# Patent

## Title
Surface proteins of Leptospira

## Abstract

The invention relates to Leptospiral surface proteins, and the nucleic acid molecules which encode them. Various uses are described, including immunoprophylactic, diagnostic and therapeutic methods.

## Background
RELATED APPLICATION
This application claims priority of application Ser. No. 60/360,566, filed Feb. 28, 2002, incorporated by reference in its entirety.


FIELD OF THE INVENTION
This invention relates to membrane associated proteins of Leptospira bacteria, Leptospira Copenhageni in particular. The proteins are useful both therapeutically, as, e.g., antisera, immunoprophylactically, as vaccines, as well as diagnostically. They can be used, for example, to detect antibodies in samples taken from subjects suspected of being infected, and also to generate antibodies which can then be used to detect the proteins, epitopic portions of the proteins, as well as the bacteria per se. Also a feature of the invention are nucleic acid molecules encoding these proteins, methods for purifying them, as well as various applications thereof.
BACKGROUND AND PRIOR ART
Leptospira is a genus of bacteria which is a member of the Spirochetes family. Other members of this family are Borrelia and Treponema. All three genuses are characterized by mobility, and a helical shape. All members of the family cause disease in animals, including humans, livestock, domesticated animals, and wild animals. For example, Borrelia is the causative agent for Lyme disease. Other diseases caused by Spirochetes include relapsing fever, syphilis and yaws.
Leptospira genus consists of a genetically diverse group of 12 species, eight of which are pathogenic, and four of which are non-pathogenic, and saprophytic. See Faine, et al., Leptospira and Leptospirosis (2nd edition, 1999, Melbourne, Australia, MediSci); Farr, Clin. Infect. Dis 21:1-8 (1995). Levett, Clin. Microbiol Rev. 14:296-326 (2001), incorporated by reference. There are over 200 known pathogenic serovars of Leptospira. It is hypothesized that structural heterogeneity in lypopolysacchacide structure accounts for this. See, Levett, supra.
Leptospirosis, the disease caused by Leptospira, is zoonotic. Transmission to humans results from contact with domestic or wild animal reservoirs, or via contact with animal urine. Infected individuals show a wide spectrum of clinical manifestations in the early phases of the illness including fever, headache, chills and severe myalgia. In 5-15% of the clinical infections, severe multisystem complications result, including jaundice, renal failure and hemorrhaging. See Farr, supra, Faine, et al., supra. Severe leptospirosis has a mortality rate of 5-40%.
There is a large spectrum of animal species which serve as reservoirs for the bacteria. As a result, human leptospirosis is found throughout the world, and is considered to be the most widespread zoonotic disease. High risk groups include military personnel, farmers, miners, sewage and waste removal workers, veterinarians and abattoir workers. See, in this regard, Levett, supra, Faine, et al., supra. New patterns of transmission of the disease have emerged; however, emphasizing the public health issues associated with this disease. In “developed” countries outbreaks have been associated with recreational diseases, such as white water rafters in Costa Rica, and sporting events involving extensive outdoor activity, such as triathlons and “Eco-challenges.” See, e.g., Levett, supra; Jackson, Pediatr. Infect. Dis. J 12:48-54 (1993); Center For Disease Control and Prevention: Outbreak of leptospirosis among white water rafters in Costa Rica (1997); Update: Leptospirosis and Unexplained Acute febrile illness among athletes participating in triathlons: Illinois and Wisconsin (1998). Update: Outbreak of Acute Illness Among Athletes Participating in Eco-Challenge—Sahah 2000—Borneo, Malaysia (2000). Underlying conditions associated with poverty have led to large, urban, epidemics of leptospirosis in Brazil and other countries, resulting in high mortality rates. See Lomor, et al., Infect. Dis. Clin. North Am 14:23-39 (2000); Ko, et al., Lancet 354:820-5 (1999).
Leptospirosis is a major issue in agriculture as well, due to its association with livestock and domestic animals. Among the manifestations of animal leptospirosis are spontaneous abortions, still births, infertility, failure to thrive, reduced milk production, and high fatality rates in diverse species such as cows, pigs, sheep, goats, horses and dogs. See Faine, et al., supra. Other manifestations of the disease are chronic infection, and shedding of pathogenic leptospires. Standard approaches to controlling this include international and national quarantines in the animal husbandry industry, with negative economic ramifications.
Clearly, there is a need to control leptospirosis; however, efforts have been hindered due to a lack of effective approaches. Long term survival of pathogenic leptospires in soil and water, as well as the abundance of animal reservoirs support long term survival of the pathogen so eradication is not a viable option. Hence, efforts have turned to approaches based upon vaccines.
Currently, available vaccines are based upon inactivated whole cell, or membrane preparations of pathogenic bacteria. These appear to induce protective responses via antibody induction. See Levett, supra, Faine, et al, supra. These vaccines do not produce long term protection against infection, and they do not confer cross protective immunity against serovars not included in the vaccines used. The number of serovars possible, and the cost of multicomponent serovar vaccines have thwarted development in this area.
