WO2007070996A2 - Liga and ligb proteins (leptospiral ig-like (lig) domains) for vaccination and diagnosis - Google Patents

Abstract

The present invention refers to isolated DNA (deoxyribonucleic acid) molecules that encode for LigA and LigB proteins. The invention further refers to the use of fragments of these proteins in the diagnosis, and formulation of vaccines for prevention of infections caused by Leptospira in humans and animals.

Description

LigA and LigB Proteins (Leptospiral Ig-like (Lig) domains) for vaccination and diagnosis Field of Invention

The present invention refers to isolated DNA (deoxyribonucleic acid) molecules that encode for LigA and LigB proteins. The invention further refers to the use of fragments of these proteins in the diagnosis, and formulation of vaccines for prevention of infections caused by Leptospira in humans and animals. Basis for the Invention

Leptospirosis is a zoonosis caused by Leptospira spp. Pathogenic bacteria, capable of affecting different animal species, including man. Rodents (rats and mice) are the main leptospirosis transmitters, although any infected animal can transmit the disease. Dogs, cats, bovines and swine are just a few examples.

Leptospires are aerobic spirochetes that are divided into two species: L. interrogans and L. biflexa. The complex L. interrogans sensu lato contains more than 200 serovars grouped in 25 serogroups . Four of these serovars account for the majority of cases of the disease in man, namely; Icterphaemorrhagiae, Canicola, Pomona and Autumnalis .

Human infection caused by Leptospira results from direct or indirect exposure to infected animal urine. There are less important transmission routes such as the handling of animal tissues and ingestion of contaminated water or food. Penetration of the Leptospira microorganism likely occurs through skin lesions in the mouth, nose and eye mucous membranes, and may occur through uninjured skin, when immersed in water for a long period.

Leptospirosis is characterized by a wide spectrum of clinical symptoms ranging from non-apparent infection to a fulminating, fatal disease. Leptospirosis is typically a biphasic disease. In the leptospiremic or septicemic phase, which lasts from four to nine days, initial symptoms are headache, generally frontal, myalgias, more intense in the calf muscles and lumbar regions, 39°C fever with chills, anorexia, nausea, and vomiting. In the second or immune phase immunoglobulin M (IgM) type antibodies appear, which determine the formation of immunocomplex circulating that may cause meningitis, uveitis and circulatory collapse, among other disorders. The duration and clinical manifestations of this phase are very variable.

Leptospirosis diagnostic confirmation is by direct detection of microorganisms in the blood or urine

(leptospiremia and leptospiruria) , by serologic tests or by microorganism isolation in an inoculated animal. Macroscopic seroagglutination a study involving IgM) and microscopic seroagglutination (a study involving IgG) are fundamental to diagnosis of the disease.

However, human and animal leptospirosis control is made difficult by the current lack of suitable diagnosis tools. The standard serologic test, known as microscopic seroagglutination test (MAT) , is inadequate for rapid identification as this test can only be carried out in reference laboratories and requires paired serum samples for achieving sufficient sensitivity. The dependence on the MAT test result causes delays in establishing the cause of the onset in several cases. ELISA (Enzyme-Linked Immunosorbent Assay) test and other serologic quick tests have been developed based on Leptospira whole-cell antigen preparations for use as an alternative method for classifying the infection by leptospires, although it is still necessary to conduct a MAT test for case confirmation. There are reports of diagnostic tests by polymer chain reaction (PCR) , based on real-time PCR, which can differentiate between pathogenic and saprophytic Leptospira species, but these tests have not yet been thoroughly evaluated. Recombinant antigen-based serologic tests are widely used for identifying infections caused by spirochetes in Lyme disease and syphilis, but the use of recombinant proteins for the serodiagnosis of leptospirosis has not been widely investigated. It is known from a state of technique that a flagella antigen immuno-capture assay was used for the serodiagnosis of bovine leptospirsosis

[Bughio, N.I., M. Lin, and O. P. Surujballi, Use of recombinant flagellin protein as a tracer antigen in a fluorescence polarization assay for diagnosis of leptospirosis. Clin Diagn Lab Immunol, 1999. 6(4): p. 599- 605] . A recombinant heat-shock protein, Hsp58, showed a high degree of reactivity in an ELISA with small number of human leptospirosis case samples [Park, S. H., B. Y. Ahn, and M.J. Kim, Expression and immunologic characterization of recombinant heat shock protein 58 of Leptospira species: a major target antigen of the humoral immune response. DNA Cell Biol, 1999. 18(12): p. 903-10]. The use of LipL32 and GroEL protein-based ELISA tests has been reported. However, the results using recombinant proteins are not encouraging [Flannery, B., et al., Evaluation of recombinant Leptospira antigen-based enzyme- linked immunosorbent assays for the serodiagnosis of leptospirosis. J Clin Microbiol, 2001. 39(9): p. 3303- 3310] .

Furthermore, there are no effective mechanisms for leptospirosis control and prevention. Control is difficult to implement due to the long-term survival of pathogenic leptospires in soil and water. Current efforts are directed toward protective immunization as an effective mechanism against leptospirosis. Currently available vaccines are based on whole-cell or membrane preparations of pathogenic Leptospira [Martinez, R., et al . , Efficacy and safety of a vaccine against human leptospirosis in Cuba [in Spanish] . Rev Panam Salud Publica, 2004. 15(4): p. 249-55] [Yan, Y., et al . , An evaluation of the serological and epidemiological effects of the outer envelope vaccine to leptospira. J Chin Med Assoc, 2003. 66(4): p. 224-30] which apparently induce protective responses, through the induction of antibodies against lipopolysaccharide [Faine, S. B., et al., Leptospira and leptospirosis. 2nd ed. 1999, Melbourne, Australia: MediSci] . However, these vaccines do not induce long-term protection against infection. Furthermore, they do not provide cross-protective immunity against serovars that are not included in the vaccine formulation [Rodriguez, A. G., et al., [Microbiological characterization of candidate vaccine strains of Ballum serogroup Leptospira interrogans] . Rev Cubana Med Trop, 2003. 55(3): p. 146-52]. The large number of pathogenic serovars (>250) and the high cost of producing a multi- serovar vaccine have been major limitations in developing efficacious vaccines through strategies based on whole-cell or membrane preparations.

The mechanism of pathogenesis in leptospirosis, as in other spirochete diseases such as Lyme disease and syphilis, relies on the pathogen's ability to disseminate widely within the host during the early stages of infection. Membrane-associated leptospiral proteins are presumed to mediate interactions that enable entry and dissemination through host tissues. Putative surface- associated virulence factors serve as vaccine candidates in strategies that induce responses to these factors, blocking dissemination in the host. Furthermore, membrane-associated proteins are accessible to the immune response during host infection and therefore constitute targets for immune protection through mechanisms such antibody-dependent phagocytosis and complement-mediated killing. Production of these antigen targets as recombinant proteins offers a cost-effective approach for protective immunization against leptospirosis using a subunit vaccine.

In addition, selection of surface-associated targets that are conserved among pathogenic leptospires can avoid the limitations encountered with currently available whole- cell vaccine preparations.

