CROSS REFERENCE TO RELATED APPLICATIONS [0002] This application claims priority under 35 U. S. C. §119 from Provisional Application Serial No. 60/463,819, filed April 17,2003, the disclosure of which is incorporated herein by reference.

TECHNICAL FIELD [0003] This disclosure provides polynucleotides and polypeptides expressed during Vibrio cholerae infection as well as the use of these polynucleotides and polypeptide in the diagnosis, treatment, and prevention of Vibrio cholerae infections.

BACKGROUND [0004] Microbial infections are complex, dynamic processes that evolve constantly within a host. In many instances, virulence gene expression is modulated in response to the changing environment encountered at the site of infection or within a particular host (Mekalanos, J. Bacteriol. 174: 1-7, 1992; Mahan et al. , Science 259 : 686-688,1993). It is unlikely that all regulated virulence determinants of a pathogen can be identified in vitro because it is technically impossible to determine and mimic all of the complex and changing environmental stimuli that occur at a site of an infection or within a particular host.

SUMMARY [0005] The disclosure provides a substantially purified polypeptide selected from the group consisting of: (i) a polypeptide comprising a sequence as set forth in SEQ ID NO : 26,28, 30, or 32; (ii) an antigenic polypeptide consisting of a fragment of SEQ ID NO : 2, 4, 6, 8, 10,12, 14,16, 18,20, 22,24, 26,28, 30, or 32; (iii) a soluble polypeptide consisting of a fragment of SEQ ID NO : 2, 4,6, 8,10, 12, 14, 16, 18,20, 22,24, 26,28, 30, or 32; and (iv) a fragment comprising a sequence as set forth in SEQ ID N0 : 2,4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30 or 32, wherein the fragment lacks a transmembrane domain. In one embodiment, the substantially purified polypeptide is an antigenic polypeptide consisting of a sequence as set forth in: (a) SEQ ID NO : 2 from about amino acid Xi to 167 ; (b) SEQ ID NO : 4 from about amino acid Xi to 224; (c) SEQ ID NO : 6 from about amino acid x2 to 578 ; (d) SEQ ID NO : 8 from about amino acid x3 to 63, or from about amino acid X4 to 197, or from about amino acid x5 to 256; (e) SEQ ID NO : 10 from about amino acid x6 to 143; (f) SEQ ID NO : 14 from about amino acid X7 to 294, or from about amino acid X8 to 644; (g) SEQ ID NO : 16 from about amino acid xg to 416, or from about amino acid xlo to 770; (h) SEQ ID NO : 18 from about amino acid xll to 214, or from about amino acid X12 to 564 ; (i) SEQ ID NO : 20 from about amino acid 1 to x13, or from about amino acid x14 to 761; (j) SEQ ID NO : 24 from about amino acid xls to 364; (k) SEQ ID NO : 28 from about amino acid 41 to x16, or from about amino acid Xi to 353; (1) SEQ ID N0 : 32 from about amino acid x18 to 219; or (m) a fragment of any of (a)- (1) ; wherein Xi is a position between 24 to 48, inclusive; X2 is a position between 34 to 38, inclusive ; x3 is a position between 27 to 33, inclusive; X4 is a position between 83 to 88, inclusive ; xs is a position between 222 to 227, inclusive; x6 is a position between 33 to 38, inclusive; X7 is a position between 36 to 42, inclusive ; x8 is a position between 310 to 314, inclusive; X9 is a position between 26 to 31, inclusive; clo is a position between 435 to 440, inclusive; x1l is a position between 32 to 37, inclusive ; Xiz is a position between 230 to 235, inclusive; X13 is a position between 73 to 78, inclusive ; x14 is a position between 297 to 302, inclusive; xls is a position between 17 to 22, inclusive; als is a position between 202 to 207, inclusive; x17 is a position between 222 to 227, inclusive; and x18 is a position between 25 to 30, inclusive. In another embodiment, the substantially purified polypeptide is a soluble polypeptide consisting of a sequence as set forth in: (a) SEQ ID NO : 2 from about amino acid Xi tot 167; (b) SEQ ID NO : 4 from about amino acid Xi to 224; (c) SEQ ID NO : 6 from about amino acid X2 to 578; (d) SEQ ID NO : 8 from about amino acid X3 to 63, or from about amino acid X4 to 197, or from about amino acid xs to 256; (e) SEQ ID NO : 10 from about amino acid x6 to 143; (f) SEQ ID NO : 14 from about amino acid X7 to 294, or from about amino acid fo8 to 644; (g) SEQ ID NO : 16 from about amino acid xg to 416, or from about amino acid x10 to 770; (h) SEQ ID NO : 18 from about amino acid x1l to 214, or from about amino acid XI. 2 to 564; (i) SEQ ID N0 : 20 from about amino acid 1 to x13, or from about amino acid x14 to 761; (j) SEQ ID NO : 24 from about amino acid x15 to 364; (k) SEQ ID NO : 28 from about amino acid 41 to x16, or from about amino acid x17 to 353; (1) SEQ ID NO : 32 from about amino acid x18 to 219; or (m) a fragment of any of (a)- (l) ; wherein Xi is a position between 24 to 48, inclusive; x2 is a position between 34 to 38, inclusive; x3 is a position between 27 to 33, inclusive; X4 is a position between 83 to 88, inclusive; Xg is a position between 222 to 227, inclusive; x6 is a position between 33 to 38, inclusive ; X7 is a position between 36 to 42, inclusive; xe is a position between 310 to 314, inclusive; xg is a position between 26 to 31, inclusive; xlo is a position between 435 to 440, inclusive; x11 is a position between 32 to 37, inclusive; x12 is a position between 230 to 235, inclusive; Xi3 is a position between 73 to 78, inclusive; x14 is a position between 297 to 302, inclusive; x15 is a position between 17 to 22, inclusive ; 16 ils a position between 202 to 207, inclusive ; x17 is a position between 222 to 227, inclusive; and Xt8 is a position between 25 to 30, inclusive.

[0006] The disclosure also provides a fusion protein comprising a substantially purified polypeptide as described in the preceding paragraph linked to a heterologous polypeptide. In one embodiment, the heterologous polypeptide is selected from the group consisting of a purification protein, a V. cholerae polypeptide, and an adjuvant polypeptide.

[0007] The disclosure also provides an isolated polynucleotide encoding a substantially purified polypeptide as described above.

In one embodiment, the isolated polynucleotide selected from the group consisting of : (i) an isolated polynucleotide consisting of a fragment of SEQ ID NO : 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21,23, 25, 27, 29 or 31, encoding an antigenic polypeptide; and (ii) an isolated polynucleotide comprising a fragment of a sequence as set forth in SEQ ID NO : 1, 3,5, 7,9, 11,13, 15, 17, 19,21, 23,25, 27,29, or 31, encoding a soluble domain consisting of a fragment of SEQ ID NO : 2,4, 6, 8, 10,12, 14,16, 18,20, 22, 24, 26,28, 30 or 32. Also provided by the disclosure are a-vector and a host cells containing such a polynucleotide.

[0008] The disclosure also provide a substantially purified antibody which selectively binds to a polypeptide consisting of a sequence as set forth in SEQ ID NO : 8,12, 26, 28, 30 or 32. The antibody can be a monoclonal or a polyclonal antibody.

[0009] The disclosure further provides a method for detecting V. cholerae in a sample. The method comprises (a) contacting the sample with the antibody of the disclosure; and (b) detecting binding of the antibody to a polypeptide in the sample, wherein binding is indicative of the presence of V. cholerae in the sample.

[0010] The disclosure provides a method comprising: (a) contacting a sample suspected of containing a V. cholerae bacterium with a nucleic acid probe that hybridizes to a polynucleotide consisting of a sequence as set forth in SEQ ID NO : 1, 3,5, 7,9, 11,13, 15,17, 19,21, 23,25, 27,29, or 31 or a complement thereof; and (b) detecting hybridization of the nucleic acid probe with the polynucleotide, wherein hybridization is an indication that a V. cholerae bacterium is present in the sample.

[0011] The disclosure also provides a recombinant method for producing a polypeptide, comprising : transfecting or transforming a host cell with a polynucleotide of the disclosure to generate a recombinant host cell; and expressing the polypeptide encoded by the polynucleotide.

[0012] The disclosure also provides a composition comprising a substantially purified polypeptide of the disclosure and a pharmaceutically acceptable carrier.

[0013] The disclosure provides a composition comprising an isolated polynucleotide as described herein and a pharmaceutically acceptable carrier.

[0014] Also provided by the disclosure is a method of eliciting an immune response in an animal, comprising introducing into the animal a composition comprising a substantially purified polypeptide of the disclosure and a pharmaceutically acceptable carrier.

[0015] The disclosure further provides a recombinant Vibrio cholerae bacterium that lacks a viable gene encoding a pathogenic factor, wherein the pathogenic factor comprises a sequence selected from the group consisting of SEQ ID Nos : 2,4, 6,8, 10,12, 14,16, 18,20, 22,24, 26,28, 30, and 32. In one embodiment, the viable gene comprises a sequence as set forth in SEQ ID NO : 1, 3, 5, 7, 9, 11,13, 15, 17,19, 21,23, 25 27,29, or 31.

[00163 The disclosure provides a method of generating an immune response, comprising administering to an animal a recombinant host cell engineered to contain a polynucleotide of the disclosure, such that the recombinant host cell expresses the polynucleotide in vivo, thereby eliciting an immune response.

100173 The disclosure also provides an antigenic formulation comprising a substantially purified polypeptide of the disclosure and a pharmaceutical carrier or adjuvant.

[0018] In yet another aspect, the disclosure provides a recombinant host cell genetically modified to contain a polynucleotide encoding a substantially purified polypeptide of the disclosure. In one aspect the recombinant host cell is selected from the group consisting of a B. subtilis, BCG, Salmonella sp. , Listeriae, Yersiniae, Streptococci, Corynebacterium diphtheriae, and an E. coli cell.

[0019] The disclosure also provides a method for immunizing an animal against V. cholerae, comprising introducing an immunizing amount of an antigenic formulation of the disclosure or a recombinant host cell expression a polypeptide of the disclosure into the animal.

BRIEF DESCRIPTION OF THE DRAWINGS [00203 FIG. 1 shows an in vivo induced antigen technology (IVIAT) scheme as described in U. S. Patent Publication 2002/0197625.

