CA2205130A1 - Immunogens for stimulating mucosal immunity - Google Patents

Abstract

This patent application relates to immunogens for stimulating mucosal immunity to a pathogen capable of infecting its host through contact with mammalian mucosal membranes. In particular, this invention discloses a number of polypeptides and genetic constructs that include a membrane binding polypeptide operably linked to a peptide from a pathogen. Methods are detailed throughout the claims and the specification for introducing these immunogens into a mammal to stimulate mucosal immune responses.

Description

CA 02205130 1997-0;i-12 Wo 96/16178 . ~~ 708 '''' F3Fi STIMULATING MUC3SAL IMMUNITY

This application relates tc methods fcr producing mucosal antibody to organisms capable of infecting their 5 host through contact with mammalian mucosal membranes. In particular, this application relates tc protein complexes and tc gene constructs suhabie for ,oroducing immunogen capable of inducing muccsal antibody as weil as to methods for introducing the immunogen into a mammal to generate a mucosal immune response.

The mucosal surfaces of the body are generally accessibie to a wide variety of infectious agents capable 10 of causing disease. These surfaces include the ~ ' ' tract, the urogenital tract and the respiratory tract.
While the external surfaces of the body are protected by a continuous layer of keratinized squamous epithelium, the mucosal surfaces of the body lack the protective keratinized layer and are more vulnsrable to invasion by adventitious organisms. Not surprisingly, these surfaces are the main portal of entry into the body for foreign ~ ' The mucosal surfaces of the body have an extensive immune system, '' , , ' ' Iymphoid tissue 15 is dispersed througho~t the mucosa of the y~ ' and genitourinary tracts as either diffuse aggregates of cells or as organized nodules. The diffuse aggregates of Iymphoid cells are dispersed throughout the lamina propria while nodules, or Peyer's patches, which include germinal centers of proliferating B cells and peripheral areas of T
cell activity, are more prevalent in some regions of the mucosa than in others.
The Iymphoid cel!s along the mucosal surfaces are capable of responding to foreign antigen. The gut 20 epithelium overlying the Peyer's patches allows transport of antigens into the Iymphoid tissue and is capable of functioning as antigen presenting cells (Bromander, et al. Scsad. 1 Imm~moL 37:45245~, 1993) Secretory IgA
(slgA~ can traverse mucosal membranes and is often the first defense that an adventitious agent encounters when contacting the mucosal surface of a mammal.
A vaccine which is effective in preventing diseases which are associated with the mvasion cf adventitious 25 organisms into~the mucosa will preferably stimulate IgG and slgA, An effective vaccine for limiting mucosal infection will likely demonstrate slgA activity.
Many sexualiy transmitted diseases such as chlamydia, gonorrhea, syphilis, chancroid and l,i ' ' ' are caused by organisms that enter the body through the mucosal membranes. While these diseases are caused by different organisms and repiicate in different ways, these sexual~y transmitted and others, all enter 30 the body primariiy via the mucosal barrier. These and most other disease causin~q organisms carry unique antigenic determinants that are known to stimulate the immune system, In addition, the mucosal surfaces are also the main portal of entry for most viruses. Thus, mucosal surface immunity to viruses including, but not iimited to influenza, ,, "' , HIV, members of the Herpesvirus family, and the like, also integrally associate with mucosal surfaces of the body during the infection stage, replication and as part of virus egress Attempts to develop vaccines for 35 these organisms have met with little success since parenteral vaccination does not generally produce significant levels of seuetory immunity, CA 0220;il30 1997-0;i-12 wo 96/16178 1 ~1, . /~708

2-The non-viral sexually transmnted diseases can usuallr bc tured if they are diagnosed early, but many of the diseases produce mild early symptoms, it any, and thus go untreated until more adYanced symptoms occur.
Chlamydia is a useful example of a sexualiy transmitted disease that infecls ils host through mucosal membranes, primariiy of the genitourinary system. Chlamvdia tr2chomatis is Ihe leading sexually transmitted 5 organism in the United States, afflicting an estimated four million pePple a year (DiYislon of STDIHIV PreYention, 1992 Annual Report, CDC, Atlanta, 1993). Chlamydia is acquired chiefly through vaginal or anal intercourse, although il can also be Iransmitted through oral seY. C. trachoma~is infection of Ihe genital tracl can cause salpingilis in women that can result in tubal blockage and infertility. Il is eslimaled thal in the United States 200,000 women per year become infertile as a resull of chlamydial salpingitis. Moreover, infected individuals are at an increased risk 1D of acquirinU HIV if exposed to the virus iWasserhiet. ~cl ~- ' Syneryy:, ' ', Belween Human ' ' , Virus Infection and Other Sexually Transmitted Diseases," SeJtusl/y ~ransmitted Diseases, 19: 61-77, 19921. Improved methods for controlling the pathological r " of this disease are seriously needed.
Chl2mvdla trachomatis isolates occur as 15 distinct serovars thal are divided jDIO three subgroups. The major outer membrane protein ~MOMP) of Chlamydia confers serovar and 3 .. , 5, " Protective immunity to Chlamydia is directed to the major outer membrane protein.
Comparalive analysis of Ihe major outer membrane protein of Chlamydia indicates thal the MOMP genes are conserved and have four variable region domains that are unique to each serovar. These domains elicit the fonmation of serovar, subspecies, and serogroup or, s` ' anthbodies. Variable domain IV ~VDIV) is the largesl of the MOMP variable domains and is located near the C-terminus of the protein. VOIV contains subspecies, serogroup antigenic determinants and a conserved species specific antigenic determinant. All of the variable domains of MOMP are external epitopes as demonstrated by their sensitiYity to trypsin and by their accessibility to antibody binding. This invemion contemplates combining determinants from MOMP or other protein from pathogens, including but not limited to HIV, hepatitis and , ~ E. coli capable of infecting a mammaliarl host through ccnlacl with mucosal membrane with a mucosal binding polypeplide.
Several agents have proved effective as carriers ind as adjuYants for stimulating mucosal immunity (for a reYiew of these agents see Bienenslock, J. "The Nalure of Immunily al Mucosal Surfaces A Brief ReYiew." In:
Bacterial Infections of ResPiratorv and G~`,. ` ' Mucosae. Eds. Ooncachie, W, et al. IRi Press, 1988. pp. 9 18.). Accessible epilopes from various infeclious agents haYe been engineered onto the surface ot attenuated liYe vectors such as Vaccinia or Sslmor~ella tyohimurium ~see Flexner, et al. ~Vaccinia as a live vector carrying cloned toreign genes. In: New Generalion Vaccines Eds. Woodrow, G.C., et al. Marcel Dekker, Publ. New York 1990, pp.
189-206 and Curtiss, R. "Attenuated Salmonella strains as live vectors for the expression of foreign antigens. ~ Ibid, pp. 161-188). Non-living carrier systems aliow i ' ~ accessible epitopes to be presented to the immune system as Ihe producl of a genelic conslruct ùr as a peptide chemically coupled lo a carrying agenl. Such non-living carrier systems include microparticles, liposomes, solid matrix ' ~ complexes, ' ' complexes ~ISCOMs) and prolein carriers including Ihe core antigen Df hepalilis B virus, polio virions, cholera loXin and Ihe heat-labile enterotoxin from E. coii.

CA 0220~130 1997-0~-12 WO 96/16178 I ~ I, . _. . .

3 The carriers themselves may have endogenous abjuvant activity or alternatively. r~xogencus adjuvant can be used with the carriers. Dx ùile has been used as an adjuvant for craliy administered immunogens such as kiiled oral vaccines against dysentery. Dlher adjuvants that have been tested for use in inducing muccsal immumty include DEAE4 dextran, Iyso/yme, po~yornithine, sodium dodecyl benzene sulfate, iipid conjugated substances, streptomycin and vitamin A (see Holmgren, et il. Vaccine 11:117D-1184, 1933). In addition, agents such as muramyl dipeptide, acridine anb cimetidine have had some positive effect as well ~see Bienenstock, et al. suora.~.
Cholera tcxin is capable of generating mucosal immunity and is also a potent adjuvan1 for augmenting the immunizing effect of orally administered vaccines. Cholera toxin is produced by Vibrio cholerae bacteria. The toxin molecule is well characterized and in its native form consists o~ five binding a subunits assembled as a ring together 1û with a single A subunit. The B subunits bind to GM1 receptors on the cell surface. The A subunit is translocated to the inside of the cell following B subunit binding. Cholera toxin has been used both as an immunogen for the oral mucosa and as a potent adjuvant for inducing secretory IgA ~Lycke, N. and Holmgren, J. Immunologv 59:301-3D8, 1986) to cholera toxin and to unrelated antigen. Similarly, the heatiabile enterotoxin of Escherichia coli also has ~ and adjuvant properties.
Cholera toxin coupled to foreign antigen has been used to stimulate immune responses in the ua~
tract. For example, chemical coupling of the cholera toxin 8 subunit to streptococcal antigen has evoked antibody responszs in the mucosal surfaces of the gut and in saiivary glands ~Czerkinsky, et al. /nfecL Immon. 571072-lD77, 1989). Cholera toxinlSendai vlrus conjugale immunrzation has resulted in the production of s, ~ in the respiratory tract.
While cholera toxin has been used to stimulate the immune response to bacterial and select viral antigens along the u~ tract, there is no successful strategy available for producing total mucosal immunity or effective immunity along the mucosal surfaces of the urogenital tract. Moreover, even where cholera toxin, or its subunits, was used to stimulate 'mmune responses to foreign antigen, the immune responses have been poor as compared with the immune responses generated to the cholera toxin carrier. For example, the addition of amino acid residues to either the N- or C terminus of CrB has in some cases interfered with the folding and assembly of the finalproduct~Sandkvist,etal.,l~ac~eriol.169:457D4576.1987andClementsJ.D.lnfect.lmmaL58:11591166, 1990). When some of these constructs were produced in V. cholerse the foreign epitopes were cleaved off, presumably by bacterial proteases ~Schddel, et al., Geoe 99:2 i5 259, 1991). Therefore, there remains a need for improved cholera toxin dernved immunogens. = ~ ==
3D Unlike the prior art, protein complexes and genetic constructs are disclosed in this invention that slimulate mucosal immunity to patbogens which cause sexually transmitted disease. In addition, genetic constructs are disclosed that provide increased stability to foreign epitopes linked to mucosal binding polypeptide. Protein complexes are disclosed that are expressed in whole or in part from ~. coli or V. cholerae.
Thus, the present invention not only discloses strategies for the production of mucosal immune responses to non-viral sexually transmitted pathogens and to a variety of viral pathogens but the invention aiso discloses strategies for improving the immune response to the foreign antigen using a vzriety of genetic constructs.

CA 0220~130 1997-0~-12 wo 96/16178 ~l, ~. . .

BRIEF ll OF THE FIGURES
Figure 1 dia3rams the strategy for producing the pML,I E. coh expressiDn vectors of the present invention.
Figure 2 provides preferreb examples o~ expressicn vectors using the fac promoter. Figure 2(a) diagrams 5 a plasmid expressing CTB from the tac promoter. Figure 2(b~ illustrates the same vector as in Figure 2(a), adiitionally carrying the ctxAB gene fragment used for the constructi~n of CTA2 protein fusions. Figure 2~c) dia~qrams a plasmid, as disclosed in Figure 2(b~, additionally containing the lacl: gene to facilitate inducible X-gal expression of ct~rAB. Figure 2(d) diagrams a plasmid carrying the modified ctxB gene for in-frame intemal epitope insertion between the unique Kpnl and Mscl sites.
Figure 3 diagrams piasmids expressing CTB from the leftwards promoter of phage (ambda for the generation of CTB fusions ~pML-LCTB,l7~ or CTA fusions ~pP~U).
Figure 4 illustrates the li~ sequence, SEQ ID N0:5, used to regenerate the ct~A sisnal peptide and the unique restriction sites used for cioning foreign epitopes into the coristruct.
Figure 5 provides synthetic: sequences, SEQ ID N0:24 and SEQ ID ND:25, used for the insertion of thlamydial T-cell and B-cell epitopes into the ctxA gene. Boxed sequences represent the chlamydial peptides.
Figure 6 provides a diagram of the sequences, SEQ ID N0:7 and SEQ lO N0:8 which were used to form the linker to join the chlamydial epitopes A8 (SEQ lO NO:b) and VDIV (SEil ID ND:1~. The adjacent Sacl and Nhel sequences for pML-LCTBtac or pML-LCTB,I and pPJVDlV are also diagramed in this Figure.
Figure 7 diagrams the construction of plasmid pCB55-84gp309 encoding an exemplary CTB::hybrid protein.
The diagram provides an illustration of the parent plasmid, pML-LCTBtac, positioned to accept foreign antigenic sequences. Plasmid pCB55-64gp309 contained the HIV antigenic sequence derived from amino acids 309-318 of gpl20. The gpl20 nucleic acid sequence positioned between the Kpnl and BssHII restriction endonuclease sites is provided in the enclosed box with insert gpl20 sequences shown in italics.
SUMMARY OF THE INVENTION
This invention relates to immunogens useful for stimulatins mucosal immunity and for protein compiexes that are useful in assays to detect the presence of antibody to mucosal binding proteins or to foreign antigen from pathogens capable of infecting a mammalian host through mucosal membranes. In addition, the invention reiates to methods for inducing mucosal surface immunity to a pathogen capable of infecting a mammaiian host through mucosal membranes.
In one embodiment of this invention a mucosal binding composition is contemplated that comprises a mucosal binding polypeptide linhed to at least one antigen from a non-viral pathogen where the pathogen causes a sexually transmrtted disease. In a preferred embodiment the mucosal binding polypeptide is the binding subunit of cholera toxin and in another embodiment the mucosal binding polypeptide additionally comprises at least a portion CA 0220~130 1997-0~-12 wo 96/16178 r~., ~. -~n7708 .5.
of the A subunit ef cholera toxin. In a preferred embodiment the antigen from the non-viral pathogen is an antisen from Chlamvd/a.
It is Eontempiated that the chlamydia antigen can be positioned at the amino terminus of the binding subunit - of the cholera toxin, at the amino terminus of the portion of the A subunit of chclera toxin or positioned internally 5 within the binding subunit of cholera toxin. It is contemplated that the antigen can be linked to the mucesal binding protein by recombinant cr chemical means.
In a preferred embodlment, the antigen cemprises a B cell stimulating antigen frem the major outer membrane pretein ef chlamydia. In a particularly preferred embodiment, the B-cell stimulating antigen is frem the VDIV regien ef the major euter membrane preteln ef chlamydia. In yet anether preferred embediment, the antigen 10 further cemprises a T-helper cell stimulating antigen and pneferably, this T-helper cell stimulating antigen is also frem the majer euter membrane pretein ef chlamydia. In ene embediment the T-helper cell stimulating antigen is frem the major euter membrane pretein ef chlamydia and in a preferred embediment is the A8 regien frem the major euter membrane pretein of chlamydia.
In anether embediment of the present invention, a method is disclesed fer generating a mucesal immune 15 response against a nen-viral sexually transmitted disease, comprising centacting the mucesa of a mammalian hest with the mucesal binding cempositien.
In yet another embodiment of the present invention, recombinant, 1~. ' '' are disclosed which comprise a first region encoding a mucosal binding pelypeptide and a secend regien enceding an antigen of a non-viral pathegen, where the pathogen causes a sexually transmitted disease. In a preferred embediment the mucesal binding 20 polypeptide is the binding subunit of cholera toxin and the pathegen is chlamydia. In this embediment, the preferred antigen includes a T-helper cell stimulating antigen and a B cell stimulating antigen frem the euter membrane pretein ef chlamydia. In a particularly preferred embediment, the B-cell stimulating antigen is from the UDIV region ef the major outer mEmbrane protein of chlamydia.
This invention also relates te metheds fer vaccinating a marnmal against chlamydia infection cemprising 25 administering to the mucesa of a mammaiian host an effective amount ef the binding subunit of chelera toxin linked to both a B-cell epitepe and a T-cell epitepe of the majer outer membrane protein ef chlamydia. In a preferred embediment the administratien is vaginai and in ether embediments, the vaccine is delivered rectally or orally.
The invention additionally relates to mucosal binding compositions cemprising a muces21 binding polypeptide linked to at least ene antigen ef a viral pathegen where the pathogen ca~ses a sexually transmitted disease. In a 30 preferred embediment the mucesal binding polypeptide cemprises the binding subunit of cholera texin and the antigen is a HIV gp120 antigEn. In yet anether embediment the antigen is a Hepatitis B virus pre-S(Z) antigen and in a further embediment, the mucosal binding polypeptide is linked to at least ene antigen frem the ST, pretein ef E. coli.
The invention additionally centemplates purified recombinant I ~ ' ' cemprising nucleic acid enceding 35 a mucosal binding pretein operabiy linked to a 3 cell stimulating antigen where the antigen is a peptide eetained frem a pathegen capable ef infecting a mammal through the mucosal membranes. In a preferr~d embediment, the mucosal CA 0220~130 1997-0~-12 wo 96/16178 ; ~.,~

