WO1996016178A1 - Immunogens for stimulating mucosal immunity - Google Patents

Immunogens for stimulating mucosal immunity Download PDF

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Publication number
WO1996016178A1
WO1996016178A1 PCT/GB1995/002708 GB9502708W WO9616178A1 WO 1996016178 A1 WO1996016178 A1 WO 1996016178A1 GB 9502708 W GB9502708 W GB 9502708W WO 9616178 A1 WO9616178 A1 WO 9616178A1
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Prior art keywords
antigen
composition
mucosal
protein
seq
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PCT/GB1995/002708
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French (fr)
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Michael Richard Lebens
Jan Roland Holmgren
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Maxim Pharmaceuticals, Inc.
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Publication date
Application filed by Maxim Pharmaceuticals, Inc. filed Critical Maxim Pharmaceuticals, Inc.
Priority to EP95937948A priority Critical patent/EP0792365A1/en
Priority to JP8516667A priority patent/JPH10509325A/en
Priority to MX9703676A priority patent/MX9703676A/en
Priority to AU38767/95A priority patent/AU3876795A/en
Publication of WO1996016178A1 publication Critical patent/WO1996016178A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (O)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This application relates to methods for producing mucosal antibody to organisms capable of infecting their host through contact with mammalian mucosal membranes.
  • this application relates to protein complexes and to gene constructs suitable for producing immunogen capable of inducing mucosal antibody as well as to methods for introducing the immunogen into a mammal to generate a mucosal immune response.
  • the mucosal surfaces of the body are generally accessible to a wide variety of infectious agents capable of causing disease. These surfaces include the gastrointestinal tract, the urogenital tract and the respiratory tract.
  • the mucosal surfaces of the body lack the protective keratinized layer and are more vulnerable to invasion by adventitious organisms. Not surprisingly, these surfaces are the main portal of entry into the body for foreign microorganisms.
  • Non-encapsulated lymphoid tissue is dispersed throughout the mucosa of the gastrointestinal and genitourinary tracts as either diffuse aggregates of cells or as organized nodules.
  • the diffuse aggregates of lymphoid cells are dispersed throughout the lamina prop ⁇ a 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 lymphoid cells along the mucosal surfaces are capable of responding to foreign antigen.
  • the gut epithelium overlying the Peyer's patches allows transport of antigens into the lymphoid tissue and is capable of functioning as antigen presenting cells (Bromander, et al. Scand. J. Immunol. 37 * 452458, 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 invasion of adventitious organisms into the mucosa will preferably stimulate IgG and slgA.
  • An effective vaccine for limiting mucosal infection will likely demonstrate slgA activity.
  • Many sexually transmitted diseases such as chlam ⁇ dia, gonorrhea, syphilis, chancroid and t ⁇ chomoniasis are caused by organisms that enter the body through the mucosal membranes. While these diseases are caused by different organisms and replicate in different ways, these sexually transmitted microorganisms, and others, all enter the body primarily via the mucosal barrier. These and most other disease causing organisms carry unique antigenic determinants that are known to stimulate the immune system. In addition, the mucosal surfaces are also the mam portal of entry for most viruses.
  • mucosal surface immunity to viruses including, but not limited to influenza, papiliomavirus, 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 these organisms have met with little success since pare ⁇ teral vaccination does not generally produce significant levels of secretory immunity.
  • the non-viral sexually transmitted diseases can usually be cured if they are diagnosed early, but many of the diseases produce mild early symptoms, if any, and thus go untreated until more advanced symptoms occur.
  • Chlamydia is a useful example of a sexually transmitted disease that infects its host through mucosal membranes, primarily of the genitourinary system
  • Chlamydia trachomatis is the leading sexually transmitted organism in the United States, afflicting an estimated four million people a year (Division of STD/HIV Prevention, 1992 Annual Report, CDC, Atlanta, 1993).
  • Chlamydia is acquired chiefly through vaginal or anal intercourse, although it can also be transmitted through oral sex.
  • C trachomatis infection of the genital tract can cause salpingitis in women that can result in tubal blockage and infertility.
  • Chlamydia trachomatis isolates occur as 15 distinct serovars that are divided into three subgroups.
  • the major outer membrane protein IMOMP of Chlamydia confers serovar and serogroup specificities Protective immunity to Chlamydia is directed to the major outer membrane protein.
  • VDIV Variable domain IV
  • variable domains of MOMP are external epitopes as demonstrated by their sensitivity to trypsm and by their accessibility to antibody binding
  • This invention contemplates combining determinants from MOMP or other protein from pathogens, including but not limited to HIV, hepatitis and enterotoxigenic E. coli capable of infecting a mammalian host through contact with mucosal membrane with a mucosal binding polypeptide
  • pathogens including but not limited to HIV, hepatitis and enterotoxigenic E. coli capable of infecting a mammalian host through contact with mucosal membrane with a mucosal binding polypeptide
  • agents have proved effective as carriers and as adjuvants for stimulating mucosal immunity (for a review of these agents see Bienenstock, J "The Nature of Immunity at Mucosal Surfaces A Brief Review " In Bacterial Infections of Respiratory and Gastrointestinal Mucosae. Eds.
  • Non-living carrier systems allow immunologically accessible epitopes to be presented to the immune system as the product of a genetic construct or as a peptide chemically coupled to a carrying agent.
  • Such non-living carrier systems include microparticles, liposomes, solid matrix antibody antigen complexes, immunostimulating complexes (ISCOMs) and protein carriers including the core antigen of hepatitis B virus, polio virtons, cholera toxin and the heat-labile enterotoxin from E coli
  • the carriers themselves may have endogenous adjuvant activity or alternatively, exogenous adjuvant can be used with the carriers.
  • Ox bile has been used as an adjuvant for orally administered immunogens such as killed oral vaccines against dysentery.
  • adjuvants that have been tested for use in inducing mucosal immunity include DEAE-4 dextran, iysoz ⁇ me, polyormthine, sodium dodecyl benzene sulfate, lipid-conjugated substances, streptomycin and vitamin A (see Holmgren, et al. Vaccine 11:1170 1184, 1993).
  • agents such as muramyl dipeptide, acndine and cimetidi ⁇ e have had some positive effect as well (see Bienenstock, et al. supra.)
  • Cholera toxin is capable of generating mucosal immunity and is also a potent adjuvant 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 of five binding B subunits assembled as a ring together 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.
  • Cholera toxin/Sendai virus conjugate immunization has resulted in the production of Sendai-specific immunoglobu n in the respiratory tract. While cholera toxin has been used to stimulate the immune response to bacterial and select viral antigens along the gastrointestinal 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 immune responses to foreign antigen, the immune responses have been poor as compared with the immune responses generated to the cholera toxin carrier.
  • protein complexes and genetic constructs are disclosed in this invention that stimulate mucosal immunity to pathogens which cause sexually transmitted disease
  • 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.
  • 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 also discloses strategies for improving the immune response to the foreign antigen using a variety of genetic constructs BRIEF DESCRIPTION OF THE FIGURES
  • Figure 1 diagrams the strategy for producing the pML ⁇ £ coli expression vectors of the present invention.
  • Figure 2 provides preferred examples of expression vectors using the tac promoter.
  • Figure 2(a) diagrams a plasmid expressing CTB from the tac promoter.
  • Figure 2(b) illustrates the same vector as in Figure 2(a), additionally carrying the AB gene fragment used for the construction of CTA2 protein fusions.
  • Figure 2(c) diagrams a plasmid, as disclosed in Figure 2(b), additionally containing the iacP gene to facilitate inducibie X-gal expression of ctxkB.
  • Figure 2(d) diagrams a plasmid carrying the modified ct-rB gene for in-frame internal epitope insertion between the unique Kpnl and Mscl sites.
  • Figure 3 diagrams plasmids expressing CTB from the leftwards promoter of phage lambda for the generation of CTB fusions (pML-LCTB ⁇ 7) or CTA fusions (pPJ ⁇ ).
  • Figure 4 illustrates the oligonucleotide sequence, SEQ ID NQ:5, used to regenerate the ctrA signal peptide and the unique restriction sites used for cloning foreign epitopes into the construct.
  • Figure 5 provides synthetic oligonucleotide sequences, SEQ ID N0:24 and SEO ID N0:25, used for the insertion of chlamydial T-cell and B-cell epitopes into the ctxk gene. Boxed sequences represent the chlamydial peptides.
  • Figure 6 provides a diagram of the oligonucleotide sequences, SEQ ID N0:7 and SEQ ID N0:8 which were used to form the linker to join the chlamydial epitopes A8 (SEQ ID N0:6) and VDIV (SEQ ID N0:1).
  • the adjacent Sad and Nhel sequences for pML LCTBt ⁇ c or pML-LCTB ⁇ and pPJVDIV are also diagramed in this Figure.
  • Figure 7 diagrams the construction of plasmid pCB55-64gp309 encoding an exemplary CTB::hybr ⁇ d protein.
  • Plasmid pCB55-64gp309 contained the HIV antigemc sequence derived from ammo acids 309 318 of gp120.
  • the gpl 20 nucleic acid sequence positioned between the Kpnl and BssHII restriction endonuclease sites is provided in the enclosed box with insert gpl 20 sequences shown in italics.
  • This invention relates to immunogens useful for stimulating mucosal immunity and for protein complexes 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.