[0021] FIG. 2 depicts an example of sequential adsorbtion of pooled serum to eliminate antibodies reactive with in vitro expressed antigens as described in U. S. Patent Publication 2002/0197625.

[00221 FIGS. 3A and 3B show the probing of a partial genomic library as described in U. S. Patent Publication 2002/0197625.

Plates containing appropriate numbers of colonies are replicated onto medium with IPTG to induce expression of the cloned fragments.

The colonies are lifted onto duplicate membranes, which are then reacted with either the pooled unadsorbed (panel A) or pooled adsorbed (panel B) sera. Reactive clones are visualized by probing with peroxidase-conjugated goat anti-human immunoglobulin as a secondary reagent followed by development with a chemiluminescent substrate and autoradiography.

[00233 FIGS. 4A and 4B show ELISA results following sequential steps in adsorption of pooled, patient sera; OD values are corrected for background and for dilution during adsorption steps. (A) ELISA plates coated with whole V. cholerae extracts ; (B) ELISA plates coated with GM1 ganglioside and CtxB. FP ; French press.

[0024] FIGS. 5A and 5B show results utilizing pooled, convalescent sera after complete adsorption. (A) Individual, purified proteins were spotted on a nitrocellulose membrane and the membrane developed using pooled convalescent sera; (B) Individual E. coli BL21 strains containing plasmids expressing specific proteins (or control plasmid, pET30a) were transferred to a nitrocellulose membrane and developed utilizing pooled patient sera after adsorption.

[0025] FIG. 6 shows a comparison of reactivity of unadsorbed sera from individual patients collected on days 0,9, and 23 following V. cholerae infection, and control patients from day 23, against purified El Tor PilA (panel A) or TcpA (panel B).

[0026) FIGS. 7A-7K show polynucleotide and polypeptide sequences of the disclosure. Bolded regions of the polypeptides are indicative of putative transmembrane and/or hydrophobic regions of the polypeptides. The italicized region denotes a putative signal peptide domain.

DETAILED DESCRIPTION [0027] As used herein and in the appended claims, the singular forms"a,""and,"and"the"include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a clone"includes a plurality of clones and reference to"the polynucleotide sequence"generally includes reference to one or more polynucleotide sequences and equivalents thereof known to those skilled in the art, and so forth.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the disclosure belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the typical methods, devices and materials are now described.

[00291 All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the proteins, polynucleotides and methodologies, which are described in the publications that might be used in connection with the presently described disclosure. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure.

[0030] In vivo-induced antigen technology (IVIAT) is a method that circumvents limitations of animal models, allowing direct identification of microbial proteins expressed during infection.

These same proteins may be important for protective immunity following both natural infection and vaccination. For example, using the IVIAT method V. cholerae genes specifically expressed during human infection were identified.

[0031] Vibrio cholerae is a gram-negative bacillus that causes a severe, dehydrating diarrhea in subjects exposed to the organism.

V. cholerae can be differentiated by the lipopolysaccharide in the outer membrane; strains of V. cholerae that produce cholera belong to serogroup 01 or 0139. V. cholerae 01 is divided into two biotypes, classical and El Tor.

[00321 A major virulence factor for pathogenic strains of V. cholerae is cholera toxin, a protein exotoxin that consists of a single A subunit non-covalently associated with 5 B subunits. A second major virulence factor of V. cholerae is the toxin co- regulated pilus (TCP), TCP appears to be essential for colonization and virulence in both mouse models of cholera and human volunteer studies. TcpA, the 20. 5 kDa major structural subunit of TCP, has homology to the type IV pili of several other bacterial pathogens.

TcpA from El Tor and classical strains of V. cholerae show approximately 80% protein homology. Monoclonal antibodies to the two strains demonstrate epitope differences in the two biotypes.

Intestinal colonization by V. cholerae is a prerequisite for the development of immune responses during cholera, and TCP has been shown to be required for intestinal colonization of humans by V. cholerae. However, in volunteer studies in North America, anti-TCP responses were not found in convalescent sera of individuals challenged with V. cholerae. Low-level anti-TCP immune responses were present in convalescent sera from 3 of 6 individuals with cholera in Indonesia. It is not clear why antibody responses to TCP are not more prominent in normal volunteers, but perhaps immune responses to this pilus require repeated intestinal exposure.

Previous studies of immune responses to TCP have also been limited by the lack of purified El Tor TcpA for use in assays; as described above, classical and El Tor TcpA differ immunologically, and assay of responses after El Tor cholera using classical TcpA as antigen may not detect immune responses appropriately.

[0033] In addition to TcpA, the V. cholerae genome encodes two other type IV pili, the mannose-sensitive hemagglutinin (MSHA) and PilA. A strain of V. cholerae deleted in mshA showed no defect in colonization of human volunteers.

[0034] Infection with V. cholerae can induce long-lasting protective immunity against subsequent disease, but the full repertoire of immune responses mediating protection is not known.

The best characterized of the immune responses induced by V. cholerae is the vibriocidal antibody; elevated vibriocidal titers correlate with protection from subsequent clinical disease in seroepidemiologic studies. Since V. cholerae is a non-invasive organism, and since there is no disruption of the intestinal epithelium during cholera, a serum complement-fixing antibody response such as the vibriocidal antibody may have minimal activity in the intestinal lumen. The vibriocidal antibody response, therefore, may be a surrogate marker for an intestinal response that is the primary mediator of protective immunity.

[00351 Immune responses following cholera have also been examined for a number of other antigens, but none of these responses has been shown to correlate with protection. Approximately 90% of individuals in Bangladesh developed an anti-MSHA response in serum and/or stool after cholera. The majority of the anti-cholera toxin (CT) immune response is directed against the non-toxic B subunit (CtxB). Serum anti-CT and anti-CtxB increase substantially after cholera, but these responses have not been shown to protect from subsequent disease.

[0036] All of the antigens described above against which immune responses have been characterized were identified utilizing in vitro grown V. cholerae. In order to identify additional microbial antigens uniquely expressed during infection, a number of newer techniques have been applied to identify gene expression specifically in vivo.

[0037] In vivo-induced antigen technology (IVIAT) is a method that circumvents limitations of animal models, allowing direct identification of microbial proteins expressed at sufficient levels during infection in the pathogen's normal host. These same proteins may be important for protective immunity following both natural infection and vaccination. The disclosure is based upon the use of IVIAT to identify V. cholerae genes specifically expressed during human infection.

[0038] The disclosure uses methods and compositions for identifying and isolating polynucleotides and polypeptides that are expressed only during in vivo growth of a microbe or pathogen. Briefly, the methods, termed in vivo induced antigen technologies (IVIAT), comprise obtaining a sample of antibodies against antigens that are expressed by the microbe in vivo and adsorbing these antibodies with cells or cellular extracts of the microbe that have been grown in vitro. An example of a sample of antibodies that can be used in the disclosure is sera from subject (s) who have been or are infected with the microbe.

[0039] A scheme depicting an IVIAT technique is shown in FIG. 1.

IVIAT can begin with serum from one or more subjects who have or recently had a disease or infection of interest. Such sera are individually tested to verify that they exhibit measurable antibody titers versus the microbial pathogen. One or more such reactive serum samples are adsorbed with cultured cells and lysates of the pathogen to remove antibodies reactive with antigens made during in vitro growth. The resulting adsorbed serum still contains antibodies reactive with immunogenic proteins produced by the pathogen only during in vivo growth. The remaining antibodies are then used to probe an expression library of the pathogen's DNA or RNA cloned in a convenient host. Reactive clones are isolated, and the cloned insert is sequenced. If more than one open reading frame is present on an insert, each is individually subcloned and retested with the adsorbed serum to identify which one encodes the reactive antigen. For independent verification, the polynucleotide is overexpressed, and the resulting recombinant protein is purified and used to raise monospecific antibodies. The antibodies are used to probe biological samples taken from subjects infected with the pathogen. Reactivity with cells of the pathogen in clinical samples, but not with cells of the pathogen grown in vitro, provides direct confirmation that the antigen is expressed only during in vivo growth. A sample of antibodies against antigens that are expressed by a microbe in vivo is collected. The sample can comprise the serum of a host or hosts infected with or previously infected with the microbe. Because a host from which serum is collected has undergone or is actively engaged in an actual infection by the microbe of interest, the host's serum contains antibodies produced against microbial antigens expressed during the in vivo infectious process. Serum from an individual host may be used or pooled sera from two or more hosts can be used. For example, sera from about 2,5, 25,100, 500, or 1,000 hosts may be pooled.

[0040] Where targets for vaccines and diagnostics are to be identified, one or several sera samples should suffice to identify those proteins that are immunogenic in most individuals. On the other hand, if the main interest is a more detailed analysis of the potential molecular mechanisms used by the pathogen, the use of a larger pool of sera would reduce the possibility of missing genes or polynucleotides of interest that are expressed during infection due to variability in a subject's immune response. In cases where the pathogen causes protracted disease, pooled serum from subjects in early, middle and late stages of infection or sequential samples taken from subjects during acute and convalescent phases of infection would offer the best chance of identifying transiently expressed in vivo induced polypeptides. Also, certain pathogens can infect subjects (e. g., humans) by more than one route (e. g., via a wound, gastrointestinal tract, respiratory tract, or skin); in such cases, selective pooling of serum from patients infected by various routes may enable the identification of route-specific in vivo induced polypeptides. In cases where different clonal variants or strains of the microbe cause disease, selective pooling of serum from subjects infected by various clonal variants or strains can enable the study of the pathogenesis of each variant or strain.

[0041] As used herein"a host"and"a subject"may be any animal.

For example, a host or a subject can comprise humans, baboons, chimpanzees, macaques, cattle, sheep, pigs, horses, goats, dogs, cats, rabbits, guinea pigs, rats, mice, chickens, ducks, fish, and shellfish.

