fliM-< , 702 fliM- / +< , 733, further designed as Wild-Type (WT), L. interrogans serogroup Icterohaemorrhagiae and L. biflexa serovar Patoc were grown at 29°C in liquid Ellinghausen-McCullough-Johnson-Harris with shaking or on EMJH agar plates (Difco). Leptospira strains were identified at the species (16S rRNA), serogroup (MAT with rabbit antisera) and genotype (MLVA) levels. When needed, spectinomycin was added to cultures to a final concentration of 50µg / ml. The strains 702 fliM-< and 702 fliM- / +< 733 were deposited at CNCM (Collection Nationale de Cultures de Micro-organismes) on September 1, 2016 and accorded Accession Nos. CNCM I-5132 and CNCM I-5133, respectively.Genomics

[0054] DNA was extracted with QiaAmp DNA Blood Midi kit (Qiagen) for whole genome sequencing and with QiaAmp DNA Mini kit (Qiagen) for PCR amplifications, according to manufacturer's protocols. The genome sequences of WT and 702 fliM< strains were determined. Sequencing reads were assembled using CLC Genomics Workbench 7 (CLCbio) and annotated with Glimmer logarithm from GenostarSuite 4 (Genostar, Montbonnot Saint Martin, France). Polymorphisms between these two strains were detected and analyzed with Genostar Suite 4 and validated by mapping sequencing reads on identified regions with CLC Genomics Workbench 7.PCR

[0055] PCR was performed on 1ng to 10ng of template DNA with 1U of Phusion High-fidelity DNA polymerase (Thermo), 1X Phusion Buffer, 200µM of each dNTP (Life), 0.5µM of each primer in 50µl of water, on Biometra TGradient thermocycler. Primers used for this study are listed in Table 1. Table 1 Primer sequencesPrimer Fw / Rv Amplification Sequence PflgAFwfusion FlgB-fliMTAATACCCGAGCTTCAAGGAAGFliMR2Rvfusion FlgB-fliMCF109FwfliM - external primersCAATCGTGCTGAAGAATCTGAAAGAGCF099RvfliM - external primersCTGTAGCACAAGCCTGATTCGCCF108FwfliM - internal primersTCCCAAGATGAAATTGACGCGCF103RvfliM - internal primersCTACTCTTTCACCGATCTGAATGGCflaB4FFwqRT-PCR flaB4CTCATATTTGCTTGTGCGAGCflaB4RRvqRT-PCR flaB4GAACGCTACTGGTTTACAATTAGTTGCrpoBFFwqRT-PCR rpoBATGGAGCGGAACGTGTAGTCrpoBRRvqRT-PCR rpoBCTTCGTTCGTTCCATGTCCT

[0056] The sequence identification numbers are as follows: SEQ ID NO:1 = PflgA, SEQ ID NO. 2 = FliMR2, SEQ ID NO:3 = CF109, SEQ ID NO:4 = CF099, SEQ ID NO:5 = CF108, SEQ ID NO:6 = CF103, SEQ ID NO:7 = flaB4F, SEQ ID NO:8 = flaB4R, SEQ ID NO:9 = rpoBF, SEQ ID NO:10 = rpoBR.Genetic complementation

[0057] The coding sequence of fliM (LIC11836) was amplified from genomic DNA of L. interrogans serovar Manilae strain L495 (100% sequence identity with FliM of Australis strains at the amino acid level) using primers FlimF (5'-CGACACATATGACAGAAATTTTAT -3'; SEQ ID NO:11) and FlimR2 (5'-TAACTTCAATTCTAATATTCTTGTTCAGAACG -3'; SEQ ID NO:12), which incorporated a restriction digestion site for NdeI (underlined bases), and cloned into PCRII-TOPO vector (Invitrogen) according to manufacturer's instructions. The fliM coding sequence was then digested with NdeI and XhoHI, purified, and inserted into the same restriction sites of pCRPromFlgB to generate a transcriptional fusion between the gene and the Borrelia burgdorferi flgB promoter. The DNA fragment containing the transcriptional fusion was released by EcoRI digestion and cloned into the corresponding site of the replicative plasmid pMaORI (Pappas CJ et al., 2015, Appl. Environ. Microbiol., Feb. 2015), to generate pCF036. The fliM complementation construct was introduced in strain 702 by conjugation with E.coli β2163 carrying pCF036 as previously described (Pappas CJ et al., 2015, supra; Lambert A et al., 2014, FEMS Microbiol. Lett., p. fnu054). Plates were incubated 5 weeks to obtain the complemented strain 702 fliM- / +< .Soft agar plate assay

[0058] Motility of Leptospira strains was evaluated onto 0.5% and 0.3% agar EMJH plates by inoculating 5µl of mid-log phase cultures (OD 450nm of approximately 0.3). Plates were incubated at 29°C and daily observed. Briefly, soft EMJH plates with 0.5% agar inoculated with 702 fliM-< , 702 fliM- / +< and WT were observed after 10 days of incubation. 702 fliM-< didn't spread from its inoculation point (Fig. 2A), contrary to 702 fliM- / +< (Fig. 2B) and WT (Fig. 2C) which formed a circle of 2.6cm and 2.2cm respectively. One representative plate of three experiments is shown. Identical results were obtained on three 0.3% agar plates.Dark field microscopy