  • the invention relates to methods for inducing mucosal surface immunity to a pathogen capable of infecting a mammalian host through mucosal membranes.
  • a mucosal binding composition comprising a mucosal binding polypeptide linked to at least one antigen from a non-viral pathogen where the pathogen causes a sexually transmitted disease.
  • 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 of the A subunit of cholera toxin.
  • the antigen from the non-viral pathogen is an antigen from Chlamydia.
  • the chlamydia antigen can be positioned at the ammo terminus of the binding subunit of the cholera toxin, at the ammo terminus of the portion of the A subunit of cholera toxin or positioned internally within the binding subunit of cholera toxin. It is contemplated that the antigen can be linked to the mucosal binding protein by recombinant or chemical means.
  • the antigen comprises a B-cell stimulating antigen from the major outer membrane protein of chlamydia.
  • the B-cell stimulating antigen is from the VDIV region of the major outer membrane protein of chlamydia.
  • the antigen further comprises a T-helper cell stimulating antigen and preferably, this T-helper cell stimulating antigen is also from the major outer membrane protein of chlamydia.
  • the T-helper cell stimulating antigen is from the major outer membrane protein of chlamydia and in a preferred embodiment is the A8 region from the major outer membrane protein of chlamydia.
  • 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 mucosal binding composition
  • recombmant polynucleotides which comprise a first region encoding a mucosal binding polypeptide and a second region encoding an antigen of a non-viral pathogen, where the pathogen causes a sexually transmitted disease.
  • the mucosal binding polypeptide is the binding subunit of cholera toxin and the pathogen is chlamydia.
  • the preferred antigen includes a T-helper cell stimulating antigen and a B-cell stimulating antigen from the outer membrane protein of chlamydia.
  • the B-cell stimulating antigen is from the VDIV region of the major outer membrane protein of chlamydia.
  • This invention also relates to methods for vaccinating a mammal 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.
  • the administration is vaginal and in other embodiments, the vaccine is delivered rectally or orally.
  • the invention additionally relates to mucosal binding compositions comprising a mucosal binding polypeptide linked to at least one antigen of a viral pathogen where the pathogen causes a sexually transmitted disease.
  • the mucosal binding polypeptide comprises the binding subunit of cholera toxin and the antigen is a HIV gpl 20 antigen.
  • the antigen is a Hepatitis B virus pre-S(2) antigen and in a further embodiment, the mucosal binding polypeptide is linked to at least one antigen from the ST, protein of enterotoxige c £ coli.
  • the invention additionally contemplates purified recombinant polynucleotides comprising nucleic acid encoding a mucosal binding protein operably linked to a B-cell stimulating antigen where the antigen is a peptide obtained from a pathogen capable of infecting a mammal through the mucosal membranes.
  • the mucosal binding protein encodes the binding subunit of cholera toxin and in another embodiment, the nucleic acid further encodes the CTA(2) subunit of cholera toxin.
  • the B-cell stimulating antigen encodes a peptide which includes the ammo acid sequence LNPTIAG.
  • the B-cell stimulating antigen is from HIV gp120.
  • 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 am o acid sequence LNPTIAG, an antigen from the gp120 protein of HIV, the Hepatitis B virus pre-S(2) protein or the ST, protein of enterotoxigemc £ coli.
  • 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.
  • 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.
  • immunoaccessible is used herein to describe antigemc sequences that are accessible to the immune system when introduced in association with a mucosal binding polypeptide.
  • T-cell stimulating antigen or "T-cell antigen” is used herein to refer to a peptide, a polypeptide, a protem or a nonprotei ⁇ aceous molecule including carbohydrate, lipid, nucleic acid or the like, which is capable of stimulating T-helper ceil activity in standard T helper cell assays, well known in the art, either alone or in combination with other protein.
  • B-cell stimulating antigen or “B-cell antigen” is used herein to refer to a peptide, a polypeptide, a protein or a nonprotemaceous molecule, including carbohydrate, lipid, nucleic acid or the like, which is capable of stimulating antibody production from B-cells.
  • ctxA and “ctxB” refer to the gene sequences encoding the cholera toxin A and B subunits respectively.
  • 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 mammalian host through the mucosal membranes of the host.
  • mucosal binding polypeptide is used herein to refer to polypeptide capable of attaching to the mucosal surfaces of a mammal.
  • mucosal binding polypeptides 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, bacterial toxin membrane binding subunits including, at a minimum, the B subunit of cholera toxin, the B subunit of the £ coli heat-labile enterotoxin, Bordetella per ⁇ ussis toxin subunits S2, S3, S4 and/or S5, the B fragment of Diphtheria toxin and the membrane binding subunits of Shiga toxin or Shiga-iike toxins.
  • bacterial toxin membrane binding subunits including, at a minimum, the B subunit of cholera toxin, the B subunit of the £ coli heat-labile enterotoxin, Bordetella per ⁇ ussis toxin subunits S2, S3, S4 and/or S5, the B fragment of Diphtheria toxin and the membrane binding subunits of Shiga toxin or Shiga-iike toxins.
  • mucosa binding subunits contemplated within the scope of this invention include bacterial fimbriae protein including E. coli fimbria K88, K99, 987P, F41, CFA/I, CFA/II (CS1 , CS2 and/or CS3), CFA/IV (CS4, CS5 and/or CS6), P fimbraiae, or the like.
  • Other fimbriae contemplated within the scope of this invention include Bordetella pertussis filamentous hemagglutinin, vibrio cholerae toxin coregulate pilus (TCP), Mannose sensitive he agglutinin
  • MSHA fucose-sensitive hemagglutinin
  • mucosal-binding molecules contemplated within the scope of this invention include viral attachment proteins including influenza and Sendai virus hemagglutinins and animal lectins or lectin-like molecules including immunoglobulin molecules or fragments thereof, calcium-dependant (C-type) lectins, selectins, collecti ⁇ s or Helix pomatia hemagglutinin.
  • Plant lectins with mucosa-binding subunits include concanavalin A, Wheat-germ agglutinin, phytohemagglutinin, abri ⁇ and ricin.
  • the invention discloses the use of mucosal binding compositions to stimulate mucosal immunity to pathogens capable of infecting a mammalian host through the mucosal membranes of the host.
  • a method for producing mucosal immunity to a pathogen is disclosed using the mucosal binding subunit of bacterial toxins.
  • £ coli heat-labile enterotoxin are coupled to antigen obtained from pathogens capable of causing a disease.
  • methods are disclosed for chemically coupling a mucosal binding polypeptide such as the mucosal binding portion of the B subunit of cholera toxin or of the heat labile enterotoxin of £ coli to antigen obtained from a pathogen capable of causing a disease.
  • membrane binding compositions of this invention will be useful for stimulating secretory immunoglobulin.
  • This secretory immunoglobulin can be used in diagnostic assays, such as
  • 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 the foreign epitope.
  • the secretory antibody collected from experimental animals can be used in topical preparations against a specific pathogen or as a general component of a topical preparation or in additional studies to assess the character of secretory immunoglobulin.
  • mucosal binding polypeptides 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 synthesized 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 polypeptide for its ability to function as a carrier and to facilitate foreign antigen presentation to the immune system.
  • mucosal binding polypeptide 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 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 provided above. For recombinant protein expression, those skilled in the art of molecular biology will isolate nucleic acid fragments encoding this gene, using standard techniques known in the art, and incorporate these fragments into expression constructs.
  • the recombinant or purified mucosal binding polypeptide is next tested for its ability to be bound by test antibody where the test antibody is known to recognize the naturally occurring mucosal binding polypeptide.
  • suitable tests for determining antibody recognition include ELISAs, immunoblots or other well known immunologic assays available in the art. Exemplary ELISA and immunoblot assays are provided in the Examples (see Example 4 and 6). These assays are well known and can be readily modified by those skilled in the art to identify other mucosal binding protein.
  • the candidate mucosal binding polypeptide is further tested for its ability to bind mucosa. Mucosal binding assays are known in the art.
  • 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 biopsy). 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).
  • 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 binding to the mucosa surface. Alternatively, 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.
  • subunits or fragments of the Vibrio cholerae 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 similarities 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.
  • LTB like CTB, can be secreted into the medium when V cholerae cultures are transformed with expression vectors expressing recombinant LTB Further, CTB and LTB bind to gangliosides o mucosal membranes (see Hirst, et al Proc. Natl Acad Sci USA 83:9174-9178, 1984 and Schodel, et al. Gen 99:255 259, 1991).
  • the Hirst, et al reference indicates that CTB sequences can be replaced with LTB sequence for recombinant expression.
  • CTB sequences can be replaced with LTB sequences and used in the methods and examples detailed below.
  • the mucosal binding polypeptide is derived from cholera toxin or the heat labile enterotoxin from £ coli.
  • the non- limitmg examples, provided below, include a variety of compositions employing mucosal binding 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 least one antigen from a pathogen that enters its host through the mucosal membranes.
  • the immunogen derived from the this pathogen can be selected from a variety of immunogens which are known to stimulate immune responses in a mammal susceptible to the disease caused by that pathogen.
  • the antigen includes at least one antibody stimulating determinant, preferably from a surface protem of the disease-causing pathogen.