A major limitation in the field of leptospirosis has been identifying surface-associated and host-expressed proteins with conventional biochemical and molecular methods . From the genome sequence of the spirochete Borrelia burgdorferi, more than 100 surface associated lipoproteins were identified. The genomes of L. interrogans serovars Lai and Copenhageni were recently sequenced by China and Brazil. [Nascimento, A. L., et al., Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J Bacterid, 2004. 186(7): p. 2164-72] [Ren, S. X., et al . , Unique physiological and pathogenic features of Leptospira interrogans revealed by whole-genome sequencing. Nature, 2003. 422(6934): p. 888-93]. Based on predicted coding sequences analysed for surface exposed domains, Leptospira have over 260 surface-associated proteins [Nascimento, A. L., et al . , Comparative genomics of two Leptospira interrogans serovars reveals novel insights into physiology and pathogenesis. J Bacterid, 2004. 186(7): p. 2164-72]. At present, 16 surface-associated proteins have been characterized through isolation of membrane extracts, purification and cloning of these target proteins [Matsunaga, J., et al., Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily. MoI Microbiol, 2003. 49(4): p. 929-45], [Cullen, P.A., et al . , LipL21 is a novel surface- exposed lipoprotein of pathogenic Leptospira species. Infect Immun, 2003. 71(5): p. 2414-21], [Haake, D.A. and J. Matsunaga, Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins. Infect Immun, 2002. 70(9): p. 4936- 4945.] and [Haake, D.A., et al., Leptospiral outer membrane proteins OmpLl and LipL41 exhibit synergistic immunoprotection. Infect Immun, 1999. 67(12): p. 6572-82].

Immunization with recombinant proteins for several identified targets, LipL32, OmpLl and LipL41, induce partial, but not complete, protective responses. [Haake, D.A., et al., Leptospiral outer membrane proteins OmpLl and LipL41 exhibit synergistic immunoprotection. Infect Immun, 1999. 67(12): p. 6572-82], [Haake, D.A., et al., The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun, 2000. 68(4): p. 2276-85], [Branger, C, et al., Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination. Infect Immun, 2001. 69(11): p. 6831-6838] e [Branger, C, et al . , Protection against Leptospira interrogans Sensu Lato Challenge by DNA Immunization with the Gene Encoding Hemolysin-Associated Protein 1. Infect Immun, 2005. 73(7): p. 4062-4069]. LigA and LigB protein leptospirosis obtained from L. interrogans sorovar Manilae (UP-MMC-NID) induced a completely protective immune response in a mouse model of lethal leptospirosis [Koizumi, N. and H. Watanabe, Leptospiral immunoglobulin-like proteins elicit protective immunity. Vaccine, 2004. 22(11-12): p. 1545-52]. However, this model is not ideal for several reasons; a large number of leptospires are necessary to cause a lethal infection, additionally the mouse lineage used contained a mutation in the TLR4 allele (C3H/HeJ) . In addition to the aforementioned literature, we also cite document WO03/098214, published on 11/27/2003, owned by the Oswaldo Cruz Foundation and document WO2004/032599, published on 04/22/2004 and owned by the Cornell Research Foundation Inc. Both patent applications claim Lig proteins for use in diagnosis and vaccines, but neither document presents evidence of protection using the Lig proteins. Additionally, these documents do not refer to Lig protein fragments, neither do they present sequences of these fragments for use in diagnosis, nor in a vaccine formulation for combating leptospirosis. Summary of the Invention

The first objective of the present invention deals with the DNA molecules of Leptospira spp. and the polypeptides encoded by these molecules, both containing Leptospiral immunoglobulin-like (Lig) repetitive domains.

A further purpose of the present invention refers to the isolated or combined use of polypeptides derived from LigA or LigB, the use of antibodies against these polypeptides and the use of polynucleotides that encode for LigA e LigB, with pharmaceutically acceptable vehicles for treating or preventing a Leptospira infection.

Another purpose of the present invention is to provide a kit for the diagnosis of Leptospira spp. based on recombinant protein fragments of LigA and LigB. Description of Figures

Figures 1 and IA shows an outline of the lig genes and the cloning strategy used. Figures 2 and 2A show the purified recombinant polypeptides from the (rLig) Lig proteins.

Figure 3A shows an ELISA evaluation of the immune response in hamsters immunized with LigA polypeptides. Figure 3B shows an ELISA evaluation of the immune response in hamsters immunized with LigB polypeptides.

Figure 3C shows an ELISA evaluation of the immune response in hamsters immunized with LigA polypeptides.

Figure 3D shows an ELISA evaluation of the immune response in hamsters immunized with LigB polypeptides.

Figures 4 and 4A show the reaction of patient sera with recombinant Lig polypeptides (rLig) .

Figure 4B shows the reaction of sera, from patients infected with Leptospira interrogans Copenhagen!, with recombinant Lig polypeptides (rLig) from Leptospira interrogans Canicola. Detailed Description of Preferred Embodiments

The present invention describes two L. interrogans- derived DNA molecules, which encode proteins, designated herein as LigA and LigB, which have a high molecular weight, 128 and 200 kDa respectively, such molecular weight being based on the amino acid sequence of the polypeptides.

Both proteins of the present invention have 12 or 13 tandem repeat sequences (in series) of approximately 90 amino acids. The repeat sequences from LigA and LigB are highly conserved, that is they have an amino acid sequence identity of >90% and belong to the family of Bacterial immuno£lobulin-like (Big) proteins. These Big regions are found in virulence factors of other pathogenic bacteria. The DNA molecules of the present invention that encode for Leptospira proteins with Lig domains, herein called "HgA" and "HgB", can be inserted as heterologous DNA into expression vectors for producing peptides and polypeptides. Recombinant polypeptides can be purified from surrogate hosts transformed with such expression vectors. LigA and LigB-derived polypeptides are serological markers for active and past infection since sera from leptospirosis patients and animals infected or immunized with pathogenic Leptospira spp. recognize isolated Lig polypeptides.

Furthermore, LigA and LigB polypeptides from recombinant or native antigen preparations are immunogenic. Antibodies obtained from experimental animals immunized with purified recombinant LigA and LigB polypeptides recognize native antigen from Leptospira spp., and are useful for detecting pathogenic spirochetes in samples from subjects with a suspected infection.

It is also important to emphasize that LigA and LigB polypeptides of the present invention induce an immune response against pathogenic spirochetes.

LigA and LigB-derived polypeptides, antibodies to these polypeptides and polynucleotides that encode for LigA and LigB may be used alone or combined with a pharmaceutically acceptable carrier to treat or prevent infection with Leptospira. Since Big domains are present in proteins associated with virulence in other bacterial pathogens, these moieties may be used to treat or prevent infections unrelated to those caused by Leptospira spp. Lig polynucleotides sequences are listed and were analysed using a bioinformatics program (AlignX - Vector

NTI V. 8, Invitrogen) to determine the level of conservation of the Hg polynucleotides in pathogenic Leptospira spp.