[0059] Leptospires were observed by dark-field microscopy using x20 to x200 magnification with Olympus BX-53 139 microscope coupled with a camera Hamamatsu Orca Flash2.8. See Figs. 1A - 1X. Pictures were taken with CellSense Software (Olympus) and videos were recorded with MicroManager 1.4 software (µManager). Images were post-treated with ImageJ software (ImageJ), and three pictures of each strain containing approximately 80 bacteria were used to manually measure bacterial length with ImageJ.Transmission electron microscopy (TEM)

[0060] Commercial 400 mesh carbon-coated copper grids were glow-discharged just before use. Samples were placed in contact with the grids for 10min. Grids with samples were then fixed with 2% glutaraldehyde in 0.1M cacodylate sodium buffer for 10min and washed 3 times for 1min in water for biosecurity requirement. Grids were dried and negatively stained with 4% uranyl acetate for few seconds. Samples were observed with a transmission electron microscope (TEM) TECNAI T12 FEI operating at 120kV. For manual length measurements of flagella, 5 fields were chosen randomly and analyzed using ImageJ software.Recombinant FliM purification and antisera production

[0061] The plasmid containing flgB-fliM fusion was digested by NdeI and BamHI and cloned into corresponding sites in pET28 vector (EMD Biosciences). The resulting plasmid pET28-(His)6FliM was introduced into E. coli BL21DE3 cells. Recombinant protein expression was induced on 2L of transformed cells at OD 600nm =0.7 by addition of 1mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4h at 37°C. FliM was highly insoluble in the growth condition used and was purified from inclusion bodies by the following manner. Cells were harvested and pellets were resuspended in buffer A (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM Imidazole, 10% glycerol) and lysed by sonication in the presence of protease inhibitor cocktail (complete mini EDTA-free, Roche) and 1 mM phenylmetylsulfonyl fluoride (PMSF). The insoluble fraction obtained after a 60 min centrifugation at 40,000 x g at 4°C was resuspended in buffer A containing 1% Triton X100 to solubilize membrane proteins. Inclusion bodies obtained after a 30 min centrifugation at 40,000 x g were solubilized in buffer B (100 mM NaH2PO4 pH 8.0, 8M urea, 10 mM Tris pH 8.0, 200 mM NaCl, 1 mM PMSF, 0.1% Triton X100), incubated for 1h at room temperature with stirring and centrifuged 30 min at 4°C at 40,000 x g. The supernatant was loaded on a nickel-nitrilotriacetic (Ni-NTA) resin (Qiagen) equilibrated with buffer B. Since recombinant (His)6-FliM could not be eluated from the column by conventional methods, the flow-through was used to further purify the protein by filtration on a 100 kDa cut-off cellulose membrane (Amicon Ultra 100K, Merck Millipores). The flow-through after filtration was dialyzed against C (50 mM Tris pH 8.0, 150 mM NaCl, 2 mM 2M urea, 2 mM DTT, 5 mM EDTA) and concentrated to 2.5mg / mL.

[0062] To produce polyclonal sera, two rabbits were immunized 4 times with 2mg of purified FliM protein, and sera were collected on day 38 (Aldevron).Preparation of periplasmic flagella

[0063] Flagella extraction protocol was adapted from Lambert (2012, supra). Three independent mid-log phase cultures at OD 450nm of approximately 0.3 (5x10 8< cells / ml) of 200ml for each strain were centrifuged at 8000 x g 20min at 4°C. Pellets were washed with 22ml PBS and then with 24ml sucrose solution (0.5 M sucrose, 0.15 M Tris pH 8). Cells were resuspended in 12ml sucrose solution and stirred on ice for 10min. Addition of 1.2ml of 10% Triton X-1000 was followed by incubation of 30min at room temperature. 120µl of 10mg / ml lysozyme were added dropwise and incubated 5min on ice. Then 1.2ml of 20mM EDTA pH8 were added dropwise. After incubation of 2h at room temperature, 240µl of 0.1M MgSO 4 were added and stirred 5min at room temperature, then 240µl of 0.1M EDTA were added and stirred again 5min at room temperature. Mixtures were then centrifuged at 17000 x g 15min at 4°C. Supernatants were mixed with 1.6ml 20% polyethylene glycol 8000 previously dissolved in 1M NaCl, and incubated 30min on ice. Suspensions were centrifuged again at 24400 x g for 45min at 4°C. Pellets were resuspended in 2.5ml of water and 100µl of 1% sodium azide in PBS, and were placed at 4°C overnight. Mixtures were then ultracentrifuged 45min at 80 000 x g at 4°C in a Beckman Optima MAX Ultracentrifuge, with rotor TLA 100.3. Flagellar preparations were resuspended in an appropriate volume of water depending on the OD of the culture (approximately 800µl) and 100µl of 1% sodium azide.Protein gels