A key mechanism in the pathogenesis of leptospirosis, as in other spirochetal diseases (such as Lyme disease and syphilis), is the ability of the pathogen to disseminate widely in the host during the early stage of infection. See Faine, et al., supra. It is presumed that surface associated leptospiral proteins, mediate interactions which facilitate entry and dissemination through host tissues. Virulence factors associated with the surface of the bacteria serve as vaccine candidates, in that any immune or other protective response would block dissemination in the host. Another important aspect of surface associated proteins is that they are, literally, “surface-associated,” rendering them accessible to immune attack. Protective mechanisms associated with such surface associated proteins include antibody-dependent phagacytosis, and complement-mediated killing.
It would be desirable to have pure, or substantially pure Leptospira surface associated proteins available. Production of such proteins, in recombinant form, for example is cost effective, and provides a method to screen proteins to determine sub-unit or epitopic fragment based vaccines. Further, availability of recombinant proteins permits one to determine which proteins are conserved, especially among different pathogenic Leptospira, to determine which are the most suitable, cross-serovar vaccines.
There have been difficulties in identifying surface associated Leptospira proteins, using conventional biochemical and molecular biological methods. The genome of the spirochete Borrelia burgdorferi has been analyzed, and more than 100 surface associated lepoproteins were identified. The large size of the Leptospira genome ( ̃4.6 Mb), and its complex life cycle suggest that a far greater number of surface associated proteins will be found. Using standard membrane extraction, isolation, and purification techniques, less than 10 Leptospira surface associated proteins have been identified and characterized. See Haake, et al., Infect. Immun 66:1579-1587 (1998); Haake, et al., Infect. Immun. 6572-82 (1999); Haake, et al., Infect. Immun 68:2276-2285 (2000); Shang, et al., Infect. Immun 63:3174-3181 (1995); Shang, et al., Infect. Immun 64:2232-30 (1996). Also see U.S. Pat. Nos. 5,091,301; 5,643,754; 5,638,757; 5,824,321; 6,140,083; 6,262,235; 6,306,623; and 6,308,641. All of these articles and patents are incorporated by reference. While Haake, et al., Infect. Immun 67:6572-82 (1999), describe immunization with recombinant protein L1pL32, OmpL1 and L1pL41 but, the response was not complete. None of these reference identify virulence associated proteins.
As has been pointed out, supra, the Leptospira genome is large. As a result, technical difficulties have prevented meaningful results in identifying surface associated proteins; however, the emerging field of bioinformatics has placed extremely powerful and valuable tools in the hands of those involved in Leptospiral research.
Via application of the techniques described supra, the inventors have discovered further Leptospira surface associated proteins useful in vaccine production, as well as in production of diagnostic kits for use in determining presence, onset, or decrease in Leptospiral infection. These, and other aspects of the invention will be clear from the disclosure which follows.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1-11 inclusive, present data obtained from Western Blotting experiments, using the recombinant proteins of the invention. In all figures, control rat serum, and serum from rats that had been immunized with Leptospira, and control human sera as well as sera from patients diagnosed as suffering from Leptospirosis were used. FIG. 1 shows work done with the protein of SEQ ID NO: 2 & 4, FIG. 2 with SEQ ID NO: 6 & 8, and so forth.
FIG. 1 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 2 and 4.
FIG. 2 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 6and 8.
FIG. 3 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 10 and 12.
FIG. 4 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 14 and 18.
FIG. 5 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 20 and 22.
FIG. 6 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 24 and 26.
FIG. 7 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 28 and 30.
FIG. 8 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 32 and 34.
FIG. 9 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 36 and 38.
FIG. 10 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 40 and 42.
FIG. 11 shows data from Western Blotting experiments using the proteins of SEQ ID NOS: 44 and 46.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