An embodiment of this invention identifies lig polynucleotides and polypeptides thereof that are: (1) recognised by sera from subjects infected with pathogenic Leptospira and (2) able to stimulate an immune response in laboratory animal models. In addition, a method is provided to identify polynucleotides, using bioinformatics software, with the potential to be recognised by sera from subjects infected with Leptospira spp. or other pathogenic bacteria. A third embodiment of the present invention is to provide pharmaceutical composition (s) for inducing protective immune responses in subjects to pathogenic spirochetes, where said compositions comprise an immunogenically effective amount (10 - 100 meg) of one or more selected antigens among the group consisting of LigA and LigB polypeptides or polypeptides with functionally equivalent sequences in a pharmaceutically acceptable vehicle. In addition, a method is provided for producing an expression vector containing HgA and HgB polynucleotides and obtaining substantially purified polypeptides derived from these sequences.

In a fourth embodiment, the present invention provides a method for identifying polypeptides with functionally equivalent sequences, which includes the stages of: (a) incubating components comprising of the compound and LigA or LigB polypeptide or polypeptides with functionally equivalent sequences under conditions sufficient to allow the components to interact; and (b) measuring the binding of the compound to the LigA or LigB polypeptide or polypeptides with functionally equivalent sequences. Preferably, the method is based on serodiagnosis utilizing serum from a subject with a suspected active or past infection with Leptospira spp. or other related bacterial pathogen.

In a fifth embodiment, the invention provides a method for detecting pathogens in a sample which includes contacting a sample suspected of containing a pathogenic spirochete with a reagent that binds to the pathogen- specific cell component and detecting binding of the reagent to the component. In a specific aspect of the method, the reagent that binds to the pathogen-specific cell component is an oligonucleotide for the identification of HgA and HgB polynucleotides. Further, in another specific aspect, the reagent that binds to the pathogen- specific cell component is an antibody against the LigA or LigB polypeptide or polypeptides with functionally equivalent sequences.

In a sixth embodiment of the present invention, a kit is provided for the detection of: (1) LigA and LigB polypeptides or polypeptides with functionally equivalent sequences; (ii) HgA and HgB polynucleotides; or, (iii) antibodies that bind to LigA or LigB polypeptides or polypeptides with functionally equivalent sequences. The present invention will be now described with reference to the Examples, which are should not be considered as limitative of the present invention. Example 1 Amplification, Cloning, Sequencing and Analysis of Hg Genes .

This Example 1 illustrates the identification and isolation of the Hg genes. Hg polynucleotides in the genome of pathogenic Leptospira spp. were amplified using PCR technique. Oligonucleotides were designed with the following sequence: ligA Forward: SEQ ID NO: 1 5 ' -CAATTAAAGATCGTTATATACGATAC

ligA Reverse: SEQ ID NO: 2 5 ' -AAGAAGAAACGATCACAAGGTC

ligB Forward: SEQ ID NO: 3

5 ' -CAAAGTTGTATGTCTTGGCCACT

ligB Reverse: SEQ ID NO: 4

5 ' -TTATTGATTCTGTTGTCTGTAAATTTTG PCR was performed with purified genomic DNA isolated from selected pathogenic Leptospira spp. The QIAquick PCR

Purification Kit (Qiagen) was used to obtain purified PCR products. PCR products were cloned using a TOPO kit

(Invitrogen) and sequenced using a MegaBase 48 capillary sequencer (Amersham) . Sequencing was carried out using vector-based primers and primer walking, each base was sequenced a minimum of two times in both the forward and reverse strands.

HgA polynucleotides were translated into amino acid sequences of LigA polypeptides and alignment of amino acid sequences was performed using the bioinformatics program,

Vector NTI v.8 (Invitrogen) , from L. interrogans serovars

Copenhagen! (SEQ ID No. ), Manilae (SEQ ID No. 5), Pomona C

(SEQ ID No. 8) and L. kirschneri serovar Grippotyphosa (SEQ

ID No. 7), which are highly conserved, with >75% amino acid sequence identity. The conservation level of LigA between individual strains is described in Table 1.

HgB polynucleotides were translated into amino acid sequence of LigB polypeptides and alignment of amino acid sequences was performed using Vector NTI v.8 (Invitrogen) from L. interrogans serovars Copenhageni (SEQ ID No. 9), Lai (SEQ ID No. 10), Manilae (SEQ ID No. 11), Pomona (SEQ ID No. 13) and L. kirschneri serovar Grippotyphosa (SEQ ID No. 12), which showed to be highly related, with >85% amino acid sequence identity. The conservation level of LigB between individual strains is described in Table 1.

Table 1. Level of identity between the Lig proteins of selected pathogenic Leptospira, spp.

LigB from JJ. interrogans serovars Copenhageni, Lai, Canicola, Pomona, Manilae and L. kirschneri serovar Grippotyphosa were highly related, with >85% amino acid sequence identity

Big domain repeat regions are conserved in all LigB polypeptides found in pathogenic Leptospira spp. The Manilae serovar sequence deposited in Genbank (AB098517), does not appear to have a carboxy-terminal domain due to a stop codon in the HgB polynucleotide sequence. It is believed that the Manilae serovar sequence deposited in Genbank is strain specific; DNA downstream 3' of the stop codon encodes for part of a carboxy-terminal domain. Example IA

Amplification, Cloning, Sequencing and Analysis of the l±g

Genes .

This example illustrates the identification and isolation of the Hg genes. The Hg polynucleotides from the genome of pathogenic Leptospira spp. were amplified by- using PCR. The oligonucleotides used were as cited in Example 1. All procedures and evaluations of the sequencing were performed in accordance with Example 1. The HgA polynucleotides were translated and the alignment of the sequences of amino acids was performed using ClustalW (AlignX, Vector NTI ver 8, Invitrogen) for the amino acid sequences of LigA polypeptides from L. interrogans serovars Copenhageni (SEQ ID NO.5), Canicola Kito (SEQ ID NO.32) and Pomona Kennewickii PO-Oβ-047 (SEQ ID NO.33), which appeared highly conserved, with >81% identity at the amino acid level. The level of conservation of LigA between the strains is described in Table IA.

The polynucleotides of HgB were translated and the alignment of the sequences of amino acids was performed using ClustalW (AlignX, Vector NTI ver 8, Invitrogen) for the amino acid sequences of LigB polypeptides from L. interrogans serovars Copenhageni (SEQ ID NO.9), Canicola Kito (SEQ ID NO.34), Pomona Kennewickii PO-06-047 (SEQ ID NO.35), L. borgpetersenii Hardjo-bovis JB197 (SEQ ID NO.36) and L. weilii Hebdomadis Ecochallenge (SEQ ID NO.37), which appeared highly conserved, with >60% of identity in the sequence of amino acids. The level of conservation of LigB and LigA between the strains is described in Table IA. Table IA: Identity level between the Lig proteins of selected pathogenic Leptospira spp.