[0064] Cultures of leptospires were centrifuged for 10min at 7000 x g, washed once in PBS and then normalized in PBS at 1 or 5 OD 450nm unit per milliliter to obtain whole cell lysates. These cell lysates and flagellar preparations were mixed with 3x lysis buffer composed of 0.2M Tris pH8, 16% 2-mercaptoethanol, 6% SDS, 30% glycerol and 0.06% bromophenol blue, and boiled at 100°C for 10min before loading on 4-20% TGX gels (SDS-PAGE, Biorad). 5µl of ladders, Precision Plus All Blue Standards (Biorad) and Magic Mark XP (Life) and different volumes of samples were separated under 150V to 200V for 30min to 1h. Coomassie gels were washed 3 times with distilled water for 5min, stained for 1h with Simply Blue stain (Invitrogen) and destained in water bath for 1h. Western Blots were performed by transferring proteins from gels to nitrocellulose membranes for 7min at 25V and 1.3mA per gel using Transblot-Turbo transfer system (Biorad). Aspecific sites on membranes were blocked after an incubation of 1h in Odyssey buffer (Li-Cor). Rabbit primary antibodies anti-FlaB (LIC11531, 1:1000), FlaA2 (1:1000), FliM (1:100), LipL41 (1: 100) were diluted in Odyssey buffer and incubated for 1h. Membranes were then washed 3 times with PBS + 0.1% Tween, and incubated with secondary anti-rabbit IgG (H&L) goat antibody IRdye800 ®< Conjugated (Rockland) diluted 1: 10000 for 45min in the dark. Three more washes with PBS + 0.1% Tween were performed before revelation using Odyssey ®< imaging system at 800nm. Relative approximate quantification was performed by measuring intensities of bands with Odyssey ®< software (Li-Cor).Immunofluorescence (IFA)

[0065] Protocol was adapted from Pinne and Haake (Pinne M et al., 2011, J. Vis. Exp., no. 53). 5x10 8< leptospira were washed in PBS + 5mM MgCl 2 and then applied to a well of a 4 well Lab-Tek ®< II CC 2< Slide (Nunc). Cells adhesion was realized by incubating leptospires for 1h at 29°C. Liquid was removed by gentle aspiration, and cells were fixed and permeabilized with 1ml ice-cold 100% methanol for 20min at -20°C. Non-specific sites were blocked with 1ml of PBS-BSA 2% for 1h. Leptospires were then incubated with 200µl of primary FlaB antibody diluted at 1:100 in PBS-BSA 2% for 1h, then washed 3 times with 1ml of PBS. Secondary antibody Cy ™< 3-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L, Jackson ImmunoResearch) diluted 1:100 in PBS-BSA 2% was added and incubated for 1h. Cells were washed twice with 1ml PBS and once with 1ml of water. Chambers were removed and slides were air-dried for several minutes. A drop of ProLong ®< Gold Antifade reagent containing 4',6'-diamidino-2-phenylindole (DAPI) for DNA counterstaining (Life Technologies) and a coverslip were added, and incubated overnight in the dark. Slides were observed with a Nikon Eclipse Ti-S fluorescence microscope (Nikon) coupled to Digital Sigth DS-5Mc camera (Nikon) and pictures were acquired with Nikon NIS Elements F Package 4.0 (Nikon) and post-treated with ImageJ software.Virulence assay

[0066] Syrian Gold female hamsters of 6 to 7 weeks old were infected with 10 8< Leptospira injected intraperitoneally. Duration of the study was first established at 21 days but protocol was stopped at day 14 given the rapid death of hamsters (see results section). 2 groups of 5 hamsters were infected by 702 fliM-< and WT and 1 group of 10 hamsters was infected with 702 fliM- / +< (see Fig. 6).Protection studies

[0067] Groups of twenty 4-week-old Syrian Gold female hamsters were vaccinated by intraperitoneal route (IP) with a single dose of 10 6< live L. interrogans serogroup Australis 702fliM -< or 10 8< live saprophytic L. biflexa serovar Patoc. As a control, a group of 10 hamsters was vaccinated with 1ml of EMJH. Fourteen days after immunization, animals were challenged via the IP route with virulent L. interrogans. Half of the group received 20 leptospires of serogroup Australis strain 733 while the other half was inoculated with 1000 leptospires of Icterohaemorrhagiae. These challenge doses have been previously determined as the minimal amount of bacteria necessary to kill at least 80% of hamsters. Animals were monitored for 21 days after infection. Moribund hamsters were euthanized by intracardiac injection of Dolethal. See Figure 7.Accession numbers

[0068] The genome sequences of L. interrogans strains 702 and 733 are available in GenBank under accession numbers NZ _LMXF00000000.1 and NZ _LMXK00000000.1, respectively.Results Isolation of a spontaneous nonmotile mutant

[0069] L. interrogans serogroup Australis strain 702 was isolated from the blood of a dog with acute leptospirosis. The dog died from its infection a few days after sampling. The positive culture was apparently clonal with non-translating leptospires under dark-field microscopy. Similarly, after plating the isolate onto solid EMJH media, all colonies were small. Assuming that this non-motile phenotype arose from a motile parental strain, the closest phylogenetic strain was selected from the collection for further phenotypic analysis.

[0070] The motile strain 733, which was also isolated from a sick dog visiting the same clinics 3 months later, exhibited the same molecular (16S rRNA and MLVA profiles) and serological (serogroup Australis) features as the nonmotile strain 702. Furthermore, 702 and 733 showed similar cell growth kinetics (not shown). Taken together, these data indicate that the two strains are very close, thus strain 733 could be considered as a WT strain to be compared with 702 for following experiments. Strain 733 was consequently named WT.