[0042] The microbe or pathogen can be any kind of a bacterium, a virus, a parasite, a prion, or a fungus. For example, the microbe can be Candida, Aspergillus, Sporothrix, Blastomyces, Histoplasma, Cryptococcus, Pneumocystis, Coccidioides, Tinea, Toxoplasma, Plasmodium, Pseudomonas, Actinobacillus, Staphylococcus, Bacillus, Clostridium, Listeria, Corynebacterium, Actinomyces, Mycoplasma, Nocardia, Bordetella, Brucella, Francisella, Legionella, Enterobacter, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Streptococcus, Yersinia, Vibrio, Campylobacter, Helicobacter, Bacteroides, Chlamydia, Borrelia, Treponema, Leptospira, Aeromonas, Rickettsia, Ascaris, Cryptosporidium, Cyclospora, Entamoeba, Giardia, Shistosoma, Trypanosoma, herpes virus, cytomegalovirus, Epstein-Barr virus, hepatitis virus, adenovirus, papillomavirus, polyomavirus, enterovirus, rotavirus, influenza virus, paramyxovirus, rubeola virus, rhabdovirus, human immunodeficiency virus, arenavirus, rhinovirus, and reovirus. The microbe or pathogen can also be any kind of veterinary microbe or pathogen. See e. g., Veterinary Microbiology, Hirsh & Zee, Eds. , Blackwell Science, Inc., Malden, MA, 1999; Clinical Veterinary Microbiology, Quinn, Ed. , Wolfe Publishing, London, (1994). As discussed more fully herein, of particular interest is the Vibrio cholerae pathogen.

[0043) Antibodies, which bind to antigens that are produced during in vitro propagation of a microbe of interest, are eliminated from the sample of antibodies thereby leaving antibodies against antigens that are expressed by the microbe in vivo. The antibodies that are produced in vitro can be removed from the sample by, for example, adsorbtion. A sample containing antibodies against antigens that are expressed by the microbe in vivo, such as a serum sample of an infected host, are contacted with in vitro grown whole cells, cell extracts, or a combination of whole cells and cell extracts of the microbe.

[0044] All or substantially all of the antibodies in the antibody sample whose corresponding antigens are expressed during in vitro growth of the microbe will bind to these antigens to form immune complexes. However, antibodies directed against antigens that are specifically expressed during the in vivo infectious process will remain uncomplexed since their corresponding antigens are not present in the in. vitro grown cells and/or cell extracts. The adsorbtion step can be performed by, for example, contacting the antibody sample with whole cells and/or cell extracts that are immobilized on a solid support, such as a nitrocellulose membrane (see, Brady & Daphtary, Infect. Dis. 158 : 965-972,1988).

Optionally, the whole cell and/or cell extract sample can be denatured before use to expose additional immunoreactive epitopes.

Several successive adsorbtions can be performed using the same or different adsorbtion methodologies.

[00451 After the adsorbtion step or steps, unbound antibodies are separated from the antibody-antigen complexes. After elimination of antibodies that form immune complexes with microbial antigens expressed in vitro, the sample will comprise antibodies produced in response to antigens expressed in vivo. This sample can then be used to screen one or more, same or different, genomic expression libraries, for example, plasmid or bacteriophage genomic expression libraries, of the microbe of interest. Methods of constructing genomic expression libraries are known in the art (see, e. g., Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. , 1994; Maniais et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1989). Optionally, DNA or RNA from more than one strain or clonal variant of the microbe of interest can be pooled for expression library construction. Methods for screening these libraries with one or more labeled or unlabeled antibody-containing samples and for identifying and isolating clones from the library that encode the reactive antigens of interest are also known in the art, e. g., colony blotting methods (see, e. g. , Ausubel, 1994 ; Maniatis, 1989).

[0046] Reactive clones identified by screening expression libraries of the microbe of interest can be characterized by conventional analysis. For example, the clone can be sequenced and the presence of open reading frames can be predicted, for example, using Fickett's, start/stop codon or other methods (see, e. g., Fickett, Nucleic Acids Res. 10: 5303-5318,1982 ; Solovyev, Nucleic Acids Res.

22: 5156-5163,1994 ; Saqi, Protein Eng. 8: 1069-1073,1995 ; Ladunga and Smith, Protein Eng. 20: 101-110, 1997; Birney, Nucleic Acids Res.

24: 2730-2739,1996).

[0047] Identification of signal sequences, ribosome binding sites and transcription termination sequences, can also be made since the information provided by these analysis can be helpful in prioritizing the subcloning of open reading frames if more than one is present on the cloned insert. Moreover, the obtained sequence information can be used to identify sequence similarities to known polynucleotides, e. g. , by BLAST analysis, and possible relationships to proven or putative virulence factors can be evaluated. In instances where more than one open reading frame is present on a cloned insert, all of them may be analyzed for their ability to express in vivo induced antigens. For example, each open reading frame can be independently subcloned and tested with the adsorbed serum to identify in vivo induced antigens. roo4sX In vivo induced antigens identified by the foregoing IVIAT method can be verified as actually being expressed by the microbe by probing biological samples taken from infected subjects. For example, a suspected in vivo antigen polypeptide that is expressed from a clone can be used to raise polyclonal antibodies. The antibodies can be detectably labeled (e. g., labeled with fluorescein isothiocyanate (FITC) ). A biological sample from a host infected with the microbe of interest and from an in vitro grown microbe are obtained and contacted with the labeled antibody. The biological sample and the matched in vitro grown sample of the microbe are assayed by, for example, immunofluorescence microscopy. The labeled antibodies will react with the microbe found in the biological sample, but will not react with in vitro grown cells. These results can provide evidence that the microbe expresses an IVIAT identified antigen during in vivo growth.

[00493 Using the techniques described above, sera from ten patients convalescing from cholera infection in Bangladesh were pooled, adsorbed against in vitro-grown El Tor V. cholerae 01, and used to probe a genomic expression library in E. coli constructed from El Tor V. cholerae O1 strain N16961. Thirty-eight positive clones were identified in the screen, encoding pili (PilA, TcpA), cell membrane proteins (PilQ, MshO, MshP, CapK), methyl-accepting chemotaxis proteins, chemotaxis and motility proteins (CheA, CheR), a quorum sensing protein (LuxP), and four hypothetical proteins. Analysis of immune responses to purified PilA and TcpA in individual subjects demonstrated that the majority seroconverted to these proteins, confirming results with pooled sera. These results suggest that PilA and its outer membrane secretin, PilQ, are expressed during human infection and may be involved in colonization of the gastrointestinal tract. This is also the first demonstration of substantial immune responses to TcpA in patients infected with El Tor V. cholerae 01.

[0050] The methods and techniques identified PilA, TcpA, PilQ, MshO, MshP, CapK, CheA, CheR, LuxP and Hypl, Hyp2, Hyp3, and Hyp4 polypeptides as being expressed by V. cholerae during in vivo infection as well as the polynucleotides encoding the polypeptides.

The polynucleotides and polypeptides corresponding to the identified molecules above have a number of specific utilities as set forth herein.

[00511 The term"isolated"and"purified"as used herein means altered"by the hand of man"from its natural state ; i. e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a naturally occurring polynucleotide or a polypeptide naturally present in a living animal, a biological sample or an environmental sample in its natural state is not"isolated"or"purified", respectively, but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is"isolated"and"purified", as the term is employed herein. Polynucleotides, when introduced into host cells in culture or in whole organisms, still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment. Similarly, the polynucleotides and polypeptides may occur in a composition, such as a media formulation (solutions for introduction of polynucleotides or polypeptides, for example, into cells or compositions or solutions for chemical or enzymatic reactions). For example, an isolated polynucleotide is a polynucleotide comprising a sequence of the disclosure such that the isolated polynucleotide is separated, in some way, from the polynucleotide sequence in the naturally occurring genome at the 5'and 3'end of the isolated polynucleotide of the disclosure. Thus, the term"isolated polynucleotide" includes any nucleic acid molecules that are not naturally occurring. The term therefore includes, for example, a recombinant polynucleotide which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences. The terms"isolated"and"purified"do not necessarily mean 100% isolated or purified from other materials, but rather means that some substantial degree of isolation or purification has taken place. For example, an isolated or purified polynucleotide or polypeptide, respectively, includes polynucleotides or polypeptides that are 20% or more free of material of which that are naturally associated.

[00523''Polypeptidell refers to any polymer of two or more individual amino acids linked via a peptide bond. The term "polypeptide"is understood to include the terms"protein"and "peptide" (which, at times may be used interchangeably herein). In addition, polypeptides comprising multiple polypeptide subunits (e. g., DNA polymerase III, RNA polymerase II) or other components (for example, an RNA molecule, as occurs in telomerase) will also be understood to be included within the meaning of"polypeptide"as used herein. Similarly, fragments of polypeptides are also within the scope of the disclosure.

[0053] Polypeptides of the disclosure can either be full-length polypeptides or fragments of polypeptides (e. g., soluble domains and/or extracellular domains of the polypeptides). For example, fragments of polypeptides of the disclosure can comprise about 5, 10,25, 50,100, 200,250, or more amino acids of polypeptides of the disclosure so long as the fragment is shorter in length than the full length of the polypeptide from which it is derived. Examples of polypeptides of the disclosure include Vibrio cholerae polypeptides as set forth in SEQ ID Nos: 2,4, 6,8, 10,12, 14,16, 18,20, 22,24, 26,28, 30, and 32. Homologous polypeptides include polypeptide sequences which are at least about 75%, typically about 90%, 95%, 98%, or 99% identical to the polypeptide sequence set forth in SEQ ID Nos: 2,4, 6,8, 10,12, 14,16, 18, 20,22, 24,26, 28,30, or 32.

[0054] Polypeptides of the disclosure further comprise biologically functional equivalents/fragments of at least about 5,10, 25,50, 100, 200,250, or more amino acids of a polypeptides shown in SEQ ID NOs : 2,4, 6,8, 10,12, 14,16, 18,20, 22,24, 26,28, 30, or 32. A polypeptide is a biological equivalent if it can be detected in an assay such as an immunohistochemical assay, an ELISA, an RIA, or a western blot assay using reagents that specifically recognize polypeptides comprising a sequence as set forth in SEQ ID Nos: 2,4, 6,8, 10, 12, 14, 16, 18,20, 22,24, 26,28, 30, or 32.

[00553 Polypeptides of the disclosure include polypeptides that comprise an antigen that is recognized by an antibody that specifically interacts with a polypeptide comprising SEQ ID Nos: 2, 4,6, 8,10, 12,14, 16,18, 20,22, 24,26, 28,30, or 32. A polypeptide antigen of the disclosure comprises one or more epitopes (or antigenic determinants) specifically recognized by an antibody that binds to a polypeptide comprising a sequence as set forth in SEQ ID Nos : 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22,24, 26, 28, 30, or 32. An epitope can be a linear, sequential epitope or a conformational epitope. Epitopes within a polypeptide of the disclosure can be identified by several methods (see, e. g. , U. S.