bindins profein encodes the binding subunit ot cholera toxin and in anDther embodiment, the nucleic acid further encodes the CTA(2) subunit of cholera toxin. In a preferred embodiment the B-cell stimulating antigen encodes a peptide which inciudes the amino acid sequence LNPTIAG. in one embodiment, the B-cell stimulating antigen us from HIV gpl20.
In yet another preferred embodiment, the nucleic acid encoding the B-cell stimulating antigen is positioned in-frame within the coding region of the nucleic acid encoding the mucosal binding protein. The B-cell stimulating antigen may alternatively include the amino acid seouence LNPTIAG, an antigen from the gpl20 protein of HIV, the Hepatitis B virus pre-S(2) protein or the ST, protein of , '~ ' E. ~oL In these embodiments it is contemplated that the nucleic acid encoding the B-cell stimulating antigen is between 21 and 150 bases in length and more preferably between 21 and 60 bases in length.
DETAILED L-~Lhlr 11 OF THE INVENTIDN
The present invention relates to immunogens useful for producing mucosal immune responses to a variety of viral and non-viral pathogens, to methods for preparing these immunogens and to methods for producing mucosal immune responses to pathogens capable of infecting a mammalian host through the mucosal membranes of the host.
The term "' '' " is used herein to describe antigenic seouences that are accessible to the immune system when introduced in association with a mucosal binding polypeptide.The term "T-cell stimulating antigen" or "T-cell antigen" is used herein to refer to a peptide, a polypeptide, a protein or a , . molecuie including carbohydrate, lipid, nucleic acid or the like, which is capable of stimulating T-helper cell activity~ in standard T helper cell assays, well known in the art, either alone Dr in combination with other protein.
The term "B-cell stimulating antigen" or "B-cell antigen" is used herein to refer to a peptide, a polypeptide, a protein or a I , . molecule, including carbohydrate, lipid, nucleic acid or the like, which is capable of stimulating ant'lbody production from B-cells.
The torms "ctxA" and ~ctxB" refer to the gene seouences encoding the cholera toxin A ani B subunits respectively.
The tenm "mucosal binding composition" is used herein to refer to compositions that include a mucosal binding polypeptide and an antigen from a pathogen capable of infecting a mammaiian host through the mucosal membranes of the host. The term "mucosal binding poiypeptide" is used herein to refer to polypeptide capable of attaching to the mucosal surfaces of a mammal.
There are a variety of mucosal binding polypeptides disclosed in the art. It is contemplated that the mucosal binding composition will include at least one mucosal binding polypeptide. Other polypeptides that associate with the mucosal binding polypeptide are also contemplated within the scope of this invention. The mucosal binding polypeptides of the present invention include, but are not limited to, bacteria~ toxin membrane binding subun'lts ~5 inciuding, at a minimum, the B subunit of cholera toxin, the B subunit of the E. coli heat-labile enterotoxin, Bordetella CA 0220~130 1997-0~-12 WO 96/16178 P~,l,. -U`~708 7- ~
pertussis toxin subunits S2, S3, S4 andlor S5, the B fragment of Diphtheria toxin and the membrane binding subunits of Shiga toxin or Shiga-like toxins.
Other mucosa binding subunits contempla1ed within the scope of this invention inciude bacterial fimbriae protein including E. coli fimbria K88, K99, 987P, F41, CFAII, CFAIII (CS1, CS2 andlor CS3), CFAIIV ~CS4, CS5 andlor 5 CSEi), P fimbraiae, or the like. Other fimbriae contemplated within the scope of this invention include Bordetella pertussis filamentous ' ' ', vibrio cholerae ~nY~ pilus ITCP), '' ' hema3glutinin IMSHA), ~ hemaûglutinin IPSHA), and the like.
Still other ' ! ' molecules contemplated within the sccpe of this irlvention include viral attachment proteins includins influenza and Sendai virus ' , ' and animal lectins or lectin-like molecules including lG ' ' ' ' molecules or fragments thereof, ' :', ' IC-type) lectins, selectins, collectins or Helix OOlal;3tid ' __' Plant lectins with ! ' ' subunils incrude concanavalin A, Wheat germ agglutinin, ~1- ' ' abrin and ricin.
The invention discloses the use of mucosal binding cDmpositions to stimulate mucosa~ immunity to pathogens capable of infecting a mammaiian host through the mucosal membranes of the host. In one preferred embodiment 15 of this invention, a method for producing muccsal immunity to a pathogen is disclosed using the mucosal binding subunit of bacterial toxins. As one aspect of this embodiment, chimeric constructs cf either the cholera toxin or the E. coli heat-bbile enterotoxin are ccupled to antigen obtained from pathogens capable of causing a disease. In another aspect of this embodiment, methods are disclosed for chemically couplmg a mucosal binding polypeptide such as the muccsal binding portion of the B subunit of cholera toxin or of the heat labile enterotoxin of f coli to antigen 20 obtained frcm a pathogen capable of causing a disease.
It is additionally contemplated that the membrane binding compositions of this invention will be useful for stimulating secretory ' ' ' ' This secretory ' ' ' can be used in diagnostic assays, such as ELlSAs, immunoblots, or the like. In addition, it is contemplated that the binding compositions themselves can be used in assays to detect the presence of antibody in the sample to either the membrane binding polypeptide or to 25 the foreign epitope. Moreover, the secretory antibody collected from experimental animals can be used in topical preparations against a specific pathogen cr as a general ccmponent of a topical preparation cr in additional studies to assess the character of secretory ' ' "
There are a variety of methods available in the art for obtaining mucosal binding polypeptides. The polypeptides, or fragments thereof, can be isolated from nature or the polypeptides can be chemically synthesi,ted 3D or produced as a recombinant product from a prokaryotic or eukaryotic expression system. Those skilled in the art will be able to select and test a mucosal binding pclypeptide fcr its ability to function as a carrier and to facilitate foreign antigen presentaticn to the immune system.
To select and determine whether a particular mucosal binding polypeptide will facilitate foreign antigen presentation to the immune system, one skilled in the art will begin by selecting a mucosal binding polypeptide from 35 the literature or from other research sources. The mucosal binding protein can then be purified from nature or derived as a product of a recombinant expression system. Exemplary mucosal binding polypeptides have been CA 0220~130 1997-0~-12 wo 96/16178 A .

prDvided above. For recDmbinant prctein expression, th2se skilled in the art of mobcular biology will isolate nucleic acid fragments encodinû this gene, using standard techniques kncwn in the art, and incorporate these tragments into expression constructs. There are a wide range of eukaryotic and prokaryDtic expression systems known in the art and it is recognized that the skilled artisan will be able to inccrporate a nucleic acid fra3ment enc3ding a mucosal binding polypeptide into a vanety of gene expression systems without undue , Exemplary gene expression constructs are provided here which are suited for gene expression in ~. coh or V. Choler~e. Alternativeiy, the mucosal binding polypeptide can be isolated from nature using known purification techniques.
The recombinant or purified mucosal binding pclypeptide is next tested for its ability to be bound by test antibody where the test antibody is known to recoynize the naturally occurring mucosal binding polypeptide. Suitable tests for determining antibody recognition include ELlSAs, immunoblots or other well known immunologic assays available in the art. Exemplary ELISA and immunoblot assays are provided in the Examples Isee Example 4 and B).
These assays are well known and can be readily modified by those skilled in the art to identify other mucosal bindins protein. Next, the candidate mucosal binding polypeptide is further tested for its ability to bind mucosa. Mucosal binding assays are known in the art. These assays can employ fixed mucosal tissue, primary mucosal cells placed in tissue culture, commercially available mucosal cell lines in culture, or assays involving mucosal cell membrane Iysates or purified mucosal cell membrane prDtein. Mucosal tissue can be obtained by biopsy, and processed into tissue cell culture or as fixed tissue sections (from an autopsy specimen or as a punch biopsyi. Mucosal cell lines are commercially available from a variety of sources. The following cell lines are available from the American Type Culture Collection (Rockville, Maryland). These include colon tissue (ATCC #'s CRL 1459, CRL 1539, CRL 1541 and CRL l790), nasal septum (CCL 30), palate (CRL 148Ei), rectal cell lines (CCL 235, CCL 234), vulva-derived cell lines (HTB 88, HT~3 117 and HTB 118), bronchal cell lines (CCL 208) and carcinoma cell lines of the mouth (CCL 17).
Additionally binding assays can be performed in test animals. These assays may involve a reporter molecule such as a radioisotope, an enzyme linked to an antibody or to the mucosal binding composition to detect bindins to the mucosa surface. Alternatnvely, the binding can be assayed using an antibody specifically recognizing the mucosal binding composition in an ELISA, a modification~thereof, a western blot assay, or the like.
In a preferred series of embodiments of this invention, subunits or fragments ot the Vibrio cholerde toxin are used to direct an immunogen to the mucosa. Embodiments are disclosed in the Examples, provided below, which detail the association of the mucosa binding polypeptide, the cholera toxin B subunit, to an immunogen derived from a non-viral pathogen causing a sexually transmitted disease.
Despite differences in the primary sequences of the proteins, there are a number of striking similarhies between the nontoxic subunit of E. coli heat-labile enterotoxin (LTB) and the B subunit of cholera toxin (CTB). The LTB gene sequence is provided in Leong, J., et al. (Infect. Immun. 48:73 77, 1985 and see Tsuji, et al. Microbial.
~thogenesis 2:381390, 1987). LTB, like CTB, can be secreted into the medium when V. choleree cultures are transformed with expression vectors expressing recombinant LTB. Further, CTB and LTB bind to gangliosides on mucosai membranes ~see Hirst, et al. Proc. ~tl Acad Sci USA 83:9174-9178, 1984 and Schodel, et al. Gene 99:255-259, 1991). The Hirst, et al. reference indicates that CTB sequences can be replaced with LTB sequences CA 0220~130 1997-0~-12 O 96116178 P~l,_. ~708 .9.
for recombinant expression. Thus, il is crlntemplated that CTB sequences can be replaced wilh LTi3 sequences and used in the methods and examples detailed below.
There are any number of constructs that one skilled in the art could prepare and test for their abaity to - promote the production of an antibody response to a non-viral pathogen causing a sexually transmitted disease where the mucosal binding polypeptide is derived from cholera toxin or the heat labiie enterotoxin from ~. coL The non-limiting examples, provided below, include a vanety of compositions employing mucosal bmding polypeptide and include antigen from four different pathogens whose primary route of infection is through mucosa membranes.
The mucosal binding compositions of the present invention contain at leist one antigen trom a pathogen that ente~s its hcst through the mucosal membranes. This invention contemplates that the immunogen derrved from the this pathogen can be selected from a variety of immunogens which are known to stimulate immune responses in a mammal susceptibie to the disease caused by that pathogen. It is contemplated that the antigen includes at least one antibody stimulating determinant, preferably from a surface protein of the disease-causing pathogen. It is also contemplated that where the antigen is a polypeptide, r~ will contaln at least one consecutive region of amino acids, preferably from an '' domain of the surface polypeptide of the pathogen. In addition, the l antibody stimulating polypeptide can optionally include one or more domains derived from the same or different proteins from the pathogen. These domains may include other antibody-stimulating amino acid sequences, multiple copies of these sequences or amino acid sequences that stimulate T-Cells or those that assist in the generation of an antibody response through the actrvation of T-Helper cells or other T-Cell populations. Further, it is contemplated that the antihody stimulating determinant can be repeated in tandem or separated by suitable linking sequences, or 2û the like, to further stimulate the antibody response.
Thus, the first step for selecting an antigen contemplated in this inverition is to Identify a non viral pathogen capable of infecting a mammal via entry through a mucosal membrane. Next, one can optionaliy determine whether those mammals who are infected with the pathogen develop an antibody response to the pathogen. This can be determined , '11 or based on the literature related to the particular pathogen.
Z5 As one example of a method for determining whether individuals who are infected with the pathogen develop an antibody response, serum samples are taken from a mammal and tested for the presence of antibody by contacling the serum sample with cell Iysates containing pathogen, or alternativeiy with intact pathogen, and detecting binding of serum antibody to the sample. Assays useful for detecting serum antibody binding include enzyme-linked ' ' assays ~ELISA), immunoblots such as Western blots, or the like. Such assays are well 3G known in the art and are detailed in a variety of methodology texts including Harlow, et aL ~Antibodies: A Laboratorv Manual, Cold Spring Harbor Press, 1988). Optionally, those skilled in the arl may elect to directly test for the presence of IgG or secretory antibody to the pathogen in a mammal. In these assays, secretory fluid is lavaged from the mucosal surfaces of a patient or test mammal and the volume is optionally reduced using any suitable reduction or concentratiolt method well recognized in the art. This sample is then tested for the presence of, ' ~ 5, ' ~' ' ' in general or sigA or IgG, specifically, using standard ,, well known in the art.

CA 0220~130 1997-0~-12 wo 96/16178 ~1,~.. ..

Alternatively, those skilled in the art can determine which protein or prDteins stimulate a neutrali2ing immune response in vitro to the pathogen. Determinants stimulating immune responses are mapped to specific protein using the well known Western blot technique, or the like. The immune system stimulating determinants can be mapped to the protein usibg any number of strategies well known in the art. As one e1ample of a method for mapping antibody 5 stimulating determinants on a protein see Geysun, et al. and Miles, et al. ~"Strategies for epitope analysis using peptide synthesis", J. ImmmunoL Methods 102:259-274, 1987 and "Multiple Peptide Synthesis for the Systematic Analysis of B and T-cell Epitopes" Parasitology Todav 5:397-400, 1989 respectively).
These screening regimes permit the identificition of determinants capable of stimuiating the immune system and are also well known in the literature for a variety ot pathogens, including chlamydia. Methods are also well 10 known in the scientific literature for selecting linear antigenic determinants that stimulate antibody production from 8-Cells or selecting determinants that stimulate T-Helper cell activity. For strategies for identifying epitopes that stimulate T-Helper cell responses to C. trachomatis see Su, et al. ~ Jtp. Med 172 203 212, 1990). To identdy epitopes that stimulate antibody production in C. trachomatis see Zhong, et al. ~InfecL znd Imm. 59:1141-1147, 1991~.
Finally, it is cnntemplated that peptide mapping sttategies, also well known in the art, can be used to identify a polypeptide that is capable of stimulating a neutralizing immune response to the pathogen. For methods for testing B-Cell stimulating determinants for their ability to stimulate neutralizing antibody in vitro see Zhang, et al. ~1 ImmunoL 43B:575581, 1g87).
All of these steps permit one skilled in the art to identify candi&te polypeptide sequences that can be linked to a mucosal binding polypeptide. These steps provide one strategy for identdying candidate polypeptide sequences which can be linked to a mucosal binding polypeptide, yet those skilled in the art will recognize that each step is not absolutely necessary for the identification of a polypeptide capable of generating an immune response.
Exemplary screening strategies are provided herein to enable those skilled in the art to determine whether a particular combination of polypeptide derived from a pathogen which enters its host through the mucosal membranes will stimulate antibody production in a mammal when linked tD a mucosal binding polypeptide. These strategies are detailed in the examples below (see Examples 4 and B).
As a first step for producing the composition of this invention, the mucosal binding protein is iinked to the antigen selected from the pathogen. There are a variety of linking strategies contemplated within the scope of this application. For example, the antigen can be linked to the mucosal binding protein by chemical coupling or through a linking member, including lipid, carbohydrate or protein. Alternatively, the antigen can be synthesized by means of a gene construct either separately or together with the mucosal binding protein.
As specdic examples of the use of the present invention to stimulate mucosal immunity to a pathogen capable of infecting its host through mucosal membranes, Example 1 details a preferred strategy for the chemical coupling of a membrane binding polypeptide to a determinant from a pathogen. In this example a fragment of the cholera toxin protein B subunit was expressed using the method of Lebens, et al. IBioTechnolog~ 15741578, 1993) and chemically coupled to an immunogen derived from the major outer membrane protein of C. ~rachomdtis.