[0071] Strain 702 presented an atypical straight morphology under dark field microscope, lacking characteristic hook and spiral-shaped extremities and missing translational and non-translational motility (Fig. 1A and 1D - 1J). Approximately 2% of total population showed a small hook on one extremity, conferring sporadic and slow rotational movements but unable to propel the cells. We confirmed this absence of motility of 702 on soft agar plates, as the strain didn't spread from its inoculation site contrary to WT (Fig. 2A). In addition, this strain showed a filamentous morphology. We measured cells length and compared to WT using different pictures at x20 magnification. Average length of WT was 9.4µm as already described for typical pathogenic Leptospira and 16.5µm for 702 (on 275 counted bacteria). Moreover, distribution of cell sizes were different between the two strains: for WT, 99% of bacteria were shorter than 15µm, and 1% comprised between 15µm and 30µm, whereas for the non-motile strain, 51% of bacteria were shorter than 15µm, 43% between 15µm and 30µm, and 6% longer than 30µm, including 2 bacteria of 70µm, indicating probable division defects.A single spontaneous mutation abolished FliM protein expression in the non-motile strain

[0072] The whole genome of strains 702 and WT were sequenced to identify mutation(s) that could result in the motility-deficient phenotype in strain 702. 50,272,184 reads and 10,974,264 reads were analyzed for strains 702 and WT, respectively, representing a depth of coverage of 1000x and 200x respectively. Reads were assembled into contigs and annotated. Comparative analysis of the genome sequences led to the identification of two polymorphisms, one DIP and one nonsense SNP. These two mutations induced premature stop codon apparition on fliM (LIC11836) and LIC10453 genes. However, LIC10453 which encodes a hypothetical protein was also truncated 50 amino acids (on a total size of 364aa) downstream in two other motile L. interrogans strains available at MERIAL, suggesting that this gene is not essential to motility. By contrast, fliM, annotated as a flagellar motor switch protein, was found to be highly conserved among Leptospira genus, with more than 99% identity amongst pathogenic L. interrogans and L. borgpetersenii and 88% identity with the saprophytic L. biflexa. See Fig. 3B, fliM original sequence (GTT TCT GAA CAA AAA AAA GTA AAG ATC TAC GAT TTT; SEQ ID NO: 13). This premature stop codon in fliM, which is the result of a 1-nucleotide deletion 105bp downstream the start codon, was then more likely implied in the motility-deficient phenotype. See Fig. 3B, fliMmutated sequence (GTT TCT GAA CAA AAA AAG TAA AGA TCT ACG ATT TTA; SEQ ID NO:14). This deletion was confirmed by PCR sequencing after amplification with primers CF109 (SEQ ID NO:3)-CF099 (SEQ ID NO:4). No polar effect due to this mutation is to be expected because no genes were present downstream of fliM (Fig. 3A). To further investigate the expression of FliM, we produced polyclonal antibodies to recombinant FliM. As expected, no FliM expression was detected in whole cell lysates of strain 702 (Fig. 5D). Mutation of fliM therefore caused absence of FliM expression and was likely responsible for loss of motility. We therefore further named this strain 702 fliM-< .Complementation with fliM restores typical cell shape, motility, and virulence

[0073] To confirm that a mutation in fliM specifically causes loss of motility, we complemented the strain 702 fliM-< in trans with the replicative vector pMaORI containing a functional fliM gene under the control of a constitutive promoter (fliM is the second gene of a putative operon (Fig. 3A), preventing complementation with native fliM promoter). FliM expression was restored in the complemented strain to WT level according to Western Blot experiments on 3 independent cultures of 702 fliM- / +< (Fig. 5D). Microscopic observations showed the restoration of wild type motility and morphology with hook and spiral-ends (Fig. 1B and 1K - 1Q). Spread of bacteria on soft agar plate was similar to WT (Fig. 2B). Moreover, no long filaments were observed and cell length was comparable to WT: on 275 measured bacteria, average length was 10.9µm, including 91% shorter than 15µm and 9% between 15µm and 30µm. Growth kinetics were similar to 702 fliM-< and WT. Taken together, these data prove that trans complementation successfully reintroduced functionalfliM in strain 702 fliM-< , and that fliM is essential for cell shapes, motility, and virulence.fliM disruption induces a defect in flagella assembly

[0074] Since FliM is one of the components of the flagellar motor, we further investigated the integrity of flagella in mutated, complemented and WT strains. Immunofluorescence assays (IFA) were performed using major filament protein FlaB antibodies (Fig. 4A - 4C). Staining of the WT strain (Fig. 4B) revealed the presence of FlaB proteins from the extremities to the middle of the cell without overlapping, consistent with previous IFA experiments and the description of the flagella in Leptospira. Staining on complemented strain 702 fliM- / +< (Fig. 4C) was similar to WT. Mean FlaB stained flagella length of these two strains was 3.5µm. However, 702 fliM-< (Fig. 4A) showed either shorter flagella of approximately 1.3µm or no flagella in 30% of the cells. In order to better characterize flagellar length, purified flagella of the 3 strains were observed by electron microscopy (Figs. 4D - 4I). For WT strains (Figs. 4E and 4H), we observed intact coiled flagella of a median size of 3.2µm in length, consistent with IFA experiment, and 20nm in diameter. Observed flagella were composed of the basal body at one extremity and a filament displaying a thinner 15nm diameter at the other extremity, probably due to the lack of sheath as already described in previous leptospiral flagella observations (14). A small number of broken flagella were observed, due to the fragility of the filaments. For 702 fliM-< (Figs. 4D and 4G), all flagella were significantly shorter, with a median length of 0.7µm. This observation was confirmed on 3 batches of flagella extracted from 3 independent cultures, excluding the fact that this shortness was only caused by flagella breaks.