Patent No. 4,554, 101; and Jameson & Wolf, CABIOS 4: 181-186, 1988).

For example, a polypeptide of the disclosure can be isolated and screened. A series of short peptides, which together comprise the entire polypeptide sequence, can be prepared by proteolytic cleavage. By starting with, for example, 100-mer polypeptide fragments, each fragment can be tested for the presence of epitopes recognized in an enzyme-linked immunosorbent assay (ELISA). For example, in an ELISA assay a microbial polypeptide, such as a 100- mer polypeptide fragment, is attached to a solid support, such as the wells of a plastic multi-well plate. A population of antibodies are labeled, added to the solid support and allowed to bind to the unlabeled antigen, under conditions where non-specific adsorbtion is blocked, and any unbound antibody and other proteins are washed away. Antibody binding is detected by, for example, a reaction that converts a colorless substrate into a colored reaction product.

Progressively smaller and overlapping fragments can then be tested from an identified 100-mer to map the epitope of interest.

[0056] Soluble domains of the polypeptides of the disclosure are particularly useful in developing antibodies and for use in generating an immune response in a subject that has been administered a soluble domain of a polypeptide comprising a sequence as set forth in SEQ ID Nos : 2, 4, 6, 8,10, 12, 14, 16,18, 20,22, 24,26, 28,30, or 32. Figure 7 provides the various polynucleotide and polypeptide sequences of the disclosure. The bolded regions of the polypeptide sequences are indicative of putative transmembrane domains and/or hydrophobic regions of the polypeptide. The italicized letters correspond to amino acids that are part of a putative signal domain. The phrase"inside to outside"or"outside to inside"is indicative of the putative direction of the first putative transmembrane region of the polypeptide (e. g., inside the cell to outside the cell). Typically soluble domains that are useful in generating vaccines or in generating antibodies will comprise a domain that is on the extracellular surface of the polypeptide. Utilizing the information provided in Figure 7, one of skill in the art can determine the orientation and extracellular domains of the polypeptides of the disclosure. In addition, one of skill in the art can also predict from the polypeptide sequences the corresponding coding region for both the transmembrane and/or hydrophobic region in the polynucleotide sequences. It should be kept in mind that the putative transmembrane region and hydrophobic regions are predicted by in silico techniques and may differ by 1-5 amino acids at either the N-terminal or C-terminal end of the putative domain. Accordingly, the disclosure provides polypeptides having a sequence as set forth in SEQ ID Nos: 2,4, 6,8, 10, 12, 14,16, 18,20, 22,24, 26,28, 30, and 32 that specifically lack a hydrophobic or transmembrane domain as identified herein.

[0057] A polypeptide of the disclosure can be produced recombinantly. A polynucleotide encoding a polypeptide of the disclosure can be introduced into a recombinant expression vector, which can be expressed in a suitable expression host cell system using techniques well known in the art. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system can be used. Optionally, a polynucleotide encoding a polypeptide can be translated in a cell- free translation system.

[0058] A"polynucleotide"refers to a polymeric form of nucleotides. In some instances a polynucleotide refers to a polymer of nucleotides that are not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5'end and one on the 3'end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e. g., a cDNA) independent of other sequences.

The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. A polynucleotide as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single-and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-and double-stranded regions.

[0059] Polynucleotides can be cloned into an appropriate vector.

The vector used will depend upon whether the DNA is to be expressed, amplified, sequenced etc. Cloning techniques are known in the art or can be developed by one skilled in the art, without undue experimentation. The choice of a vector will also depend on the size of the polynucleotide and the host cell to be employed in the methods of the disclosure. Thus, a vector used in the disclosure may be a plasmid, a phage, a cosmid, a phagemid, a virus (e. g. retroviruses, parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof. For example, cosmids and phagemids are typically used where the specific polynucleotide to be cloned is large because these vectors are able to stably propagate large polynucleotides.

[0060] Polynucleotides of the disclosure can also comprise other nucleotide sequences, such as sequences coding for linkers, signal sequences, heterologous signal sequences, TMR stop transfer sequences, transmembrane domains, or encode ligands useful in protein purification ("purification proteins") such as glutathione- S-transferase, histidine tag, and staphylococcal protein A or adjuvants useful in promoting an immune response upon expression.

More than one polypeptide of the disclosure can be present in a fusion protein.

[0061] A polynucleotide of the disclosure comprises a sequence as set forth in SEQ ID No: 1,3, 5,7, 9,11, 13,15, 17,19, 21, 23, 25,27, 29, or 31; a polynucleotide complementary to SEQ ID NO : 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27,29, or 31; and a polynucleotide wherein the deoxynucleotides A, G, C, and T of SEQ ID NO : 1, 3,5, 7,9, 11, 13, 15, 17, 19,21, 23, 25, 27, 29 or 31 are replaced by ribonucleotides A, G, C, and U, respectively. Also included in the disclosure are fragments of the above-described polynucleotides that are at least 15 bases in length (e. g., 15, 30, 50,100, 200 or more bases in length), which is sufficient to permit the fragment to specifically hybridize to a polynucleotide sequence of SEQ ID NO : 1, 3,5, 7,9, 11,13, 15,17, 19,21, 23,25, 27,29, or 31. The term"specifically hybridize"refers to hybridization under moderately or highly stringent conditions that excludes non- related nucleotide sequences. Also encompassed by the disclosure are polynucleotides that encode a polypeptide comprising SEQ ID NO : 2, 4,6, 8,10, 12, 14, 16, 18, 20,22, 24,26, 28,30, or 32, as well as a fragment of any of the foregoing. Such fragments are useful for encoding antigenic epitopes for use in developing antibodies and the like.

[0062] A particular amino acid sequence of a given polypeptide (i. e. , the polypeptide's"primary structure, "when written from the amino-terminus to carboxy-terminus) is determined by the nucleotide sequence of the coding portion of an mRNA, which is in turn specified by genetic information, typically genomic DNA (including organelle DNA, e. g. mitochondrial or chloroplast DNA). Thus, determining a nucleotide sequence of a polynucleotide assists in predicting the primary sequence of a corresponding polypeptide and more particular the role or activity of the polypeptide encoded by that polynucleotide.

[0063] Once a polynucleotide of the disclosure is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector. This is referred to as clonal amplification.

[0064] The vector containing the cloned polynucleotide is amplified by plating or transfecting a suitable host cell with the vector (e. g., a phage on an E. coli host). By"transformation"is meant a permanent (e. g., stable) or transient genetic change induced in a cell following incorporation of new polynucleotide (e. g., DNA exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the polynucleotide into the genome of the cell. By"transformed cell"or"recombinant host cell"is meant a cell (e. g., prokaryotic or eukaryotic) that has been genetically modified. A recombinant host cell includes a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques a polynucleotide of the disclosure or fragment thereof, or which has been modified to lack a viable gene comprising a polynucleotide sequence as set forth in SEQ ID No : 1, 3,5, 7,9, 11,13, 15,17, 19,21, 23,25, 27,29, and/or 31, or which lacks an active or viable gene product (e. g., a polypeptide comprising a sequence as set forth in SEQ ID N0 : 2,4, 6,8, 19,12, 14, 16, 18, 20,22, 24, 26,28, 30, and/or 32).

[0065] Polynucleotides can be cloned into an expression vector comprising, for example, promoters, enhancers, or other regulator elements that drive expression of the polynucleotides of the disclosure in recombinant host cells. An expression vector can be, for example, a plasmid, such as pBR322, pUC, or ColEl, or an adenovirus vector, such as an adenovirus Type 2 vector or Type 5 vector. Other vectors that can be used with the polynucleotides of the disclosure include, but are not limited to, Sindbis virus, simian virus 40, alphavirus vectors, poxvirus vectors, cytomegalovirus and retroviral vectors, such as murine sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and Rous sarcoma virus. Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes, virus particles, virus-like particles, cosmids (plasmids into which phage lambda cos sites have been inserted) and replicons (genetic elements that are capable of replication under their own control in a cell) can also be used.

[0066] In general, a polynucleotide is cloned into a vector at an appropriate restriction endonuclease site (s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art. A typical cloning scenario may have the DNA"blunted"with an appropriate nuclease (e. g., Mung Bean Nuclease), methylated with, for example, EcoRI Methylase and ligated to EcoRI linkers GGAATTCC (SEQ ID NO : 33). The linkers are then digested with an EcoR I Restriction Endonuclease and the DNA size fractionated (e. g., using a sucrose gradient). The resulting size fractionated DNA is then ligated into a suitable vector for sequencing, screening or expression (e. g., a lambda vector and packaged using an in vitro lambda packaging extract).

10067] A polynucleotide in the expression vector is operatively linked to an appropriate expression control sequence (s) (e. g., a promoter) to direct mRNA synthesis. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp.

Eukaryotic promoters include CM ?-immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.

[00681 In addition, the expression vectors typically contain one or more selectable marker genes to provide a phenotypic trait for selection of recombinant host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

[0069] A vector comprising a polynucleotide of the disclosure can be transformed into, for example, bacterial, yeast, insect, or mammalian cells so that the polypeptides of the disclosure can be expressed from the polynucleotides and purified from cell culture.

Any technique available in the art can be used to introduce polynucleotides into the host cells. These include, but are not limited to, transfection with naked or encapsulated polynucleotides, cellular fusion, protoplast fusion, viral infection, and electroporation.

[0070] Transformation of a cell with a vector or polynucleotide of the disclosure to generate a recombinant host cell may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of polynucleotide uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method by procedures well known in the art.

Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.

[0071] When the host is a eukaryote, methods of transfection or transformation with DNA include calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors, as well as others known in the art, may be used.

Eukaryotic cells can also be cotransfected with a second foreign DNA molecule encoding a selectable marker, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzinan ed., 1982). The eukaryotic cell may be a yeast cell Saccharomyces cerevisiae) an insect cell (e. g., Drosophila sp. ) or may be a mammalian cell (e. g., human cells, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and38).

[0072] Polypeptides and polynucleotides that are homologous to the polypeptide and/or polynucleotides of the disclosure can be identified using database search algorithms and genomic databases. A number of source databases are available that contain either genomic sequences and/or a deduced amino acid sequence for use with the disclosure in identifying or determining the activity encoded by a particular polynucleotide sequence or identifying homologs of the polynucleotides and/or polypeptides of the disclosure.