  • the antigen is a polypeptide
  • it will contain at least one consecutive region of ammo acids, preferably from an immunoaccessible domain of the surface polypeptide of the pathogen
  • the 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 ammo acid sequences, multiple copies of these sequences or am o acid sequences that stimulate T-Cells or those that assist in the generation of an antibody response through the activation of T-Helper cells or other T-Cell populations
  • the antibody stimulating determinant can be repeated in tandem or separated by suitable linking sequences, or the like, to further stimulate the antibody response.
  • the first step for selecting an antigen contemplated in this invention is to identify a non-viral pathogen capable of infecting a mammal via entry through a mucosal membrane.
  • a non-viral pathogen capable of infecting a mammal via entry through a mucosal membrane.
  • serum samples are taken from a mammal and tested for the presence of antibody by contacting the serum sample with cell lysates containing pathogen, or alternatively with intact pathogen, and detecting binding of serum antibody to the sample
  • Assays useful for detecting serum antibody binding include enzyme-linked immunosorbent assays (ELISA), immunoblots such as Western blots, or the like.
  • ELISA enzyme-linked immunosorbent assays
  • immunoblots such as Western blots
  • those skilled in the art may elect to directly test for the presence of IgG or secretory antibody to the pathogen in a mammal.
  • 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 concentration method well recognized in the art
  • This sample is then tested for the presence of pathogen-specific immunoglobulin, in general, or slgA or IgG, specifically, using standard immunoassays well known in the art.
  • those skilled in the art can determine which protein or proteins stimulate a neutralizing 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 using any number of strategies well known in the art.
  • a method for mapping antibody 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 Today 5:397400, 1989 respectively).
  • peptide mapping strategies 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.
  • B-Cell stimulating determinants for their ability to stimulate neutralizing antibody in vitro see Zhang, et al. (J Immunol 438:575-581 , 1987).
  • 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 to a mucosal binding polypeptide. These strategies are detailed in the examples below (see Examples 4 and 6).
  • the mucosal binding protein is linked to the antigen selected from the pathogen.
  • the antigen can be linked to the mucosal binding protein by chemical coupling or through a linking member, including lipid, carbohydrate or protein.
  • the antigen can be synthesized by means of a gene construct either separately or together with the mucosal binding protein.
  • Example 1 details a preferred strategy for the chemical coupling of a membrane binding polypeptide to a determinant from a pathogen.
  • a fragment of the cholera toxin protein B subunit was expressed using the method of Lebens, et al. (Bio Technology 1 1.1574-1578,
  • the polypeptide derived from the major outer membrane protein is the VDIV peptide (SEQ ID NO: 1 ) corresponding to a portion of the fourth variable domain of the major outer membrane protein of C. trachomatis.
  • the polypeptide is a linear chime ⁇ c polypeptide that comprises the A8 domain and the VDIV domain from the major outer membrane protein of C. trachomatis (SEQ ID NO:2). Both the A8 and the VDIV domain as well as the chimenc peptide are described by Su, et al. [Vaccine 11:1159-1166, 1993).
  • a polypeptide from a protein derived from a non-viral pathogen causing a sexually transmitted disease is expressed as a recombmant protein with a subunit or fragment of a mucosal binding protein.
  • the mucosal binding protein is a portion of the cholera toxin and the antigemc determinant is derived from C. trachomatis.
  • the antigemc determinant or determinants from the pathogen could be placed at the ammo or carboxy terminus of the B subunit, at the am o or carboxy terminus of the A subunit, or at the ammo terminus of a portion of the A subunit.
  • the A subunit is operatively linked to the B subunit.
  • the antigemc determinant is incorporated in frame into an internal portion of either the A or B subunit, or a portion thereof.
  • constructs could also be derived from the B subuntt of the heat labile enterotoxin of £ coli without undue experimentation. 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-LCTBt*c2 has an identical restriction pattern to pML LCTBtac but incorporates the pUC19 origin of replication (see Lebens, et al. Biotechnology, supra). Thus, the use of this plasmid is also contemplated within the scope of this invention.
  • a chtme ⁇ c protein is produced that includes a polypeptide linked at its carboxy terminus to the ammo terminus of the B subunit (CTB) from cholera toxin.
  • CTB ammo terminus of the B subunit
  • the HIV epitope IQRGPGRAFV is incorporated into the ammo terminus of the B subunit.
  • the antigen from the pathogen is incorporated onto the ammo terminus of a polypeptide which is a fusion protein of a fragment of the A subunit of cholera toxin fused to CTB and expressed in either V. cholera or £ coli.
  • the antigemc peptide is the VDIV sequence from the major outer membrane protein of C. trachomatis and in another preferred embodiment the antigemc peptide is the A8 sequence from the major outer membrane protein.
  • the peptide is a chimenc protein containing both the antigemc peptide sequences of A8 and VDIV separated by a linker (see Figure 6).
  • this same linker introduced into, for example, pML- LCTB/1J would facilitate the construction of multiple copies of either epitope
  • the linker, or other linkers could be used to insert the A8 and VDIV sequences into other constructs or the linker could facilitate the transfer of inserted sequences between vectors.
  • the order of the antigemc sequences can be switched and that multiple determinants of A8 or VDIV can be linked either in tandem or within the recombinant construct.
  • the literature describes a variety of antigemc peptide sequences for C. trachomatis that are serovar specific, subgroup specific or are broadly conserved among serovars.
  • other antigemc peptides from the major outer membrane protein or from other proteins of C. trachomatis would also be useful in stimulating antibody production along the mucosal surfaces.
  • Job ng, et al. (Infect, and Imm. 60:49154924, 1992) have produced fusion proteins in which entire bacterial proteins were linked via the ammo terminus to the A subunit fragment, CTA2.
  • Job ng, et al. demonstrated that chimenc toxin could be produced from a cholera toxin construct, both portions of the chimera were bacterial protein and the signal sequences for chimenc expression were derived from the inserted bacterial protein.
  • the present invention demonstrates that antigemc sequences which are foreign to the bacteria can be inserted into CTA2.
  • the CTA2 fusion proteins with foreign antigemc sequences were coexpressed with CTB from V. Cholera to give assembled products which were excreted extracellularly and were detectable in a GM1 -ELISA.
  • the gene products produced by Job ng et al. were not excreted extracellularly.
  • the polypeptide is incorporated, in frame, into an internal portion of the mucosal binding polypeptide, preferably at an immunoaccessible site, in one example of this strategy, it is contemplated that a short foreign polypeptide is introduced into an internal region of the cholera B subunit in place of CTB am o acids 56-63.
  • this peptide includes the VDIV epitope, LNPTIAG, and in another embodiment, illustrating that this strategy can be used for other peptide sequences, the peptide is the HIV neutralizing epitope IQRGPGRAFV (aa 309-318 of HIV 1 isolate HTLV-ltIB).
  • 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 enterotoxigemc £ coli. Plasmids were constructed encoding CTB hybrid proteins which had either an eleven ammo acid peptide form Hepatitis B Virus (HBV) pre-S(2) (SEQ ID NO: 28) or one of two peptides (SEQ ID N0:30 and SEQ ID NO: 31 , see Table 1 ) related to the heat-stable toxin (ST.) of enterotoxigemc £ coli.
  • HBV Hepatitis B Virus
  • 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 products stimulated strong serum antibody responses against CTB.
  • Example 4 Data is provided in Example 4 to indicate that the CTB::foreign antigen hybrids of the present invention retain all of the important characteristics of native CTB, such as folding, pentamerization, extra-cellular secretion when produced in V. cholerae and GMI-binding. Moreover, many of the inserted peptide constructs were resistant to cleavage by V. cholerae proteases. The data in this example indicated that the CTB::foreign antigen hybrids reacted with monoclonal antibody directed against the foreign antigen, both in the denatured and non-denatured forms of the protein.
  • the environment of the inserted peptide can be modified to improve the immunogenicity.
  • 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 sequence and that these modifications do not limit the scope to this invention.
  • mucosal binding complex of binding protein and antigen Once 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.
  • Methods for detecting hybrid protein expression are provided in Examples 4 and 6.
  • In vitro assays testing mucosal binding are useful for demonstrating the intactness of the complex. Such assays include binding assays to intact mucosal epithelium cell cultures or mucosal membrane sections. If the mucosal receptor is known, specific ELISAs or receptor/iiga ⁇ d chromatographic assays, or the like, can be used to ascertain mucosal binding.
  • the complex will be formulated as a mucosal binding composition that 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, rectall ⁇ , nasally, intramuscularly, intraperitoneally or intravenously.
  • Sera or mucosal immune responses are monitored in the experimental mammal or in clinical trial by ELISA, immunoblot, or by western blot to detect antibody to either the mucosal binding polypeptide or the foreign polypeptide.
  • antibodies in serum or antibody in mucosal 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.
  • the mucosal secretions are collected through wicking action using an absorbent pack, such as a tampon, an absorbent plug, or the like.
  • biopsy of mucosal tissue during the testing phases of this invention will be used to assay, by histopathology or by cell activity, for immune responses specific to the mucosal binding polypeptide.
  • Example 7 indicates that immunization of mice with the intracham 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.
  • CTB Cholera toxin B subunit
  • the polypeptides were covalentl ⁇ conjugated to CTB using N succ ⁇ n ⁇ m ⁇ dyl(3-[2-pyr ⁇ dyl] d ⁇ th ⁇ o) propio ⁇ ate (SPDP, Pharmacia) as a bifunctional coupling reagent according to the manufacturer's instructions
  • CTB was derivatized with SPDP at a molar ratio of 1 :5 in 0.1M phosphate buffer/0.1 NaCl pH 7.5. After incubation at 23 °C for 30 mm., excess SPDP was removed by gel filtration through Sephadex G 25 columns (Pharmacia) and eluted with PBS. The absorbance of the modified protein was measured at 280 nm.