CA 0220~130 1997-0~-12 Wo 96/16178 ln one preferred embodiment employins chemical coupling, the polypeptide derived from the major oùter membrane protein is 1he VDIV peptide ~SEQ ID N0:1 ) corresponding to a portion of the fourth variable domain of the major outer membrane protein 3f C. ~r~chomstis. In anolher preferred embodiment, the polypeplide is a linear chimeric polypeptide that comprises the A8 domain and the VDIV domain from the major outer membrane protein of C. trachomatis (SEQ ID N0:2). Both the A8 and the VDIV domain as well as the chimeric peptide are described by Su, st al. (Vaccine 11:1159-1166, 1990.
As another embodiment of this invention, and as an example of alternative iinkinp strategies, a polypeptide from a protein derived from a non viral pathogen causing a sexually transmitted disease is expressed as a recombinant protein with a subunit or fragment of a mucosal binding protein. As one example of the recombinant expression of 1D this complex, the mucosal binding protein is a porrion of the cholera toxin and the antigenic determinant is dernved from C trr~chom~tis.
It is contemplated that there are a variety of construct designs that could b2 used to express protein having a portion of the cholera toxin and a poiypeptida from a protein derived from a pathogen capable of infecting its host through mucosal membranes. For example, the antigenic determinant or determinants from the pathogen could be placed at the amino or carboxy terminus of the B subunit, at the amino or carboxy terminus of the A subunit, or at the amino terminus of a portion of the A subunit. In a preferred example, the A subunit is operatively linked to the B subunit. In another contemplated embodiment, detailed in the Examples below, the antigenic determinant is incorporated in frame into an internal portion of either the A or B subunit, or a portion thereof.
It is contemplated that these constructs, provided in the examples below, could also be derived from the B subunit of the heat labile enterotoxin of E. co/; without undue , It is further contemplated that variations in the plasmids are also within the scope of this invention. For example, this work has also demonstrated that the high copy number derivative pML-LCTBtac2 has an identical restriction pattern lo pML-LCT8t~c but incorporates the pUC19 origin of replication (see Lebens, et al. ~iotechnology, supr~). Thus, the use of this plasmid is also contemplated within the scope of this invention.
In one construct detailed in Example 2, a chimeric protein is produced that includes a polypeptide linked at its carboxy terminus to the ammo terminus of the B subunit (CTBi from choiera toxin. As another example to indicate that this construct is similarly useful for producing stimulating antibody to other antigens, the HIV epnope IQRGPGRAFV is incorporated into the amino terminus of the B subunit.
In another example of this embodiment, detailed in Example 3, the antigen from the pathogen is incorporated onto the amino terminus of a polypeptide which is a fusion protein of a fragment of the A subunil of cholera toxin fused to CT8 and expressed in either V cholera or E. coli. In one aspecl of this embodiment, the antigenic peptide is the VDIV sequence from the major outer membrane protein of C. tr~chomrJtis and in another preferred embodiment the antigenic pepfde Is the A8 sequence from Ihe major outer membrane protein. In a third aspect of this embodiment, the peptide is a chimeric proteln containmg both the anti~qenic peptide sequences of A8 and VDIV
separated by a linker (see Figure 61. It is also contemplated that this same linker introduced into, for example, pML
LCTB,17, would facilitate Ihe construction of multiple copies of either epitope. Alternativelv, the linker, or other :

CA 0220~130 1997-0~-12 WO96/16178 r~I.. ..

linkers, couid be used to insert the A8 and VDIV sequences into other constructs or the linker could faciGtate the transfer of inserted se3uences between vectors. It is further contemplated wilhin the scope of this embodiment that the order of the antigenic sequences can be switched and that multipie determinants of A8 or VDIV can ba linked either in tandem or within tha recombinant construct.
The literature describes a variety of antigenic peptide sequences f or C. frachoma~is that are serovar spechic, subûroup specific or are broadly conserved amonU serovars. Thus, it is also contemplated within the scope of these embodiments that other antigenic peptides from the major outer membrane protein or from other proteins of C.
trachomatis wouid also be useful in stimulatins antibody production alons the mucosal surfaces.
Joblin3, et al. ~Infect. and Imm. 60:49154924, 199Z) have produced fusion prote`ins in which entire bacterial proteins were linked via the amino tarminus to the A subunit fragment. CTA2. Whereas Jobling, et al.
demonstrated that chimeric toxin could be produced from a cholera toxin construct, both portions of the chimera ware bacterial protein and the signal seguences for chimeric expression were derived from the inserted bacterial protein.
The present invention demonslrates that antiSenic sequences which are foreign to the bacteria can be inserted into CTA2. The CTA2 fusion proteins with foreign antigenic seguences were coexpressed with CTB from i~. Cholera to give assembled prDducts which were excreted extracellularly and were detectable in a GM1-ELISA. The gene products produced by Jobling et al. ware not excreted extracellularly.
In a third example employing genetic constructs, contemplated within the scope of this invention and detailed in Example 4, the polypeptide is incorporated, in frame, into an internal portion of the mucosal binding polypeptide, preferably at an i ' ' site. In one example of this strategy, it is contemplated that a short foreign polypeptide is introduced into an internal regiorl of the cholera B subunit in place Df CTB amino acids 56 63.
In one embodiment, this peptide includes the VDIV epitope, LNPTIAG, and in another embodimant, illustrating that this strategy can be used for other peptide sequences, the peptide is the HIV neutralizing epitope IORGPGRAFV laa 309-318 of HIV 1 isolate HTLV-1116~.
Six additional plasmids were constructed, encoding internal HIV::CTB hybrid proteins with ten to fourteen amino acids from the V3 loop of gp120 genetically inserted at different positions between amino acids 52 and 65, with deletions of different CTB (Cholera toxin B subunit) amino acids ISee Table 1). The plasmids which encoded proteins with peptides inserted between 55 and 64, 53 and 64 or 56 and 57 in CTB gave rise to the synthesis of proteins which reacted with CTB specific nnonoclonal antibodies (mAb) and bound to GM1 gangliosides. This indicated that insertions at these sites did not alter the functional conformation or the receptor binding properties of native CTB.
In yet another example, the peptide is a portion of the pre-S~2) protein from Hepatitis B and in a further example the peptide is one of two peptides from the ST, protein from `~ ' ~. coL Plasmids were constructed encoding CTB hybrid proteins which had either an eleven amino acid peptida form Hapatitis B Virus IHBV) pre S:2) ISED ID N0: 28) o~ one of two peptides ~SED ID N0:30 and SE0 ID N0: 31, see Table 1~ related to the heat stable toxin ~ST,) of, ~ . coli. This work demonstrated that an internal permissive site could be used to insen peptides of several different amino acid compositions.

CA 0220;il30 1997-0;i-12 wo 96/16178 J

'" antibcdies were used in GM1-ELISA and immun3blot assays to screen the 3rctein hybrids fcr their . ~l ' integrity, for their abili~y to bind to membrane determinants or to be recognized by the foreign epitope specific antibody. Example 4 provides data relating to this screening. All of the CTB:foreign antigen products induced low levels of serum antibodies in mice against the full length foreign protein. In addition the 5 products stimulated strong serum antibody responses against CTE.
Cata is provided in Example 4 to indicate that the CT3..foreign antigen hybrids of the present invention retain all of the important 1' ' of native CT3, such as folding,, ' ' extra-cellular secretion when produced in V. choleree and GM1-binding. Moreover, many of the inserted peptide constructs were resistant to cleavage by V. choler~e proteases. The data in this example indicated that the CTE.::foreign antigen hybrids 10 reactedwhthmonoclonalantibodydirectedagainsttheforeignantigen,bothinthedenaturedandnon-denaturedforms of the protein. This indicated that the substitution of eight amino acids from a region in the native molecuie with amino acids from an unrelated protein, such as gp120, VDIV, pre-S(Z), and ST, produces a construct with foreign antiyen that is accessible to the immune system of its host.
It is also contemplated within the scope of the invention that the environment of the inserted peptide can 15 be modified to improve the ' ~ . For example, it is contemplated that flanking residues can be added to the inserted peptides or that the position of the insert can be shifted within the insert permissive region of the CTB
seouence and that these modifications do not limit the scope to this invention.
Dnce the mucosal binding complex of binding protein and antigen has been generated it will be helpful to test the integrity of the complex using antibody to the foreign antigen and antibody to the mucosal binding protein.
Z0 Methods for detecting hybrid protein expression are provided in Examples 4 and 6. Ia vitro assays testing mucosal binding are useful for l' ' . ' ~ the intactness of the compiex. Such assays include binding assays to intact mucosal epithelium cell cultures or mucosal membrane sections. If the mucosal receptor is known, specific ELlSAs or , ;li~ , ' ' assays, or the like, can be used to ascertain mucosal binding.
Next, it is contemplated that the complex will be formulated as a mucosal binding composnion that 25 optionally includes physiologic buffers, additional adjuvant, or the like, to facilitate the production of an immune response in a mammal. The composition is then introduced into a mammal either vaginally, orally, rectally, nasally, ' 1~, i"l,, ' 'I~ or intrivenously. Sera or mucosal immune responses are monitored in the experimental mammal or in ciinical trial by ELISA, immunoblot, or by western blot to detect antibody to either the mucosal binding polypeptide or the foreign polypeptide. It is conlemplated that antibodies in serum or antibody in 30 mucosai secretions can be assayed for the presence of IgA, IgG or total antibody reactivity to a particular peptide, polypeptide or pathogen preparation using assays well known in the art. The mucosal secretions are harvested as a lavage, an aspirate or as a wash preparation. Alternatively, the mucosal secretions are collected through wicking action using an absorbent pack, such as a tampon, an absorbent piug, or the like. Similarly, biopsy of mucosal tissue during the testing phases of this invention will be usea to assay, by i , ' ' ~, or by celi activity, for immune 35 responses specific to the mucosal binding polypeptide. For exempiary methods for preparing biopsy tissue see Eriksson, et al. or Quiding, et al. ("Perfext: A Simple Perfusion-Extraction Procedure for iiuantitative Ana~yses of wo 96/16178 . ~l,. . .

Antibodies and Cytokines in Gistinct Anatomicai r~ , i ' Abstract presented at the 1 2th European Immunology Meeting. ~iune 14-17, 1994 and J. crn~ Invest. 88(11:143-148, 1991 respectivelyl. Strategies for detecting IgG
and IgA in a mammal are known in the art. rhese incluie the measurement of antibodies in serum or mucosal secretions using ELlSAs, immunoblots, or the like.
This invention details methods for introducing the compositions of the present invention into a mammal for stimulating antibody production. Methods are provided for stimulating vaginal, oral and rectal immune responses ~see Examples 7-9).
Results detailed in Example 7 indicated that immunization of mice with the intrachain CTB::HIV hybrid protein gave rise to very strong serum antibody responses to the CTB moiety. Antibody to the foreign epitope was also observed. Example 10 indicates that the combination of a mucosal binding polypeptide with a foreign antigen from a pathogen produced mucosal antibody specific to the pathogen.
All references cited herein are expressly incorporated by reference in their entirety. Particular embodiments of the invention are discussed in detail below and reference has been made to pcssible variations within the scope of the invention. There are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit cne tD successfully perform the intended inventiDn.

Chemical Coupling of Polypeptide to a Membrane Binding Protein In this example, Cholera toxin B subunit (CTB) was produced in a mutant strain of Vibtio rholerae deleted of the cholera toxin genes and transfected with a plasmid encDding CTB (as disclDsed by Lebens, et al. supr~. In this expressiDn system, CTB was recDvered as secreted prDtein in yields at or abDve 1 911. Bacterial cultures were centrifuged at 8000 rev per min fDr 220 min and the supernatants were adjusted tD pH 4.5 withdilute HCI. After precipit'atiDn w'lth ' , ' , ' (final CDnCentratiDn Df 2.5 9111 fDr 2 hr at 23C fDllDwed by centrifugatiDn at 8000 rev per min, the pellets were dissDlved with 0.1M sDdium phDsphate buffer, pH 8.0 and dialyzed against 0.01 M, ' , ' ' '' ' saiine (PBS), pH 7.2. The dialysate was then centrifuged at 15,000 reY per min tD remDve insDluble material and the supernatant was further clarified by filtratiDn thrDugh 0.22 ,um filters IMillipDre, BedfDrd, MA). CTB was purified by standard gel filtration ~ thrDugh cDlumns Df Sephadex G 100 (Pharmacia, Upsala, Sweden).
The pDlypeptides were cDvalently cDnjugated to CTB using N ' '1~:;3-~2r,.iJ~:jr '' ` prDpiDnate (SPDP, Pharmacia) as a bifunctiDnal cDupiing reagent accDrding to the rnanufacturer's instructions. CTB was derivatized with SPDP at a molar ratio of 1:~ in 0.1M phDsphate bufferlO.1M NaCI pH 7.'i. After incubatiDn at 23 C fDr 30 min., excess SPDP was remDved by gel filtratiDn thrDugh Sephadex G 25 cDlumns (Pharmacia) and eluted with PBS. The absDrbance Df the mDdified prDtein was measured at 280 nm. To estimate the degree of substitution with 2-pyridyl disulphide residues, the absorbance at 343 nm of 100 ,ul protein solution in 4DO yl was measured 3'i after incubatiDn with 50 ,vl dithiDthreitDI ~0.1M) fDr 15 minutes (mDlar extinctiDn coefficient at 343 nm - 8.08 x 103 M''cm'l~. This concentration was eouivalent to the concentration of 2 pyridyl disulphide residues in the protein.
Since the 2 pyridyl disulphide groups contribute to the absorbance at 280 nm, a correctiDn was appiied to the wo 96~16178 1 ~I, .
.15 calculation of protein A2,3 due tD the protein - A2," - ~B x s.1X1031 where 5 is the molar concentration of pyridine-2-thione released. From thsse results the number of moles of Z-pyndyl disulphide substituted per mole of protein were calcuiated. The substitution ratio obtained with these conditions ranged between 2-3 mo~es of SSPY per mole of CTB pentamer.
S Peptides and SPDP-derivetized CTB were mixed at a ratio of 5 mol peptidel1 mol SSPY and incubated for 24 hr at 23C. The resulting CTB-peptide conjusates were purified by gel filtratiDn through columns of Sephadex G-25. Conjugates were purified over GM1-columns of Sephadex G-25. Purified cDnjugates were shown to retain GM1-binding capacity and to retain both CTB and peptide specrfic serolDgical reactivities by means of a solid phase ELISA using GM1 as a capture system and , ! antibodies to CTB or to the foreign polypeptide sequence coupled tc CTB. An exemplary ELISA using GM1 is provided in Examples 4 and 5, below.
In this emùodiment, three different peptides were conjugated to CTB. The first, Peptide 166 ~SED ID
N0:13,A8-VDlV),isthecolinearpeptidedisclosedbySu,etal.(V~ccinell:11591i66, 1993j. Itcontainsasingle cysteine residue. Peptide 172 ISE0 ID ND:14) has a free extra cysteine coupled to the aminD end and Peptide 173 (SE0 ID NO:15~ has a free extra cysteine coupled to the carboxyl end Df the A8-VDIV sequence.
The CTBIA8-VDIV complexes were introduced into C57 BL~6J female mice ~obtained from the Animal Care Facility of the Deparlment of Medical Microbioiogy and Immunology, University of Goteborg~. Mice were 6-8 weeks of age. The immuni~aticn protocol for generating mucosal antibody is provided in Example 8.