Representative portions (or the full length) of the sequences (e. g., SEQ ID Nos: 1-32) are used to search a sequence database (e. g., GenBank, PFAM or ProDom), either simultaneously or individually. A number of different methods of performing such sequence searches are known in the art. The databases can be specific for a particular organism or a collection of organisms. For example, there are databases for V. cholerae, C. elegans, Arabadopsis. sp. , M. genitalium, M. jannaschii, E. coli, H. influenzae, S. cerevisiae and others. The sequence of SEQ ID NO : 1-31, or 32 is aligned to sequence (s) in the database or databases using algorithms designed to measure homology between two or more sequences.

[00733 Such sequence alignment methods include, for example, BLAST (Altschul et al. , 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), and FASTA (Person & Lipman, 1988). The probe sequence (e. g., a sequence as set forth in SEQ ID Nos: 1-32) can be any length, and will be recognized as homologous based upon a threshold homology value. For example, the sequence used to probe the database can be a fragment of the full-length sequence or the full-length sequence.

In one aspect, the percent identity is determined based upon a comparison to the full sequence and not a fragment of the sequence.

The threshold value may be predetermined, although this is not required. The threshold value can be based upon the particular polynucleotide length. To align sequences a number of different procedures can be used. Typically, Smith-Waterman or Needleman- Wunsch algorithms are used. However, faster procedures such as BLAST, FASTA, PSI-BLAST can be used.

[0074] For example, optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Theor Biol, 1981 ; Smith and Waterman, J Mol Biol, 1981 ; Smith et al. , J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch, 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr. , Madison, Wis. , or the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin, Madison, Wis.), or by inspection, and the best alignment (i. e. , resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. The similarity of the two sequences (i. e. , the probe sequence and the database sequence) can then be predicted.

[0075] Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications. The terms"homology"and"identity"in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.

[0076] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences (i. e. , probe sequences) are compared. When using a sequence comparison algorithm, probe and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the probe sequences relative to the reference sequence, based on the program parameters.

[oO773 A"comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions (e. g., nucleotides or amino acids in a polynucleotide or polypeptide, respectively) selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a probe sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can be the full-length sequence as set forth in SEQ ID NO : 1-31, or 32.

[0078] One example of a useful algorithm is BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. , Nuc. Acids Res.

25: 3389-3402,1977 ; and Altschul et al., J. Mol. Biol. 215 : 403-410, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the internet at www-ncbi. nlm. nih. gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.

T is referred to as the neighborhood word score threshold (Altschul et al. , supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0). The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands.

[0079] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e. g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90: 5873,1993). One measure of similarity provided by BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a probe sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the probe sequence to the reference sequence is less than about 0. 2, typically less than about 0.01, and more commonly less than about 0.001.

[0080] Sequence homology or identity means that two polynucleotide sequences are homologous (i. e. , on a nucleotide-by-nucleotide basis) over the window of comparison. A percentage of sequence identity or homology is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid bases (e. g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i. e. , the window size), and multiplying the result by 100 to yield the percentage of sequence homology. Thus, substantial homology denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 60 percent sequence homology, typically at least 70 percent homology, often 80 to 90 percent sequence homology, and most commonly at least 99 percent sequence homology as compared to a reference sequence of a comparison window of at least 25-50 nucleotides, wherein the percentage of sequence homology is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

[0081] Sequences having sufficient homology can then be further identified by any annotations contained in the database from which a reference sequence is derived, including, for example, species and activity information.

10082] Antibodies can be developed and purified that specifically interact with a polypeptide or antigenic epitope of the disclosure.

Antibodies of the disclosure are antibody molecules that specifically and stably bind to a microbial polypeptide of the disclosure or fragment thereof. An antibody of the disclosure can be a polyclonal antibody, a monoclonal antibody, a single chain antibody (scFv), or a part of an antibody. Parts of antibodies include Fab and F (ab) 2 fragments. Antibodies can be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. Means for preparing and characterizing antibodies are well known in the art (see, e. g., Dean, Methods Mol. Biol. 80: 23-37, 1998 ; Dean, Methods Mol. Biol. 32: 361-79,1994 ; Baileg, Methods Mol.

Biol. 32: 381-88,1994 ; Gullick, Methods Mol. Biol. 32: 389-99,1994 ; Drenckhahn et al., Methods Cell. Biol. 37: 7-56,1993 ; Morrison, Ann.

Rev. Immunol. 10 : 239-65, 1992 ; and Wright et al., Crit. Rev.

Immunol. 12: 125-68,1992). For example, polyclonal antibodies can be produced by administering a polypeptide of the disclosure (e. g., a polypeptide having a sequence as set forth in SEQ ID NO: 2,4, 6, 8, 10, 12,14, 16, 18,20, 22,24, 26,28, 30, or 32) to an animal, such as a mouse, a rabbit, a goat, or a horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by chromatography, typically affinity chromatography.

Techniques for producing and processing polyclonal antibodies are known in the art.

[0083] Additionally, monoclonal antibodies directed against epitopes present on a polypeptide of the disclosure can also be readily produced. For example, normal B cells from a mammal, such as a mouse that has been immunized with a polypeptide of the disclosure can be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomas producing microbe- specific antibodies can be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution.

Clones producing microbe-specific antibodies are isolated by another round of screening. Techniques for producing and processing monoclonal antibodies are known in the art.

[0084] The antigen polypeptides of the disclosure (e. g., comprising a sequence as set forth in SEQ ID NO : 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, or 32) can be used to screen biological samples (e. g., sera, tissue, urine, feces, vomitus and the like) of subjects for reactive antibodies. For example, a biological sample can be obtained from a subject suspected of having a V. cholerae infection. The sample can be probed for reactive antigen with a labeled, purified antibody. Alternatively, the sample may be contacted with an antigen to determine the pressure of reactive antibodies produced by the subject. If antibodies are present this is also indicative of an infection.

10085] Similarly, antibodies, monoclonal and/or polyclonal, which are directed against polypeptide antigens of the disclosure, are useful for detecting the presence of microbes or microbial antigens in a sample, such as a serum sample, tissue sample, urine, feces, vomitus and the like from a microbe-infected subject (e. g. , a subject having a V. cholerae infection). Although the following description describes immunoassays utilizing labeled antibodies, similar assays (known in the art) can be performed utilizing labeled polypeptide antigens of the disclosure (e. g., antigens comprising a sequence as set forth in SEQ ID NO : 2,4, 6,8, 10,12, 14, 16, 18, 20,22, 24,26, 28,30, or 32).

100863 An immunoassay for a polypeptide antigen of the disclosure may utilize one antibody or several antibodies. An immunoassay for a polypeptide antigen may use, for example, a monoclonal antibody directed towards a microbial epitope, a combination of monoclonal antibodies directed towards epitopes of one polypeptide antigen, monoclonal antibodies directed towards epitopes of different polypeptide antigens, polyclonal antibodies directed towards the same polypeptide antigen, polyclonal antibodies directed towards different polypeptide antigens, or a combination of monoclonal and polyclonal antibodies. Immunoassay protocols are based, for example, upon competition, direct reaction, or sandwich type assays using, for example, a labeled antibody. The labels may be, for example, fluorescent, chemiluminescent, or radioactive.

Immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection can be used to detect polypeptide antigens of the disclosure in a biological sample.

[0087] The antibodies (or fragments thereof) useful in the present disclosure may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of V. cholerae polypeptide antigens. In situ detection may be accomplished by removing a histological specimen from a subject thought to be infected by V. cholerae (e. g., a serum sample, intestinal sample, stool sample and the like), and applying thereto a labeled antibody of the disclosure. The antibody (or fragment) is applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of a V. cholerae polypeptide antigen, but also its distribution in the examined tissue or other biological sample. Using the disclosure, those skilled in the art will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[oO88} Immunoassays as described above, generally, comprise incubating a biological sample such as a biological fluid including, but not limited to, blood, plasma, or blood serum, a tissue extract, freshly harvested cells, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody that binds to a V. cholerae polypeptide antigen of the disclosure, and detecting the bound antibody by any of a number of techniques well known in the art.

[0089] Monoclonal antibodies directed toward a V. cholerae polypeptide antigen of the disclosure are useful for the in vivo detection of V. cholerae and/or V. cholerae antigen. The detectably labeled monoclonal antibody is given in a dose that is diagnostically effective. The term"diagnostically effective"means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of V. cholerae and/or an antigen for which the monoclonal antibodies are specific.

(00901 The concentration of detectably labeled monoclonal antibody, which is administered to a subject, should be sufficient such that the binding of the antibody to its respective antigen is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.

10091] For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting a given radioisotope. The radioisotope chosen must have a type of decay that is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 key range, which may be readily detected by conventional gamma cameras.

[00923 For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional cheating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.

Typical examples of metallic ions which can be bound to the monoclonal antibodies of the disclosure are l n, 97RU, 57Gar 68Ga, As,"Zr, and Tl.

[0093] The monoclonal antibodies of the disclosure can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include 157Gd, 55Mn, 162Dy, 52Cr, and 56Fe.

[0094] The monoclonal antibodies of the disclosure can be used to monitor the course of amelioration of V. cholerae infection. Thus, by measuring an increase or decrease of a V. cholerae polypeptide antigen of the disclosure in various body fluids, feces or tissues, it would be possible to determine whether a particular therapeutic treatment aimed at ameliorating the infection is effective. t00957 Polyclonal or monoclonal antibodies developed against polypeptide antigens comprising a sequence as set forth in SEQ ID NO : 2,4, 6,8, 10,12, 14, 16, 18,20, 22,24, 26,28, 30, or 32 may also be used to isolate microbes or microbial antigens by immunoaffinity columns. The antibodies can be affixed to a solid support by, for example, adsorbtion or by covalent linkage so that the antibodies retain their immunoselective activity. Optionally, spacer groups may be included so that the antigen-binding site of the antibody remains accessible. The immobilized antibodies can then be used to bind microbes or microbial antigens from a sample, such as a biological sample including saliva, plaque, sputum, blood, urine, vomitus, feces, cerebrospinal fluid, amniotic fluid, wound exudate, or tissue. The bound microbes or microbial antigens are recovered from the column matrix by, for example, a change in pH.