Cop = L. interrogans Copenhageni Fiocruz Ll-130 (deposited in Genbank)

Gri = L. kirschneri Grippotyphosa RM52 (deposited in

Genbank)

Man = L. interrogans Manilae (deposited in Genbank)

Can = L. interrogans Canicola Kito

Pom = L. interrogans Pomona Kennewickii

PomC = L. interrogans Pomona (deposited in Genbank)

00

Cop = L. interrogans Copenhageni Fiocruz Ll-130 (deposited in Genbank)

Can = L. interrogans Canicola Kito (UFPeI)

Lai = L. interrogans Lai (deposited in Genbank)

Pom = L. interrogans Pomona Kennewickii (UCLA)

PomC = L. interrogans Pomona (deposited in Genbank by Cornell grp)

Man = L. interrogans Manilae (deposited in Genbank)

Gri = L. kirschneri Grippotyphosa RM52 (deposited in Genbank)

Heb = L. weilii Hebdomadis Ecochallenge (UCLA)

Har = L. borgpetersenii Hardjo-bovis JB197 (deposited in Genbank)

Example 2

Construction of vectors for expression of recombinant

LigANI and LigBNI Protein

Identified polypeptide regions from LigA. and LigB as described in Example 1 and the corresponding Hg polynucleotides were amplified, cloned into E. coli expression vectors, expressed and purified as described herein.

Oligonucleotides were designed for amplification of specific regions of the sequences encoding regions HgA (SEQ ID NO. 14) and HgB (SEQ ID NO. 15) of L. interrogans. The region within LigA, including the 8th to 13th repeat domains, corresponding to positions 625-1224 (SEQ ID NO.9) of the amino acid sequence. The region within LigB, including the 8th to 12th repeat domains corresponded to positions 625-1259 (SEQ ID NO.9) of the amino acid sequence. Oligonucleotides were designed based upon sequence of Jj. interrogans (as described in patent application W003/098214 (PCT/BR02/00072) and their sequences are: SEQ ID No. 16

LigANIF 5 ' -CACCTCCTCTAATACGGATATT SEQ ID No. 17 LigANIR 5 ' -GGTCTAGATTATGGCTCCGTTTTAATAGAGG SEQ ID No. 18

LigBNIF 5 ' -CACCTCCTCTAATACGGATATT

SEQ ID NO. 19

LigBNIR 5 ' -TTACACTTGGTTTAAGGAATTAC These oligonucleotides were used in a PCR reaction to amplify polynucleotide fragments corresponding to the

LigANI and LigBNI regions. These 1,800 bp (SEQ ID NO. 20) and 1,905 bp (SEQ ID NO.21) PCR products were ligated into a commercial vector, for example pETIOOtop.

The cloned insert was sequenced to confirm that the polynucleotide was cloned in the correct orientation and reading frame for protein expression. The predicted positions of these regions, designated LigANI and LigBNI, are shown the schematic of the Hg genes in Figures 3a, 3b, 3c e 3d and Table 2. Example 2A.

Construction of vectors for expression of recombinant LigB fragments BigI3 and pRGY Protein Oligonucleotides were designed for amplification of specific regions of the sequences encoding regions of LigB L. Interrogans. One of the regions to be amplified included the 2nd — 6th Big repeat domains corresponding to positions 131-649 (SEQ ID NO.9) of the L. interrogans LigB amino acid sequence. The other region to be amplified included the Tttt to 12th repeat domains corresponding to positions 579-1126

(SEQ ID NO-.9} of the L_^ Interrogans LigB amino acid sequence. Oligonucleotides were designed based upon sequence of L_* interrogans fas described in. patent application WO03/098214 (PCT/BRQ2/00072) and their sequences are: Bigl.3:

SEQ IB NO..24 BigL3 -45B-1 S'-ATGGGACTCGAGATTACCGTTACACCAGCCATT-S' SEQ ID NO. 25

BigL3 -45B-2 5'-ATTCCATGGTTATCCTGGAGTGAGTGTATTTGT-S' pRoy2 :

SEQ ID NO. 26

BigL3(Xb) 5'-TTTTCTAGAACTGCTGCAAAACTTGTTGAAATAC - 3^X

SEQ ID NO. 27

BigL3 (Hd) 5 ' -CGGAAGCTTATTATTGAACCGTCGGAGCTATCGTGTC -3 ^N

These oligonucleotides were used in PCR reactions to amplify polynucleotide fragments corresponding to BigL3 and pRoy region. The 1,557 bp(SEQ ID NO.28) and 1,644 bp (SEQ ID NO.29) PCR products were digested and ligated into the expression vector pAE, digested with the appropriate restriction enzymes. The cloned inserts were sequenced to confirm that the polynucleotides were inserted in the correct orientation and reading frame for protein expression. The predicted positions in amino acid sequence of these regions, designated BigL3 and pROY, are shown in the schematic of the Ligs genes in Figure 1 and Table 2.

Table 2 : Recombinant; Ligs proteins

a) Repeat domains are in brackets b) Amino acid coordinates in L. interrogans (Copenhageni Ll-130) and amino acid size in brackets. c) Molecular weight in recombinant protein Daltons Example 2B Expression and Purification of Recombinant Lig Proteins .

Plasmids based on pAE vector containing lig polynucleotide sequences were used to transform BL21(DE3) cells; plasmids based on pETlOO containing lig polynucleotide sequences were used to transform BL21star cells. Expression clones were selected after approximately 16 hours growth in Petri dishes containing LB Agar (Lucia Bertani) at a temperature of 37⁰C. The expression of lig polynucleotides in mid-log growth phase LB cultures was induced by addition of IPTG (isopropyl-beta-D tiogalactopiranoside) to a final concentration of 1 mM. The cultures were incubated for an additional 2-4 hours and pellets harvested by centrifugation. Pellets were resuspended and stored at -20⁰C until purification.

The pellet containing recombinant protein was solubilised in 6-8 M urea/wash buffer (20 mM NaH₂PO₄, 5 mM imidazole, 0.5 M NaCl, pH 8.0) and Hisδ-tagged recombinant protein purified by employing the immobilised metal chelate affinity chromatography technique (IMAC) on Sepharose columns (Amersham) chelated with Ni²⁺ ions. After binding to the column, unbound proteins were removed by washing with 6-8 M urea in wash buffer (20 mM NaH₂PO₄, 0.5 M imidazole, 0.5 M NaCl, pH 8.0). Fractions were screened for Lig recombinant protein by SDS/PAGE analysis. Reactions containing the protein of interest were pooled and slowly dialysed with a PBS solution, during a 15-day period to remove urea and imidazole. Purified recombinant Lig (rlάg) protein polypeptides are represented in Figure 2. Example 2C Construction of vectors for the expression of recombinant proteins LigA domain 7-11 (625-945aa) , LigB domains 7-11 (625-947aa) and LigB domains 11-12 (945-1257aa) .

The regions of polypeptides identified from LigA and LigB, as described in Example 1, and the corresponding polynucleotide Hg were amplified, cloned in vectors of expression of E. coll, expressed and purified as described herein.