A strain of Leptospira, referred to as “Fiocruz L1-30,” was used. The strain was isolated from a patient with severe leptospirosis, contracted during an epidemic in 1996. See Ko, et al., Lancet 354:820-5 (1999), incorporated by reference. Leptospires were detected during dark field microscopy examination of a culture of tween-albumin media that had been inoculated with patient blood, following “Guidelines for the Control of Leptospirosis.” WHO Offset Publ. (1982) incorporated by reference. The strain was identified as Leptospira interrogans, serovar Copenhageni, via biochemical and sera typing analysis. See Ko, et al., supra, Barocchi, et al., J. Clin. Microbiol 39:191-195 (2001). A culture of the organism was then prepared in media with 10% glycerol, and stored at −70° C. Virulence capacity was determined by inoculating 28 day old weaning hamsters. Kidneys were removed from anesthetized animals, approximately 7 days after infection, macerated and were used to inoculate Tween-albumin media. Any cultures for which growth was detected were used to infect additional hamsters, as part of a second passage step. In all, three passage steps were performed. Isolates from the final passage were used to prepare large scale, two liter cultures, again in Tween albumin media. Following centrifugation (10,000×g for 20 minutes), pellets were stored at −70° C., prior to use in DNA purification steps. The genomic DNA was extracted using standard methods, as set forth in, e.g., Sambrook and Maniatis, Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor, 1989).
Once the genomic DNA was isolated, it was partially digested with MboI, in order to generate fragments which ranged from 30 to 60 kilobase pairs. These were cloned into a cosmid vector “Lawrist 4,” described by Hanke, et al., Biotechniques 21(4):686-8 and 690-3 (1996) incorporated by reference to produce a cosmid library. Any dephosphorylated DNA fragments were ligated to the cosmid vector, in the presence of T4 DNA ligase. The resulting, recombinant DNA was then packed into phage particles, using a commercially available product, and was then transfected into E. coli DH5*. Transfected cells were plated onto solid, Luria-Bertani medium supplemented with kanamycin, and were grown, overnight, at 37° C.
Following culture, the transfectants were used in a shotgun library construction protocol. To elaborate, genomic DNA was purified, in accordance with Fleischmann, et al., Science 269:496-512 (1995), incorporated by reference, and the purified DNA was sheared into fragments via nebulization.
The resulting fragments were repaired by end filling, using the Klenow fragment of E. coli DNA polymerase, or phage T7 DNA polymerase, or both, before ligation into the SmaI BAP site of pUC18. The resulting, recombinant DNA was transformed into E. coli DH10B cells, via electroporation. The transformants were plated, on Luria-Bertani agar medium containing ampicillin, and grown overnight at 37° C.
Single bacterial colonies resulting from the culture were inoculated in 96 well microtiter plates containing nutrient broth and ampicillin. These were grown, overnight, with shaking, at 37° C. DNA was purified via standard alkaline lysis, with one modification. Specifically, at the end of the procedure, supernatant was passed through a multi-screen filter prior to precipitation. Precipitated DNA was resuspended in water, and used as described in the following example.
EXAMPLE 2
The purified DNA described in example 1 was sequenced, using commercially available reagents, and well known methods.
Specifically, commercially available products were used to carry out Taq dye deoxy terminator cycle sequencing reactions, using M13 reverse and forward matching primers, which flanked the inserts of the clones. Reaction products were analyzed on a commercially available genetic analyzer.
There were a total of 289,963 shotgun genomic sequences. Open reading frames were obtained from these, and assembled using phred/phrap software, as described by Ewing, et al., Genome Res 8(3):186-194 (1998) incorporated by reference. The assembly yielded 2,042 contigs. The “Glimmer” program of Delcher, et al., Nucl. Acids Res 27:4636-4641 (1999), incorporated by reference, was applied to the contigs, and yielded 5826 putative open reading frames. Each of these ORFs contained at least 90 base pairs, and overlapped other ORFs by 30 base pairs, or 10% of the ORF size, at most. Any ORFs with “N” or “X,” which indicated either poor quality, or repetitive regions were discarded. The “PSORT” program described by Nakai, et al., Proteins: Structure, Function and Genetics 11:95-110 (1991), incorporated by reference, was used to predict protein location within the bacterium. Any ORF, with a PSORT score of 0.1 or more (outer membrane or periplasmic space localization), were considered to be potentially useful.
Protein coding genes were identified using known algorithms GeneMark and Glimmer, as described by Delcher, et al., Nucl. Acids Res. 27:4636-4641 (1999), incorporated by reference, after which the “PSORT” program, described by Nakai, et al., Proteins: Structure, Function and Genetics 11:95-110 (1991), incorporated by reference, was used to predict protein localization within the bacterium. The sequences chosen for further analysis were those which the program predicted to encode surface associated proteins. Such proteins are ideal candidates for generation of antibodies.
Once the relevant nucleotide sequences were identified, protocols were developed to amplify them for further work.
In each case, a pair of oligonucleotide primers were developed to hybridize specifically to the target sequence of interest. In general, primers from 18-28 nucleotides long were used. In each case, the forward primer was then modified to add the sequence “CACC” at the 5′ end. This facilitates directional cloning in the vector used. As a general principle, the primers were designed to hybridize from 10-25 amino acids away from the start codon, so as to avoid hydrophobic regions that are characteristic of signal peptide sequences. The primer sequences follow