Genetic Fusion of ForciDn polypeptide to the aminc terminus of CTB
In cne embodiment, the starting plasmid was pPL-lambda obtained from Pharmacia AB, Sweden. The Seneration of the pMU plasmids is diagramed in Figure 1. pPL-lambda was digested with Smal and BamHI.
Digested DNA was resolved on an agarose gel and a 1,217 bp fragment carrying the lambda promDter regi3n was recovered by !-1 More of the same plasmid was digested with BamHI and Pvull and again resolved on an agarose gel. This time a 2,305 bp BamHllPvull fragment carrying the pBR322 origin of replication and the ampicillin resistance gene was recovered by band extraction. The two extracted bands were ligated together and ligated DNA was transformed into E. ~oD strain N483D l, the Gal-P2 transductant of N4830 ~Pharmacia. Sweden;.
In the resulting plasmid, pMU1, the orientation of the phage lambda DNA within the plasmid has been reversed and 1,689 bp of pBR322 DNA has been removed.
pMU1 was digested to completion with BamHI and subsequently partially digested with Haelll. The digested DNA was religated and used to transtorm E. coli strain N4830-1. Transformants were screened on the basis of restriction anaiysis for piasmids in which the iambda N gene and tL l terminator had been removed. The resulting plasmid was pMU2 and carried the promoter ,IP, with unique downstream restriction sites 8amHI, Smal and EcoRI that could be used for cloning of recombinant genes. The powerful trpA transcription terminator (derived from the IrpA cassette, Pharmacia~ was introduced between the unique EcoRI and Aatll sites within pMU2. At the CA 0220~130 1997-0~-12 ~o 96/16178 1 ~1, ~ ,.

same time, an addilional unique Xhol site and a Hindlll site that could be used in clonina procedures were introduced into the plasmid (see Figure 1).
The unique Ndel site within the plasmid was removeb by digestion with Ndel, blunt end repaired with the Klenow fragment of DNA polymerase and religated. The Sspl site in the vector was removed by insertion of an EcoRI linker whith generated BspEI sites on either side of the introduced EcoRI site. The resuhing plasmid was digested with BspEI and religated. This plasmid was used for recombinant expression in E. coL
Fusions of peptides to the amino terminus of CTB were generated by inserting synthetic liL
between unique Sacl and Sspl sites in the expression vector pML-LCTB,i7 (see Figure 3~. For exampie, the HIV
RP335 peptide was placed at the amino terminus of CTB in pML-LCTB,f7 between the Sacl and Sspl sites using lD two SED ID N0:9 and SE0 ID N0:10. In a second construct, the insertion was placed within the structural gene at position ~ 3 of the CTB sequence generating a Hpal she and at the same time destroying the Sspl site. nliL corresponding to SE0 ID N0:11 and SE0 ID N0:12 were used for this vector construction.
The ctxB gene in this vector is under the control of the inducible ,iP, promoter and was constructed essentially as described previously ILebens. M. et al EioTechnology 11:1574-1578, 1993~. The vector was chosen in order to allow the construcdon of genetic fusions, the products of which may be deleterious to the host cells since the inducible system only allows expression under inducing condnions. Thus, using these constructs, cultures can be maintained under conditions in which expression is not switched on and the recombinaot protein does not accumulate to harmful levels. Expression at high levels can be induced for a short time immediately before the cells are harvested at which point survival of the culture is no longer an issue.
In another embodiment, a second construct was produced that employed pML~r derivative plasmids. This permitted expression of the resuiting protein fusions in V. cholerae. The starting plasmid in this case was the expression vector pKK223 3 obtained from Pharmacia AB ~Sweden~. In order to obtain the plasmid used as the basis of the . , system for ~TB and its derivatives, the 1689bp PvulllBamHI fragment was removed. This was achieved by digestion of a plasmid into which the cr,~B gene had been inserted with Pvull and BamHI, followed by 25 blunt end repair of the plasmid with Klenow enzyme and suobsequent religation. This procedure regenerates the BamHI site shown in Figure 2.
In the course of exchange of different ctxB derivatives between the two expression systems, the rmB T1T2 transcription term~nator was replaced with the trpA terminator described above.
A futther development of the vector was to introduce the /~cl gene upstream of the tac promDter to make 30 the vector produce cloned gene products jD an inducible manner. This was done using PCR. The Ptac-based expression plasmids of this invention ~see Figure 2~ were amplified using SE0 ID ND:16 and SE0 ID ND:17. The /~clq gene was obtained by PCR fragment amplification of expression plasmid pMMB66 (Furste, et al. Gene 48:119-131 19û6~ using SED ID N0:18 and SED ID ND:19. The /aclq gene was introduced between BamHI and Bglll restriction sites so that it could be removed easiiy to generate a plasmid giving constitutive expression from the t~c promoter 35 Isee Figure 2~.

-Wo 96116178 ln a third embodiment, the same gp12U peptide was fused to the N-terminus ~f ~TB by inserting ~ synthelk encoding Ihe peplide between the S~cl and Xrnal restrictiDn srtes at the junction between the leader peptide and the malure CT8 in Ihe expression plasmid pJS752-3 (ApR), a derhrathve of pJS162 ~Sanche2, et al. Proc a9f~ Ac~rl. ScL USA 86: 481485, 1989). This plasmid is essentially the same as 5 pML-LCT~t~c excepl that the Ec3RllHindll fragment carrrin3 the recombinanl ctx8 gene is derived from pJS~62.
The parent plasmid is the expression Yector pKK223 3 ~Pharmacia~. Plasmid JS752-3 carries the gene encoding CTB
under t~c promoter control. In plasmid pJS54 Isee Example 4, below), an oligo If ormed from the hybridkation and ligation of corresponding to SEn ID N0:20 and SEQ ID N0:21) was inserted between unique Sacl and Smal siles in p iS752-3.
The recombinant V. rholerde strain produciny ths N-lerminal CTB::HIV hybrid protein was shown t3 secrete Ihe chimeric protein inlo the cuilure medlum using Ihe GM-1 ELISA provided in Example 4. The cullured cells secreted protein reactiYe with mAb r_ _ ~,120 when analyzed in a GMl-ELISA. The GM1-ELISA was useful as a tool to quantilale Ihe amounl of expressed prolein and lo monilor the exlenl of proleolytic degradation of the added peptide. Culture conditions required to produce the constructs are provided in Example 5.
In another embodimenl, another construcl whh an amino lerminus substhtution was made in whi h the gpl2D epitope was placed al the N-lerminus of Ihe CTB protein wilh two extra amino acids, corresponding lo Ihe two first amino acids of CTB, placed N-lerminaliy to the gpl 20 peptide, and Ihis extended epilope was linked direclly o Ihe complete mature CTB. The resulling plasmid, pCB2gp309-318, was expressed in v choler~e JS1SB9 ~strain 644). When cultured at 37~C, a protein which bound to GM1 reacted with the CTB-specific mAb LT39 and was 2D produced within the periplasm. Unlike native CTB, il was nol activsly secreted into the medium. However, the protein produced by slrain 644 reacled wilh mAb P4~D10 increasingly wilh lime. The fused gpl20 peplide (SEQ
ID ND: 291 in Ihis protein was more resistanl lo proleolylic degradalion Ihan Ihe construc' iacking the N-terminal amino acids of CTB. The plasmid pCB2gp309-31B was also expressed in E. coli HBlD1 (strain 5D4) and could be purified from the periplasmic space by osmolic shock Iysis of the cells followed by precipitation Wilh 80% ammonium sulfate and extensive dialysis against PBS. This protein ran at a higher molecular weighl than CTB in SDS-PAGE, indicating Ihe formalion of aggregates, but the protein reacled well wilh bolh mAb P41D10-anli-gp120 and with mAb LT39 and CT6 againsl penlameric and monomeric CTB.

6enetic Fusiun of Foreign polypeptide to the r~mino terminus of CTA-2 The plasmid used for the expression of CTA-2 was construcled so Ihat the CTA-2 fusion and CTB were co expressed to obtain assembly of a hol3protein in viw. The ct,rA2 and ct,rB genes were obtained from PCVD30 ISee Kaper, J. et al. BioTechnologv 2, 345-349, 1984~ as an XballHindlll fragment which was cloned into pUC19 (ranisch-Perron. C., et al. Gene 33, 1D3-109, 19851. The ctxA ribosome binding site and signai peplide sequence were reintroduced by the insertion of synlhetic between the Xba' sile and a unique EcoRI sile upstream from it in the vector (see Figure 4). The resulting cccRllHindlll fragment Carrring the crr genes contained :: , CA 0220~130 1997-0~-12 wo 96/16178 anique Sacl and Xbal sitss between which synthetic 'jL ' ' can be inserted to generate amino terminal fusions between cr,rA2 and the added epitope of interest. This fragment was then transferred to each of the expression plasmids illustrated in Figures 2 and 3. In each case,, ' l ~' ' expression of CTB could be demonstrated. Plasmids cartyiny the ct~ genes onder the control of the ~ac promoter were maintained in V. cholerae since the levels of CTB generated in this example were too high to be tolerated by E. coli. This construct lacks the recognition site for the proteo~ytic cleavage inv3ived in the maturation of CTA.The antigens used in this work were those f rom Chlannvdia trachomatis strains ident'died by Su and Caldweli ~Su, H. et al. Vaccine 11, 1159-1166, 1393). These include a T-cell epitope A8 SEQ ID NO:6, situated within a 25 amino ac'ld peptide from serovat A MOMP and a B-cell epitope VDIV SEQ ID NO:1, from serovar B MOMP containing 17 amino acids. Within the VOIV sequence a septapeptide seDuence has been mapped as an epitope reacting whh the monoclonal antibody OIII-A3. The amino acid sequences of the two peptides are shown in Figure 5 together with the synthetic nucleotide sequences used for the generation of gene fusions with CTB. The " ' '' cottesponding to VOIV and the ' ' ' corresponding to A8 are identified.
The first of the fusions were made to the amino terminus of CTAZ. The synthetic IjL
corresponding to the foreign antigen were cloned info the vectors. It was possible to express assembled protein complexes in which the VDIV peptide was associated with CTE and detectable in a GM1 ELISA assay using Mab Dlll-A3 as the primary antibody. In this case the assembled protein was produced in E. coh under the control of the AP1 promoter. In V. cholerae, assembled protein was detected in the periplasm when the construct was under control of the tac promoter. The A8 peptide was cloned independently into similar vectors.
2D In order to produce a fusion between the A8 and VDIV peptides attached to CTA2, the linker shown in Figure 6 was inserted into the Sacl site of plasmid pPJ-VDlV,i using SEQ ID NO:7 and SEQ ID ND:8. This allowed the addition of the A8 sequence to the amino end of the VOIV sequence by cloning of a BglllXbal fragment from pPJA8,1 into BglllNhel digested pPJVDlV,11. pPJA8~1 is a CTA2 fusion veclor into which the oligo encoding the A8 T-cell epitope was inserted.

Insertion of D IlleuttDlizin~q Antigen in a Surface Exposed InternDI Re~qion of the Gholera Tuxin El-Subunit In this example, novel intrachain CTB fusion protein were prepared w'nh foreign peptide inserted into an internal region of CT3. The resulting chimeric protein relained important functional Ll.c, ~L;~ ,;L~ of the native CT3 including: 1 ) I ' , 21 GMl ganglioside receptor binding: and 3) resistance to proteolytic degradation during production of the protein in Vibrio choler~e. The inserted epitopes were detected with antibody known to bind the epitopes using ELISA and immunoblot assays to demonstrate that the epitope was present and accessible on the surface of the protein. Immunization of mice with the test hybrid protein elicited antibody responses to the mucosal binding polypeptide and the insened antigen.

CA 0220~130 1997-0~-12 WO 96116178 1'~
19.
In cne example, the forsisn polypeptide was inserted at the position of an internal loop structure between L~4 and a2 in CTB, extendin3 with a few residues into the a-helix, as predicted by a comparison of the resolved crystal structure of LTB (Sixma, et al. A/~ture 351:371-377, 19911. Here, the VDIV fragmenl TTLNPTIAGAG is - incorporated into the internal CTB site using the two restriction endonuciease recognition shes Kpal and Mscl.
S n~ ' ''' cor~esponding to SEQ ID NO: 22 and SEL ID NO:23 ara hybridized, digested with the restriction enzymes Kpnl and Mscl, purified and ligated to p~asmid pCB5E-64.
In a second example, expression vectors were constructed having HIV-1 epitopes positioned inteMally in-frame within CTB, the CTB expression plasmid pML-LCTBt~c (Ap~l was mutagenized using the polymerase chain reaction (PCR), employing a modified protocol of that disclosed by Schddel et al. (~Hybrid hepatitis 8 virus corelpre-S
10 particlesexpressedinattenuatedSdlmoneJldefororalimmunrzation." In: Brown,i'.,etal.(Eds),Vaccines'91. Cold Spring Harbor Laboratory Prsss, Cold Spring Harbor, NY, 1991, pp. 319-325.). In the first construct, the 'il ' '' used in the PCR raactions are provided as SEQ IO NQ:3 and SEQ ID NO:4. These primers incorporated a sequence encoding ten amino acids from ~he central portion of the V3 loop in HIV gpl20 between resibue 55 and 64 in CTB. Two unique restriction enzyme sites, ~ssHII and Kpnl, ware also imroduced, with the ''~ ' '' primers, into the final plasmid pCB55-64gp309 (see Figure 7).
The PCR reaction was run in the presence of 1.5 mM MgCI2 and 1.25 mM d~ . ' ' under the following reaction conditions: denaturation at 94C for 1 min, anrleaiing of primers at 65C for 2 min and elongation of ONA by T~q DNA polymerasa ~Boehringer Mannheim, Indianapolis, Indiana) at 72C for 4 min. The reaction was repeated for 30 cycles, increasing the elongation step by 2 sec with each cycle. To complete all synthssized DNA strands, a final incubation step at 72C for 10 min was performed. After ,' " '' extraction, the PCR product was digested with ~ssHII (60ehringer Mannheim) according to the manufacturer's instructions,, ' " '' '~, extracted again and raligated using T4 DNA ligaæ (Pharmacla, Llpsala, Sweden) at 16C for 3 h. The ligated plasmid was ' ~ into IE choler2e Istrain JS1569, c~xA, ctxe') by the method of Lebens et al. (SUprd). The GNA sequence was confirmad by the dideoxy chain termination method of Sanger et al. IProc. /VarL ~Ic~d. Scl. USA 74:5463-5467, 1977).
The CTB region was selected for substitution because it had been reported that this region reacted with antibodies recognrzing primary protein structure rather than CTB, ' ' ' epitopes IJacob et al., ~M~O J.