Oligonucleotides were designed for the amplification of specific regions from the sequences that encode for the regions HgA (SEQ ID NO. 14) and HgB (SEQ ID NO. 15) of L. interrogans. The region within HgA, including from the seventh to the eleventh repetitive domain, corresponding to the positions 625-945 (SEQ ID NO.38) of the sequence of amino acids. The regions within HgB, including from the seventh and eleventh to the twelfth repetitive domains, corresponding to the positions 625-947 (SEQ ID NO.39) and 945-1257 (SEQ ID NO.40) of the LigB amino acid sequence. The oligonucleotides designed were based upon the sequence of L. interrogans, as described in the patent application WO03/098214 (PCT/BR02/00072) and their sequences are: SEQ ID No. 41

LigA (7-1I)F 5' -CACCCTTACCGTTTCC&ACACAAAC SEQ ID No.42 LigA (7-11) R 5'- TTAAGCTATTCTTGCCGGAGTAAC SEQ ID No . 43

LigB ( 7 -1I ) F 5 ' - CACCTCCTCTAATACGGATATT

SEQ ID NO . 44

LigB (7-1I)R 5'- TTAAATGGAATCTAACGTGGCAG SEQ ID No. 45

LigB (11-12)F 5'~ CACCATTAAAATCAATCCAGTCAAC SEQ ID NO. 46

LigB (11-12)R 5'~ TTACACTTGGTTTAAGGAATTAC

These oligonucleotides were used in accordance with the steps of Example 2 to amplify the fragments of polynucleotides corresponding to the regions of HgA for the domains 7-11 and HgB domains 7-11 and 11-12. These PCR products were 945 bp (SEQ ID NO.47), 972 bp (SEQ ID NO.48) and 910 bp (SEQ ID NO.49) respectively, and the following procedures were performed as described in Example 2.

The aforementioned positions in the amino acid sequences of this region, designated LigA 7-11 (SEQ ID

NO.40), LigB 7-11 (SEQ ID NO.41) and LigB 11-12 (SEQ ID

NO.42), are shown in the schematic of the Hg genes in Figures IA and Table 2A.

The Expression and Purification of Recombinant Lig Proteins were performed in accordance with the stages and procedures described in Example 2B. Table 2A

Recombinant LigA and LigB proteins

Molecular

Protein³ Amino acids^b Vector Weight⁰

LigA (7-ll) 625-945 (320 ) 32 , 525 pETlOO

LigB ( 7-ll ) 625-947 ( 322 ) 33 , 428 pETlOO

LigB ( 11-12 ) 945-1257 (312 ) 32 , 932 pETlOO

a) Repetitive domains are in parentheses b) Coordinates of amino acid in L. interrogans (Copenhageni Ll-130) and size of amino acid in parentheses. c) Molecular weight in Daltons of the recombinant protein Example 2D

Construction of a vector for the expression of polypeptide Fragments of LigA and LigB from L. Interrogans serovar Canicola and the recognition of these rLig fragments by antibodies of patients infected with L. interrogans sorovar Copenhageni

This example illustrates and reinforces the data of Example IA that shows the high level of identity between the LigA and LigB proteins of Leptospira spp. through the recognition of epitopes common to the recombinant polypeptides LigBrep, LigANi and LigBNi from different serovars by antibodies in the sera of patients infected with L. interrogans Copenhageni. The cloning and expression were performed in accordance with the stages and procedures described in Example 2. The polynucleotides related to the sequences of HgANi (HgANIC) and HgBNi [HgBNiC) of L. interrogans Canicola were obtained by PCR using the oligonucleotides for LigANI (SEQ ID NO.16 and 17), and for LigBNI (SEQ ID NO.18 and 19). The oligonucleotides for amplification of the region of bigL3rep [bigL3repC) were outlined based upon the sequence of L. interrogans serovar Copenhageni, as described in patent application WO03/098214 (PCT/BR02/00072) and their sequences are: SEQ ID No. 50 BigL3 F δ'-CACCATTACAGTTACACCΑGCCACT SEQ ID No. 51 BigL3 R 5'- CTATCCTGGAGTGAGTGTATTTGTAAT

These products of PCR: HgANiC of 1,803 bp (SEQ ID NO.52), HgBNiC of 1,899 bp (SEQ ID NO.53) and bigL3repC of 1,560 bp (SEQ ID NO.54) were cloned into a commercial vector, for example the pETlOO/TOPO. The insert was sequenced to confirm its identity with the sequences of the HgA (SEQ ID NO.55) and HgB (SEQ ID NO.56) genes of L. interrogans Canicola, and also its orientation and reading frame. The aforesaid positions and size in sequences of amino acids of these regions, designated as LigANIC (SEQ ID NO.57), LigBNIC (SEQ ID NO.58) and BigL3repC (SEQ ID NO.59) are shown in the scheme of the Hg genes in Figures 1, Table 2B. Table 2B

Recombinant Canicola LigA and LigB proteins

Protein^a Amino acids^b Molecular Vector Weight⁰

BigL3 (1-6) 131-650 (519) 53.722 PETlOO

LigBNi (8-12) 625-1257 (632) 66.296 pETlOO

LigANI (8-13) 625-1224 (599) 62.913 PETlOO The expression and purification of the recombinant Lig proteins were performed in accordance with the stages and procedures described in Example 2B.

The purified rLig polypeptides (ca. 1.5 meg/band) obtained in Example 2C were submitted to electrophoresis in 12% sodium dodecyl sulphate/polyacrylamide gel (SDS/PAGE) by using an intermittent buffer system, and were transferred to nitrocellulose membranes (Osmomics) as described [Guerreiro, H., et al., Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans. Infect Immun, 2001. 69(8): p. 4958-4968] . The membrane of nitrocellulose was blocked with TBST (100 mM Tris, 0.9% NaCl, 0.5% Tween20) , 5% skimmed milk, and then it was incubated for 1 hour with serum from patients with laboratory confirmed leptospirosis. As control experiments, incubations with the serum of healthy Brazilian individuals were performed. The sera were diluted to 1:25 or 1:100 before use. After washing with TBST, the membranes were incubated with secondary antibody, anti-IgG and/or IgM conjugated with alkaline phosphatase (Sigma) , diluted at 1:1,000 or 1:40,000 for 1 hour. Antigenic- antibody complexes were detected by colour reaction with an alkaline phosphatase detection kit (Biorad) . The finding of any visible coloration of the band corresponding to a rLig polypeptide in the immunoblot test was considered as a positive reaction. The pooled sera from patients infected with leptospirosis highly recognized the LigANI, LigBNI and BigL3 rep proteins from L. interrogans Canicola when compared to the same rLig polypeptides from L. interrogans

Copenhageni, as shown in Figure 4A.

Example 3

Induction of a Protective Immune Response against Leptospira, in Animals immunized with recombinant Lig polypeptides in Freund Adjuvant

This example illustrates that a protective immune response can be induced by recombinant LigA NI and LigB NI polypeptides derived from L. interrogans by immunization with recombinant Lig proteins (rLig) .

In this example, Golden Syrian hamsters were used as a model of lethal leptospirosis.