TABLE 1







Oligonucleotide primer sequences employed to amplify the target


nucleic sequences by PCR methodology. Molecular weights are


from the recombinant proteins obtained in E. coli expression


systems.











Molecular

SEQ



Weight

ID



(KDa)
Primers
NO:















LpL53
53.8
LpL53F:
CACCACCAATGTGTTTGGTATAGCG
47




LpL53R:
CAGCGTTTTGTGATAAAATTAAC
48


OMPL55
53.8
OMPL55F:
CACCGATGCTTACTACGGACTGGATG
49




OMPL55R:
CAGGAGTGTGATCGAGCTTG
50


OMPL16
15.9
OMPL16F:
CACCCCGTGTTCTTTTGGTTTAGAT
51




OMPL16R:
TTCCAACAAATCGAATCATCT
52


OMPL31
32
OMPL31F:
CACCAAGAAGGATTCCAACGATGATG
53




OMPL31R:
TCTCCTGCTTGACAGCCGAC
54


OMPL15
15.2
OMPL15F:
CACCATGCGTGCTGTCAGTAGAGAAAC
55




OMPL15R:
GTCGACATTGGCAGAATTTACG
56


OMPL20
21.2
OMPL20F:
CACCTTTGCACAATCCAAAGAGAAATG
57




OMPL20R:
TCATTTCCGAACCGGATGAC
58


LpL23
18.5
LpL23F:
ACCATGGGATCCGCTCTTTTGGTTGATCCAGAG
59




LpL23R:
GAATTCCTAACAACCAGGACCTTCACAT
60


LpL40
39
LpL40F:
ACCATGGGACTCGAGACGCCTCCTCCTAAAGATCC
61




LpL40R:
CTCCATGGTCATTTCAAAACTTCTAGGGGC
62


LpL22
22.8
LpL22F:
CACCCCTTCGAGGTTGGAAATCG
63




LpL22R:
AATCGATGGATCACGTTACG
64


OMPL17
18.4
OMPL17F:
CACCAAACCTGGATATGGAATGGC
65




OMPL17R:
TACAGAGGTAGAAGCGTTAGAAG
66


OMPL30
29.2
OMPL30F:
CACCAATCGACTTTTCACTGAGTTTCTT
67




OMPL30R:
CGAAAGTATCAAGAAGAACCGTA
68


OMPL27
27.5
OMPL27F:
CACCCAGGAAACGGAAAACGCTAA
69




OMPL27R:
CTTATTGTTTGCCGTAGGTTTC
70


OMPL21
21.9
OMPL21F:
CACCATGGGCGCTTTTAATCGG
71




OMPL21R:
CGGAACTAGGGAACTTTTCAAC
72


OMPL22
20.6
OMPL22F:
CACCATCATTCCTTCGGGAAGTGAC
73




OMPL22R:
CCATTCTCTGTTGTTTGATCCC
74


MPL17
15.4
MPL17F:
CACCGAAAGTCCCGTAAGGTTCAAA
75




MPL17R:
TGCAGGAGTTCCCACATTTTA
76


MPL21
31.9
MPL21F:
CACCACGTCTCAAAGTTACGCTTCAG
77




MPL21R:
TTCTCACCATCCAGCTCGG
78


OMPAL21
20.6
OMPAL21F:
CACCGAGCCTTCAACGCAAGAGCAA
79




OMPAL21R:
AACGTAAGACGTTGAGTTGCCACA
80


OMPL63
64.3
OMPL63F:
CACCACGATGATTCAGCCTACTTGG
81




OMPL63R:
GAAGTAGAACCGGAAGATTTATTT
82


OMPL14
18.6
OMPL14F:
CACCGGATGCAAACAAGATCCAGTAG
83




OMPL14R:
GAAACGCCACTAAGTTAGATCAC
84


MPL36
35.1
MPL36F:
CACCACGTCTTGTGCGTCGGTAGAG
85




MPL36R:
CCAAGTATTCTATTTATACGTCCGAG
86


MPL39
30.8
MPL39F:
CACCTCAGTAACTACTGGTCAGTGTAATG
87




MPL39R:
CACGTGTTAGTTCTTTGGTTG
88


MPL40
43.2
MPL40F:
CACCTTGTTTTTAAAAAAAAGGAAAGC
89




MPL40R:
AACTAAGGAACCGGAGTTGC
90


MPL21
18.6
MPL21F:
CACCGACATGCTTCCTACTTATTCCC
91




MPL21R:
CGCTAAAAGTATCACAATGGTAA
92









The amplification was carried out by combining template DNA (1.0 ng), nucleotide triphosphates-(0.4 mM), Pfx polymerase (0.5 units, taken from a stock solution of 1 unit/μl),and 0.2 μM primers, at a pH of 8.0. The total reaction volume was 50 μl.
PCR was then carried out in a thermocycler, where the DNA denaturation step was carried out for 3 minutes at 94° C., followed by primer-DNA template annealing at 55° C., for 20 seconds, and nucleotide polymerization for 4 minutes at 68° C. This cycle was repeated, 45 times, except that in the repeats, denaturation was for 20 seconds.
Following amplification, the sequences were cloned into a commercially available vector, “pENTR.TOPO.” This vector includes a kanamycin resistance sequence useful in E.coli selection a pUC ORI, two “attL1” and “attL2” sites for site-specific recombination, and a TOPO cloning site for directional cloning of blunt end PCR products. Specifically, this is the sequence “CCCTT”, which serves as a topoisomerase I binding site.
Cloning was carried out according to manufacturer's instructions, which call for 15 ng of the PCR product, and 5 ng of pENTR.TOPO vector, an 5mM Tris buffer, pH 7.4, at a total volume of 10 μl. The reaction proceeded for 10 minutes, at room temperature, i.e., about 25° C.
Positive clones were obtained via culture in kanamycin containing Luria Broth.
Following the reaction, the PCR products were integrated into the pENTR.TOPO vector, and were transferred to specific expression vectors very easily, via reaction at the forementioned attR1 and attR2 sites, in the presence of LR Clonase.
Expression was facilitated by use of an expression vector derived from T7, the commercially available pDEST17 vector. This vector includes an ORI, a promoter sequence to facilitate transcription of inserted sequences, a ribosomal binding site, and an ATG start codon.
In the practice of the invention, 2 μl of the cloning mixture described supra, 15 ng of pDEST17, & 4 μl of LR Clonase enzyme mix were combined in Tris-EDTA (10MM Tris-HCl, 0.1 mM EDTA) buffer, at pH 8, for 60 minutes, at room temperature. After 60 minutes, proteinase K (4 μg) was added for 30 minutes, at 37° C., to inactivate enzymes. Positive clones were isolated from Luria Broth agar plates, which contained 20 μg/ml of ampicillin. DNA sequences, when cloned, included a sequence encoding six His residues, at the N terminus of the protein. This well known technique was used to facilitate the purification of protein via metal affinity chromatography.
EXAMPLE 3