4:3339-3343, 1985 and Kazemi, et al., MDL ImmunoL 29:865-876, 1991). In addition, based on the crystal structure of LTB, a substitution in this reyion would presumably not affect the beta-sheet or alpha-halix structures essential for the correct folding of the molecule. Further, based on the crystal structure of LTB, a substitution in this area would: not affect the assembly of pentamers lsee Sixma et al., 1991, SUprd). Finally, a substitution is this araa would likely not interfere with the site responsibla for GM1 receptor binding on the mucosal surface.
The gpl20 peptide used for these studies was takan from amino acids 309-318, having the sequance IQRGPGRAFV ~SEQ ID N0:29), representing a portion of the V3 IODP of Hlv'-1 isolate HTLV-IIIB. The sequence numbering of gp120 is based on the Los Alamos database sequence for gpl20 ~Los Alamos National Laboratories, Los Alamos, New Mexico~. The seouence contains a principal neutralizing B-cell detarminant of HIV-1. Ths ssouence CA 0220~130 1997-0~-12 WO 96/16178 ~ ~ m7708 GPGR within the peptide is conserved between several HIV-l iso~ates and the peptide is of great interest in the development of a peptide-based vaccine asainst HIV-1 ~Javaherian et al., Proc. Alat~ Acad Sci USA 86:6768-677Z, 19891. The gpl20 peptide replaced amins acids 56-63 of the recombinant CT8, thus adding two amino acids to the net number of residues in the protein.
s r, ~ gel ~ ., ' (PAGEI and immunDblot analyses of partialhy purified proteins frcm cuhure supernatants of V. chalerde carrying the plasmid pCB55-64gp309 revealed that the internal hybrid protein was synthesized and actively secreted from the cell into the culture medium like native CTB, accumulating to approximately 5-15 mg per liter medium~ Cultures were yrown in 50ml modified syncase medium in 250 ml Erlenmeyer fiasks shaken at 250 rpm overnight at 37 C. These plasmids can also be grown in E. coli without adjustments in vector construction.
A number of additional plasmids were prepared which incorporated foreign antigen inta the CTB seguence between positions 54 and 64 of the CTB protein. The features of the different plasmids are listed in Table 1 Isee page 38). pCB55-64gp309 (Ap) refers to the CTB:HIV hybrid protein described in the preceding paragraphs of this example. The constructs of Table 1 were prepared using the methods described above. Plasmid pCB55-64gp309-318 used SE0 ID N0: 32 and SEO ID N0:33 as ' ' ' to incorporate the gp120 epitope into the CT8 protein. Plasmid FCB56gp309 318 used SEn ID N0: 34 and SE0 ID N0: 35; pC852-58gp309-318 used SED ID N0:
36 and 37, pCB53-64gp3D9-318 used SED ID NO: 38 and SED ID N0: 39, pCB53-64gp307-318 used SEO ID ND:
4D and SED ID ND: 41, pCB55-64gp309-322 used SEQ ID ND: 42 and SEQ ID N0: 43, pCB55 64STdeca used SE0 ID N0: 44 and SE0 ID ND: 45, pCB55-64 st, used SED ID ND: 46 and SE0 ID N0: 47, pCB55 64ps133-143 used SEQ ID N0: 48 and SE~ ID N0: 49, and pCB56psl33-143 used SE0 ID ND: 50 and SE0 ID ND: 51.
Plasmids rrr~C~"'lnq-318 and pCB53-84gp307-318 were made by the cloning of ,' synthetic li~ oligos~ between the Kpnl and BssHII sites in pCB55-84gp309318 with the introduction of a BamHI site in the latter. In order to construct the plasmid pCB55-64gp308-322, r , ' synthetic oligos encoding amino acids 314-322 from sp120, carrying a BssHII site, were cloned in between the unique llsA and Mscl sites fianking the peptide insened between 55 and 84 in the plasmid pCB5-643p12. A
BssHlllHinolll fragment of around 1 kb, encoding amino acid 315-322 of the insened epitope together with amino acids 64 to 1û3 of the gene encDding CTB (c~xB) and sequence up to the terminator sequence was obtained from the intermediate plasmid and subcloned between the assHII and Hindlll sites in plasmid pCB55 64gp309-318.
The other HIV::CTB gene fusions were made by oligo directed PCR mutagenesis of the expression vector pML-LCTB~ac, introducing nucleotides coding for amino acid 309-318 from gp120, and containing a BssHII site, either as a straight insertion at position 56, resulting in plasmid pCB56GP309318, Ot with deletions of the CTB amino acids 56-64 ~pCB55-65GP309-318) or amino acids 5357 ~pCB5258gp309-318).
To make the ST::CT8 plasmids, two pairs Df synthetic oliqos were synthesized which encode either a ST,-related decapeptide comaining a neutralizing B cell epitope, or the whoie ST, of l9 aa, with a Kpnl site at the 5'-snd a Mscl site at the 3' end. A Sphl site was introduced with the o~igos, which were cloned into the plasmid pCB55-64gpl2, to obtain the plasmids pCB55-645T~,", and PC355 64ST, respectively.

Wo 96116178 }

The plasnnids encoding the HBV::CTB hvbrid proteins were also constructed by oli~o dire~ted PCR
mutaqenesis of pML LCTBf~c, imroducinq DNA encoding amino acids 133143 frDm pre-5~2~ together with an J~hol site, either between amino acids 55 and 64 of CTB, yie~din3 the plasmid pCB55-64psl33-143, or as a strai~qht insertion after position 56 in the plasmid pCB56psl33-143.
All hybrid genes were sequenced by the dideoxy chain termination method o~ San~qer et al. (1977).

,~

CA 0220~130 1997-0~-12 wo 96116178 .

Table 1. Propertios of the differ~nt plasmids and the corTesponding hybrid proteins Recombin Unique ~estricticn ant CTB aa Aa in inserted Aa sequence of enzyme sites Plasmid l~ ~holeNe deleted epitope inserted epitope introduud with the strain insert number'

5 pCB55-34gp309-310i 407 5B-63 gp120 309 318' IQRGPGRAFV t7ssHII, Kpnl pCB55-65gp309 318 40B 56-64 gp120 309-318 IQRGPGRAFV ~ssHII, Kpnl pCe56sp309-31a 439 gpl20 309-31a IORGPGRAFV 9ssHlI
pCB525agp309-31a 440 5357 gp120 309-31a IORGPGRAFV 2ssHlI
FrR~q E' ,' 31a 460 54-63 gp120 309-31a IORGPGRAFV dssHII, Kpnl 10pCB53-64gp30731a 5a6 54-63 gp120 307-31a IRIQRGPGRAFV 8ssHII, Kpnl, e8mH
pr~ El ,~nq322 550 56-63 gpl20 309-322 IQRGPGRAFVTIGK 8ssHII, Kpnl pCB2gp309-31B 644 ~504) gpl20 309-31a IORGPGRAFV ~ssHII
pJS54 2a5 gp120 309-317 lORGPGRAFPGYAHGr Xmel pCB55-64STdeu 551 56 63 ST decapeptide CAELCCNPAC Sohi 15pCB55-64st, 557 56-63 ST 1-19 NSSNYCCELCCNPACT Sphl GCY
prr~ E~fl 1~ t i43 395 56-63 pre-5~21 133-~43 i~PRVRGLYFPA Xhol pCB56ps133-143 549 preS121133-143 DPRVRGLYFPA Xhoi al The maternal strain is V. choler~e JSl569 20 bl The nomenciature used for th2 plasmids is as follows: CB indicates that the maternal protein is CTB: the first number indicates the position in CTB of the inserted peptidY (a striight inseninn without delotjons is ncted with the aa which the insen is placed aiter the two numbers (eg. 55-641 indicate that the aa between the numbcrs are deietedl; the ne tt two (etter describes thr, origin of the inserted aa, gp, for gpl20, ps for pre Si2~ and ST for STa, and the last numbers or letters indicated the aa insened 25 cl Numbering of gp120 aa acccrdingl to the Los Aiamos database d; Strain number 504 is an E. coli HB101 strain carryins the p(asmid pCB2gp309 31B
el Amino acids in bold ccnstitutes 1he inlervening (inker sequence SUBSTITUTE SHEET(RULE 26) CA 0220~130 1997-0~-12 ~o 96116178 1'~1,- ;

EXAMPLE ~
BDcteriel Growth CondiUons for Hybrid Pnlypeptide Produttion S ~ ~
The recombinant V. chole~ae strains producing th& hybrid prctein polypeptides were cultured in a modified Syncase 0edium (Lebens et al., Biotechnology 11:1574-1578, 1993) wilh 100 ~glml Ampicillin at 37C with shaking. Samples were taken at 3, 6, 8, 13 and 24 h after inoculation and both the tclls and the culture supernatant were analyzed in a GM1-ELISA (see Example 4 above and 6 below~ with the mAb LT39-anti-CTB and F581H3-anti-gpl2D. The major part of the produced hybrid proteins were found in the supernatant. The amount of CTB produced was calculatad using a standard curve with purifled recombinant CTB. The titers of F581H3 binding were defined as the interpolated dilution giving an A45D of OA above background.
Cultures of E. co~i were grown in the same way using L-broth rather than syncase medium.

Screenfng for Expression of the Hybrid Proteins:
~ ; of the different internsl hybrid proteins The internal HIV::CTB, HBV::CTB and ST::CTB hybrid proteins were synthesized in the V. cholerae strain JSl569 (CtXA; CtX.1 which was transformed with the different plasmids by !~ L", The resulting recombinant bacterial strains were cultured at 37C in the presence of 1ûO ,uglml Ampicillin.
Hybrid protein was found in the culture medium which could be precipitated by acidification in the presence of , , ILebens et al., EioTechniq~les 19931. Precipitates were redissolved and dialyzed extensively against PBS and then analyzed in SDS PAGE and immunoblot.
r.~ ~ Gel AnDlysis rlnd 13 ~9 of purified recombinant CTB and hybnd proteins were partially purified from culture supernatants by precipitatitn with sodium , , as described by Lebens et al. /n ~ ., , 1993~. Samples were prepared in Laammli sample buffer with ,e and were loaded both boiled and unboiled onto 15-17% 1 ~ ~: gels containing 0.1% sodium dodecyl sulfate (SDS). This separation provides visualization of bands corresponding to both the monomeric and pentameric forms of the proteins. The ~ ~ , was performed at a constant voltage of 200 V for approximately 1 h.
GBIS were sithsr stainsd with Coomassie Brilliant Blue or L'~ transferrsd to nitrocslluloss membranes for immunoblot analyses. After blocking with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS), the membranes were incubated wilh either the two CTB specific mAb LT39 and CT6, ~ reacting with penlameriC and monomeric CTB respectively, mAb P41D10 againsl gp120, mAb ST1:3 against ST, or mAb 5520 against pre-S(2). Horse-radish peroxidase labelled ~ ~ i IgG ~Jackson) was used as secondary antibody and the membranes were developed with the chromogenic substrate 4chloro-1-naphthol ~Bio-Rad).

CA 0220~130 1997-0~-12 wo 96/16178 1 ~1, ~_ ~17708 The CTB::HIV fusion protein migrated like natiYe CTB in standard r~ !' stained PAGE
in both pentameric and monomeric forms. The inser7ed heterologous gp120 epitope in the hybrid protein was detected in standard immunoblots using mAb F581H3, directed against the V3 loop of gp120. The gp120 epitope was recognized both when the hybrid protein was present in the assembled pentameric form of the protein and after dissociation into morlomers. This also indicated that there was a significant retention of i' ,' ' structure in the assembied pentamers.
The internal CTB::HIV hybrid protein showed an increasing titer with mAb F581H3 proportional to the amount of protein produced and setreted. The peptide was resistant to proteolytic degradation when placed internally in CTB.
The additional HIV epitope - contairling plasmids producing HIV::CTB hybrid proteins, pCB55-65gp309-318, pCB52-58gp3D9 318 and pCB55 64gp309 322, did not produce detectable levels of CTB
hybrid protein. The reason for this is likely due to instability of the resulting proteins and degradation of thepolypeptidechains. Thestructurald'lfferencesbetweenthe proteinswhichwereproducedindetectable levels and the proteins which were not produced at detectable levels were minimal.
The ST::CTB hybrid proteins, encoded by the plasmids pCB5564ST~", (strain 5511 and pCB55 64ST, (strain 557) were both produced in slightly higher amounts than the HIV::CTB and HBV::CTB
chimeras, even in those proteins where the entire ST, 19 aa fragment was inserted between amino acids 55 and B4 of CTB. Comparative expression levels were 15 3D ~glml for pCB55-64ST"" (strain 551) and pCB55-64ST, (strain 557) as compared with 515 I~glml for the HBV::CTB and the HIV::CTB chimeras encoded by the plasmids pCB55 64gp309 318, pCB53-64gp309 318 and pCB53 84gp307 318. Thus, the amino acid composition of the foreign insert is also important. The ST se4uences include several cysteines which may form internal disulfide bridges that potentially stabilize the structure of the inserted peptide and of the entire chimeric protein.
The HIV::CT8 protein encoded by the plasmid pCB56gp309 318 (strain 439), having a straight insertion of the gp120 epitope without any deletions from CTB, was produced at a much higher level than the other proteins ~up to 300 ~glml after 24 hours). When analyzed in SDS-PAGE and immunoblot assays, Ihe protein migrated as a sharp band of the size of pentameric CTB when analyzed unboiied. However, the main part of the protein fell apart when boiled in sample buffer containing SiiS and ~
The HBV::CTB hybrid protein from strain 549, with a peptide from pre-S(2) inserted at the same pnsition, behaved in e~actly the same way. Resistance to proteolytic cleavage is probably dependent on the conformation and accessibdity of the peptide in each individual hybrid protein.
It is worth notmg that proteins can maintain a conformation which resembles CTB enough to migrate as CTB pentamers on a, 1~ ' gel, bind to GM1 gangliosides and be recognized by mAb LT39 anti-CT3, even after they have been cleaved at the position of the inserted peptide. Thus, further analysis of some clones may be necessary to ensure that they are not unduiy susceptible to cleavage.

CA 0220~130 1997-0~-12 Wo 96116178 l ~ 7a~

Analysis of the hyborid protein in GM1-ELISA ASSDYS
ChDiera toxin prDIein was detected using a GM1-ELISA thal has been disclosed in the an Isee Sanchez et al., FEBS ~et~. 241:110-114, 1988 and Svennerholm et al., J. Clin. Mi~ro~iol. 24:585-590, 1886). Microtiter welis were coated overnisht at room temperature with D.3 nmnl GM1-sansboside ISigma, St. Louis, Missouri) in 100 IJI PBS and blocked with 0.1% BSA in PBS ~PBS-BSA) for 30 min at 37C.
After three washinss with PBS, samples were diluted in PBS BSA and incubated for Dne hDur.
RecDmbinant CTB was also used at startin~q at 0.5 ~glml. This and all subsequentincubations were performed at ambient temperature. After subsequent washinss with 0.05% Tween-20 in PBS (PBS T), enher mAb LT39-anti-CTB, or foreiyn epitope specific antibody such as mAb F571H3-anti-Y3 IDOP of gp120, TB mAb P41D10-anti-gp120, ST1:3-anti-STa or 5520-anti-pre-S12), were adbed in PBS with 0.1% BSA and 0.05% Tween 20 (PBS-BSA-T) and incubated fDr one hour. HDrse-radish perDxidase labelled -~ iacksDn LabDratDries) in PBS-BSA-T was added as secDnd antibody to the wells after washings wnh PBS T, and after one hour the plates were washed again and then developed with the chromogenic substrate u, ~ OPD) in citrate buffer with 0.012% H202. The absorbance was measured at 450 nm after 10-20 min. The amounts Df each hybrid protein added in the first well ot the microtiter plate was adjusted tD 10 ~rg as estimated frDm CDDmassie stained I 11. ~: ' gels with recDmbinant CTB as a slandard and were alsD cDnfirmed by the reaction with mAb LT39-anti-CTB.
Titers were defined as the interpDlated serum dilutiDn givin~o a A~so Df 0.2 above backgrDund.
Analysis of the internal gp120 epitope hybrid prDtein in a 6M1-ELISA indicated that 6M1 binding was retained tDgelher with affinity fDr the CTB, s~ '' mAb, LT39. Under the same cDnditions, the CTB::HIV protein also reacted with the anti gpl20 mAb F581H3, indicating that the sp120 epitDpe was cxpDsed Dn the surface of the molecule. The EL)SA assays fDr GM1 expressiDn used the mAb LT39 and included a standard curve with recombinant CTB starting at 0.5 ~g~ml, and reactivity with mAb P41D10, which was defined as the interpDlated serum dilutiDn giving a ~tlro of D.4 above backgrDund.
AntiDenic propertl'es of the gp1ZO epitope in the HIY::CTB proteins The ten amino acid-peptide from sp120 laa 309-318) yielded a relatively strong signal whan placed N-terminally in CTB (see Example 3), or between aa 55 and 64, indicatin~q that the epitope was surface expDsed in these tWD prDteins ~from strain 504 and 407~. When increasins the length Df the inserted epitDpe tD twelve aa laS in strain 586), the reactiDn of the epitDpe with antibody was weaker.
Either the epitope was less well expDsed Dn the surface or it adopted a conformation which the mAb did not recognize.
The HIV::CT8 hybrid proteins were also analyzed in immunobiot wlth the same anti-gp120-mAb.
When run in the unboiied pentameric form, the inserted sp120 epitope was cnly detected in the constructs with substitutions within ammo acid positions 55-64. In all the other HIV::CTB proteins, the epitope could only be detected in the boiled monomenc form. This could imply that the epitope was eYposed in the monomers form, but became more buried or distorted when pentamers were fDrmed. At the same time, CA 0220~130 1997-0~-12 WO96116178 1?~l,_,,9' the reacticn in GMl-ELISA assars indicated that aft&r binding to 6M1, the pentameric forms of sDme of the proteins exposed the inserted HIV epitope enough to he re~ognized by the anti-gp120 monoclonal antibody. It is possibie that the binding to GM1 may therefore facilitate the accessibility of the inserted HIV epitope on the pentamer surface.
The protein from strain 439, which is cleaved after it has been synthesized, was also analy2ed in immunoblot with monoclonal P41D10. The weak monomeric band of around 11 kD, which presumably is still uncieaved, reacts with the mAb, but the majority of the pFotein, which was degraded, showed no anti-gpl2D reactivity.
Antigenio properties of the ST and pre-S(2) epitopes in the ST::CTB and HBV::CTB hybrid proteins When analyzed in GMl-ELISA, the ST, epitopes inserted between amino acids 55 and 64 in CTB
strains 551 and 557 were detected with the ST-specific neutralizing mAb STl:3 Isee Svennerholm et al., 1 ~lin. MicrobioL 24: 585-590, 1986~, and reacted more strongly than when these ST peptides were placed N or C-terminally in CTB ~see Sanchez et al., ~es. MicrobioL 47:971-979, 1990~. In immunoblot assays, the intrachain ST, peptides were only detected in the monomeric protein form. This is consistent with what was saan with most of the HIV::CTB proteins. The same was true for the HBV::CTB protein, where the pre-S~2~ sequence could not be detected with mAb 552D-anti-pre-S~2) ~see Milich et al., J.
Immunol. 137: 2703-2710, 1986) ehher in GM1 ELISA or in the unboiled pentameric form in in~munoblot assays, but were readily obsarved in the boiled monomeric form, These results indicated that when foreign antiyen was inserted into internally in CTB within the 56-64 amino acid region, Ala-64 should be positioned C-terminally to the insert to gct production of the protein. This observation is consistent with other scientists who have reported that Ala 64 is imponant for the stability of pentameric CTB. Likewise, the N-terminal position of Pro 53 was important to the insen since when delated, as in pCB52-5Bgp309-318, the corresponding hybrid protein was not produced at detectable levels.
Based on ELISA assays to detect antibody binding to the toxin or to the foreign antigen, it was determined that the level of foreign epitope reactivity was somewhat iess than the level of reactivity of the CTB epitopes. Since the intrachain fusion protain was resistant to proteolytic cleavage during production in V. cholerse, degradation of the foreign peptide epitope is probably not the explanation for the reduced i ~ r of the foreign epitope in the hybrid protein as comparad with level of CTB
reactivity. It is more likely that the surface density of the insened foreign epitope is low compared to the several different, strong, mainly , CTB epitopes. Thus, this invention also contemplates that multipla copies of the foreign antigen will by useful for promoting an increased immune response.
It is additionally contemplated that the length of the insert will aiso affect stability. Thus, those skilled in the art should also contemplate varying the length of the insen in those substitutions where stability is potentially a problem.