  • the absorbance at 343 nm of 100 ⁇ l protein solution in 400 ⁇ l was measured after incubation with 50 ⁇ dithiothreitol (0.1 M) for 15 minutes (molar extinction coefficient at 343 nm - 8.08 x 10 3 M 'c 1 ). This concentration was equivalent to the concentration of 2-pyr ⁇ dyl disulphide residues in the protein.
  • the resulting CTB eptide conjugates were purified by gel filtration through columns of Sephadex 6-25. Conjugates were purified over GM1 -columns of Sephadex G-25. Purified conjugates were shown to retain GM1 -binding capacity and to retain both CTB and peptide specific serological reactivities by means of a solid phase ELISA using GM1 as a capture system and enzyme-labelled antibodies to CTB or to the foreign polypeptide sequence coupled to CTB.
  • An exemplary ELISA using GM1 is provided in Examples 4 and 6, below.
  • Peptide 166 (SEQ ID N0:13, A8-VDIV)
  • A8-VDIV is the colinear peptide disclosed by Su, et al. (Vaccine 11:1159-1 166, 1993). It contains a single cysteine residue.
  • Peptide 172 (SEQ ID NO: 14) has a free extra cysteine coupled to the amino end and Peptide 173 (SEQ ID NO: 15) has a free extra cysteine coupled to the carboxyl end of the A8-VDIV sequence.
  • the CTB/A8-VDIV complexes were introduced into C57 BL/6J female mice (obtained from the Animal Care
  • Example 8 The immunization protocol for generating mucosal antibody is provided in Example 8.
  • the starting plasmid was pPL-lambda obtained from Pharmacia AB. Sweden.
  • the generation of the pML ⁇ 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 promoter region was recovered by band-extraction. More of the same plasmid was digested with BamHI and Pvull and again resolved on an agarose gel. This time a 2,305 bp BamHI/Pvull 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 £ coli strain N4830-1, the Gal P2 transductant of N4830 (Pharmacia, Sweden).
  • pML ⁇ I the orientation of the phage lambda DNA within the plasmid has been reversed and 1 ,689 bp of pBR322 DNA has been removed.
  • pML ⁇ 1 was digested to completion with BamHI and subsequently partially digested with Haelll. The digested DNA was religated and used to transform £ coli strain N4830-1. Transformants were screened on the basis of restriction analysis for plasmids in which the lambda N gene and tL 1 terminator had been removed.
  • the resulting plasmid was pML ⁇ 2 and carried the promoter ⁇ P, with unique downstream restriction sites BamHI, Smal and EcoRI that could be used for cloning of recombinant genes.
  • the powerful trpk transcription terminator (derived from the trpk cassette, Pharmacia) was introduced between the unique EcoRI and Aatll sites within pML ⁇ 2.
  • an additional unique Xhol site and a Hindlll site that could be used in cloning procedures were introduced into the plasmid (see Figure 1 ).
  • the unique Ndel site within the plasmid was removed 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 which generated BspEI sites on either side of the introduced EcoRI site.
  • the resulting plasmid was digested with BspEI and religated. This plasmid was used for recombinant expression in £ coli.
  • Fusions of peptides to the ammo terminus of CTB were generated by inserting synthetic oligonudeotides between unique Sad and Sspl sites in the expression vector pML-LCTB ⁇ 7 (see Figure 3).
  • the HIV RP335 peptide was placed at the ammo terminus of CTB in pML-LCTB ⁇ 7 between the Sa and Sspl sites using two oligonudeotides: SEQ ID N0:9 and SEQ ID N0:10.
  • the insertion was placed within the structural gene at position +3 of the CTB sequence generating a Hpal site and at the same time destroying the Sspl site.
  • Oligonudeotides corresponding to SEQ ID N0:11 and SEQ ID N0:12 were used for this vector construction.
  • the ctxB gene in this vector is under the control of the inducible AP, promoter and was constructed essentially as described previously (Lebens, M. et al BioTechnology 1 1 :1574-1578, 1993).
  • the vector was chosen in order to allow the construction of genetic fusions, the products of which may be deleterious to the host cells since the inducible system only allows expression under inducing conditions.
  • cultures can be maintained under conditions in which expression is not switched on and the recombinant protein does not accumulate to harmful levels.
  • a second construct was produced that employed p Ltac derivative plasmids. This permitted expression of the resulting protein fusions in V. cholerae.
  • the starting plasmid in this case was the expression vector pKK223 3 obtained from Pharmacia AB (Sweden).
  • the 1689bp Pvull/BamHI fragment was removed.
  • a further development of the vector was to introduce the lac ⁇ gene upstream of the tac promoter to make the vector produce cloned gene products in an inducible manner. This was done using PCR
  • the Pt ⁇ r-based expression plasmids of this invention were amplified using SEQ ID N0 16 and SEQ ID NO: 17.
  • the /aclq gene was obtained by PCR fragment amplification of expression plasmid pMMB66 (Furste, et al. Gene 48:1 19 131 1986) using SEQ ID NO: 18 and SEQ ID N0:19.
  • the lac ⁇ t gene was introduced between BamHI and Bglll restriction sites so that it could be removed easily to generate a plasmid giving constitutive expression from the tac promoter (see Figure 2).
  • the same gpl 20 peptide was fused to the N-terminus of CTB by inserting complementary synthetic oligonudeotides encoding the peptide between the Sad and Xma ⁇ restriction sites at the junction between the leader peptide and the mature CTB in the expression plasmid pJS752-3 (Ap R ), a derivative of pJS162 (Sanchez, et al. Proc. Natl. Acad. Sci. USA 86: 481485, 1989).
  • This plasmid is essentially the same as pML-LCTfltar except that the EcoRI/Hindll fragment carrying the recombinant ctxB gene is derived from pJS162.
  • the parent plasmid is the expression vector pKK223-3 (Pharmacia).
  • Plasmid JS752-3 carries the gene encoding CTB under tac promoter control.
  • pJS54 see Example 4, below, an oligo (formed from the hybridization and ligation of oligonudeotides corresponding to SEQ ID N0:20 and SEQ ID N0:21) was inserted between unique Sacl and Smal sites in pJS752-3. The recombinant V.
  • cholerae strain producing the N-terminal CTB::HIV hybrid protein was shown to secrete the chimeric protein into the culture medium using the GM-1 ELISA provided in Example 4.
  • the cultured cells secreted protein reactive with mAb F58/H3-a ⁇ tr-gp 120 when analyzed in a GM1-ELISA.
  • the GM1-ELISA was useful as a tool to quantitate the amount of expressed protein and to monitor the extent of proteolytic degradation of the added peptide.
  • Culture conditions required to produce the constructs are provided in Example 5.
  • strain 644 reacted with mAb P4/D10 increasingly with time.
  • the fused gp120 peptide (SEQ ID NO: 29) in this protein was more resistant to proteolytic degradation than the construct lacking the N-terminal amino acids of CTB.
  • the plasmid pCB2gp309-318 was also expressed in £ coli HB101 (strain 504) and could be purified from the periplasmic space by osmotic shock lysis of the cells followed by precipitation with 80% ammonium sulfate and extensive dialysis against PBS.
  • This protein ran at a higher molecular weight than CTB in SDS-PAGE, indicating the formation of aggregates, but the protein reacted well with both mAb P4/D10-anti-gp120 and with mAb LT39 and CT6 against pentameric and mono eric CTB.
  • the plasmid used for the expression of CTA-2 was constructed so that the CTA-2 fusion and CTB were co-expressed to obtain assembly of a hoioprotein in vivo.
  • the ct/A2 and ctx_ genes were obtained from PCVD30
  • the resulting EcoRI/Hindlll fragment carrying the ctx genes contained unique Sad and Xbal sites between which synthetic oligonudeotides can be inserted to generate ammo terminal fusions between ctxkl 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, plasmid-denved expression of CTB could be demonstrated. Plasmids carrying the ctx genes under the control of the tac promoter were maintained in V. cholerae since the levels of CTB generated in this example were too high to be tolerated by £ coli. This construct lacks the recognition site for the proteolytic cleavage involved in the maturation of CTA.
  • the antigens used in this work were those from Chlamydia trachomatis strains identified by Su and C aid well (Su, H. et al. Vaccine 11, 1159-1166, 1993). These include a T-cell epitope A8 SEQ ID N0:6, situated within a 25 ammo acid peptide from serovar A MOMP and a B-cell epitope VOIV SEQ ID N0:1 , from serovar B MOMP containing 17 am o acids. Within the VDIV sequence a septapeptide sequence has been mapped as an epitope reacting with the monoclonal antibody DIII-A3.
  • ammo acid sequences of the two peptides are shown in Figure 5 together with the synthetic nudeotide sequences used for the generation of gene fusions with CTB.
  • the oligonucleotide corresponding to VDIV and the oligonudeotides corresponding to A8 are identified.
  • the first of the fusions were made to the ammo terminus of CTA2.