The expression vectors resulting from the preceding examples were used to express the relevant proteins. E. coli strains BL21(DE3) or BL21SI were used under inducing conditions, including 1 mM IPTG, or 300 mM NaCl, respectively, using standard methods. Proteins were then analyzed on 10-20% SDS-PAGE gels, under denaturing conditions. Each of FIGS. 1-22 presents an SDS-PAGE pattern for the proteins.
In addition to the SDS-PAGE work, the proteins were purified, by using Ni2+ chelating sepharose. Either proteins were mixed with charged beads, or were applied onto columns in a balanced salt buffer (0.1M Tris/0.3 M NaCl, pH 8.0). Any impurities were washed away using the same buffer, with a low concentration of imidazole (20-60 mM). Proteins of interest were then eluted, using a buffer containing imidazole at a concentration of from 0.75 to 1.0M.
EXAMPLE 4
Once the proteins were purified, they were tested for their ability to bind to antibodies in the sera of infected subjects.
Proteins were purified on 10-15% SDS-PAGE, and then blot transferred to nitrocellulose membranes. Protein was visualized with Ponceau staining.
The proteins were then contacted with either pooled sera obtained from patients who had been diagnosed with leptospiroses, or with rats immunized with Leptospira interrogans serovar Copenhageni. In the case of rats, dilutions varied from 1/500 to 1/1000. For patients, dilutions ranged from 1/100 to 1/1000. As controls, sera from healthy individuals and non-immunized rats were used.
After contact with the sera, a second antibody, either anti-rat or anti-human IgG, labeled with peroxidase was added, followed by O-phenyl diamine benzidine and hydrogen peroxide.
The results, shown in FIGS. 1-22, inclusive, show that all of the proteins reacted with sera from both sources.
In the discussion of the proteins which follows, signal and transmembrane regions were based upon the presence of hydrophobic amino acids, including Ile, Tyr, Val, Phe, Leu, Met and Ala, and Nielsen, et al., Protein Engineering 12:3-9 (1999), incorporated by reference. Each description is followed by reference to where the sequence appeared in the provisional application.
SEQ ID NO: 1 sets forth a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 2 with a predicted molecular weight of 52.7 KDa. It is a new Leptospiral lipoprotein, LpL53, according to the rules described in Haake, Microbiology 146:1491-1504 (2000), incorporated by reference. It has a hydrophobic region of a signal peptide from amino acid 4 to 10 characterized by the presence of Tyr, Leu, Ile, Phe, Leu, Phe, Ile, the lipoprotein signal peptidase cleavage site from amino acid 11-14, with Leu, Phe, Ser, Asn, and the cysteine to be lipidated at position 15 of the polypeptide sequence. (3909 & 3910)
SEQ ID NO: 3 sets forth a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 4 with a predicted molecular weight of 55 KDa. It has a signal peptide cleavage site from amino acid 1 to 28. (3531 & 3532)
SEQ ID NO: 5 sets forth a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 6 with a predicted molecular weight of 15.8 KDa. It has a signal peptide cleavage site from amino acid 1 to 38. (3489 & 3490)
SEQ ID NO: 7 sets forth a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 8 with a predicted molecular weight of 30.9 KDa. It has a signal peptide cleavage site from amino acid 1 to 19. (3871 & 3872)
SEQ ID NO: 9 sets forth a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 10 with a predicted molecular weight of 15 KDa. No match with any deposited protein in gene bank was found with this amino acid sequence. It has a signal peptide cleavage site from amino acid 1 to 19 and a transmembrane segment from amino acid 7 to 29, partially overlapping the signal peptide sequence. (3973 & 3974)
SEQ ID NO: 11 is a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 12 with a predicted molecular weight of 20.1 KDa. It has a signal peptide cleavage site from amino acid 1 to 20. (3679 & 3680)
SEQ ID NO: 13 corresponds to a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 14 with a predicted molecular weight of 23 KDa. This polypeptide sequence is a newly identified Leptospiral lipoprotein, according to Haake, supra. It has a signal peptide hydrophobic region from amino acid 6 to 15 (Ile, Val, Tyr, Val, Ile, Tyr, Leu, Phe, Leu, Ile), characterized by a higher proportion of hydrophobic amino acids, a lipoprotein signal peptides from amino acid 16 to 19 (Ser, Leu, Tyr, Gly) and a cysteine to be lipidated at position 20 of the polypeptide sequence. (81 & 82)
SEQ ID NO: 15 sets forth a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 16 with a predicted molecular weight of 40.6 KDa. This polypeptide sequence is a newly isolated Leptospiral lipoprotein, according to the rules described in Haake, supra. It has a signal peptide hydrophobic region from amino acid 7 to 16 (Ile, Leu, Phe, Val, Leu, Thr, Gly, Phe, Ile, Phe), characterized by a higher proportion of hydrophobic amino acids, a lipoprotein signal peptides from amino acid 1 to 20 (Phe, Val, Ser, Ala) and a cysteine to be lipidated at position 21 of the polypeptide sequence. (43 & 44)