[0096] Antibodies of the disclosure can also be used in immunolocalization studies to analyze the presence and distribution of a polypeptide of the disclosure during various cellular events or physiological conditions. Antibodies can also be used to identify molecules involved in passive immunization and to identify molecules involved in the biosynthesis of non-protein antigens.

Identification of such molecules can be useful in vaccine development.

[00973 Antibodies of the disclosure can also be used to treat, prevent, or ameliorate a V. cholerae infection, to diagnose or detect the presence or absence of V. cholerae, and to purify an antigen or antigens to which the antibody specifically binds.

[0098] Polynucleotides of the disclosure can be used, for example, as probes or primers to detect the presence of V. cholerae polynucleotides in a sample, such as a biological sample. The ability of such probes to specifically hybridize to a polynucleotide sequence of the disclosure consisting of SEQ ID NO : 1, 3,5, 7,9, 11,13, 15,17, 19,21, 23,25, 27,29, 31, complements thereof, or fragments thereof will enable a technician to detect the presence of complementary sequences in a given sample. Polynucleotide probes of the disclosure can hybridize to complementary sequences in a sample such as a biological sample, including plaque, saliva, sputum, blood, urine, vomitus, feces, cerebrospinal fluid, amniotic fluid, wound exudate, or tissue. Polynucleotides from the sample can be, for example, subjected to gel electrophoresis or other size separation techniques or can be dot blotted without size separation.

The polynucleotide probes are typically labeled. Suitable labels, and methods for labeling probes are known in the art and include, for example, radioactive labels incorporated by nick translation, or kinase, biotin, fluorescent probes, and chemiluminescent probes.

The polynucleotides from the sample are then contacted with a probe under hybridization conditions of suitable stringencies.

[009'R1 Depending on the application, varying conditions of hybridization can be used to achieve varying degrees of selectivity of the probe towards a target polynucleotide in a sample. For applications requiring high selectivity, relatively stringent conditions can be used, such as low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50°C to about 70°C. In one aspect, the conditions include 0.5 M salt at 60°C. For applications requiring less selectivity, less stringent hybridization conditions can be used. For example, salt conditions from about 0.14 M to about 0.9M salt, at temperatures ranging from about 20°C to about 55°C. The presence of a hybridized complex comprising the probe and a complementary polynucleotide from the sample indicates the presence of a V. cholerae polynucleotide in the sample.

[00100] Polypeptides or polynucleotides of the disclosure can be used to elicit an immune response in a subject.'Elicitation of an immune response can be used, inter alia, to provide model systems to optimize immune responses to V. cholerae and to provide prophylactic or therapeutic treatment against V. cholerae infection. Elicitation of an immune response can also be used to treat, prevent, or ameliorate a disease or infection caused by a microbe or pathogen.

An immune response includes humoral immune responses and cell mediated immune responses, or a combination thereof. Typically, an immune response is a humoral immune response.

[00101] The generation of an antibody titer by an animal against a microbe can be important in protection from infection and clearance of infection. Detection and/or quantification of antibody titers after delivery of a polypeptide or polynucleotide can be used to identify epitopes that are particularly effective at eliciting antibody titers. Epitopes responsible for a strong antibody response to a microbe can be identified by eliciting antibodies directed against microbial polypeptides of different lengths.

Antibodies elicited by a particular polypeptide epitope can then be tested using an ELISA assay to determine which polypeptides contain epitopes that are most effective at generating a strong response.

Polypeptides or fusion proteins which contain these epitopes or polynucleotides encoding the epitopes can then be constructed and used to elicit a strong antibody response.

[00102] A polypeptide or a polynucleotide of the disclosure can be administered to an animal, such as a mouse, rabbit, guinea pig, macaque, baboon, chimpanzee, human, cow, sheep, pig, horse, dog, cat, chicken, and duck, to elicit antibodies in vivo. Injection of a polynucleotide has the practical advantages of simplicity of construction and modification. Further, injection of a polynucleotide results in the synthesis of a polypeptide in the host. Thus, the polypeptide is presented to the host immune system with native post-translational modifications, structure, and conformation. Naked DNA is typically used for in vivo delivery of a polynucleotide.

[00103] Administration of a polynucleotide or a polypeptide can be by any means known in the art, including intramuscular, intradermal, intraperitoneal, or subcutaneous injection, intranasal, mucosal, topical, and oral, including injection using a biological ballistic gun ("gene gun"). Typically, a polynucleotide or polypeptide is accompanied by a pharmaceutically acceptable carrier for administration. A combination of administration methods may also be used to elicit an immune response.

1001041 Administration of polypeptides or polynucleotides can elicit an immune response in the animal that lasts for at least 1 week, 1 month, 3 months, 6 months, 1 year, or longer. Optionally, an immune response can be maintained in an animal by providing one or more booster injections of the polypeptide or polynucleotide at 1 month, 3 months, 6 months, 1 year, or more after the primary injection.

[00105] A composition of the disclosure comprising a polypeptide, polynucleotide, or a combination thereof is administered in a manner compatible with the particular composition used and in an amount which is effective to elicit an immune response as detected by, for example, an ELISA, as described above. A polynucleotide is typically injected intramuscularly to a large mammal, such as a baboon, chimpanzee, or human, at a dose of 1 ng/kg, 10 ng/kg, 100 ng/kg, 1000 ng/kg, 0. 001 mg/kg, 0.1 mg/kg, or 0. 5 mg/kg. A polypeptide is typically injected intramuscularly to a large mammal, such as a human, at a dose of 0. 01, 0.05, 0. 5, 0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg. Polypeptides and/or polynucleotides can be administered either to an animal that is not infected with V. cholerae or can be administered to a V. cholerae-infected animal.

The particular dosages polynucleotides or polypeptides in a composition will depend on many factors including, but not limited to, the species, age, and general condition of the mammal to which the composition is administered, and the mode of administration of the composition. An effective amount of the composition of the disclosure can be readily determined using routine experimentation.

In vitro and in vivo models can be employed to identify appropriate doses. If desired, co-stimulatory molecules or adjuvants can also be provided before, after, or together with the compositions (e. g., as fusion proteins).

[00106] A pharmaceutically acceptable carrier should not itself induce the production of antibodies harmful to the host.

Pharmaceutically acceptable carriers are known to those in the art.

Such carriers include, but are not limited to, large, slowly metabolized macromolecules, such as proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads and the like; polylactic acids, polyglycolic acids ; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers ; peptoids; lipitoids; and inactive, virulent virus particles or bacterial cells.

[00107] Pharmaceutically acceptable salts can also be used in compositions of the disclosure, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates. Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art. Compositions of the disclosure can also contain liquids or excipients, such as water, saline, glycerol, dextrose, malodextrin, ethanol, or the like, singly or in combination, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes can also be used as a carrier for a composition of the disclosure.

[001083 If desired, co-stimulatory molecules, which improve immunogen presentation to lymphocytes, such as B7-1 or B7-2, or cytokines such as MIPla, GM-CSF, IL-2, and IL-12, can be included in a composition of the disclosure. Adjuvants can also be included in a composition including, but are not limited to, MF59-0, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N- acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637), referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethyla mine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

[00109] The compositions of the disclosure can be formulated into ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, injectable formulations, mouthwashes, dentifrices, and the like. The percentage of one or more polypeptides or polynucleotides of the disclosure in such compositions and preparations can vary from 0. 1% to 60% of the weight of the unit.

[00110] The disclosure includes various compositions that can be used to induce an immune response in a mammal (e. g. , a human) against infection or progression of a V. cholerae. In one aspect, a composition useful for inducing an immune response includes a cell or virus expressing a polynucleotide of SEQ ID NO: 1,3, 5,7, 9, 11,13, 15,17, 19,21, 23, 25,27, 29,31, or a fragment thereof, e. g., a live vaccine cell. Examples of suitable cells that can be genetically modified (i. e.,, a recombinant cell) to contain a polynucleotide of the disclosure include B. subtilis, BCG, Salmonella sp., Vibrio cholerae, Listeriae, Yersiniae, Streptococci, Corynebacterium diphtheriae, and N. coli. A live vaccine cell can be a naturally virulent live microorganism, or a live microorganism with low or attenuated virulence, that expresses a polypeptide of SEQ ID N0 : 2,4, 6, 8, 10,12, 14,16, 18, 20, 22,24, 26, 28, 30, and/or 32. In one aspect, a V. cholerae bacterium is genetically modified to lack one or more viable genes comprising a sequence as set forth in SEQ ID NO : 1, 3,5, 7,9, 11,13, 15,17, 19,21, 23, 25,27, 29, and/or 31. As described herein, the polynucleotides and the encoded polypeptides of SEQ ID Nos: 1-32, respectively, have been shown to be expressed during in vivo infection. Accordingly, a V. cholerae lacking one or more viable genes encoding a polypeptide of SEQ ID NO : 2,4, 6,8, 10,12, 14,16, 18, 20,22, 24, 26, 28,30, and/or 32 (e. g. , a pathogenic factor) can be designed to have reduced pathogenicity and thus be suitable as a live vaccine (i. e. , useful for eliciting an immune response while having limited to no pathogenicity).

[00111] Methods of modifying V. cholerae to lack pathogenicity include mutations to any of the naturally occurring sequences in the V. cholerae genome comprising the sequence as set forth in SEQ ID NO : 1, 3,5, 7,9, 1,13, 15,17, 19,21, 23,25, 27,29, and/or 31 by methods known in the art. Such mutations can include deletions, insertions, substitutions that result in a non-functional gene or gene product. A deletion may include a deletion of a full coding sequence as set forth in any of SEQ ID Nos, 1,3, 5,7, 9,11, 13, 15,17, 19, 21, 23,25, 27,29, and 31, or a fragment of any of the foregoing. The deletion may comprise a single nucleotide deletion resulting in a frame shift rendering the gene product non- functional. A substitution includes a substitution of a sequence that replace or disrupts a gene comprising a sequence as set forth in SEQ ID Nos, 1,3, 5,7, 9,11, 13, 15,17, 19, 21,23, 25,27, 29, and 31.

(001121 The following are provided for exemplification purposes only and are not intended to limit the scope of the disclosure described in broad terms above.