  • the synthetic oligonudeotides corresponding to the foreign antigen were cloned into the vectors. It was possible to express assembled protein complexes in which the VOIV peptide was associated with CTB and detectable in a GM1 ELISA assay using Mab DI1I-A3 as the primary antibody. In this case the assembled protein was produced in £ coli under the control of the ⁇ P1 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. 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 Sad site of plasmid pPJ-VDIV ⁇ using SEQ 10 N0:7 and SEQ ID N0:8. This allowed the addition of the A8 sequence to the am o end of the VDIV sequence by cloning of a Bgll/Xbal fragment from pPJA ⁇ into Bgll/Nhel digested pPJVDIV , 1.
  • pPJAJM is a CTA2 fusion vector into which the oligo encoding the A8
  • novel mtrachain CTB fusion protein were prepared with foreign peptide inserted into an internal region of CTB.
  • the resulting chimeric protein retained important functional characteristics of the native CTB including: 1 ) pentamerization; 2) GM1 ganglioside receptor binding; and 3) resistance to proteolytic degradation during production of the protem in Vibrio cholerae.
  • 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 inserted antigen.
  • the foreign polypeptide was inserted at the position of an internal loop structure between /?4 and al in CTB, extending with a few residues into the ⁇ -helix, as predicted by a comparison of the resolved crystal structure of LTB (Sixma, et al. Nature 351:371-377, 1991 ).
  • the VDIV fragment TTLNPTIAGAG is incorporated into the internal CTB site using the two restriction endonuclease recognition sites Kpnl and Mscl Oligonudeotides corresponding to SEQ ID NO: 22 and SEQ ID N0:23 are hybridized, digested with the restriction enzymes Kpnl and Mscl, purified and ligated to plasmid pCB56-64.
  • expression vectors were constructed having HIV-1 epitopes positioned internally in- frame within CTB, the CTB expression plasmid pML LCTBtac (Ap fl ) was mutage zed using the polymerase chain reaction (PCR), employing a modified protocol of that disclosed by Sch ⁇ del et al. ("Hybrid hepatitis B virus core/pre-S particles expressed in attenuated Salmonellae for oral immunization." In: Brown, F.. et al. (Eds), Vaccines '91. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1991, pp. 319-325.).
  • the oligonudeotides used in the PCR reactions are provided as SEQ ID N0:3 and SEQ ID N0:4. These primers incorporated a sequence encoding ten ammo acids from the central portion of the V3 loop in HIV gp120 between residue 55 and 64 in CTB Two unique restriction enzyme sites, Bssl ⁇ ll and Kpnl, were also introduced, with the oligonucleotide primers, into the final plasmid pCB55-64gp309 (see Figure 7).
  • the PCR reaction was run in the presence of 1.5 mM MgCI 2 and 1.25 mM deoxynucleotides under the following reaction conditions: denaturation at 94°C for 1 m , annealing of primers at 65°C for 2 mm and elongation of DNA by Tag DNA polymerase (Boehrmger Mannheim, Indianapolis, Indiana) at 72°C for 4 m .
  • the reaction was repeated for 30 cycles, increasing the elongation step by 2 sec with each cycle To complete all synthesized DNA strands, a final incubation step at 72°C for 10 mm was performed.
  • the PCR product was digested with BssrlW (Boehrmger Mannheim) according to the manufacturer's instructions, phenol/chloroform extracted again and religated using T4 DNA hgase (Pharmacia, Upsala, Sweden) at 16°C for 3 h.
  • the ligated plasmid was electroporated into V. cholerae (strain JS1569, ctxA , ctxB') by the method of Lebens et al. (supra)
  • the DNA sequence was confirmed by the dideoxy chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977).
  • the CTB region was selected for substitution because it had been reported that this region reacted with antibodies recognizing primary protein structure rather than CTB conformational epitopes (Jacob et al., EMBO J. 4:3339-3343, 1985 and Kazemi, et al., Mol. Immunol. 29:865-876, 1991).
  • a substitution in this region would presumably not affect the beta-sheet or alpha-helix structures essential for the correct folding of the molecule.
  • a substitution in this area would not affect the assembly of pentamers (see Sixma et al., 1991, supra).
  • a substitution is this area would likely not interfere with the site responsible for GM1 receptor binding on the mucosal surface.
  • the gpl 20 peptide used for these studies was taken from ammo acids 309-318, having the sequence IQRGPGRAFV (SEQ ID N0:29), representing a portion of the V3 loop of HIV-1 isolate HTLV-IIIB
  • the sequence numbering of gp120 is based on the Los Alamos database sequence for gp120 (Los Alamos National Laboratories, Los Alamos, New Mexico).
  • the sequence contains a principal neutralizing B-cell determinant of HIV 1
  • the sequence GPGR within the peptide is conserved between several HIV-1 isolates and the peptide is of great interest m the development of a peptide-based vaccine against HIV-1 (Javahe ⁇ an et al., Proc. Natl. Acad. Sci.
  • the gpl 20 peptide replaced ammo acids 56-63 of the recombinant CTB, thus adding two ammo acids to the net number of residues in the protem.
  • Polyacrylamide gel electrophoresis (PAGE) and immunoblot analyses of partially purified proteins from culture supernatants of V. cholerae 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 grown in 50ml modified syncase medium in 250 mi Erlenmeyer flasks shaken at 250 rpm overnight at 37 °C. These plasmids can also be grown in £ coli without adjustments in vector construction.
  • Plasmid pCB55-64gp309 318 used SEQ ID NO: 32 and SEQ ID NO:33 as oligonudeotides to incorporate the gp120 epitope into the CTB protein.
  • Plasmids pCB53-64gp309-318 and pCB53-64gp307-318 were made by the cloning of complementary synthetic oligodeoxynucleotides (oligos) between the Kpnl and BssHll sites in pCB55-64gp309 318 with the introduction of a BamHI site in the latter.
  • a BssHlllHmdlW fragment of around 1 kb, encoding ammo acid 315-322 of the inserted epitope together with ammo acids 64 to 103 of the gene encoding CTB (ctxB) and sequence up to the terminator sequence was obtained from the intermediate plasmid and subcloned between the BssHll 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-LCTBf ⁇ , introducing nucleotides coding for am o acid 309 318 from gp120, and containing a BssHll site, either as a straight insertion at position 56, resulting in plasmid pCB56GP309 318, or with deletions of the CTB ammo acids 56-64 (pCB55-65GP309-318) or ammo acids 53-57 (pCB52-58gp309-318).
  • a Sphl site was introduced with the oligos, which were cloned into the plasmid pCB55-64gp12, to obtain the plasmids pCB55 64ST I
  • the plasmids encoding the HBV::CTB hybrid proteins were also constructed by oiigo directed PCR mutagenesis of pML-LCTBtac, introducing DNA encoding amino acids 133-143 from pre-S(2) together with an Xhol site, either between amino acids 55 and 64 of CTB, yielding the plasmid pCB55-64ps133-143, or as a straight insertion after position 56 in the plasmid pCB56ps133-143. All hybrid genes were sequenced by the dideoxy chain termination method of Sanger et al. (1977).
  • the maternal strain is V. cholerae JS1569 b)
  • the nomenclature used for the plasmids is as follows: CB indicates that the maternal protein is CTB; the first number indicates the position in CTB of the inserted peptide la straight insertion without deletions is noted with the aa which the insert is placed after the two numbers (eg. 55-64) indicate that the aa between the numbers are deleted); the next two letter describes the origin of the inserted aa, gp, for gpl 20, ps for pre-S(2) and ST for STa, and the last numbers or letters indicated the aa inserted c) Numbering of gpl 20 aa accordingl to the Los Alamos database d! Strain number 504 is an £ coli HB101 strain carrying the plasmid pCB2gp309-318 e) Am o acids in bold constitutes the intervening linker sequence EXAMPLE 5
  • the recombinant V. cholerae strains producing the hybrid protein polypeptides were cultured in a modified Syncase medium (Lebens et al., Biotechnology 11:1574-1578, 1993) with 100 ⁇ g/ml Ampicillin at 37° C with shaking. Samples were taken at 3, 6, 8, 13 and 24 h after inoculation and both the cells and the culture supernatant were analyzed in a GM1 -ELISA (see Example 4 above and 6 below) with the mAb LT39-anti-CTB and F58/H3-anti-gp120. The major part of the produced hybrid proteins were found in the supernatant. The amount of CTB produced was calculated using a standard curve with purified recombinant CTB. The titers of F58/H3 binding were defined as the interpolated dilution giving an A ts0 of 0.4 above background.
  • the internal HIV::CTB, HBV::CTB and ST::CTB hybrid proteins were synthesized in the V. cholerae strain JS1569 (ctxA, ctx,) which was transformed with the different plasmids by electroporation.
  • the resulting recombinant bacterial strains were cultured at 37° C in the presence of 100 ⁇ g/ml Ampicillin.
  • Hybrid protein was found in the culture medium which could be precipitated by acidification in the presence of hexametaphosphate (Lebens et al., BioTechniques 1993). Precipitates were redissolved and dialyzed extensively against PBS and then analyzed in SDS-PAGE and immunoblot. Polyacr ⁇ lamide Gel Analysis and Immunoblotti ⁇ g
  • the internal CTB::HIV hybrid protein showed an increasing titer with mAb F58/H3 proportional to the amount of protein produced and secreted.
  • the peptide was resistant to proteolytic degradation when placed internally in CTB.