SEQ ID NO: 17 corresponds to a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 18 with a predicted molecular weight of 22.6 KDa. This polypeptide sequence is a new Leptospiral lipoprotein, according to Haake, supra. It has a signal peptide hydrophobic region from amino acid 8 to 18 (Ile, Asn, Ile, Leu, Phe, Phe, Phe, Leu, Val, Tyr, Phe), a lipoprotein signal peptidase from amino acid 19 to 22 (Leu, Leu, Phe, Gly) that conforms to the rules described in Haake and a cysteine to be lipidated at position 23 of the polypeptide sequence. (125 & 126)
SEQ ID NO: 19 corresponds to a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 20 with a predicted molecular weight of 17.3 KDa. It has a signal peptide cleavage site from amino acid 1 to 20. (3991 & 3992)
SEQ ID NO: 21 sets forth a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 22 with a predicted molecular weight of 30.9 KDa. It has a signal peptide cleavage site from amino acid 1 to 27. (3521 & 3522)
SEQ ID NO: 23 sets forth a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 24 with a predicted molecular weight of 27.1 KDa. It has a signal peptide cleavage site from amino acid 1 to 20. It has some similarity to proteins belonging to cytochrome c family. Examples are: di-haem cytochrome c peroxidase and cytochrome c, class I found in bacteria, such as Bacillus subtilis. (3533 & 3534)
SEQ ID NO: 25 corresponds to a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 26 with a predicted molecular weight of 20.9 KDa. It has a signal peptide cleavage site from amino acid 1 to 20. (3561 & 3562)
SEQ ID NO: 27 corresponds to a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 28 with a predicted molecular weight of 21.2 KDa. It has a signal peptide cleavage site from amino acid 1 to 34. It has some similarity to prokaryotic N-terminal methylation site found in bacterial general secretion pathway protein G and bacterial type II secretion system protein I/J. Examples of bacteria are E. coli, Xanthomonas campestris and Pseudomonas aeruginosa. (3675 & 3676)
SEQ ID NO: 29 corresponds to a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 30 with a predicted molecular weight of 16.6 KDa. It has a signal peptide cleavage site from amino acid 1 to 36. (3819 & 3820)
SEQ ID NO: 31 corresponds to a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 32 with a predicted molecular weight of 20.5 KDa. It has a signal peptide cleavage site from amino acid 1 to 22. (3829 & 3830)
SEQ ID NO: 33 is a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 34 with a predicted molecular weight of 20.9 KDa. It has a signal peptide cleavage site from amino acid 1 to 22. This polypeptide has some similarity to proteins having an outer membrane domain (from amino acid 77 to 182) that belong to the OmpA family. Most of these bacterial outer membrane proteins in this group are porin-like, integral membrane proteins, but some are small peptidoglycan-associated lipoprotein (such as pal). Escherichia coli is an example of a bacterium expressing a protein and having this domain. (3793 & 3794)
SEQ ID NO: 35 is a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 36 with a predicted molecular weight of 63.5 KDa. It has a signal peptide cleavage site from amino acid 1 to 29. This polypeptide has some similarity to outer membrane efflux protein identified in other bacteria. Examples include the E. coli TolC outer membrane protein; the Rhizobium nodulation protein; and the Pseudomonas FusA protein. (4007 & 4008)
SEQ ID NO: 37 is a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 38 with a predicted molecular weight of 14 KDa. It has a signal peptide cleavage site from amino acid 1 to 23. (3883 & 3884)
SEQ ID NO: 39 is a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 40 with a predicted molecular weight of 35.6 KDa. It has a signal peptide cleavage site from amino acid 1 to 36. This polypeptide sequence has some similarity to bacterial lipoproteins family identified in bacteria, such as, Escherichia coli. (2019 & 2020)
SEQ ID NO: 41 is a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 42 with a predicted molecular weight of 39.8 KDa. This protein has a transmembrane region from amino acid 68 to 90. This sequence has similarity to DshA protein of Leptospira with unknown function. (1031 & 1032)
SEQ ID NO: 43 is a polynucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 44 with a predicted molecular weight of 40 KDa. It is a cytoplasmic membrane protein, with one transmembrane segment from amino acid 63 to 79. This polypeptide sequence has some homology to the MoxR protein identified in Borrelia burgdorferi. The protein family (St. Louis Pfam Web site) (http://pfam.wustl.edu/) predicted two domains in this protein: (i) ATPase family domain associated with various cellular activities (AAA), such as chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. (2517 & 2518)
SEQ ID NO: 45 corresponds to a polynucleotide segment encoding an amino acid sequence as set forth in SEQ ID NO: 46 with a predicted molecular weight of 21 KDa. This protein has a signal peptide cleavage site from amino acid 1 to 40 and a transmembrane domain from amino acid 20 to 40. The polypeptide sequence has some similarity to bacterial signal peptidases identified in bacteria, such as, Sinorhizobium meliloti and Bacillus subtilis. This is a Leptospiral membrane protein MPL21. (1991 & 1992)