EXAMPLES [00113] Strains, plasmids and media. Strains and plasmids used are listed in Table 1. Strain N16961 was used to construct the genomic library described below; the genome sequence of this strain has been published (Heidenberg et al., Nature, 406: 477-483, 2000). Bacterial strains were grown in vitro in Luria-Bertani (LB) or AKI media (Murley et al.. Infect. Immun. 68: 3010-3014, 2000), and maintained at-70°C in LB broth containing 15% glycerol.

[00114] Genetic methods and strain constructions. Oligonucleotides used for PCR and DNA sequencing were obtained from Operon Technologies (Alameda, CA). PCR was performed with TaKaRa Taq polymerase (New England Biolabs, Beverly, MA) or Pfx DNA polymerase (Stratagene, La Jolla, CA), and an MJ Research Thermocycler (model PTC 100). PCR templates were prepared by boiling a single colony of V. cholerae strain N16961 in distilled H20, followed by centrifugation and recovery of supernatant. DNA sequencing was performed at the DNA Sequencing Core Facility, Department of Molecular Biology, Massachusetts General Hospital. DNA sequences were assembled and open reading frames assigned with Gene Works Analysis software package (The Institute for Genomic Research, Rockville, MD). Plasmids pLHEABl, pLHEAB2, pLHEAB3, pLHMAI, and pLH1 were used for expressing intact PilQ, PilA, TcpA, TcpF, and MshA from V. cholerae strain N16961, respectively, as shown in Table 1.

Table 1. Bacterial strains and plasmids used.

Strains Genotype and/or phenotype M16961 Wild-type E1 Tor biotype strain. Genomic sequence available; se E1 Tor Isolate from a patient in Bangladesh. clinical isolate Plasmids pET30 (abc) Expression vectors allowing cloning of fragments in each of three reading frames; Kanr pLHEAB1 pET30 (a) with the intact pilQ gene amplified by PCR from N16961Sm (1731 bp), and cloned in the Nadel and Mol restriction enzyme sites in the vector; Kanr pLHEAB2 pET30 (a) with the intact pilA gene amplified by PCR from N16961Sm (459 bp), and cloned in the Ndel and Xhol restriction enzyme sites in the vector; Kanr pLHEAB3 pET30 (a) with the intact tcpA gene amplified by PCR from N16961Sm (673 bp), and cloned in the Ndel and XhoT restriction enzyme sites in the vector; Kanr pLHMAl pET30 (a) with the intact tcpF gene amplified by PCR from N16961Sm (1014 bp), and cloned in the Nadel and Xhol restriction enzyme sites in the vector; Kan" pLHI pET30 (a) with the intact mshA gene amplified by PCR from N16961Sm (534 bp), and cloned in the EcoRV and Xhol restriction enzyme sites in the vector ; Kan' Smr, streptomycin-resistant,; Kanr, kanamycin-resistant; Apr, ampicillin- resistant.

[00115] Patient and control sera. Serum was collected from patients at the International Centre for Diarrhoeal Disease Research in Dhaka, Bangladesh (ICDDR, B). Each patient had culture-confirmed acute diarrheal illness due to El Tor V. cholerae 01, and each demonstrated a 4-fold or greater rise in vibriocidal antibody titer between acute and convalescent sera. Patients seen at the ICDDR, B during the same time period but who were not infected with V. cholerae (by culture and paired serum vibriocidal assay) provided control sera. For both groups, sera were obtained on day 0,9, and 23 following presentation, and stored at-80°C until use. Patients were entered following informed consent and the study was approved by the Institutional Review Boards at both the ICDDR, B and Massachusetts General Hospital.

[00116] Adsorption of sera. Equal volumes of convalescent sera were pooled (both day 9 and day 23) from ten patients infected with V. cholerae and this pool was extensively adsorbed against an El Tor V. cholerae strain isolated from one of the patients in the study.

Pooled sera were serially adsorbed against in vitro-grown V. cholerae whole cells, then French-press extracts and finally heat- denatured extracts, as described (Handfield et al., Trends Microbiol. 8: 336-339, 2000). Resultant adsorbed serum was aliquoted and stored at-80°C.

[00117] The pool of sera was assayed at various steps of the adsorption process utilizing an ELISA reaction against either whole V. cholerae extracts, or the in vivo-expressed protein, CtxB. To assay antibodies in the serum reactive with whole V. cholerae extracts, a French-pressed lysate was diluted 1 : 2 in carbonate buffer pH 9.6, and 100 ml of this lysate was added per well to an ELISA plate. After blocking and washing, serum samples were added at dilutions of 1: 200 to 1: 25,600, and binding quantitated by addition of peroxidase conjugated goat anti-human affinity purified immunoglobulin (ICN Biomedicals, Inc. , Aurora, OH), which is reactive with all classes of human immunoglobulins. Reactivity was measured using the substrate, 2, 2'azino- bisethylbenzthiazolinesulfonic acid (Sigma Chemical Co. , St. Louis, MO), and optical density readings at 405 nm recorded using a Vmax <BR> <BR> microplate reader (Molecular Devices Corp. , Sunnyvale, CA). To assay antibodies specific for CtxB, wells in an ELISA plate were sequentially coated with ganglioside (1 mg in 110 ml carbonate buffer, pH 9.6) and 100 ng of purified CtxB (List Biological Laboratories, Inc., Campbell, CA). After blocking and washing, pooled sera were serially diluted from 1: 50 to 1: 25,600 and added to each well. Reactivity was measured as above.

[00118] Pooled sera from patients convalescing from cholera in Bangladesh was successively adsorbed against in vitro grown V. cholerae. As shown in Figure 4A, there was a progressive decrease in reactivity of the pooled sera with in vitro grown V. cholerae, particularly after the French press lysate adsorption step. In contrast, there was substantially less removal of antibodies recognizing CtxB (Figure 4B). Cholera toxin is not significantly expressed by El Tor V. cholerae during growth in LB broth, but is expressed during human infection.

[00119] Retention of immunoreactivity to in vivo-expressed V. cholerae proteins in pooled convalescent sera following adsorption was further assessed utilizing individual purified proteins. In addition to CtxB, purified MshA and TcpF, a protein in the TCP gene cluster that is secreted by V. cholerae, was included. As mentioned above, genes in the TCP cluster in E1 Tor V. cholerae are not expressed during growth in LB broth, but are expressed during human infection. As shown in Figure 5A, substantial reactivity to in vivo expressed V. cholerae proteins was retained after adsorption of convalescent sera.

[00, 1201 Construction of a genomic expression library of V. cholerae 01 strain N16961. An expression library was constructed using pETabc expression vectors (Novagen, Madison, WI). These vectors allow cloning of inserts in each of three reading frames under transcriptional control of a T7 promoter. Vector DNA was digested with BamHI, gel-purified using the QIAEX IT Gel Extraction Kit (Qiagen, Valencia, CA), and treated with shrimp alkaline phosphatase. Genomic DNA from V. cholerae strain N16961 was partially digested with Sau3A, and two pools of genomic DNA fragments ranging in size from 0.5-1. 5 and 1.0-3. 0 kbp were gel purified. Vector and genomic DNA fragments from each pool were ligated to create two genomic libraries, and each was electroporated into competent E. coli DH5a and spread on LB plates containing kanamycin. After overnight incubation at 37°C, growth on plates was collected by scraping, and plasmid DNA was recovered from an aliquot of the library and electroporated into E. coli BL21 (DE3).

[00121] Screening for proteins uniquely expressed in vivo by V. cholerae. To screen the genomic library with adsorbed pooled sera, an aliquot of the library in expression host BL21 (DE3) was diluted and spread on LB plates containing kanamycin to produce approximately 300 colonies per plate. After overnight growth at 37°C, colonies were lifted using nitrocellulose membranes, replica plated onto LB plates containing kanamycin and isopropyl-b-D- thiogalactoside (lmM), and incubated overnight at 37°C to induce expression of genes in cloned inserts. After overnight incubation, plates were exposed to chloroform for 15 minutes to partially lyse bacteria and release induced proteins, then overlaid with a nitrocellulose membrane for 15 minutes at room temperature. Each membrane was removed, blocked with 10% non-fat skim milk, and reacted with a 1: 100 dilution of adsorbed pooled sera at room temperature for one hour with mild agitation. Clones reacting with antibody in adsorbed sera were detected using peroxidase-conjugated goat, anti-human immunoglobulin at a 1: 5,000 dilution, and developed using an ECL chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ). Reactive clones were identified by their position on the master plate; each positive clone was purified at least two additional times and confirmed as reactive with adsorbed sera.

Plasmids from individual reactive clones were purified, and the V. cholerae DNA inserted in the vector was sequenced in both directions using pET30-specific primers (Novagen). PCR with these same primers was used to estimate the size of inserted DNA. Altogether, approximately 30,000 clones from the two differently sized DNA expression libraries were screened, and 38 positive clones were identified, confirmed on multiple occasions, and sequenced.

Proteins encoded in the cloned insert fragments were identified using the genomic sequence of V. cholerae strain N16961.

[001221 Screening of the two expression libraries with the adsorbed convalescent sera yielded 38 clones that were persistently reactive after at least three rounds of purification. The identity of proteins encoded by clones reactive with convalescent sera is shown in Table 2; these include proteins encoding two of the type IV pili in V. cholerae (PilA and TcpA), a minor component of the MSHA pilus (MshO) and a related outer membrane protein (MshP), a cell membrane protein (PilQ) that acts as an outer membrane secretin for PilA in other organisms, a cell membrane protein (CapK) that is involved in biofilm-associated exopolysaccharide synthesis, three methyl- accepting chemotaxis proteins, two proteins involved in chemotaxis and motility (CheA and CheR), a quorum sensing protein (LuxP), and four hypothetical open reading frames. Most of the reactive clones contained only a portion of the coding sequence of the relevant protein product. A number of reactive clones expressed V. cholerae proteins previously detected by screens in infant mice using RIVET and STM screening methods, including TcpA (which was identified 8 times in the screen), three different methyl-accepting chemotaxis proteins, and CapK.