In this example purified recombinant proteins obtained in Example 2A were used. Groups of 6-10 hamsters were immunized subcutaneously with 80 meg of rLigANI and rLigBNI polypeptide prepared in

Freund' s complete adjuvant (Sigma). Two weeks after the first immunization a booster dose of 40 meg of the same polypeptide in Freund' s incomplete adjuvant (Sigma) was administered subcutaneously to each animal (hamsters) . A control group of hamsters were immunized with Freund' s complete adjuvant, containing no protein, two weeks after the first immunization.

One week after the second immunization, the animals were challenged with 1,000 virulent leptospires

(representing 20 times the number of the leptospires required to cause death in 50% (LDs₀) of the infected animals) . Blood was collected from the animals before the first immunization (pre-immune sample) and after the boost with each rLig polypeptide (immune sample) . The study proceeded for up to 28 days after challenge with pathogenic L. interrogans. The primary end-point was animals' morbidity.

Table 3 below shows the ability of rLig polypeptides to induce a protective immune response. Table 3 shows the statistical representation of the protective immune response.

Table 3 - Vaccinal Efficacy of rLig polypeptides in Golden Syrian hamsters model for leptospirosis

From Table 3 above, it is observed that rLigA NI and rLigB NI polypeptides induced a significant protection in animals challenged with pathogenic Leptospira spp. compared to control groups immunized with Freund' s adjuvant only. The challenge was carried out using homologous L. interrogans spp._r in other words, with leptospires from which the Hg nucleotides were obtained. Immunization with a Lig polypeptide induced significant protection, ranging from 67-100% (Table 3), in the Golden Syrian hamster model described herein, compared to hamsters immunized with Freund' s adjuvant only. Example 3A

Induction of Protective Immune Response against Leptospira in Animals immunized with recombinant Lig polypeptides in Aluminium Hydroxide

This example illustrates that a protective immune response can be induced against recombinant LigA NI and LigB NI (rLig) polypeptides, by immunization with recombinant Lig proteins (rLig) , prepared with aluminium hydroxide adjuvant. In this Example 3A, (Golden Syrian) hamsters were used as a model of lethal leptospirosis. In this example purified recombinant proteins were obtained as described in Example 2B.

Groups of 6-10 hamsters were immunized subcutaneously with 80 meg of recombinant rLigANI and rLigBNI polypeptide prepared with aluminium hydroxide adjuvant. Two weeks after the first immunization a booster dose of 40 meg of the same polypeptide in aluminium hydroxide adjuvant was administered subcutaneously to each group of animal

(hamsters) . A control group of hamsters were immunized with aluminium hydroxide adjuvant with addition of negative control protein BSA (Bovine albumin) and the booster with aluminium hydroxide adjuvant with addition of a negative control protein BSA (Bovine albumin) two weeks after this first immunization. One week after the second immunization, the animals were challenged with 1,000 virulent leptospires

(representing 20 times the number of the leptospires required to cause death in 50% (LD₅₀) of the infected animals) .

Blood was collected from the animals before the first immunization (pre-immune sample) and after the boost with each rLig polypeptide (immune sample) . The study proceeded for up to 28 days after the challenge with pathogenic L. interrogans. The primary end-point was animals' morbidity.

Table 3A below shows the ability of rLig polypeptides to induce a protective immune response in challenged animals with pathogenic Leptospira spp. compared to control groups immunized with aluminium hydroxide only. Table 3B shows the statistical representation of the protective immune response. The challenge was carried out using homologous L. interrogans spp., in other words, with leptospires from which the Hg nucleotides were obtained. Immunization with a Lig polypeptide induced significant protection, 78% (Table 3B) , in the Golden Syrian hamster model described herein, compared to hamsters immunized with aluminium Hydroxide only.

Table 3A - Vaccinal Efficacy of rLig polypeptides prepared in aluminium hydroxide adjuvant in a Golden Syrian hamster model for leptospirosis

Example 3B

Induction of a Protective Immune Response against Leptospirosis in Animals immunized with recombinant Lig polypeptides in Aluminum Hydroxide and Polygene Adjuvants This example illustrates Example 3A with a greater number of formulations and combinations between the polypeptides, and reinforces and adds data that confirm the capacity of the recombinant LigA NI, LigB NI and BigL3 rep polypeptides (rLig) in inducing a protective immune response when they are separated or combined, in accordance with all procedures and stages described in Example 3A.

Table 3B below shows the capacity of rLig polypeptides and combinations among them to induce sterilizing immunity in the animals challenged with pathogenic Leptospira spp. compared with the control groups, which were immunized with Aluminum Hydroxide or Polygene adjuvants only. Table 3B shows the statistic significance of the protective immune response. The challenge was homologous, using L. interrogans spp., i.e., by using leptospires from which lig polynucleotides were obtained. Immunization by a Lig polypeptide induced significant protection in animals, 78- 100% (Table 3B) , in the Golden Syrian hamster model, which is described and compared with that of hamsters immunized with Aluminum Hydroxide only. Table 3B - Vaccinal efficacy of the polypeptides of rLig prepared in adjuvant of aluminum hydroxide and Polygene in a model of Golden Syrian hamsters for leptospirosis

Example 3C

Passive Protection Test

This example illustrates that the protective immune response stimulated by recombinant Lig polypeptides against Leptospira spp. is based upon the transfer of hyperimmune antibodies produced in rabbits, which were immunized with 03 intramuscular doses of 80 meg of recombinant LigA NI and LigB NI polypeptides, formulated and prepared in Freunds complete and incomplete adjuvant (Sigma) . Hamsters (Golden Syrian) were used in this example 3C as a model of lethal leptospirosis .

The purified recombinant proteins obtained in Example 2B were used in this example.

Groups of 10 hamsters were immunized intraperitoneally with 1 ml of anti-Lig hyperimmune serum from rabbits.

Twenty-four hours later the animals were challenged with

1,000 virulent leptospires (that represents 20 times the number of leptospires necessary to cause the death of 50%

(DL₅₀) of the infected animals) . The study continued until the twenty-eighth (28^th) day after the challenge with pathogenic L. interrogans . The primary endpoint was the mortality of the animals. Table 3C below shows the capacity of the anti-Lig hyperimmune sera from rabbits to induce a protective immune response based upon the transfer of antibodies. Table 3D shows the statistic representation of the protective immune response. Table 3C

It can be observed in Table 3D above that the hyperimmune sera produced by immunization with recombinant Lig polypeptides induced significant protection in the animals challenged with pathogenic Leptospira spp. compared with the control groups immunized with normal serum of rabbits. Each challenge was made using homologous L. interrogans spp., i.e. by using leptospires from which the lig polynucleotides were obtained. The passive immunization induced significant protection in animals, varying between 60-80% (Table 3D) in the Golden Syrian hamster model described in example 3A. Example 4

Immunogenicity of Recombinant: Lig Polypeptides in Immunized Hamsters This example demonstrates the level of antibodies induced by Lig polypeptides in subjects. Golden Syrian hamsters (4 weeks old) were immunized as described in Example 2. Animals' blood was collected before the first immunization (pre-immune sample) , and 7 days after the final immunization. Flat-bottomed polystyrene microtitre plates were coated at 4⁰C overnight with rLigB polypeptide