The foregoing disclosure set forth various aspects of the invention, including the isolated, Leptospira surface proteins, the amino acid sequences of which are set forth as an attachment hereto, and isolated nucleic acid molecules which encode these proteins such as those set forth herein. It will be understood that once an amino acid sequence is known, various degenerate nucleotide sequences can be provided which encode that sequence. All of those are encompassed by this invention.
“Surface protein” as used herein, refers to proteins which are associated and/or exposed to the surface of the organism.
The proteins of the invention may be used, alone or in combination with each other, as or as components of vaccines. Further, they can be used as immunogens, so as to generate an antibody response. The antibodies thus generated can be used, e.g., as diagnostic tools to determine Leptospira infection or presence, as well as components of vaccines used to generate passive immunity. Proteins and antibodies may be administered “neat” or “compounded” with other standard materials used in preparing vaccines, such as carriers, adjuvants, and other materials. Intravenous formulations are one embodiment and, it will be understood that other formulations of vaccines are possible, including intradermal, subcutaneous, oral, such as sublingual forms, and others. The vaccines may be in liquid or “dry” form, such as in lyophilized form. This type of vaccine is especially suitable when it must be carried “in the field,” and used at some point in time later than when carried.
The nucleic acid molecules of the invention, as will be understood by the skilled artisan, can be used to produce the proteins described supra, via any of the recombinant methodologies well known to the skilled artisan. They can be placed in, e.g., expression vectors, under control of a promoter or other regulatory element, or they can be used “as is” to transfect or to transform cells. Eukaryotic cells, such as yeast cells, CHO cells, fibroblasts, insect cells, etc., are among the eukaryotes which can be transformed or transfected. Prokaryotes, such as E. coli or other bacteria can also be used. The choice of host cell will depend upon many factors, including whether or not glycosylation is desired, and to what end the transformant or transfectant will be used. One way these recombinant cells can be used is as in the form of a cell based vaccine, such as a whole cell vaccine. As the recombinant proteins of the invention are all surface proteins, it is to be expected that they will be expressed on the surface of host cells. If the cells are then processed so as to become non-proliferative, the cells present an ideal vaccine, especially if the host cell is one that is not normally the target of immune surveillance in the host.
Another aspect of the invention is the use of membrane preparations, or cellular “ghosts” of transformants or tranfectants. Such approaches have been used with other bacterial species, so preparation of these is well known. Transformants or transfectants which express the surface proteins of the invention can be used to prepare these materials in a fashion taught by the art.
The proteins have been discussed as vaccines, supra. It is to be understood that the vaccines of the invention can be formulated for any subject. Human vaccines are, of course, included, but so are vaccines for livestock animals, such as sheep, bovine animals, goats, pigs and so forth, and domesticated animals such as pets, confined zoo animals, etcetera. The vaccines may be used prophylactically, e.g., by administering them prior to possible exposure to Leptospira , and may also be used post exposure, in order to treat a pre-existing infection.
As will be recognized by the skilled artisan, the use of the proteins of the invention, or portions thereof, constitutes vaccination, and the protein or portion of the protein constitutes the vaccine. Upon administration to the individual or subject, in any of the ways described supra, an immune response results which protects the individual or subject when confronted with the pathogen. Such an approach, i.e., the immunization with one or more proteins or portions of proteins, can be used therapeutically or prophylactically. Passive immunization, as described supra, can also be used. In such a case, antibodies are developed against the proteins or protein portions, and via passive transfer serve a role in, e.g., prophylaxis.
It is to be understood that, while immunization with the proteins or portions of proteins can serve to stimulate an antibody response, cellular immune responses are also a part of the response of the subject to the immunization. It is well within the skill of the artisan to determine which proteins or portions of proteins function as antibody or cellular immune vaccine agents and that artisan can then formulate, e.g., “cocktails” of appropriate mixes of proteins which have the desired, immune effect.