  • the ST sequences include several cysteines which may form internal disulfide bridges that potentially stabilize the structure of the inserted peptide and of the entire chimenc protein.
  • strain 439 The HIV::CTB 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 ⁇ g/ml after 24 hours).
  • the protein migrated as a sharp band of the size of pentameric CTB when analyzed unboiled.
  • proteins can maintain a conformation which resembles CTB enough to migrate as CTB pentamers on a polyacryla ide gel, bind to GMI gang osides and be recognized by mAb LT39-ant ⁇ -CTB, even after they have been cleaved at the position of the inserted peptide.
  • further analysis of some clones may be necessary to ensure that they are not unduly susceptible to cleavage Analysis of the hybrid protein in GM1 -ELISA Assays
  • PBS-BSA-T 0.1 % BSA and 0.05% Tween-20
  • PBS-BSA-T horse-radish peroxidase labelled goat-anti-mouse-lgG (Jackson Laboratories) in PBS-BSA-T was added as second antibody to the wells after washings with PBS T, and after one hour the plates were washed again and then developed with the chromogenic substrate orthophenylenediamme (OPD) in citrate buffer with 0.012% H 2 0 2 .
  • OPD chromogenic substrate orthophenylenediamme
  • each hybrid protein added in the first well of the microtiter plate was adjusted to 10 ⁇ g as estimated from Coomassie stained potyacrylamide gels with recombmant CTB as a standard and were also confirmed by the reaction with mAb LT39-ant ⁇ -CTB. Titers were defined as the interpolated serum dilution giving a A 450 of 0.2 above background.
  • the ELISA assays for GM1 expression used the mAb LT39 and included a standard curve with recombinant CTB starting at 0.5 ⁇ g/ml, and reactivity with mAb P4/D10, which was defined as the interpolated serum dilution giving a A ti0 of 0.4 above background.
  • Antigenic properties of the g 120 epitope in the HIV::CTB proteins included a standard curve with recombinant CTB starting at 0.5 ⁇ g/ml, and reactivity with mAb P4/D10, which was defined as the interpolated serum dilution giving a A ti0 of 0.4 above background.
  • the ten am o acid-peptide from gp 20 yielded a relatively strong signal when placed N-term ⁇ ally in CTB (see Example 3), or between aa 55 and 64, indicating that the epitope was surface exposed in these two proteins (from strain 504 and 407)
  • the reaction of the epitope with antibody was weaker. Either the epitope was less well exposed on the surface or it adopted a conformation which the mAb did not recognize.
  • the HIV::CTB hybrid proteins were also analyzed in immunoblot with the same ant ⁇ -gp120-mAb.
  • the inserted gpl 20 epitope was only detected in the constructs with substitutions within ammo acid positions 55-64
  • the epitope could only be detected in the boiled monomeric form. This could imply that the epitope was exposed in the monomers form, but became more buried or distorted when pentamers were formed.
  • the reaction in GM1-ELISA assays indicated that after binding to GM1, the pentameric forms of some of the proteins exposed the inserted HIV epitope enough to be recognized by the ant ⁇ -gp120 monoclonal antibody. It is possible 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 analyzed in immunoblot with monoclonal P4/D10. The weak monomeric band of around 11 kD, which presumably is still uncieaved, reacts with the mAb, but the majority of the protein, which was degraded, showed no antt-gp 120 reactivity.
  • the ST, epitopes inserted between ammo acids 55 and 64 in CTB strains 551 and 557 were detected with the ST-specific neutralizing mAb ST1:3 (see Svennerholm et al., J Clin. 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., Res. Microbiol. 47:971-979, 1990).
  • the mtrachain ST, peptides were only detected in the monomeric protein form. This is consistent with what was seen with most of the HIV::CTB proteins.
  • Ala-64 should be positioned C-term ⁇ nally to the insert to get production of the protein. This observation is consistent with other scientists who have reported that Ala-64 is important for the stability of pentameric CTB. Likewise, the N-terminal position of Pro-53 was important to the insert since when deleted, as in pCB52-58gp309-318, the corresponding hybrid protein was not produced at detectable levels.
  • the length of the insert will also affect stability.
  • those skilled in the art should also contemplate varying the length of the insert in those substitutions where stability is potentially a problem.
  • the ELISA screening methods in combination with polyacr ⁇ lamide gel electrophoresis staining and immunoblot analysis serve to guide those skilled in the art to prepare appropriate combinations and to screen these combinations for expressed protein.
  • the results provided here indicated that CTB can be modified without loss of synthesis of the desired protein.
  • mice Female C57B1/6 mice were immunized intraperitoneally (i.p.) with three doses of 10 ⁇ g of the
  • the first dose was given with Freund's complete adjuvant (DIFCO Laboratories, Detroit, Michigan) and the subsequent doses with Freund's incomplete adjuvant.
  • mice were immunized using the same scheme with a CTB hybrid protein with an irrelevant foreign epitope or with Freund's adjuvant alone. Serum samples were collected before the first dose and seven days after the second and third dose. Sera were analyzed in ELISA, using either CTB (bound to GM1 in GM1 -coated microwells using the methods of Example 6) or recombinant gpl 20 (Bolmstedt et al., J. Gen. Virol. 73:3099-3105, 1992) as antigen.
  • mice Female mice were given 10 mg of progesterone subcutaneously 10 and 3 days before the first immunization and were then treated once a week with progesterone. Groups of mice were immunized either four times intravaginally (at 1-2 week intervals between each dose) or with three intraperitoneal doses followed by an intravaginal dose. Each i ⁇ travaginal immunization dose consisted of ca. 0.5 mg CTB-peptide conjugate (see Example 1). Each dose was estimated to contain approximately 0.4 mg CTB and 0.1 mg A8-VDIV peptide and additionally included 5 meg cholera toxin as extra adjuvant and each intraperitoneal dose contained one third this amount. EXAMPLE 9
  • the CTB fusion proteins are isolated from bacterial culture supernatants using the expression system described by Lebens, et al. (BioTechnology, supra).
  • the fusion proteins are isolated by GM1 affinity purification as disclosed by Tayot, et al. (Europ. J. Biochem. 113. 249-258) and dialyzed against PBS.
  • the specific protein concentrations of the samples used for immunization are determined by ELISA using a CTB standard.
  • the chimenc 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 mg for repeated immunizations.
  • 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 3-10 ml for rectal or vaginal immunization.
  • the oral immunization is likely to be given together in a pharmaceutical acceptable buffer containing about 25-200 ml liquid containing about 2 grams of sodium bicarbonate or equivalent acid buffering agent.
  • Serum, mesente ⁇ c lymphnodes, spleen, cervix, vagina, small intestine, colon and rectum were collected from mice sacrificed (and perfused with heparin containing buffer) one week after the last immunization dose with the preparation of Example 1 using the immunization protocol of Example 8.
  • the organs were frozen and then extracted with 2% (WfV) of saponm using the PERFEXT method (Quidmg, et al. J. Clin. Invest. 88(11:143-148, 1991 ).
  • the sapon extracts were tested for IgA and IgG anti-CTB and anti-VDIV antibodies by ELISA.
  • ELISA plates were coated with GM1 (0.3 nmol/ml) or A8 VDIV peptide (1 ⁇ g/ml) respectively.
  • immunized mice were of the C57/B1 strain and were 8-10 weeks old at the onset of immunizations.
  • the immunization protocol was 1 mg medroxyprogesterone acetate (Depoprovera; UpJohn Company, Kaiamazoo, Ml) subcutaneously 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.
  • mice of strain C57B1/6 were immunized intraperitoneally (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 chimenc proteins, with Freund's adjuvant. All mice responded with high titered serum anti-CTB IgG-responses. As noted above, the ten ammo acid HIV epitope induced serum IgG-respo ⁇ ses against gp120 when placed between ammo acids 55 and 64 of CTB.
  • a weak response against g l 20 was also induced by the gp 20 moiety in the mice immunized with the internal HIV::CTB hybrids from strain 460 (ten gpl 20 ammo acids between 53 and 64) and 586 (twelve gpl 20 ammo acids between 53 and 64) and by the N- terminally placed gp120-epitope in the protein from strain 504. in fact, all these proteins showed immunogenic properties similar to the previously described HIV::CTB protein.
  • the ST decapeptide 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 i munological reactivity of the inserted peptide was modest in vitro.
  • the ST::CTB protein from strain 557 which represented the entire
  • ST peptide
  • the HBV::CTB chimeric protein from strain 395 was given either i.p. or perorally (p.o.) with cholera toxin (CT) as adjuvant to either BALB/c or C57B1/6 mice.
  • CT cholera toxin
  • High serum-anti-CTB titers where obtained in both p.o. and i.p. immunized mice, whereas the most significant levels of a ⁇ ti-pre-S(2)-ab were induced with i.p. immunizations using CT as adjuvant.
  • Table 3 illustrates the various immunization protocols and their results.
  • MOLECULE TYPE peptide
  • HYPOTHETICA NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (ix) FEATURE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (Vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: ATTCAGCGTG GTCCGGGTCG TGCTTTTG 28
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA (ill)
  • HYPOTHETICAL NO (iv)
  • ANTISENSE NO (v)
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (Vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
  • MOLECULE TYPE CDNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • FRAGMENT TYPE (vi) ORIGINAL SOURCE:
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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

IMMUNOGENS FOR STIMULATING MUCOSAL IMMUNITY
FIELD OF THE INVENTION
This application relates to methods for producing mucosal antibody to organisms capable of infecting their host through contact with mammalian mucosal membranes. In particular, this application relates to protein complexes and to gene constructs suitable for producing immunogen capable of inducing mucosal antibody as well as to methods for introducing the immunogen into a mammal to generate a mucosal immune response.