(as obtained in Example 2), 1 - 100 ng/well, in 0.1 M sodium carbonate and in pH 9.6. The plates were washed three times with PBST solution (PBS, 0.5% Tween20) . Each well was incubated with blocking buffer (PBST, 1% BSA) for 1 hour at room temperature of approximately 23⁰C. The wells were washed three times with PBST solution and stored at - 20⁰C temperature until use. Sera was serially diluted from 1:400 to a maximum in blocking buffer and incubated for 1 hour at 37⁰C with shaking. The wells were washed three times with PBST solution and the secondary antibody conjugate, anti-hamster IgG conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories) , at a dilution of 1:25,000, added to each well. Plates were incubated at 37⁰C for 1 hour with shaking. Wells were washed twice with PBST solution followed by a wash with PBS solution at room temperature approximately of 23⁰C with shaking. Wells were incubated with 50 mcl of 0.01% tetramethylbenzidine in substrate buffer (0.03% hydrogen peroxide, 25 mM citric acid, 50 mM Na₂HPO₄, pH 5.0) for 20 minutes in the dark at room temperature of approximately 23°C. Colour development was stopped with the addition of 25 mcl 1 M H₂SO₄. Colour development was assayed by measuring absorbance at 450 nm in a microplate reader (e.g. Tecan Genios) .

In the assays 100 ng/well of recombinant Lig polypeptide was used to coat the plate. Hamster sera titrations were performed at 1:400, 1:1,600, 1:6,400 and 1:25,600 dilutions.

Sera from individual hamsters immunized with the relevant recombinant Lig polypeptide were tested in duplicate and the mean absorbances at 450 nm were used in subsequent analyses. From Figure 3 we can observe that immunization with rLig fragments induces high antibody titres in hamsters. In Figure 3, Chart A represents seroreactivity for rLigA after immunization with this protein; Chart B represents seroreactivity for rLigB after immunization with this protein; Chart C represents hamster seroreactivity to Freund's adjuvant only (no protein) for the rLigA control; and Chart D illustrates hamster seroreactivity to Freund' s adjuvant only (no protein) for the rLigB control. Pre- immune sera (triangles) and immune sera (squares) were serially diluted to determine IgG response titration. Lig protein stimulated a response with a titre >l:25,600 in all tested sera. Recombinant Lig protein was used to coat ELISA wells (100 ng/well) . Mean absorbance values (OD 450nm) and standard deviations are represented in Charts A-D.

Next, examples of antibody detection against recombinant Liq proteins will be presented. The examples below illustrate two methods among several that utilize rLiq polypeptides to detect antibodies in animal samples. Furthermore, this example provides methods for a serodiagnostic kit for identifying infection in subjects suspected of harbouring infection. Example 5A Immunoblot Detection of Antibodies to Recombinant Lig Polypeptides in Samples from patients with Leptospirosis

Purified rLig polypeptide (ca. 1.5 meg/lane), obtained in Example 2C, was analysed in a 12% sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) using a discontinuous buffer system and the proteins transferred to nitrocellulose membranes (Osmomics). , as described in [Guerreiro, H., et al., Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans. Infect Immun, 2001. 69(8): p. 4958-4968] . The nitrocellulose membrane was blocked with TBST solution (100 mM Tris, 0.9% NaCl 0.5% Tween20) , 5% skimmed milk, and incubated for 1 hour with sera from patients with laboratory confirmed leptospirosis. As control experiments, incubations were performed with sera from healthy individuals from Brazil. Sera were diluted

1:25 or 1:100 prior to use. After washing with TBST solution, membranes were incubated with secondary antibody anti-IgG and/or IgM conjugated to alkaline phosphatase

(Sigma), diluted 1:1,000 or 1:40,000, for 1 hour. Antigen- antibody complexes were detected by colour reaction with an alkaline phosphatase detection kit (Biorad) . The finding of any visible colorization of the band of recombinant Lig polypeptide in the immunoblot was considered a positive reaction. Pooled sera from leptospirosis patients strongly recognized purified recombinant LigANI, LigBNI and BigL3 Rep protein as shown in Figure 4.

The microagglutination test is the gold standard test to confirm the diagnosis of leptospirosis in patients with clinically suspected disease. Sera from leptospirosis patients was collected during surveillance for leptospirosis in the city of Salvador, Brazil. Leptospirosis is endemic in locales within Salvador and the patients from which the sera was derived represent an at risk population for urban leptospirosis. The collection of sera from control individuals was obtained from preexisting serum banks of hospitalized patients and healthy individuals from Salvador, Brazil.

Data shown in Table 4 demonstrate that more than 90% of hospitalized patients with leptospirosis react to recombinant fragment of rLigB during active infection. About 98% of leptospirosis patients react to rLig during the convalescent-phase of their illness.

Table 4. Detection of BigL3 Rep IgG and IgM anti- recombinant antibodies in patient sera

^a paired sera samples were evaluated from 95 patients with confirmed leptospirosis. Acute and convalescent phase samples were collected 9.0 (+/- 3.8 days) and 35.3 (+/- 26.8 days) days after onset of symptoms. ^b Acute phase samples were tested from 40 patients with laboratory confirmed leptospirosis, who had negative reactions in ELISA-IgM, based on whole cell. Next, Table 5 compares seroreactivity for standard diagnosis tests (MAT, IgM-ELISA) . It can be observed in Table 5 that rLigB seroreactivity was higher during the initial disease phase than in those observed for standard diagnosis tests. Together these results illustrate that the method of the present invention can be employed as serological marker of active infection and is the basis for a kit that can be used for diagnosis of leptospirosis.

Table 5. Comparison of the recombinant LigB-based ixπmunoblot with standard diagnostic tests for leptospirosis .

Furthermore, again with reference to Table 4, seroreactivity results for rLigB are shown in endemic regions having high leptospirosis risk. 2-3% of the population living in these endemic regions demonstrate IgG seroreactivity to rLigB, indicating that this reaction is a useful marker to identify past infection. Among patients with confirmed leptospirosis, 4-13% were seroreactive against rLigB in a two-year period after infection with leptospirosis (see Table 5) . 2 to 4 years after infection with leptospirosis, 6-8% of patients demonstrated rLigB seroreactivity. These results, as a whole, illustrate that a kit based on the immunoblot method can be used with efficacy to detect an already occurred infection by leptospirosis . Example 5C

Immunoblot Detection of Antibodies to rLig Polypeptides in Serum Samples from Individuals

This example illustrates the use of a dotblot format based on the Western Blotting technique to detect antibodies against recombinant Lig polypeptides in subjects infected with pathogenic Leptospira spp.