[0075] To further characterize the short filaments on 702 fliM-< , purified flagella were analyzed by electrophoresis. Coomassie staining revealed a different flagellar proteins content of 702 fliM-< compared to WT and complemented strains (Fig. 5A). In particular, we noticed less protein with an apparent molecular mass of about 30-40 kDa, corresponding to the size of previously described flagellar proteins (Liao, 2009, supra). Production of two major 30kDa filament proteins, FlaA2 (LIC10787) and FlaB (LIC11531), was followed on Western Blot (Figs. 5B and 5C). FlaA2 (Fig. 5B) and FlaB (Fig. 5C) protein levels were approximately 2.5 fold lower in 702 fliM-< flagella compared WT and 702 fliM- / +< strains, confirming an abnormally low quantity of flagellar proteins consistent with a shorter filament. The same reduction of FlaA2 and FlaB proteins expression was seen in whole cell lysates but not by qPCR, indicating that flagellar proteins content is lower in nonmotile strains than in WT but genes expression is not impaired. All together, these data show that fliM disruption leads to incomplete flagella assembly, and that these short flagella are not able to confer motility.Motility is essential for virulence

[0076] To investigate the role of motility in virulence, groups of five to ten hamsters were infected by intraperitoneal route with 10 8< counts of 702 fliM-< , 702 fliM- / +< or WT strains and followed for 14 days. While all 702 fliM-< infected hamsters survived, those receiving WT and 702 fliM- / +< died within six days (Fig. 6). These data indicate that the non-motile 702 fliM-< is not virulent even at high infection dose, and that restoration of fliM and motility correlates with virulence restoration. Motility is therefore essential to virulence.Live 702 fliM-< Vaccine Protects Against Homologous and Heterologous Challenge

[0077] To investigate whether the 702 fliM-< strain may constitute a promising live canine vaccine candidate, we assessed protection of hamsters against homologous and heterologous challenges. Groups of 20 hamsters were vaccinated by intraperitoneal route with a single dose of 10 6< live L. interrogans serogroup Australis 702 fliM-< , or 10 8< live saprophytic L. biflexa serovar Patoc or EMJH medium. Fourteen days after immunization, animals were challenged with virulent L. interrogans Australis or Icterohaemorrhagiae serogroup. and monitored for 21 days.

[0078] While 80 to 100% of EMJH or Patoc vaccinated animals died, 90% of the 702 fliM-< vaccinated hamsters survived in both challenge groups (see Fig. 7). These data indicate that the live non-motile 702 fliM-< virulence attenuated Serogroup Australis strain, but not the avirulent Patoc, used as live vaccine, is able to confer cross protective immunity against L. interrogans serovar icterohaemorrhagiae.Discussion

[0079] Spirochetes are highly invasive bacteria, probably due to their remarkably efficient motility. In Leptospira, propulsion of the cells varies between 6µm / s and 30µm / s depending on viscosity and is conferred by only 2 flagella (Xu J, et al., 2015, Microbiol. Res., vol. 171, pp. 21-25; K. Takabe K, et al., 2013, Microbiol. Immunol., vol. 57, no. 3, pp. 236-239; Cameron CE, 2015, Curr. Top. Microbiol. Immunol., vol. 387, pp. 21-41). B. burgorferi swims at similar speed but contains 7 to 11 PFs, and Critispira has at least 100 PFs (Malawista SE et al., 2008, PLoS ONE, vol. 3, no. 2, p. e1633). The performance of the 2 Leptospira flagella may be due to a more complex structure provided by a higher number of genes related to motility. While 50 genes are referenced on T. pallidium and B. burgorferi, approximately 90 genes are present in Leptospira genome (A. L. T. O. do Nascimento ALTO et al., 2004, Braz. J. Med. Biol. Res., vol. 37, no. 4, pp. 459-477; M. Picardeau M et al., 2008, PLoS ONE, vol. 3, no. 2, p. e1607). Nonetheless, we showed in this study that a single nucleotide deletion in one of these genes, fliM, can destabilize the whole flagellar structure and suppress motility. This deletion was identified by whole genome sequencing and comparative genomics of strains 702 fliM-< and WT. A spontaneous deletion occurred in a homopolymer region close to the start of fliM gene, preventing expression of the protein. Complementation with functional fliM restored wild type phenotype, showing that this only mutation was responsible for 702 fliM-< defects. fliM is annotated as flagellar motor switch protein by inference with homologous genes in other bacterial species, but no studies confirmed this function so far on Leptospira. The attenuated mutant identified in this study is thus of great interest.

[0080] Phenotypic observations of the mutant revealed an atypical morphology. Cell extremities were impacted: the mutant strain lacked hook and spiral shaped ends. The main feature of the mutant is its motility loss. No translational shiftings were observed and only a minor population (approx. 2%) displayed slow and sporadic extremity movements not able to propel the cell. These findings indicate the impairment of flagella and motor rotation. We further characterized flagella and found that they were either absent or present in a truncated form in one or both extremities of our fliM-deficient mutant. Measures of their length by IFA and TEM revealed a reduced size of 2.5 to 4.5 fold compared to WT, consistent with a similar decrease of filament proteins expression noticed on western blots. However, when formed, flagella presented typical diameter and curvature compared to WT and previously published works (Nauman RK et al., 1969, J. Bacteriol., vol. 98, no. 1, p. 264), meaning that core and sheath of the flagella are not directly impaired. fliM is then required for flagella full-length and rotation, both essential for translational motility.

[0081] To further characterize the implication of flagella and motility in Leptospira pathogenicity, animal model (hamsters) were infected with the mutant strains. Infection experiments revealed the lack of virulence of the non-motile strain and restoration of full infectivity of complemented strain. This is the first report that we know of that direct implication of absence of motility and pathogenicity in Leptospira is shown, without possible polar effects. This finding is coherent with the idea that motility is essential for breaching the mucosal membranes, entering the tissues though damaged skin, dissemination in the host and organ penetration (Murray 2015, supra).