BACKGROUND OF THE INVENTION
The mucosal surfaces of the body are generally accessible to a wide variety of infectious agents capable of causing disease. These surfaces include the gastrointestinal 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 vulnerable to invasion by adventitious organisms. Not surprisingly, these surfaces are the main portal of entry into the body for foreign microorganisms.
The mucosal surfaces of the body have an extensive immune system. Non-encapsulated lymphoid tissue is dispersed throughout the mucosa of the gastrointestinal and genitourinary tracts as either diffuse aggregates of cells or as organized nodules. The diffuse aggregates of lymphoid cells are dispersed throughout the lamina propπa 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 lymphoid cells along the mucosal surfaces are capable of responding to foreign antigen. The gut epithelium overlying the Peyer's patches allows transport of antigens into the lymphoid tissue and is capable of functioning as antigen presenting cells (Bromander, et al. Scand. J. Immunol. 37*452458, 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 invasion of adventitious organisms into the mucosa will preferably stimulate IgG and slgA. An effective vaccine for limiting mucosal infection will likely demonstrate slgA activity.
Many sexually transmitted diseases such as chlamγdia, gonorrhea, syphilis, chancroid and tπchomoniasis are caused by organisms that enter the body through the mucosal membranes. While these diseases are caused by different organisms and replicate in different ways, these sexually transmitted microorganisms, and others, all enter the body primarily via the mucosal barrier. These and most other disease causing organisms carry unique antigenic determinants that are known to stimulate the immune system. In addition, the mucosal surfaces are also the mam portal of entry for most viruses. Thus, mucosal surface immunity to viruses including, but not limited to influenza, papiliomavirus, 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 these organisms have met with little success since pareπteral vaccination does not generally produce significant levels of secretory immunity. The non-viral sexually transmitted diseases can usually be cured if they are diagnosed early, but many of the diseases produce mild early symptoms, if any, and thus go untreated until more advanced symptoms occur.
Chlamydia is a useful example of a sexually transmitted disease that infects its host through mucosal membranes, primarily of the genitourinary system Chlamydia trachomatis is the leading sexually transmitted organism in the United States, afflicting an estimated four million people a year (Division of STD/HIV Prevention, 1992 Annual Report, CDC, Atlanta, 1993). Chlamydia is acquired chiefly through vaginal or anal intercourse, although it can also be transmitted through oral sex. C trachomatis infection of the genital tract can cause salpingitis in women that can result in tubal blockage and infertility. It is estimated that in the United States 200,000 women per year become infertile as a result of chlamydial salpingitis. Moreover, infected individuals are at an increased risk of acquiring HIV if exposed to the virus (Wasserhiet. "Epidemiological Synergy: Interrelationships Between Human Immunodeficiency Virus Infection and Other Sexually Transmitted Diseases," Sexually Transmitted Diseases, 19: 61- 77, 1992). Improved methods for controlling the pathological manifestations of this disease are seriously needed.
Chlamydia trachomatis isolates occur as 15 distinct serovars that are divided into three subgroups. The major outer membrane protein IMOMP) of Chlamydia confers serovar and serogroup specificities Protective immunity to Chlamydia is directed to the major outer membrane protein.
Comparative analysis of the major outer membrane protein of Chlamydia indicates that the MOMP genes are conserved and have four variable region domains that are unique to each serovar These domains elicit the formation of serovar, subspecies, and serogroup or species-specific antibodies Variable domain IV (VDIV) is the largest of the MOMP variable domains and is located near the C-terminus of the protein VDIV contains subspecies, serogroup antigenic determinants and a conserved species-specific antigentc determinant. All of the variable domains of MOMP are external epitopes as demonstrated by their sensitivity to trypsm and by their accessibility to antibody binding This invention contemplates combining determinants from MOMP or other protein from pathogens, including but not limited to HIV, hepatitis and enterotoxigenic E. coli capable of infecting a mammalian host through contact with mucosal membrane with a mucosal binding polypeptide Several agents have proved effective as carriers and as adjuvants for stimulating mucosal immunity (for a review of these agents see Bienenstock, J "The Nature of Immunity at Mucosal Surfaces A Brief Review " In Bacterial Infections of Respiratory and Gastrointestinal Mucosae. Eds. Doncachie, W, et al IRL Press, 1988. pp. 9 18.). Accessible epitopes from various infectious agents have been engineered onto the surface of attenuated live vectors such as Vaccinia or Salmonella typhtmυrium (see Flexner, et al "Vaccinia as a live vector carrying cloned foreign genes. In- New Generation 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 allow immunologically accessible epitopes to be presented to the immune system as the product of a genetic construct or as a peptide chemically coupled to a carrying agent. Such non-living carrier systems include microparticles, liposomes, solid matrix antibody antigen complexes, immunostimulating complexes (ISCOMs) and protein carriers including the core antigen of hepatitis B virus, polio virtons, cholera toxin and the heat-labile enterotoxin from E coli The carriers themselves may have endogenous adjuvant activity or alternatively, exogenous adjuvant can be used with the carriers. Ox bile has been used as an adjuvant for orally administered immunogens such as killed oral vaccines against dysentery. Other adjuvants that have been tested for use in inducing mucosal immunity include DEAE-4 dextran, iysozγme, polyormthine, sodium dodecyl benzene sulfate, lipid-conjugated substances, streptomycin and vitamin A (see Holmgren, et al. Vaccine 11:1170 1184, 1993). In addition, agents such as muramyl dipeptide, acndine and cimetidiπe have had some positive effect as well (see Bienenstock, et al. supra.)
Cholera toxin is capable of generating mucosal immunity and is also a potent adjuvant 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 of five binding B subunits assembled as a ring together 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. Immunology 59.301 308, 1986) to cholera toxin and to unrelated antigen. Similarly, the heat-labile enterotoxm of Eschenchia coli also has immunomodutatmg and adjuvant properties Cholera toxin coupled to foreign antigen has been used to stimulate immune responses in the gastrointestinal tract. For example, chemical coupling of the cholera toxin B subunit to streptococcal antigen has evoked antibody responses in the mucosal surfaces of the gut and in salivary glands (Czerk sky, et al. Infect. Immun. 57.1072-1077, 1989) Cholera toxin/Sendai virus conjugate immunization has resulted in the production of Sendai-specific immunoglobu n in the respiratory tract. While cholera toxin has been used to stimulate the immune response to bacterial and select viral antigens along the gastrointestinal 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 immune 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 ammo acid residues to either the N or C terminus of CTB has in some cases interfered with the folding and assembly of the final product (Sandkvist, et al., J Bacterioi. 169:4570-4576, 1987 and Clements J.D. Infect Immun 58.1 159 1 166, 1990). When some of these constructs were produced in V. cholerae the foreign epitopes were cleaved off, presumably by bacterial proteases (Schόdel, et al., Gene 99:255 259, 1991 ). Therefore, there remains a need for improved cholera toxin derived immunogens. Unlike the prior art, protein complexes and genetic constructs are disclosed in this invention that stimulate mucosal immunity to pathogens 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 also discloses strategies for improving the immune response to the foreign antigen using a variety of genetic constructs BRIEF DESCRIPTION OF THE FIGURES
Figure 1 diagrams the strategy for producing the pMLΛ £ coli expression vectors of the present invention.
Figure 2 provides preferred examples of expression vectors using the tac promoter. Figure 2(a) diagrams a plasmid expressing CTB from the tac promoter. Figure 2(b) illustrates the same vector as in Figure 2(a), additionally carrying the AB gene fragment used for the construction of CTA2 protein fusions. Figure 2(c) diagrams a plasmid, as disclosed in Figure 2(b), additionally containing the iacP gene to facilitate inducibie X-gal expression of ctxkB. Figure 2(d) diagrams a plasmid carrying the modified ct-rB gene for in-frame internal epitope insertion between the unique Kpnl and Mscl sites. Figure 3 diagrams plasmids expressing CTB from the leftwards promoter of phage lambda for the generation of CTB fusions (pML-LCTBΛ7) or CTA fusions (pPJΛ).
Figure 4 illustrates the oligonucleotide sequence, SEQ ID NQ:5, used to regenerate the ctrA signal peptide and the unique restriction sites used for cloning foreign epitopes into the construct.
Figure 5 provides synthetic oligonucleotide sequences, SEQ ID N0:24 and SEO ID N0:25, used for the insertion of chlamydial T-cell and B-cell epitopes into the ctxk gene. Boxed sequences represent the chlamydial peptides.
Figure 6 provides a diagram of the oligonucleotide sequences, SEQ ID N0:7 and SEQ ID N0:8 which were used to form the linker to join the chlamydial epitopes A8 (SEQ ID N0:6) and VDIV (SEQ ID N0:1). The adjacent Sad and Nhel sequences for pML LCTBtøc or pML-LCTBΛ and pPJVDIV are also diagramed in this Figure. Figure 7 diagrams the construction of plasmid pCB55-64gp309 encoding an exemplary CTB::hybrιd protein.