wo 96/16178 p~ "~, _ 5 Cl ~708 The ELISA screening melhods in combination with, 1~ ' ' gel ' , ' staining and immunohlot analysis serve to guide those skilled in Ihe art to prepare appropriate ccmbinations and to screen these combinations for expressed protein. Importantly, the results provided here inditated that CTB
can be modified without ~oss of synthesis Df the desi~ed prDtein.
S

' " Protocol for Optfmizing Serum Antibody Levels Female C57B11û mice were immuni2ed , 'I~ (i.p.~ with three doses of 10 ~9 of theCTB::HIV hybrid protein partially purified from JS1559 supernatant. The first dose was given with Freund's complete adjuvant ~OIFC0 Laboratories, Detroit, Micbigan) and Ihe subsequent doses with Freund's incomplete adjuvant. As negatiYe controls, mice were immunized using the same scherne with a CTB hybrid protein with an irrelevant foreiûn epitope or with Freund's adjuvant alone. Serum samples were collected before the first dose and seven days atter the second and third dose. Sera were analy2ed in ELISA, using either CTB (bound to GM1 in GM1-coated microwells using the methods of Example 6~ or recombinant gp1Z0 (Bolmstedt et aL, J. Gea Virol. 73:3D99-3105, 1992~ as antigen.
A strong serum antibody response to the CTB moiety was detected in all mice immunized with either of two hybrid proteins. There was also a significant serum antibody response against the HIV moiety in the majorhty of the mice tested. The mice receiving only Freubd~s adluvant gave no antibody responses against either GTB or gp120.
EXAMPLE û
Protocol for Producing Vaginnl Immune response Femals mice were given 10 mg of progesterone ' `~ 10 and 3 days before the firstimmunization and were therl treated once a week with progesterone. Groups of mice were immunized either four times intravaginally (at 1-2 week intervals between each dose~ or wlth three ;..;i, ' doses followed by an intravaginal dose. Each intravaginal immunization oose ccnsisted of ca. 0.5 mg CTB-peptide conjugate (see Example 11. Each dose was estimated IO contain approximately 0.4 mg CTB and 0.1 mg A8-VOIV peptide and additionally included 5 mcg cholera toxin as extra adjuvant and each i dose contained one third this mount.

WO96/16178 }~-,~. .

Immunizrltion tu produce Mucosol Antibody The CTB fusion proteins are isolated from bacterial culture supernatants usinS the expression system described by Lebens, et al. (8ioTechnologv, supre). The fusion proteins are isolated by GM1 affinity purification as disciosed by Tayot, et al. I~uro,o. J. ~iochem. 113: 249-2581 and dialy2ed ayainst PBS. The specific protein, ' of the samples used for immunizati3n are determine~ by ELISA using a CTB
standard.
~n an experimentai model, male monkeys are immunized with 40-250 ~9 of CTB equivalents of protein in each immunization. In total, 3-5 injections were give every 34 weeks, the first three with the antigen suspended in complete Freunb adjuvant and subsequent immunizations in incomplete adjuvant. Sera prepared from the bleedings taken before the start of the immunizations and those taken 2 weeks after the third or fourth vaccination were assayed for spec'rfic antibodies against CTB and the foreign antigen by ELISA. In addition, the ability of the sera to neutralee the pathogen was assayed in the infant mouse test. Serum from immunked monkeys in a dilution of 115 was mixed with an equal volume of patho6en and introduced into the mouths of infant mice. Mice were monnored for the presence of the disease over time.
For human immunrzation it is contemplated that the chimeric protein will be administered once or on repeated occasions by the oral, rectal, vaginal or nasal routes using between 0.1-2 mg of construct for single immunizations and between 0.01 0.2 mQ for repeated The protein may be given either in a liquid form or dispersed in an inert gel with estimated volumes of inoculum of between 0.1-1 ml for injection, 0.5 - 2 ml for nasal and between 310 ml for rectal or vaginal immunization. It is contemplated that the oral immunization is likely to be given toyether in a I ' ' acceptable buffer containing about 25-200 ml liquid containing about 2 Srams of sodium bicarbonate or equivalent acid buffering agent.

Experinnentol Screening for IgA snd IgG Mucosol Antibody Ssrum, mesenteric Iymphnodes, spleen, cervix, vagina, small intestine, colon and rectum were collected from mice sacrificed (and perfused with heparin containing buffer) one week after the last immunrzation dose with the preparation of Example 1 usinS the immunization protocol of Example 8. The organs were frozen and then extracted with 2Y~ (WIV) of saponin using ths PERFEXT method ~Quidin3, et al. J. ~lln. Invest. 88~ 143-148, 1991). The saponin extracts were tested for IgA and 196 anti-CTB and anti-VDlV antibodies by ELISA. ELISA plates were coated with GM1 10.3 nmollml) or A8 ~1DIV peptide 11 //6lml) respectively, CA 02205130 1997-0~-12 wo 96/~6178 In this example, immuneed mice were of the C571B1 strain and were ~ lD weeks old at the onset of ~ The immunization protocol was I my ' ! ,. . _ ' acetate (Depoprovera; UpJohn Company, Kalamazoo, Mll ' `~ ten and three days before the first immunization and then once weekly through the course of the immunization period. Immunizations were given by the indicated routes with an interval of two weeks between the first and second dose and then one week between the following doses.

CA 0220Sil30 1997-OSi-12 WO96/16178 P~, .v; . ,.

TABLE 2. IMMUNE RESPONSES IO THE FEMALE GENITAL TRACT AND SERUM OF MICEIMMUNIZED 8Y VARIOUS ROUTES WITH A CHEMICALLY PREPARED CONJUGATE BET~VEEO

ELISA antibody tiler to A8-VDIV
Immunization Vagina Cervix Fallopian Serum uteri tubes IgA IgG IgA IgG IgA IgG IgA IgG
None <.5 <.5 ~ <.5 <.5 <.5 <.5 <.5 <.5 10 Intravaginal x 4 290 .5 920 .5 50 .5 3 43 Oral x 4 <.5 <.5 <.5 <.5 22 <.5 <.5 3 ' x 3 + 14 99 40 130 38 nd 2 3500 intravaginal x 1 Oral x 4 + intravagirial x 2 <.5 2 <.5 <.5 <.5 <.5 <.5 This table indicates that vv'lth repeated intravaginal immunization or a combination of repeated ' priming f ollowed by a vaginal booster immunization, the CTB A8-VDIV conjugate could induce substantial specific genital mucosal IgA antibody formation to the Chbmvdl~ tra~hom~tis A8 YOIV antigen in test samples comprising the vaginal, cervical and Fallopian tube mucosae. After multiple ' , the IgA response was also associated with a substantial IgG response iri both the genital tract tissues and in serum.
of the fureiDn ser~uences in the CTB hybrid proteins To determine the ability of the internally inserted peptides to induce immune responses, groups of mice of stranl c57all8 were immunized i"i" i 'll (i.p.) with either the internal HIV::CTB hybrid proteins from strain 407, 460 or 586, the N terminal HIV::CTB hybrid protein from strain 504, or the two ST::CTB chimeric proteins, w'nh Freund's adjuvant. All mice responded with high titered serum anti CTB-lgA , As noted above, the ten amino acid HIV epitope induced serum IgG-responses against gp120 when placed between amino acids 55 and 64 of CTB. A weak response against gpl20 was also induced by the gpl20 moiety in the mice immunized with the internal HIV::CTB hybrids from strain 460 (ten gpl20 amino acids between 53 and 64) and 588 ~twelve gpl20 amino acids between 53 and 64) and by the N

CA 0220~130 1997-0~-12 W096/16178 I~, ~._. ,.

terminally placed 3p12D-epitope in the protein frcm strain 504. In fact, all thes2 proteins showed immunogenic propertles sirnilar to the previously bescribed HIV::CT3 protein.
The ST becapeptide in the ST::CTB hybrid protein from strain 551 induced anti-ST-serum - antibodies of relatively high titer in some of the animals, even though the immunological reactivity of the inserted peplide was modest jD virt~. The ST::CTB protein from strain 557, which represented the entire ST, peptide, was also immunogenic in terms of the ST moiety, but induced lower tners of antibody than the decapeptide in the protein from strain 551, The HBV::CTB chimenc protein from strain 395 was 3iven either i.p. or perorally ~p.o.) wrth cholera toxin ~cn as adjuvant to erther BALBlc or C57B116 mice. Hish serum-anti-CTB titers where obtained in both p.o. and i.p. immunized mice, whereas the most si3nificant levels of anti pre-S(2) ab were induced with i.p. immunieations usln3 CT is adjuvant. Table 3 iliustrates the various immuneation protocDls and their results.

,, CA 0220~130 1997-0~-12 WO 96/16178 .

Table 3. Serum IgG titcrs aRer i with the HBV: CTB hybrid protein.
Mouse strain CT- RouteD anti-CTB' anti-pre-S~2)' 2 doses 3 doses 2 doses 3 doses C57BU6 (H-2b) - p.o. ~ 53 000- 23 400 0 0 S BALB/c (H-2~) - p.o. 0 2 400 0 0 C57BU6 + p.o. 102 400 256 400 0 0 BALB/c + p.o. 4 000 38 800 0 0 1û C57BL/3 - i.p. =170 000 546 000 0 1 060 BALBk - i.p. 32 000 153 600 0 0 C~7BU6 ~ i.p. 10 682 000 8 500 3 300 BALB/c + i.p. 10~ 461 000 6 400 3 600 a) Mice were immunized with or without 5 pg (p.o.) or 1.5 pg (i.p.) cholera to:cin (CT) as adjuvant.
b) The animals were given does of 30 pg (p.o.) or 7.5 pg (i.p.) of the HBV::CTB hybnd protein from strain 395 on day 0, 14 and 24.
c) Recombinant LTB bound to GM1 was used as antigen for anti-CTB-lgG .'~:
d) A synthetic pepOde containing aa residues 133-153 from pre-S(2) (Schbdel et al., 1990) was used as antigen for anti-preS(2)-lgG ,' e) Sera were collected on day 7 after does 2 and 3 and sera from mice irom the same group (2 or 3 mice per group were pooled before analyses. Mean reciprocal senum dilutions yielding an A~G2>3 x the A~92 of preimmune sera are indicated as titens.

CA 0220~130 1997-0~-12 WO 96/16178 F~,l.. _. .

While particular embDbiments of the invention haYe been descnbed in detail, it will be a~parent to those skilled in the art that these embobiments are exemplary rathùr than limitins, and the true scDpe of the inYention is that defined In the followin~o claims.

CA 0220~130 1997-0~-12

6~78 r~

SEQUENCE LISTING
( 1 ) GENERAL INFORMATION
~i) APPLICANT: Holmgren, Jan Lebens, Michael ..
ii) TITLE OF THE INVENTION: IMMUNOGENS FOR STIMULATING MUCOSAL
IMMUNITY
iii) NUMBER OF SEQUENCES: 51 ( iv) CORRESPONDENCE ADDRESS:
(A~ ~nnRFC:CRR: Knobbe, Martens, Olson ana Bear (B) STREET: 620 Newport Center Drive 16th Floor (C) CIT-~: NewDort Beach ( D ) STATE: CA
( E ) COI~NTRY: USA
(F) ZIP: 92660 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM ~ ,~ t;hle (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii~ ATTORNEY/AGENT INFORMATION:
(A) NAME: Kaiser, AnneMarie (B) REGISTRATION NUMBER: 37,649 (C) REFERENCE/DOCKET NUMBER: HOLMG. 00lVPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPBONE: 619-235-8550 (B) TELEFAX: 619-235-0176 ( C ) TELEX:
( 2 ) INFORMATION FOR SEQ ID NO :1:
(i) SEQUENCE CBARACTERISTICS:
(A) LENGTH: 17 amino acids _ .
(B) TYPE: amino acids ( C ) STR ~NnR17NR .c .~: S ingle (D) TOPOLOGY: li~ear ii) MOLECULE TYPE: peptide i i i ) ~. Y ~U L r~ CAL: NO
. iv) ANTISENSE: NO
v) FRAGMENT TYPE: internal ,vi ) ORIGINAL SOURCE:

CA 0220~130 1997-0~-12 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:l: ~
Phe Asp Val Thr Thr~ Leu Asn Pro Thr Ile Ala Gly Ala Gly As~ Val 5 ~ 10 15 Lys , ~2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A ) LENGTH: 42 amino acids ( B ) TYPE: amino acids (C) sT~ANnRr~NR.cc single ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: pept,ld ( i i i ) Il Y ~S~ L 1 CAL: NO
( iv) ANTISENSE: NO
~v) FRAGMENT TYpE: in~er~al (vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala 5 l0 lS
Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr T~r Leu Asn Pro Thr Ile Ala Gly Ala Gly Asp Val Lys 3 5 ~ 4 0 (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CE:ARACTERISTICS:
(A LENGTU: Sl base pairs (B TYPE: nucleic acid - ~ ~
(C sTT~ANTl~nNF~c single ~-(D TOPOLOGY: linear (ii) MOLECULE TYPE: c3~A
(iii) }~YPOTHETICAL ~NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: ~:
GGGGCGCGCT lL~ ~A TrrAAAr.f~AT l-.AA~r.ATArr CTGAGGATTG C Sl ( 2 ) INFORMATION FOR SEQ ID NO: 4:
(i) 3EQUENCE ~ A~ArTR~T~TIcs: ~ -(A LENGTH: 56 ~ase pairs (B,l TYPE: ~ucleic acid . ~
(C sTR~Nl~Rn~TRcc: single . ~ - -(D TOPOLOGY: linear ii) MOLECUL E: cDNA
E TYP

iv) ANTISENSE: NO
v ) FRAGMENT TYPE:
, vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

CA 0220~130 1997-0~-12 wo 96/16178 . ~1,~.~. ..