[0082] 702 fliM-< is a non-virulent strain that was isolated from a sick dog. It is likely that the animal has been originally infected by a virulent strain and that a point mutation have occurred following isolation with the mutated non-motile strain outgrowing the WT population in vitro. On the contrary, the Patoc strain belongs to a saprophytic, noninfectious species of Leptospira. Surprising, the results show that cross protection can only be achieved by using a virulence attenuated strain such as 702 fliM-< , directly issued from a fully virulent isolate but not with an avirulent saprophytic strain.

[0083] In summary, genomic sequencing identified a spontaneous point mutation responsible for motility loss probably conferring selective advantage to Leptospira in in vitro media. This mutation in the fliM gene - which occurred during the culturing procedure - results in destabilization of the motor switch complex C-ring, leading to incomplete flagella assembly and defective rotation regulation, both essential for translational motility. In addition, apart from motility loss, improper flagella formation affects cell morphology and division. Additionally, with genetic complementation, we validate the direct implication of motility in pathogenicity of Leptospira, delivering knowledge on virulence mechanisms of these emerging zoonotic bacteria. Finally we demonstrated that the immobile mutant, directly issued from an originally virulent strain, is a suitable cross protective live vaccine candidate, with unique features that cannot be found using an avirulent saprophytic strainExample 2 Safety and Efficacy Study of Live Attenuated L. interrogans Vaccine in Homologous Challenge in Dogs

[0084] The goal of the study is to evaluate the serological response (immunogenicity), safety / diffusibility and efficacy of a live attenuated L. interrogans vaccine in a homologous vaccination-challenge model in dogs.

[0085] The study was divided in 2 parts: vaccine phase from D0 to D37, and challenge phase from Tpre (=D38) to T26 (=D69) with challenge on T0 (2 weeks after the second vaccination). Sixteen six-month old, commercial source beagles were randomized to three groups as shown in Table 2 below. Dogs in Group A were vaccinated twice subcutaneously, 25 days apart on D0 and D25, with 1 mL of the live L. interrogans vaccine containing around 5x10 7< lepo / ml of the attenuated 702 fliM< strain. Dogs in Groups B (control group) and C (shedding control group) were not vaccinated. Dogs from groups A and C were housed together in order to determine potential shedding of the vaccinal strain. Dogs from group B were housed separately from the two other groups. The vaccinated dogs were monitored for injection site reactions (swelling, pain upon palpation), general condition (apathy, prostration) and body temperature. Blood samples were collected for serum ELISA (BS dry tube), hematobiochemical analysis (BS Hep-Li and BS EDTA), qPCR analysis of diffusibility or cellular immunity analysis (BS Hep-Na). Urine samples were collected for qPCR analysis of diffusibility. Dogs in group C were euthanized on D39 (T0) for post mortem analysis of the presence of bacteria and tissue lesions in liver and kidneys. The schedule for sample collection in the vaccination phase is detailed in Table 2.

[0086] On D39 (T0), dogs in groups A and B were challenged by intraperitoneal route with a virulent strain L. interrogans serogroup Australis at dose of 5x10 9< lepo. Dogs from group C were not challenged but euthanized on D39. During the post challenge period the dogs were monitored daily for mortality, clinical signs (general condition, dehydration, ocular signs, vomiting, diarrheoa and cutaneo-mucosal signs), body temperature and weighing. Blood samples were collected for serum ELISA (BS dry tube), hematobiochemical analysis (BS Hep-Li and BS EDTA) and for qPCR analysis of leptospiremia (BS Hep-Na). Urine was collected for qPCR analysis of leptospiruria. Liver and kidneys were collected post mortem for qPCR analysis of the presence of bacteria as well as for tissue lesions observations. Surviving dogs in Group A and B were euthanized on T26. The schedule for sample collection in the vaccination phase is detailed in Table 2.1. Table 2 Experimental design - vaccine phaseGroup# of dogVaccine SCserum ELISA (BS dry tube)BS Hep-Li* + BS EDTA***qPCR (BS Hep-Na** + Urine)BS Hep-Na (cellular immunity)ChallengeD39=T0A vaccine8D0 & D25 1ml / dogD0, D7, D14, D25, prior to challengeD0, D7, D14, D25, prior to challengeD0, D2, D4 & D7D7, D14, D35D39B No vaccine6NAprior to challengeNAD39C Control sheddin g2D0, D7, D14, D25, prior to challengeD0, D2, D4 & D7NABS Hep-Li*: Blood collected in Lithium heparinised tubes for biochemistry (urea, creatinine, ALT, AST, ALP). BS Hep-Na**: Blood in Sodium heparinised tubes for detection of leptospires by qPCR and exploration of cellular immunity. BS EDTA***: samples used for platelet count. Table 2.1 Experimental design - challenge phase Group# of dogChallenge 4.5x10 8< lepo / ml IPClinical follow upBlood samplingUrineEuthanize at the end of challengeA Vaccinates8T0 (D39)Clinical examination Daily T0 to T26BS dry tube : T26T3, T5, T7, T11, T14, T21, T26T2610 ml / dogB Challenge controls6T0 (D39)10 ml / dogBS Hep-Li and BSBody temperature Daily T0 to T7EDTA: T3, T4, T5, T7 and T11Weight T0, T7, T14, T21, T26BS Hep-Na: T3, T4, T5, T7 and T11