[00123] IVIAT also identified several clones containing genes not detected in previous screens in infant mice, including mshO, mshP, cheA, cheR, luxP, and four hypothetical open reading frames. LuxP is a periplasmic protein that is a component of one of three quorum- sensing systems in V. cholerae (Miller, et al., Cell 110: 303-314, 2002). Interestingly, three of the four hypothetical open reading frames identified are encoded on the smaller chromosome II of V. cholerae (Trucksis et al., Proc. Natl. Acad. Sci. USA 95: 14464-14469, 1998). Chromosome II has many fewer genes required for growth of V. cholerae in laboratory media, but contains many genes necessary for adaptation and growth of V. cholerae in unique environments (Heidelberg et al., Nature 406: 477-483, 2000). The fact that three of four hypothetical open reading frames identified by IVIAT are encoded on chromosome II indicates that these genes encode functions specifically required for growth in human intestine.

Table 2. Proteins encoded in clones identified by IVIAT Functional Name of Gene Encoded on Number of Size Size and classes of encoded design. chromosome clones polynuc. location encoded protein in V. I or II identified In BP of gene protein product cholerae in IVIAT (a.a.) fragment products (SEQ ID NO) genome screen in clone sequence identified by IVIAT Pili pilA (1) VC2423 I 17 501 34-501 PilA (2) (167) tcpA (3) VC0828 I 8 672 1-672 TcpA (4) (224) Cell pilze (5) VC2630 1 1 1734 416-1347 membrane PilQ (6) (578) proteins mshO* (7) VC0412 1 1 768 23-768 MshO (8) (256) mshP* (9) VC0413 1 429 1-429 MshP (10) (143) capK (11) VC0924 1 1 1338 396-908 CapK (12) (446) Methyl-mcp (13) VCA0176 II 1 1932 410-1050 accepting MCP (14) (640) chemotaxis mcp (15) VCA1056 II 1 2310 410-1100 proteins MCP (16) (760) mcp (17) VC0216 1 1 1695 763-1016 MCP (18) (564) Chemotaxis cheA (19) VC1397 1 1 2286 22-580 and CheA (20) (762) motility cheR (21) VC1399 1 1 864 269-432 proteins CheR (22) (228) Quorum luxe (23) VCA0737 II 1 1092 1-572 sensing LuxP (24) (364) protein protein hypl (25) VCA0536 II 1 864 1-864 proteins Hypl (26) (288) hyp2 (27) VCA0884 II 1 1059 523-1059 Hyp2 (28) (353) hyp3 (29) VCA0931 II 1 1380 1-267 Hyp3 (30) (460) hyp4 (31) VC1431 1 1 657 127-657 Hyp4 (32) (219) *These two genes were encoded on the same recovered fragment.

[00124] The V. cholerae gene sequence identified most frequently by IVIAT was that encoding PilA, the structural subunit of a third type IV pilus in V. cholerae (identified 17 separate times in the IVIAT screen). In addition, IVIAT identified antibodies in convalescent sera to PilQ. Sera from control patients did not recognize clones encoding PilA or PilQ. The fact that PilA and PilQ are expressed and immunogenic during human infection, but that mutation of pilA has no effect on colonization of infant mice by V. cholerae, suggests that PilA and its assembly apparatus may play a specific role in colonization of human intestine.

[00125] To confirm the results of the IVIAT screen, plasmids were constructed that specifically express TcpA, TcpF, MshA, PilA, and PilQ. Reactivity of adsorbed patient sera with E. coli containing each of these plasmids was then examined. As shown in Figure 5B, each selected clone showed prominent reactivity with adsorbed convalescent sera, whereas a strain containing the control plasmid did not.

[00126] Because of the importance of type IV pili in colonization of the gastrointestinal tract, and because adsorbed, convalescent sera from patients specifically recognized all three V. cholerae type IV pili, further analysis of immune responses to these pili was examined. Whether TcpA and PilA reactivity was present at high level in a subset of patients from whom sera was pooled, or if most or all of the individual patients recovering from cholera had antibody reactive to these proteins, was examined. For this, immunoscreening with purified TcpA and PilA and unadsorbed sera from days 0,9, and 23 following V. cholerae infection was used, comparing results to control patients who did not have V. cholerae infection. Because the quantities of purified TcpA and PilA were limited, a dot-blot assay was used for analysis. Results in Figure 6 show that between day 0 and day 23, six patients increased immunoreactivity to PilA and four patients (numbers 2,8, 9, and 10) showed immunoreactivity that did not change or decreased, suggesting infection with V. cholerae may have begun earlier than study entry.

For TcpA, six of ten patients showed increasing reactivity, three patients (numbers 8, 9, and 10) showed immunoreactivity that did not change (or decreased) and one patient (patient 7) did not show immunoreactivity to TcpA, despite having culture-documented E1 Tor V. cholerae 01 infection and antibody to PilA. Control patients did not demonstrate significant immunoreactivity with either protein.

[00127] Type IV pili share homology at the amino terminus (Patel et al., Infect. Immun. 59: 4674-4676,1991). The possibility that the serologic responses might be directed to epitopes shared between TcpA and PilA was considered. However, the histidine-tagged form of TcpA used in the assay is missing the first 28 amino acids of the mature protein, the area of highest homology between type IV pili.

Therefore, it is unlikely that the results represent cross- reactivity between these two different pilus proteins. In addition, IVIAT identified reactivity with other components of each of the type IV pilus systems of V. cholerae, and these components do not share the same homology as the mature pilus subunits. This is the first time strong human immune responses to TcpA and PilA following El Tor V. cholerae 01 infection has been shown.

[00128] Purification of proteins. A derivative of TcpA from El Tor V. cholerae 01, with a histidine tag fused at the amino-terminus of residue 29 of mature TcpA, and a glutathione-S-transferase derivative of TcpF from classical V. cholerae 01, were over- expressed and purified. Purified MshA from El Tor V. cholerae O1 was purified as described (Qadri et al. , Clin. Diagn. Lab. Immunol.

4: 429-434,1997). PilA from N16961 was over-expressed with a carboxyl terminal hexa-histidine tail and purified using a combination of nickel affinity chromatography and non-denaturing preparative gel electrophoresis.

[001291 To further confirm specific recognition of PilA, PilQ, TcpA, TcpF, and MshA by pooled, adsorbed convalescent sera from cholera patients, expression plasmids for each intact protein in a dot-blot hybridization assay identical to the IVIAT screen of the library above, to confirm immunoreactivity was used. As a control, results were compared to those with empty plasmid vector.

[00130] Analysis of RNA transcription of selected genes in vitro by RT-PCR. After overnight culture of strain N16961 in LB broth or AKI media, RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. RNA was purified with an RNeasy Mini Kit (Qiagen) and treated with an RNase-Free DNase kit (Qiagen) according to manufacturer's instructions. RNA was eluted in DEPC treated water and treated with DNase I (Ambion, Austin, TX).

Control PCR reactions were performed on the purified RNA preparations to ensure absence of amplification from contaminating DNA. Reverse transcription to cDMA was done using a Reverse Transcription System (Promega, Madison, WI). Primers specific for tcpA, pilA, pill, and rpoB (as a control) were used in PCR reactions on cDNA ; primers were obtained from the Tufts University Core Facility (Boston, MA). PCR products were detected on a 1.5% agarose gel with ethidium bromide.

[001311 Expression of selected genes in vitro were assessed utilizing RT-PCR. Transcription of tcpA was not detected following growth in LB broth but was present following growth in AKI media, as previously reported (Lee et al., Cell 99: 625-634, 1999). Expression of pilA was detected after overnight growth in both LB broth and AKI media, even though antibodies reactive with PilA were not removed during the adsorption process. Expression of pilQ could not be detected following growth in either media. The control gene, rpoB, was expressed in both media as expected.

[00132] Since the RT-PCR results suggested no transcription of pilQ in LB broth, one possibility is that pilA is transcribed, but PilA is not assembled on the cell surface because of the absence of PilQ (and perhaps other components of the assembly machinery). This might lead to premature degradation of PilA when V. cholerae is grown in LB broth.

[001333 Xu et al. (Proc. Natl. Acad. Sci. USA 100: 1286-1291,2003) analyzed genes specifically expressed by El Tor V. cholerae during growth in a rabbit ileal loop, utilizing a gene microarray. Xu et al. demonstrated that genes for mshABCD are highly expressed in rabbit ileal loop compared to in vitro, as are genes for motility and chemotaxis. Merrell et al. (Nature 417: 642-644,2002) demonstrated that V. cholerae recovered directly from human stool are hyperinfectious for infant mice compared to organisms grown in LB broth. Transcriptional profiling of V. cholerae in human stool suggested induction of genes involved in nutrient acquisition and motility, perhaps correlating with enhanced infectivity to subsequent hosts. Bina et al. (Proc. Natl. Acad. Sci. USA 100: 2801- 2806,2003) examined gene expression in V. cholerae in stools of cholera patients rapidly frozen at the bedside. Their results showed that several genes involved in pathogenesis were more highly expressed in stool compared to growth in LB broth ; these more highly expressed genes included ctxAB, mshK, msh0, mshP, pilE, the pilOPQ operon, and cheA ; of these genes, cheA and the pilOPQ operon were particularly strongly expressed during human infection. The genes for tcpA and mshA were less strongly expressed.

[00134] IVIAT identified many of the genes that were strongly expressed in both rabbit ileal loops and human stool, particularly genes encoding cell surface proteins, such as PilQ, MshO, MshP, CapK, and the methyl-accepting chemotaxis proteins. However, IVIAT did not identify other genes strongly expressed in human stool, particularly those whose protein products are not surface localized.

This may in part be due to the fact that the screen of the V. cholerae genome is not saturated, as demonstrated by the fact that clones for cholera toxin were not identified, despite the fact that its in vivo expression is well established. It is also of interest that all of the genes from chromosome Il of V. cholerae identified as expressed during human infection by IVIAT were expressed poorly in human stool utilizing microarray technology (Bina et al.). One possibility is that genes on chromosome Il turn off expression particularly quickly during the transit from the upper gastrointestinal tract to stool. IVIAT appears to preferentially identify proteins that are surface localized and highly immunogenic during human infection with a mucosal pathogen such as V. cholerae.

Since these same proteins are likely involved in generating protective immune responses, IVIAT may provide data that enhances microarray technology to identify relevant antigens expressed in vi vo.

[00135] The fact that convalescent sera from cholera patients specifically recognizes PilA and PilQ suggests that this pilus may be uniquely expressed during human infection, and may play a role in V. cholerae pathogenesis not suspected by previous animal model experiments. IVIAT may provide a new technology to detect genes specifically expressed during human infection.