GGGGCGCGCC CCGGACCACG CTGGATACTA CCTGGTACCT CTACTTGA~A AGTTGC 56 (2~ INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGT~: 125 base pairs (B) TYPE: nucleic acid (C) s~ Nn~nNFcs: double (D) TOPOLOGY: linear ii) MOLECULE TYPE: cDNA
iii) ~Y~UlS~LlCAL: NO
iv) ANTISENSE: NO _ =
v) FRAGMENT TYPE:
vi ) ORIGINAL SOURCE:
( ix) FEATURE:
(A) NAME/~CEY: Coding Sequence ~ ~
(B) LOCATION: 33 125 ~ :
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Me~ Val Lys Ile Ile Phe Val TTT TTC TTA TCA TCA TTT TCA TAT GCA AAT GAT GAT
Phe Phe Leu Ser Ser Phe Ser Tyr Ala Asn Asp Asp Lvs Leu Gly Ala 101 CCT GAT TCT AGA GCG ATG AGT AAT
Pro Asp Ser Arg Ala Met Ser Asn ~ 125 ( 2 ) INFORMATION FOR SEQ ID NO: 6: ~ --~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2S amino acids -- -~B) TYPE: amino acids (C) STR~NnFnNRCC siugle (D) TOPOLOGY: li~ear ~=
ii ) MOLECULE TYPE: I~eptide i i i ) ~ ~ ~u ~ CAL: NO
:iv) ANTISENSE: NO
, v) FRAGMENT TYPE: internal ,vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6- ~ -Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala 5 ~ 10 15 Thr Thr Gly Tyr Leu Lys Gly Asn Ser ( 2 ) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE ~ h~ 'F~T cTICS:
(A) LENGT~: lS base pairs (B) TYPE: nucleic acid (C) s~ Nn~nN~C~ singIe CA 02205130 1997-0~-12 wo 96/16178 1 ~1/~,~ . , .
~7-(D) TOPOLOGY: linear ;~ ~
(ii) MOLECULE TYPE: cDNA
(ii' ) ~Y~L)l~llCAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
- (~.ri ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CCGGCTAGCG CAGCT . ~ 15 (2) .INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHAR~ACTERISTICS:
(A) LENGTH: lS base pairs ~ =
(B) TYPE: nucleic acid (C1 STRANnpnNpqs: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( i i i ) II Y ~O l ~ ~ CAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

( 2 ) INFORMATION FOR SEQ ID NO: 9:
( i ) S EQUENCE CHARACTER I STI CS:
(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) sTR~NnPnNP.qS: single ( D ) TOPOLOGY: l inear ii ) MOLECULE TYPE: cDNA
iii) ~LY~ul~;llC~L: NO
iv) ANTISENSE: NO
v) FRAGMENT TYPE:
vi ) OR~GINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - = ~
CCTATTCAGC ~ c~,~G GCGCGCTTTT GTTGCTCCTC AAAAT 4S
(2) INFORMATION FOR SEQ ID NO:lO:
(i) SEQUENOE ~'TTARA(-Ts'RT.qTICS:
(A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) sTRANnpnNpcq: sin~le (D) TOPOLOGY: linear-(ii) MOLECULE TYPE: cDNA
( i i i ) ~ Y ~ CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

CA 0220~130 1997-0~-12 wo 96/16178 r~l. ~.. . ..

ATTTTGACCA GCAACAI~AAG ~ ,CC----~ ACCACGCTGA ATAGGAGCT 4 9 ( 2 ) INFORMATION FOR SEQ ID NO
~i) SEQUENCE CHARACTERISTICS
(A LENGT~}: 3 9 base pairs ~B TYPE: nucleic acid : .
(C STRANDEDNESS: sinale (D l TOPOLOGY: linear ii) MOLECULE TYPE: cDNA
~iii) ~Y~u~ lCAL: NO
iv) ANTISENSE: NO
,v) FRAGMENT TYPE:
vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
CCTCAAATTC A~ ,lc~ GGGGCGCGCT TTTGTTAAC = . ~ 39 (2) INFORMATION FOR SEQ ID NO:12:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STR~ T)NF~cc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( i i i ) ~ y ~J ~ cAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GTTAACTTTA GCCGCGCCCC GGACCACGCT G}~ATTTGAGG AGCT . 44 ( 2 ) INFORMATION FOR SEQ ID NO :13:
(i) SEQUENCE CHARACTERISTICS:
(A LENGTH: 42 amino acids (B TYPE: amino acids (C I STR~Nn~nN~c~ single (D TOPOLOGY: linear _ (ii) MOLECULE TYPE: peptide ( i i i ) II Y ~ l CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal :
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
la Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu~Gly Ala Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr Leu Asn 20 25 - 3 0 ~:
Pro Thr Ile Ala Gly Ala Gly Asp Val Lys (2) INFORMATION FOR SEQ ID NO-14:
(i) SEQUENCE CHARACTERISTICS:

CA 0220~130 1997-0~-12 WO96/16178 .~1, ~ ,.

(A) LENGTP: 43 amino acids (B) TYPE amino acids -(C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE: peptide (iii) liY~uL~llCAL: NO
( iv) ANTISENSE NO
(v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE
(xi) SEQUENCE DESCRIPTION: SEQ ID NO 14 ys Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly S -== 10 15 Ala Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr leu 20 ~ 25 - 30 -Asn Pro Thr Ile Ala Gly Ala Gly Asp Val Lys 35 ~ 40 ( 2 ) INFORMATION FOR SEQ ID NO 15 (i) SEQUENCE r~ R~TFRT~TIcs ~ ~ =
(A) LENGTU 43 amino acids (B) TYPE amino acLds (C~ STR~NnFnNFc~ single -----(D) TOPOLOGY: linear ii) MOLECULE TYPE ~eptide iii) ~YI~u~ CAL NO
iv) ANTISENSE NO
v) FRAGMENT TYPE: interrLal vi ) ORIGINAL SOaRCE
(xi) SEQUENCE DESCRIPTION SEQ ID NO 15:
la Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala 1 0 , 1 5 Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr Leu Asn 20 25 ~ 30 ~
Pro Thr Ile Ala Gly Ala Gly Asp Val Lys Cys (2) INPORMATION FOR SEQ ID NO 16 (i) SEQUENCE ~r~RA~-TFRTcTICs (Al LENGT-u~: 33 base pairs (B TYPE nucleic acid ~ _ (Cl STR~NnFnNPCS: single ~ ~-(D ~ TOPOLOGY: linear - --i i ) MOLECULE TYPE cDNA
iii) Ily~uLrL~ llcAL NO
iv) ANTISENSE: NO
v) FRAGMENT TYPE
vi ) ORIGINAL SOURCE
(xi) SEQ~ENCE DESCRIPTION SEQ ID NO:16 ~,Gr.~ ATc ~ , lL~ ~L~AT GAC ~ 33 ( 2 ) INFORMAI'ION FOR SEQ ID NO :1~7 -(i) SEQUENCE rUAR~'TFRT~TICS

CA 0220~130 1997-0~-12 wo 96/16178 . ~1,. , 4c-(A~ LENGTH 36 base pairs (S) TYPE: nucleic acid (C) STRANnFnNRcq single (D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA
( i i i ) ~ Y ~ CAL NO
( iv) ANTISENSE NO
(v) FRAGMENT TYPE
(vi ) ORIGINAL SOURCE
(xi~ SEQUENCE DESCRIPTION SEQ ID NO 17 GGGGGAGATC TCTGA~ATGA G~l~l l~A~A ATTATC 36 (2) INFORMATION FOR SEQ ID NO 18:
(1) SEQUENCE CHARACTERISTICS
(A) LENGTH 33 })ase pairs ( 8) TYPE nucleic acid (C) ST~NnFnNRqc single (D) TOPOLOGY linear ) MOLECULE TYPE: cDNA
iii) liYs~ llcAL: NO
iv) ANTISENSE NO
v) FRAGMENT TYPE:
vi) ORIGINAL SOURCE
(xi) SEQUENCE DESCRIPTION SEQ ID NO: 18 r.r.r,r.r.~r.ATC Tçrr~r~Arr GTTATGATGT CGG 33 ( 2 ) INFORMATION FOR SEQ ID NO 19:
HARACTERISTICS
(A) LENGTH: 31 base ~airs (B) TYPE: nucleic acid (C) sTRANnEnNFcq single ( D ) TOPOLOGY 1 inear ii) MOLECULE TYPE cDNA
iii) ~Y~ b;llCAL: NO
iv) ANTISENSE NO
v) FRAGMENT TYPE
vi ) ORIGINAL SOURCE
(xi) SEQUENCE DESCRIPTION SEQ ID NO:19:
çGr~rr~r~ Tc rrr.~ArçcrA GCAAAGACGT A ~ 31 (2) INFORMATION FOR SEQ ID NO 20 ( i ) SEQUENCE CE~RACTERISTICS:
(A LENGT~ 28 J~ase ~airs (S~ TYPE: nucleic acid ( C l sTRANnRnNRcq singIe ~=
(D I TOPOLOGY: linear ~ _ ~ ii) MOLECULE TYPE cDNA
l iii ) ~Y ~ CAL: NO
iv) ANTISENSE: NO
v) FRAGMENT TYPE
vi ) ORIGINAI SOURCE

CA 0220~130 1997-0~-12 wo 96116178 1 ~1, ~ , (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
ATTCAGCGTG ~ ~ TGCTTTTG ~ - 2 8 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS: ~ :
(A) LENGTH: 36 base pairs - ~ -(B) TYPE: nucleic acid =
(C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ ~ --(ii) MOLECULE TYPE: cDNA
( i i i ) ~ Y ~ CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOlIRCE: ~ --(xi) SEQIJENCE DESCRIPTION: SEQ ID NO:21:
rrr.~r.AAAAr GACGACCCGG ACCACGCTGA ATAGCT =~ 36 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single :
( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: cDNA
( iii ) ~r ~ ;l lCAL: NO
( iv) ANTISENSE: NO - -(v) FRAGMENT TYPE: . _- :
(vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GCGTAGCGGT ArrArrArTC Tr~ rrr~Ar TATTGCTGGA GCTGGCTGGC CAGCACG 51 (2) INFORMATION FOR SEQ ID NO:23:
( i ) S EQUENCE rTTA ~ 2, rT~R T .':TI CS:
(A) LENGT~: 58 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~-~C single (D) TOPOLOGY: linear ~:
ii) MOLECULE TYPE: cDNA
iii) H~Olsl~llCAL: NO
iv) ANTISENSE: NO
v) FRAGMENT TYPE:
vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CL:~l~ ,C rArrrz~r.rTC CAGCAATAGT TGGGTTCAGA ~ ~lAC CGCTACGC 58 (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE r~ARArT~:RTcTIcs (A) LENGT~: 61 ba6e pairs (B) TYPE: nucleic acid - -(C) STRANn~nN~cc single =-CA 0220~130 1997-0~-12 wo 96/16178 1 ( D ) TOPOLOGY 1 inear MOLECULE TYPE: CDNA
(iii) HYPOTHETICAL: NO
(iV) ANTISENSE: NO
(V) FRAGMENT TYPE
(Vi) ORIGINAL SOURCE:
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CCATTTGATA TTACCACTTT AAATCCAACA A~ ~11.i(.7L~i CTGGTGATGT TAAACCCGGG 60 (2) INFORMATION FOR SEQ ID NO:25:
( i ) ;,EQUENCE CEARACTERISTICS
(A LENGTH 75 base pairs (B TYPE nucleic acid (C STR~NnFnN~C: single (D TOPOLOGY linear - :
(ii) MOLECULE TYPE CDNA
(iii) ~Y~V~11CAL NO
(iV) ANTISENSE: NO
(V) FRAGMENT TYPE
(Vi ) ORIGINAL SOURCE
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

ATATCAAATG GAGCT . 75 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE ~F~A~T~T.~TICS:
(Al LENGTH 76 base pairs (B TYPE nucleic acid (C I STRANDEDNESS single (D TOPOLOGY linear ) MOLECULE TYPE CDNA
(iii) ~Y~VL~I~L1CAL. NO
(iV) ANTISENSE NO
(V) FRAGMENT TYPE
(Vi) ORIGINAL SOURCE:
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
~V--1~:1L1AA ATATTTGGGA l~=LL1~AT ~LLLLLL~=LA CATTAG

(2) INFORMATION FOR SEQ ID NO:27:
( i ) ~EQUENCE CHARACTERISTICS
(Al LENGTH a4 base pairs (B TYPE nucleic acid ( C ST~ ~NnFnN~ c .5 - s in~le (D TOPOLOGY: lirear ) MOLECULE TYPE CDNA
) ~Y~VLrl~ L1CAL: NO
V) ANTISENSE NO
I V) FRAGMENT TYPE
Vi ) ORIGINAL SOURCE:

wo 96/16178 ~1. _. . ..
~3-~xi) SEQUENCE DESCRIPTION: SEQ ID-NO:27:- ~
~TAr.AArTAT TArrAAAATA ACCAGTGGTA GCACCTAATG TACATTTAAC ATCAAAACGA 60 TCCCAAATAT TTAAAGCGGG AGCT . ~- 8 4 (2) INFORMATION FOR SEQ ID NO:28: ~
(i) ~EQUENCE C~ARACTERISTIC5: ~~ ~ -(A LENGTH: ll amino acids (B TYPE: amino acids (C STRANDEDNESS: single (D~ TOPOLOGY: linear .
(ii) MOLECULE TYPE: De~tide (iii) HYPOTHETICAL: NO
( iv~ ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala ~ :

(2) INFORMATION FOR SEQ ID NO:29: . ==~
(i) SEQUENCE r~ ArT~l7TcTIcs (A) LENGTH: 10 amino acids (B) TYPE: amino acids (C) sTRANnRn-~R.c.c single (D) TOPOLOGY: lirear :~
(ii) MOLECULE TYPE: peptide (iii) ~Y~-JLli llCAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal (vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Ile Gln Arg Gly Pro Gly Arg Ala Phe Val (2) INFORMATION FOR SEQ ID NO:30:
( i ) SEQUENCE CHARACTERISTICS:
(A LENGTH: 10 amino acids (B TYPE: amino acids ( C I ST~ A~nRnNR c .c: s ingle --(D TOPOLOGY: linear ii ) MOLECULE TYPE: peptide , i i i ) ~ Y ~ l CAL: NO
iv) ANTISENSE: NO
v) FRAGMENT TYPE: internal vi ) ORIGINAL SOURCE: ~ -(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Cys Ala Glu Leu Cys Cys Asn Pro Ala Cys ~2) INFORMATION FOR SEQ ID NO:31: :