[0087] The diffusibility / safety results are shown in Table 3 below. The results show that no leptospiruria were detected in vaccinated group (group A) and shedding dogs (group C). No adverse local or general signs were observed after vaccination in the vaccinated dogs. Table 3 Diffusibility / safety resultsGroupDog No.LeptospiruriaD0D2D4D7A vaccine1NegativeNegativeNegativeNegative2NegativeNegativeNegativeNegative3NegativeNegativeNegativeNegative4NegativeNegativeNegativeNegative5NegativeNegativeNegativeNegative6NegativeNegativeNegativeNegative7NegativeNegativeNegativeNegative8NegativeNegativeNegativeNegativeB Control No vaccine1 / / / / 2 / / / / 3 / / / / 4 / / / / 5 / / / / 6 / / / / C Control shedding1NegativeNegativeNegativeNegative2NegativeNegativeNegativeNegative

[0088] Cellular mediated immunity results are shown in Figure 10. The Leptospira specific IL-17 response was detected in vaccinated dogs seven days after the 2 nd< vaccine injection. No other cytokines (such as IFNg, TNFa, IL-6, IL8, IL10) were detected in vaccinated group in the in vitro PBMCs re-stimulation assays.

[0089] The efficacy results are shown in Table 4 below. The results demonstrated the excellent protection in the vaccinated dogs (group A) in terms of clinical signs: no dogs in group A (0 / 8) showed any clinical signs and five out of six dogs in group B (5 / 6) showed severe clinical signs. Also as shown in Table 4, no leptospiruria were detected in vaccinated dogs (group A) with only two dogs that were marginally positive at one time point, while the only dog in group B which did not show any clinical signs was positive in leptospiruria. Table 4 Efficacy resultsGroupDog NoLeptospiruriachallengeT3T5T7T11T14T21T26A Vaccine1NegNegNegNegNegNeg / / 2NegNegNegNegNegNeg / / 3NegNegNegNegNegNeg / / 4NegNegNegNegNegNeg / / 5NegNegNegNegNegNeg / / 6NegNegNegNegNeg+ / / 7NegNegNegNegNegNeg / / 8NegNeg+NegNegNeg / / B Control No vaccine1NegNeg++++++++ / / 2Neg / / / / / / / 3NegNegEuthanized on T44NegEuthanized on T35NegEuthanized on T36NegEuthanized on T3C Control shedding1 / Euthanized on T02 / Euthanized on T0

[0090] The results of hematobiological parameters (see FIGs. 11-16) showed that during the vaccination phase, there was no difference between controls (group B), vaccinated (group A) or shedding (group C) dogs. After challenge, significant effect on hematobiological parameters was observed in controls (group B) while there was no effect for vaccinated dogs (group A). In the control group (group B), all dogs died after challenge except one dog. The uremia and creatinemia parameters remains affected after T5 for this surviving dog in group B, which indicate that the dog's kidney was affected as reflected by leptospiruria.

[0091] The challenge was validated and was severe since five of the six control dogs were clinically diseased with severe clinical signs (prostration, digestive and cutaneo-mucosal signs) and alteration of biological parameters (urea, creatinine, AST, ALT, ALP and platelets) and had to be euthanized for ethical reasons on day 3 or 4 after challenge. These dogs had positive one or two blood samples, positive kidney samples and kidney and liver lesions.

[0092] After challenge, all vaccinated dogs remained in good general condition without any clinical signs or hyperthermia. No alteration of blood parameters was observed. Leptospira were not detected in blood and kidneys. Two vaccinated dogs had one marginally positive urine sample only, all urine samples remaining negative in the six other vaccinated dogs. No kidney or liver lesions were evidenced in all vaccinated dogs.

[0093] Protection against mortality, signs of disease (clinical signs, bodyweight, rectal temperature, biological parameters), infection, bacterial excretion, renal carriage and renal lesions was demonstrated in vaccinated dogs when compared to control dogs.Conclusion

[0094] The results demonstrated that the live attenuated L. interrogans vaccine containing the attenuated 702 fliM< strain is safe to use. When injected in the animals, it does not show any local or general signs, and does not have any effect on hematobiological parameters. There is no sign of shedding after the live attenuated vaccine is administered to the animals. The live attenuated L. interrogans vaccine (702 fliM< strain) also prevented mortality, clinical signs of the disease, infection, urinary excretion, renal carriage and renal lesions against a very virulent L. interrogans serogroup Australis challenge performed two weeks after the 2 nd< injection of primary vaccination.Example 3 Safety and Efficacy Study of Live attenuated L. interrogans Vaccine against Heterologous Challenges in Dogs

[0095] The goal of the study is to evaluate the efficacy against heterologous challenge (onset of immunity) of a live attenuated L. interrogans vaccine in dogs.

[0096] Thirteen six-month old, commercial source beagles are randomized to two groups. Dogs in Group A are vaccinated twice subcutaneously, on D0 and D28, with 1 mL of the live L. interrogans vaccine containing 10 7< lepo / ml of the attenuated 702 fliM< strain. Dogs in Group B (control group) are not vaccinated.

[0097] On D42 (T0), each dog in groups A and B is challenged with a virulent strain L. interrrogans serogroup Icterohaemorrhagiae at a dose of 10 6< lepo / ml. During the post challenge period the dogs are monitored daily for mortality, clinical signs (general condition, dehydration, ocular signs, vomiting, diarrheoa and cutaneo-mucosal signs), body temperature and weighing. Blood and urine are collected regularly throughout the vaccination phase for serological, hematobiochemical, leptospiremia, and leptospiruria analysis. Liver and kidneys are collected post mortem for analysis of the presence of bacteria as well as for tissue lesions observations. Surviving dogs in Group A and B are euthanized on T28.