In addition to the use of the proteins as vaccines, it is to be understood that the nucleic acid molecules described herein can also be used as vaccines. Via targeted delivery of nucleic acid molecules, one can assure an “in vivo” supply of the desired protein or protein portion molecules at a site of relevance. The artisan of delivery systems, such as liposomes, adenoviruses, retroviruses, and other formats for the delivery of DNA as immunoprophylactic or a therapeutic agents, and all are envisioned as methods for administering the vaccine. It should be kept in mind that the nucleic acid molecules of the invention include those which encode the proteins of the invention, but differ in nucleotide sequence due to codon degeneracy. Indeed, due to patterns of codon usage, which vary from organism to organism, it may be desirable to alter the sequence to maximize expression of the desired vaccine in the treated subject.
To prepare a vaccine the purified polypeptide can be isolated, lyophilized and/or stabilized, as described supra. The protein may then be adjusted to an appropriate concentration, optionally combined with a suitable vaccine adjuvant, and packaged for use. Suitable adjuvants include but are not limited to: surfactants and pluronic polyols; polyanions, e.g., pyran, dextran sulfate, poly IC; polyacrylics, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, oil emulsions, vitamins, cytokines, hormones, aluminum, calcium salts, and mixtures thereof, bacterial and plant products, e.g., Bacillus Calmette-Guerin (BCG), complete Freund's adjuvant, and threalose. Alternatively, the immunogenic protein may be incorporated into liposomes for use in a vaccine formulation, may be fused to other immunogenic proteins, or may be conjugated to polysaccharides or other polymers. See Edelman, in New Generation Vaccines (Marshall Decker, N.Y. 1997), incorporated by reference.
The weight of the immunogenic protein included in a given dosage of vaccine can vary widely, e.g., from 5 ug-300 mg., and depends on: the age, weight, and physical condition of the animal or the human subject considered for vaccination.
In addition, the nucleic acid molecules of the invention can be used as vaccines. The rational of this approach is that the DNA will produce the protein following injection thus in turn inducing the desired immune response.
For such vaccines, a pharmaceutical composition can include either a mammalian recombinant expression vector, such as pTARGET, or a construct such as an expression vector, which includes a nucleic acid molecule encoding the protein, operatively linked to transcription control/terminator sequences, combined with a pharmaceutically acceptable carrier. These may include, but are not limited to, aqueous physiologically balanced solutions, artificial lipid-containing substrates, natural lipid-containing substrates, oils, esters, and glycols. Pharmaceutically acceptable carriers can also include a suitable delivery vehicle, such as liposomes, micelles, and cells. Adjuvants for DNA based vaccines can be used, such as CpG oligonucleotides and cytonkines. The vaccines can also be delivered by attenuated bacteria, such as Salmonella.
Another feature of the invention is the use of the proteins, or portions of the proteins of the invention, as well as nucleic acid molecules and portions of nucleic acid molecules, in the manufacture of kits useful for diagnosis of Leptospira infection, either “in the field” or in the laboratory. Such kits involve, e.g., the protein, protein portion, nucleic acid molecule, or nucleic acid molecule portion, in combination with, e.g., a solid phase, such as a bead, mutltiwell plate, etc., to which the component is affixed. The kit can then be used by contacting a sample of interest thereto, followed by a second component, which can also be included in the kit, such as a labeled protein or proteins portion, or labeled nucleic acid molecule or portion of a nucleic acid molecule. When nucleic acid molecules are used in the diagnostic kits of the invention, it is desirable that each portion hybridize to a separate portion of a target nucleic acid molecule.
It may be desirable to screen the proteins and nucleic acid molecules of the invention in, e.g., and animal or cellular model prior to administration to large animals or humans. Standard practice tests a potential vaccine in, e.g., a hamster, mouse, rat or other rodent model to determine it efficacy and strength, and the molecules of this invention may be so tested as well. Testing of the DNA molecules in, e.g., microorganisms such as E. coli or other prokaryotes or eukaryote organisms can be carried out to deter mine which are, in fact, good producers, i.e., molecules which produce high yields, and/or produce transformants which react well to culture. Other aspects of the inventions will be clear to the skilled artisan, and need not be reiterated here.

## Claims

1. An isolated Leptospiral protein consisting of amino acid sequence of SEQ ID NO: 8.

2. Immunogenic composition comprising the isolated Leptospiral protein of claim 1 and a pharmaceutically acceptable adjuvant or carrier.