CA 0220~130 1997-0~-12 WO 96/16178 I'~
44' (i) SEQUENCE r~P.rT~:~T.qTICS:
(A LENGTH: l9 amino ~cids (B ' TYPE: amino acids (Cl STRANDEDNESS: single (D TOPOLOGY: linear (ii) MOLECULE TYPE: peptide = _ _ =
( iii ) HYPOTHETICAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal ~:
(vi) ORIGINAL SOURCE:
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 3 l:
Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys C~ys Asn Pro Ala Cys Thr Gly Cys Tyr (2) INFORMATION FOR SEQ ID NO:32: =.
( i ) S EQUENCE rT~ rTF:~ T QTI cs:
(A) LENGTH 5l base pairs (B) TYPE: nucleic acid ( C ) STT~ ANnPnNR Q S: s ingl e (D) TOPOLOGY: linear ( ii ) MOLECUEE TYPE: cDNA
( i i i ) ~ Y ~ CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
~vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: .
GGGGCGCGCT T~ ~A Trr-~AAr~r~T GAAGGATACC CTGAGGATTG C = 51 (2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ii) MOLECULE TYPE: cDNA
i i i ) ~ Y ~ ~ I l CP L: NO
iv) ANTISENSE: NO
v ) FRAGMENT TYPE:
vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: :
GGGGCGCGCC rrr,r.~rr~rr. cTr.r.~T~rT~ CCTGGTACCT C TGAAA
TTCTTAAAAG TACT l~GTTGC~CCA 6 O
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE r~ rT~TQTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STl~AND~nN~:Qs single (D) TOPOLOGY: lin~ear CA 0220~130 1997-0~-12 WO96/16178 ~1 MOLECULE TYPE CDNA
tiii~ ~Y~UL~LLCAL NO
(iV) ANTISENSE: NO
(V) FRAGMENT TYP E
(Vi ) ORL-GINAL SUURCE
(Xi) SEQUENCE DESCRIPTION SEQ ID NO 34 GGGGCGCGCT TTCGTTCATA TAGATTCACA AAaAAAAGCG AT 42 (2) INFURMATION FOR SEQ ID NU 35 ( i ) SEQUENCE r~AR ArTFR T CTICS
(A) LENGTH 45 base pairs (B) TYPE nucleic acid :
(C) STRANnFnN~SS single (D) TUPULOGY linear ( ii ) MOLECULE TYPE CDNA
( i i i ) }~Y POTHET I CAL NO
(iV) ANTISENSE NO
(V) FRAGMENT TYPE ~-(Vi) ORIGINAL SOURCE: - ~=
(Xi) SEQUENCE DESCRIP~ION: SEQ ID NO:35: ~ ~
GGGGCGCGCC CCGGACCACG CTGGATTTGA CTACCTGGTA CTTCT_ 45 ( 2 ) INFORMATION FOR SEQ ID NO: 3 6 EQUENCE r~TTARArTF~TSTICS
(A LENGTH 3 9 base pairs (B TYPE nucleic acid (Cl sTRANnFnNFcc single (D I TOPOLOGY linear ) MOLECULE TYPE CDNA _ - -1 ) ~ Y ~.~ L S1 L 1 CAL NO
VI ANTISENSE NO
V) FRAGMENT TYPE
Vi ) ORIGINAL SOURCE:
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

(2) INFORMATION FUR SEQ ID NO:37:
( i ) SEQUENCE CHARACTERISTICS
(A LENGTH 48 base pairs (B TYPE nucleic acid (C STRANnEnNFCS singlé
(D I TOPOLOGY linear (ii) MOLECULE TYPE CDNA
(iii) ~Y~JL I~;L1CAL NO
(iV) AWTISENSE: NO
( V ) FRAGMENT TYPE
(Vi) ORIGINAL SUURCE:
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
7e~ rrr,r.ArrArr. CTGGATTACT TCTACTTGAA AAGTTFCA - 48 CA 0220~130 1997-0~-12 ( 2 ) INFORMATION FOR SEQ ID NO: 3 8:
( i ~ .8EQUENCE CHARACTERISTICS: :~ :
(A1 LENGTH: 20 base ~airs (B~ TYPE: nuclelc acid (C sTRANnFnNTc~ single (D TOPOLOGY: linear ii~ MOLECULE TYPE: cDNA
iii~ HYPOTHETICAL: NO
iv) `ANTISENSE: NO
v) FRAGMENT TYPE:
vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
CAATCCAGCG ~ 20 ( 2 ) INFORMATION FOR SEQ ID NO: 3 9:
(i) SEQIJENCE CHARACTERISTICS:
(A) LENGT~:: 28 base pairs (B) TYPE: nucleic `acid (C~ ST~ NnRT)NRcc: single ~ :
(D~ TOPOLOGY: linear ~ ~ =
(ii~ MOLECULE TYPE: cDNA
( i i i ) ~ Y ~ l CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

(2) INFORMATION POR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A LENGT~I: 26 base pairs (B TYPE: nucleic acid (C1 STRANDEDNESS: single ::
(D TOPOLOGY: linear ii) MOLEC~LE TYPE: cDNA
:iii) ~Y~Lrl~:llCAL: NO
:iv) ANTISENSE: NO
v) FRAGMENT TYPE:
vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: == =
CAATCCGGAT CC~LGCGTGGT CCGGGG ~ 2 6 (2) INFORMATION E'OR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRI~NnRT)NR.'~C: single (D) TOPOLOGY: linear : ~ =
(ii) MOLECULE TYPE: cDNA
( iii ) ~lY l:'OL~'l lCAL: NO

CA 0220~130 1997-0~-12 W096/16178 ~1,. _. ..

iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
, ACCACGCTGG ~TCCGGATTG GTAC . 3 4 (2) INFORMATION FOR SEQ ID NO:42: : -( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - ==
(ii) MOLECULE TYPE: cDNA
(iii) dY~ul~;LlcAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL 'SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: ~ : ~
~T TQTAACGAAA GCGCGCCCCG GACCACGCTG GATCAAAAAT GCA 5 3 (2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE ~ TFI~T.cTICS:
~A) LENGT'd: 48 base pairs (B) TYPE: nucleic acid ( C ~ STl? ~Nl~FnNE c~c: s ingle (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
( i i i ) d Y ~U l d~ CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
TTTTTGATCC AG~ ~ GGGGCGCGCT TTCGTTACAA TAGGAAAA 48 (2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A LENGTH: 3 9 base pairs (B l TYPE: nucleic acid .
( C ST~ ~NnFnNF.c c: s ingle (D I TOPOLOGY: linear : i i ) MOLECULE TYPE: cDN~
:iii) dY~ULd~llCAL: NO
: iv) ANTISENSE: NO
v) FRAGMENT TYPE:
vi) ORIGINAL SOIJRCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

CAGGTAGTTG CGCTGAATTG TGTTGTAATC CTGCATGCG ~ ~ 3 9 (2) INFORMATION FOR SEQ ID NO:45:

CA 0220~130 1997-0~-12 wo 96/16178 ~_,,~.. ..

(i) .SEQUENCE CHARACTERISTICS~
(A LENGTH: 43 base Dairs (B TYPE: nucleic acid ( C ~ sTR~NnFnNFqc single (D I TOPOLOGY: linear ii) MOLECULE TYPE: ~DNA
CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi ) ORIGINAL SOURCE:
xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

(2) INFORMATION FOR SEQ ID NO:46:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C) sTR~NnFnNFqs single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) ~Y~ul~llcAL: NO
(iv~ ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

GTTACG ~ : 6 6 (2) INFORMATION FOR SEQ ID NO:4Z:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 70 base pairs (B) TYPE: nucleic acid (C) sTR~NnFn~Fqq single (D) TOPOLOGY: linear ii ) MOLECULE TYPE: cDNA
iii) ~Y~Ul~l~;llCAL: NO
iv) ANTISENSE: NO
v) FRAGMENT TYPE:
vi ) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ -ID NO:47:
CGTAACATCC AGTGC~TGCA GGATTACAAC ACAATTCACl~ GCAGTAATTG CTGCTATTAC 60 .TACCTGGTAC . . . 70 (2) INFORMATION FOR SEQ ID NO 4a:
(i) SEQUENCE C~ARACTERISTICS: .__ _ (A) LENGTH: 57 base Dairs (B) TYPE: nucleic acid = : ~ =
(C) STR~NnFnNFcq single (D) TOPOLOGY: li~ear =
(ii) MOLECULE TYPE: cDNA
(iii) IIY~Ul~ llCAL: NO

CA 0220~130 1997-0~-12 ( iv) ANTISENSE: NO
(~) FRAGMENT TYPE:
(viJ ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: ~ ~
GGGCCTCGAG TCCGAGGCCT ATACTTTCCG GCGATTGAAA GGATGAAGGA TACCCTG ~7 (2) INFORMATION FOR SEQ ID NO:49:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nuclelc acld (C) STR~Nn~nNR.qS: single (D) TOPOLOGY: linear (ii) MOhECULE TYPE: cDNA
(iii) nY~1n~LlCAL: NO
(iv) ANTISENSE: NO :
(v) FRAGMENT TYPE:
(vi ) ORIGINAL SOURCE:
(x~ ) SEQUENCE DESCRIPTION: SEQ ID NO:49:
GGGCTCGAGG GTCACTACCT GGTACCTCTA CTTGAAA~GT TG 42 (2~ INFORMATION FOR SEQ ID NO:50:
( i ) S EQUENCE rT~ ~ R ~rT~R T sTI CS:
(A LENGTH: 42 base pairs (B TYPE: nucleic acid (Cl STR~Nn~nNF:qq: single (D I TOPOLOGY: linear ( ii ) MOLECULE TYPE: cDNA
(iii) nY~ n~ CAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:~0.

(2) INFORMATION FOR SEQ ID NO:~l:
( i ) SEQUENCE C~ARACTERISTICS:
(A LENGTH: 64 base pairs (B TYPE: nucleic acid (C s~rR~NnPnN~qs: single _ (D TOPOLOGY: linear (ii) MOLEC~JLE TYPE: cDNA : :
(iii) nY~ n~llCAL: NO
( iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SQURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:Sl:

GGGCTCGAGT 1~1~ , TACTTCCCGG CTCATATAGA TTCACAAAAA AAAGCGATTG 6 0

Claims (59)

49a

1. A. mucosal binding vaccine composition comprising a mucosal binding polypeptide linked to at least one antigen of a non-viral pathogen, wherein said pathogen causes a sexually transmitted disease.

WHAT IS CLAIMED IS:

2. The composition of Claim 1, wherein said mucosal binding polypeptide is the binding subunit of cholera toxin.

3. The composition of Claim 2, wherein said composition additionally comprises the A
subunit of cholera toxin or a portion thereof.

4. The composition of Claim 1, wherein the antigen is a chlamydia antigen.

5. The composition of Claim 4, wherein the chlamydia antigen is linked to the amino terminus of the binding subunit of cholera toxin.

6. The composition of Claim 4, wherein the chlamydia antigen is linked to an internal portion of the binding subunit of cholera toxin.

7. The composition of Claim 4, wherein said mucosal binding polypeptide is the binding subunit of cholera toxin and said antigen comprises a B-cell stimulating antigen from the major outer membrane protein of chlamydia.

8. The composition of Claim 4, wherein said B-cell stimulating antigen is from the VDIV
region of the major outer membrane protein of chlamydia.

9. The composition of Claim 8, wherein said antigen further comprises a T-helper cell stimulating antigen from the major outer membrane protein of chlamydia.

10. The composition of Claim 9, wherein said T-helper cell stimulating antigen is from the A8 region.

11. The composition of Claim 1, wherein said antigen is chemically linked to said binding polypeptide.

12. The composition of Claim 1, wherein said antigen is linked to said binding polypeptide as a genetic fusion protein.

13. A method for generating a mucosal immune response against a non-viral sexually transmitted disease, comprising contacting the mucosa of a mammalian host with the composition of Claim 1.

14. The method of Claim 13, wherein the composition is the composition of Claim 2.

15. The method of Claim 13, wherein the composition is the composition of Claim 6.

16. A recombinant polynucleotide, comprising a first region encoding a mucosal binding polypeptide and a second region encoding an antigen of a non-viral pathogen, wherein said pathogen causes a sexually transmitted disease.

17. The polynucleotide of Claim 16, wherein said mucosal binding polypeptide is the binding subunit of cholera toxin and said pathogen is chlamydia.

18. The polynucleotide of Claim 17, wherein said antigen is a T-cell helper cell stimulating antigen from the major outer membrane protein of chlamydia.

19. The polynucleotide of Claim 18, wherein said T-cell helper stimulating antigen is from the A8 region.

20. The polynucleotide of Claim 19, wherein said antigen further comprises a B-cell stimulating antigen from the major outer membrane protein of chlamydia.

21. The polynucleotide of Claim 20, wherein said B-cell antigen is from the VDIV region of the major outer membrane protein of chlamydia.

22. A method for vaccinating against chlamydia infection, comprising administering to the mucosa of a mammalian host an effective amount of the binding subunit of cholera toxin linked to both a B-cell epitope and a T-cell epitope of the major outer membrane protein of chlamydia.

23. The method of Claim 22, wherein said administration is vaginal.

24. The method of Claim 22, wherein said administration is rectal.

25. The method of Claim 22, wherein said administration is oral.

26. A mucosal binding composition comprising a mucosal binding polypeptide linked to at least one antigen of a viral pathogen, wherein said pathogen causes a sexually transmitted disease.

27. The composition of Claim 26, wherein said mucosal binding polypeptide further comprises the binding subunit of cholera toxin.

28. The composition of Claim 27, wherein said antigen is a HIV gp120 antigen.

29. The composition of Claim 28, wherein said antigen is the peptide corresponding to SEQ
ID NO: 29.

30. The composition of Claim 27, wherein said antigen is a Hepatitis B virus antigen.

31. The composition of Claim 30, wherein said antigen is from the Hepatitis B virus pre-S(2) protein.

32. The composition of Claim 31, wherein said antigen is the peptide fragment corresponding to SEQ ID NO: 28.

33. A mucosal binding composition comprising a mucosal binding polypeptide linked to at least one antigen from an E. coli.

34. The composition of Claim 33, wherein said antigen is from the STa protein of enterotoxigenic E. coli.

35. The composition of Claim 34, wherein said antigen is the peptide fragment corresponding to SEQ ID NO: 30.

36. The composition of Claim 34, wherein said antigen is the peptide fragment corresponding to SEQ ID NO: 31.

37. A purified recombinant polynucleotide comprising nucleic acid encoding a mucosal binding protein operably linked to a B-cell stimulating antigen, wherein said antigen is a peptide obtained from a pathogen capable of infecting a mammal through the mucosal membranes of that mammal.

38. The polynucleotide of Claim 37, wherein said nucleic acid encoding a mucosal binding protein encodes the binding subunit of cholera toxin.

39. The polynucleotide of Claim 38, wherein said nucleic acid further comprises nucleic acid encoding the CTA(2) subunit of cholera toxin.

40. The polynucleotide of Claim 39, wherein the nucleic acid encoding the B-cell stimulating antigen is positioned 5' to the nucleic acid encoding the CTA(Z) subunit.

41. The polynucleotide of Claim 40, wherein the nucleic acid encoding the B-cell stimulating antigen encodes a peptide which includes the amino acid sequence LNPTIAG.

42. The polynucleotide of Claim 40, wherein the nucleic acid encoding the B-cell stimulating antigen encodes a peptide from HIV gp 120.

43. The polynucleotide of Claim 38, wherein the nucleic acid encoding the B-cell stimulating antigen is positioned in-frame within the coding region of the nucleic acid encoding said mucosal binding protein.

44. The polynucleotide of Claim 43, wherein said nucleic acid encoding the B-cell stimulating antigen encodes a peptide which includes the amino acid sequence LNPTIAG.

45. The polynucleotide of Claim 43, wherein said nucleic acid encoding the B-cell stimulating antigen encodes a peptide from HIV gp 120.

46. The polynucleotide of Claim 45, wherein said nucleic acid encoding a peptide from HIV
gp 120, encodes the peptide IORGPGRAFV.

47. The polynucleotide of Claim 43, wherein said nucleic acid encoding the B-cell stimulating antigen is from the Hepatitis B virus pre-S(2) protein.

48. The polynucleotide of Claim 47, wherein said nucleic acid encoding the B-cell stimulating antigen encodes peptide having the amino acid sequence of SEQ ID NO: 28.

49. The polynucleotide of Claim 43, wherein said nucleic acid encoding the B-cell stimulating antigen is from the STa protein of enterotoxigenic E. coli.

50. The polynucleotide of Claim 49, wherein said nucleic acid encoding the B-cell stimulating antigen encodes peptide having the amino acid sequence of SEQ ID NO: 30.

51. The polynucleotide of Claim 49, wherein said nucleic acid encoding the B-cell stimulating antigen encodes peptide having the amino acid sequence of SEQ ID NO: 31.

52. The polynucleotide of Claim 43, wherein the nucleic acid encoding the B-cell stimulating antigen is between 21 and 150 bases in length.

52a

53. Use of the composition of Claim 1 in a medicament for generating a mucosal immune response against a non-viral sexually transmitted disease.

54. The use of claim 53, wherein the composition is the composition of claim 2.

55. The use of Claim 53, wherein the composition is the composition of Claim 6.

56. Use of a composition comprising the binding subunit of cholera toxin linked to both a B cell epitope and a T cell epitope of the major outer membrane protein of chlamydia in a medicament for generating a mucosal immune response.

57. The use of Claims 56, wherein the medicament is adapted for vaginal administration.

58. The use of Claim 56, wherein the medicament is adapted for rectal administration.

59. The use of Claim 56, wherein the medicament is adapted for oral administration.