[0098] The efficacy results show that the global clinical scores are significantly lower in the vaccinated dogs versus controls. Similarly the global biochemical and haematological scores, number of positive blood cultures per dog (leptospiremia), number of positive urine cultures per dog (leptospiruria), number of dogs with positive kidney culture and the number of dogs with kidney lesions are significantly lower in vaccinated groups when compared to controls.Conclusion

[0099] The results demonstrate the live attenuated L. interrogans vaccine (702 fliM< strain) reduces mortality, clinical signs of the disease, infection, urinary excretion, renal carriage and renal lesions upon heterologous virulent L. interrogans serogroup Icterohaemorrhagiae challenge performed two weeks after the 2 nd< injection of primary vaccination and provide cross protection against heterologous challenge.

[0100] The following Examples 4 and 5 are not according to the invention and are present for illustration purposes only.Example 4 Deletion of fliM gene

[0101] The goal of the study is to generate a recombinant and attenuated L. interrogans. by deleting the FliM gene encoding fliM protein (SEQ ID NO: 17) from Leptospira interrogans genome.

[0102] FliM gene encoding fliM protein (SEQ ID NO: 17) is deleted from Leptospira interrogans using two sequential steps of transformation.

[0103] A markerless mutation system for the pathogen L. interrogans is generated by developing tools allowing the performance of two sequential steps of transformation. By the first homologous recombination, the target gene fliM is replaced by a kanamycin resistance cassette, using an allelic exchange system as previously shown for ligB (Croda, J., et al., 2008, Infect Immun 76, 5826-5833). For the second transformation, the Flp recombinase system, a protein that promotes both inter- and intramolecular recombination (Hoang, T. T., et al., 1998, Gene 212, 77-86), is used as an efficient tool for the excision of DNA fragments inserted into the Leptospira interrogans bacterial chromosome.

[0104] Alternatively, the S. cerevisiae mitochondrial endonuclease I-SceI can be used to produce markerless deletion of fliM gene from Leptospira interrogans chromosome (Pósfai, G., et al., 1999, Nucleic Acids Res 27, 4409-4415)

[0105] The generated recombinant Leptospira interrogans where the fliM gene is deleted is stable and reliable, and is used as a live attenuated vaccine to in the efficacy studies against homologous and heterologous challenges in dogs as described in Example 5.Example 5 Safety and Efficacy Study of Live attenuated L. interrogans Vaccine against Homologous and Heterologous Challenges in Dogs

[0106] The goal of the studies is to evaluate the efficacy against homologous and heterologous challenge (onset of immunity) of a live attenuated L. interrogans vaccine in dogs. The live attenuated L. interrogans vaccine is generated in Example 4 where the FliM gene encoding fliM protein is deleted.

[0107] Commercial source beagles are randomized to two groups. Dogs in Group A are vaccinated twice subcutaneously, on D0 and D28, with 1 mL of the live L. interrogans vaccine containing 10 7< lepo / ml of the attenuated L. interrogans strain. Dogs in Group B (control group) are not vaccinated.

[0108] In one study, on D42 (T0), each dog in groups A and B is challenged with a virulent strain L. interrogans serogroup Australis (homologous). In another study, on D42 (T0), each dog in groups A and B is challenged with a virulent strain L. interrrogans serogroup Icterohaemorrhagiae (heterologous). During the post challenge period the dogs are monitored daily for mortality, clinical signs (general condition, dehydration, ocular signs, vomiting, diarrheoa and cutaneo-mucosal signs), body temperature and weighing. Blood and urine are collected regularly throughout the vaccination phase for serological, hematobiochemical, leptospiremia, and leptospiruria analysis. Liver and kidneys are collected post mortem for analysis of the presence of bacteria as well as for tissue lesions observations. Surviving dogs in Group A and B are euthanized on T28.

[0109] The efficacy results show that the global clinical scores are significantly lower in the vaccinated dogs versus controls. Similarly the global biochemical and haematological scores, number of positive blood cultures per dog (leptospiremia), number of positive urine cultures per dog (leptospiruria), number of dogs with positive kidney culture and the number of dogs with kidney lesions are significantly lower in vaccinated groups when compared to controls.Conclusion

[0110] The results demonstrate the live attenuated L. interrogans vaccine (fliM gene deleted) reduces mortality, clinical signs of the disease, infection, urinary excretion, renal carriage and renal lesions upon homologous virulent L. interrogans serogroup Australis and heterologous virulent L. interrogans serogroup Icterohaemorrhagiae challenges performed two weeks after the 2 nd< injection of primary vaccination and provide protection against homologous challenge and cross protection against heterologous challenge.

Claims

1. An isolated attenuated Leptospira interrogans, wherein the Leptospira interrogans comprises a mutated fliM gene comprising SEQ ID NO: 14.

2. A composition or vaccine comprising the attenuated Leptospira interrogans of claim 1.

3. The composition or vaccine of claim 2, wherein the composition or vaccine further comprises one or more pharmaceutically or veterinarily acceptable carrier, vehicle, or excipient.

4. The attenuated Leptospira interrogans of claim 1, or the composition or vaccine of claims 2 or 3, for use in a method of vaccinating an animal, or for inducing an immunogenic or protective response against Leptospira infection, comprising at least one administration of the composition, the vaccine or the attenuated Leptospira interrogans, optionally wherein the animal is a canine or feline.