The rLig protein (LigANI, LigBNI and BigL3 rep) was spotted onto nitrocellulose membrane strips, 400 ng/3 mcl spot, and dried. Membranes were blocked overnight in PBST (PBS, 0.05% Tween20, pH 7.4), and 4% non-fat dried milk at 4°C. Blocked membranes were washed twice in PBST and patient sera was added to the membranes in PBST, 0.25% BSA at a dilution of 1:250. Membranes and sera were incubated at room temperature, approximately 23⁰C, for 2 hours with shaking. Membranes were washed four times with PBST at room temperature with shaking, 5 minutes/wash. Anti-IgG human conjugated to alkaline phosphatase (Sigma) was added at a dilution of 1:40,000 and incubated at room temperature for 1 hour with shaking. Membranes were washed four times in PBST at room temperature with shaking, 5 minutes/wash. A final wash with PBS was followed by incubation of the membranes with an alkaline phosphatase detection solution (Biorad) for 15 minutes . The colour development was stopped with the addition of water. The strips were dried and evaluated for colour development. The finding of any visible colorization of the band of rLig polypeptide in the immunoblot was considered a positive reaction.

Table 6 below summarizes the results that demonstrate that more than 95% of hospitalized patients react to rLig (LigANi, LigBNI and BigL3 rep) proteins during active infection. 100% of leptospirosis patients reacted to rLig proteins during the convalescent-phase of their illness. These results illustrate that the method has utility as a serological marker of active infection and is the basis for a kit that can be used for diagnosis of leptospirosis. Table 6 : Double Blind study with 80 human serum samples with recombinant Lig proteins (Lig&NI, LigBNI and BigL3 rep) using Dot-blotting.

Thus, the description above demonstrates that isolated DNA (deoxyribonucleic acid) molecules that encode for LigA and LigB proteins, as well as fragments of these proteins are optimum molecules in the diagnosis, and formulation of vaccines for prevention of infections caused by Leptospira in humans and animals. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention should be apparent to those skilled in the art from the detailed descriptions presented above.

Claims

1. Oligonucleotides for amplification of Lig polynucleotides characterized by said oligonucleotides having SEQ ID NO. 1; SEQ ID NO. 2; SEQ ID NO. 3; and SEQ ID NO. 4 sequences.

2. Oligonucleotides for amplification of polynucleotides characterized by said oligonucleotides having SEQ ID NO. 16; SEQ ID NO. 17; SEQ ID NO. 18; and SEQ ID NO. 19; SEQ ID NO. 41; SEQ ID NO. 42; SEQ ID NO. 43; SEQ ID NO. 44; SEQ ID NO. 45 and SEQ ID NO. 46 sequences.

3. Oligonucleotides for amplification of BigL3 and pROY polynucleotides characterized by said oligonucleotides having SEQ ID NO. 24; SEQ ID NO. 25; SEQ ID NO. 26; and SEQ ID NO. 27 sequences.

4. Oligonucleotides for amplification of BigL3rep polynucleotide characterized by said oligonucleotides having SEQ ID NO. 50 and SEQ ID NO. 51 sequences.

5. Lig polynucleotides in Leptospira spp. characterized by said polynucleotides having SEQ ID NO.14 and SEQ ID NO.15 sequences.

6. LigANI, LigBNI and LigA(7-ll), LigB(7-ll) and LigB (11-12) polynucleotides characterized by said polynucleotides having SEQ ID NO. 20; SEQ ID NO. 21; SEQ ID NO. 47; SEQ ID NO.48; and SEQ ID NO. 49 sequences.

7. Polynucleotides according to claim 6 characterized by SEQ ID NO. 20 sequence having 1802 bp and SEQ ID NO. 21 having 1900 bp.

8. Polynucleotides according to claim 6 characterized by SEQ ID NO. 47 sequence having 945 bp and SEQ ID NO. 48 having 910 bp.

9. BigL3 and pROY polynucleotides characterized by said polynucleotides having SEQ ID NO.28 and SEQ ID NO.29 sequences .

10. Polynucleotides according to claim 9 characterized by SEQ ID NO. 28 sequence having 1557 bp and SEQ ID NO. 29 having 1644 bp.

11. BigL3rep polynucleotide from L. interrogans canicola characterized by said polynucleotide having SEQ ID NO.54 sequence.

12. Polynucleotides according to claim 11 characterized by SEQ ID NO.54 sequence having 1560 bp.

13. LigANI and LigBNI polynucleotides from L. interrogans canicola characterized by said polynucleotides having SEQ ID NO.52 and SEQ ID NO.53 sequences.

14. Polynucleotides according to claim 13 characterized by SEQ ID NO.52 sequence having 1803 bp and SEQ ID NO.53 sequence having 1899 bp.

15. Polypeptides characterized by having SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 30 and SEQ ID NO. 31; SEQ ID NO.40; SEQ ID NO.41 and SEQ ID NO.42 sequences and their sequences functionally equivalent.

16. LigANI, LigBNI and LigBrep polynucleotides from L. interrogans canicola characterized by said polynucleotides having SEQ ID NO.57; SEQ ID NO.58; and SEQ ID NO.59 sequences and their sequences functionally equivalent.

17. Polypeptides characterized by having the sequences of the 13 domains from Lig A SEQ ID NO.5 and any combination among them; domains: 1 position, 52-135aa; 2 position, 137-222; 3 position, 226-308; 4 position, 312- 398aa; 5 position, 402-487aa; 6 position, 491-576aa; 7 position, 582-β68aa; 8 position, 672-757aa; 9 position, 7βl-847aa; 10 position, 852-937aa; 11 position, 943-1028aa; 12 position, 1034-1119aa; 13 position, 1125-1212aa and their sequences functionally equivalent.

18. Polypeptides characterized by having the sequences of the 12 domains from LigB SEQ ID NO.9 and any combination among them; domains: 1 position, 52-135aa; 2 position, 137- 222; 3 position, 226-308; 4 position, 312-398aa; 5 position, 402-487aa; 6 position, 491-576aa; 7 position, 582-669aa; 8 position, 673-756aa; 9 position, 760-845aa; 10 position, 851-936aa; 11 position, 942-1028aa; 12 position, 1032-1118aa; and their sequences functionally equivalent.

19. Pharmaceutical composition to induce protective immune response in subjects for pathogenic spirochetes characterized by said composition comprising an amount in the 10-lOOmcg range of one or more selected antigens among the group consisting of defined polypeptides in claim 9 or their functionally equivalent sequences and a pharmaceutically acceptable vehicle.

20. Composition according to claim 10 characterized by said composition having fragment rLigANI or rLigBNI .

21. Method to identify polypeptides with functionally equivalent sequences, characterized for including stages of: (a) incubating components comprising the compound and one LigA or LigB polypeptide or polypeptides with functionally equivalent sequences under sufficient conditions to allow components to interact; (b) measuring the compound bound to LigA or LigB polypeptide or polypeptides with functionally equivalent sequences .

22. Method to detect pathogens in a sample characterized for including the stage of contacting the sample suspected of containing pathogenic spirochete with a reagent that binds to the pathogen specific cell component and detect the bound of the reagent to the component.

23. Diagnosis kit for detection of (i) LigA and LigB polypeptide or polypeptides with functionally equivalent sequences; (ii) ligA and ligB polynucleotides; or (iii) antibodies binding to LigA or LigB polypeptide or polypeptides with functionally equivalent sequences characterized by said diagnosis kit consisting of primary human or animal antibodies; and antibodies marked with enzymes.

24. Kit diagnosis for the diagnosis of Leptospira spp. characterized for using recombinant fragments of LigA and LigB proteins.