The diagram provides an illustration of the parent plasmid, pML-LCTBtøc, positioned to accept foreign antigemc sequences. Plasmid pCB55-64gp309 contained the HIV antigemc sequence derived from ammo acids 309 318 of gp120. The gpl 20 nucleic acid sequence positioned between the Kpnl and BssHII restriction endonuclease sites is provided in the enclosed box with insert gpl 20 sequences shown in italics.
SUMMARY OF THE INVENTION
This invention relates to immunogens useful for stimulating mucosal immunity and for protein complexes 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 relates to methods for inducing mucosal surface immunity to a pathogen capable of infecting a mammalian host through mucosal membranes.
In one embodiment of this invention a mucosal binding composition is contemplated that comprises a mucosal binding polypeptide linked to at least one antigen from a non-viral pathogen where the pathogen causes a sexually transmitted 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 of the A subunit of cholera toxin. In a preferred embodiment the antigen from the non-viral pathogen is an antigen from Chlamydia.
It is contemplated that the chlamydia antigen can be positioned at the ammo terminus of the binding subunit of the cholera toxin, at the ammo terminus of the portion of the A subunit of cholera toxin or positioned internally within the binding subunit of cholera toxin. It is contemplated that the antigen can be linked to the mucosal binding protein by recombinant or chemical means.
In a preferred embodiment, the antigen comprises a B-cell stimulating antigen from the major outer membrane protein of chlamydia. In a particularly preferred embodiment, the B-cell stimulating antigen is from the VDIV region of the major outer membrane protein of chlamydia. in yet another preferred embodiment, the antigen further comprises a T-helper cell stimulating antigen and preferably, this T-helper cell stimulating antigen is also from the major outer membrane protein of chlamydia. In one embodiment the T-helper cell stimulating antigen is from the major outer membrane protein of chlamydia and in a preferred embodiment is the A8 region from the major outer membrane protein of chlamydia.
In another embodiment of the present invention, a method is disclosed for generating a mucosal immune response against a non-viral sexually transmitted disease, comprising contacting the mucosa of a mammalian host with the mucosal binding composition
In yet another embodiment of the present invention, recombmant polynucleotides are disclosed which comprise a first region encoding a mucosal binding polypeptide and a second region encoding an antigen of a non-viral pathogen, where the pathogen causes a sexually transmitted disease. In a preferred embodiment the mucosal binding polypeptide is the binding subunit of cholera toxin and the pathogen is chlamydia. In this embodiment, the preferred antigen includes a T-helper cell stimulating antigen and a B-cell stimulating antigen from the outer membrane protein of chlamydia. In a particularly preferred embodiment, the B-cell stimulating antigen is from the VDIV region of the major outer membrane protein of chlamydia.
This invention also relates to methods for vaccinating a mammal 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. In a preferred embodiment the administration is vaginal and in other embodiments, the vaccine is delivered rectally or orally.
The invention additionally relates to mucosal binding compositions comprising a mucosal binding polypeptide linked to at least one antigen of a viral pathogen where the pathogen causes a sexually transmitted disease. In a preferred embodiment the mucosal binding polypeptide comprises the binding subunit of cholera toxin and the antigen is a HIV gpl 20 antigen. In yet another embodiment the antigen is a Hepatitis B virus pre-S(2) antigen and in a further embodiment, the mucosal binding polypeptide is linked to at least one antigen from the ST, protein of enterotoxige c £ coli.
The invention additionally contemplates purified recombinant polynucleotides comprising nucleic acid encoding a mucosal binding protein operably linked to a B-cell stimulating antigen where the antigen is a peptide obtained from a pathogen capable of infecting a mammal through the mucosal membranes. In a preferred embodiment, the mucosal binding protein encodes the binding subunit of cholera toxin and in another 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 includes the ammo acid sequence LNPTIAG. In one embodiment, the B-cell stimulating antigen is from HIV gp120. 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 am o acid sequence LNPTIAG, an antigen from the gp120 protein of HIV, the Hepatitis B virus pre-S(2) protein or the ST, protein of enterotoxigemc £ coli. 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 DESCRIPTION OF THE INVENTION
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 "immunoaccessible" is used herein to describe antigemc sequences 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 protem or a nonproteiπaceous molecule including carbohydrate, lipid, nucleic acid or the like, which is capable of stimulating T-helper ceil activity in standard T helper cell assays, well known in the art, either alone or 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 nonprotemaceous molecule, including carbohydrate, lipid, nucleic acid or the like, which is capable of stimulating antibody production from B-cells. The terms "ctxA" and "ctxB" refer to the gene sequences encoding the cholera toxin A and B subunits respectively.
The term "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 mammalian host through the mucosal membranes of the host. The term "mucosal binding polypeptide" 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, bacterial toxin membrane binding subunits including, at a minimum, the B subunit of cholera toxin, the B subunit of the £ coli heat-labile enterotoxin, Bordetella perτussis toxin subunits S2, S3, S4 and/or S5, the B fragment of Diphtheria toxin and the membrane binding subunits of Shiga toxin or Shiga-iike toxins.
Other mucosa binding subunits contemplated within the scope of this invention include bacterial fimbriae protein including E. coli fimbria K88, K99, 987P, F41, CFA/I, CFA/II (CS1 , CS2 and/or CS3), CFA/IV (CS4, CS5 and/or CS6), P fimbraiae, or the like. Other fimbriae contemplated within the scope of this invention include Bordetella pertussis filamentous hemagglutinin, vibrio cholerae toxin coregulate pilus (TCP), Mannose sensitive he agglutinin
(MSHA), fucose-sensitive hemagglutinin (PSHA), and the like.
Still other mucosal-binding molecules contemplated within the scope of this invention include viral attachment proteins including influenza and Sendai virus hemagglutinins and animal lectins or lectin-like molecules including immunoglobulin molecules or fragments thereof, calcium-dependant (C-type) lectins, selectins, collectiπs or Helix pomatia hemagglutinin. Plant lectins with mucosa-binding subunits include concanavalin A, Wheat-germ agglutinin, phytohemagglutinin, abriπ and ricin.
The invention discloses the use of mucosal binding compositions to stimulate mucosal immunity to pathogens capable of infecting a mammalian host through the mucosal membranes of the host. In one preferred embodiment of this invention, a method for producing mucosal immunity to a pathogen is disclosed using the mucosal binding subunit of bacterial toxins. As one aspect of this embodiment, chimeric constructs of either the cholera toxin or the
£ coli heat-labile enterotoxin are coupled to antigen obtained from pathogens capable of causing a disease. In another aspect of this embodiment, methods are disclosed for chemically coupling a mucosal binding polypeptide such as the mucosal binding portion of the B subunit of cholera toxin or of the heat labile enterotoxin of £ coli to antigen obtained from 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 immunoglobulin. This secretory immunoglobulin can be used in diagnostic assays, such as
ELISAs, im unoblots, 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 the foreign epitope. Moreover, the secretory antibody collected from experimental animals can be used in topical preparations against a specific pathogen or as a general component of a topical preparation or in additional studies to assess the character of secretory immunoglobulin.
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 synthesized 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 polypeptide for its ability to function as a carrier and to facilitate foreign antigen presentation 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 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 provided above. For recombinant protein expression, those skilled in the art of molecular biology will isolate nucleic acid fragments encoding this gene, using standard techniques known in the art, and incorporate these fragments into expression constructs. There are a wide range of eukaryotic and prokaryotic expression systems known in the art and it is recognized that the skilled artisan will be able to incorporate a nucleic acid fragment encoding a mucosal binding polypeptide into a variety of gene expression systems without undue experimentation. Exemplary gene expression constructs are provided here which are suited for gene expression in £ coli or V. Cholerae. Alternatively, the mucosal binding polypeptide can be isolated from nature using known purification techniques.
The recombinant or purified mucosal binding polypeptide is next tested for its ability to be bound by test antibody where the test antibody is known to recognize the naturally occurring mucosal binding polypeptide. Suitable tests for determining antibody recognition include ELISAs, immunoblots or other well known immunologic assays available in the art. Exemplary ELISA and immunoblot assays are provided in the Examples (see Example 4 and 6). These assays are well known and can be readily modified by those skilled in the art to identify other mucosal binding 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 lysates or purified mucosal cell membrane protein. 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 biopsy). 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 It's CRL 1459, CRL 1539, CRL 1541 and CRL 1790), nasal septum (CCL 30), palate (CRL 1486), rectal cell lines (CCL 235, CCL 234), vulva-derived cell lines (HTB 88, HTB 1 17 and HTB 1 18), 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 binding to the mucosa surface. Alternatively, 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 of the Vibrio cholerae 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 similarities 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. 4873 77, 1985 and see TSUJI, et al. Microbial. Pathogenesis 2:381-390, 1987). LTB, like CTB, can be secreted into the medium when V cholerae cultures are transformed with expression vectors expressing recombinant LTB Further, CTB and LTB bind to gangliosides o mucosal membranes (see Hirst, et al Proc. Natl Acad Sci USA 83:9174-9178, 1984 and Schodel, et al. Gen 99:255 259, 1991). The Hirst, et al reference indicates that CTB sequences can be replaced with LTB sequence for recombinant expression. Thus, it is contemplated that CTB sequences can be replaced with LTB 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 ability 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 labile enterotoxin from £ coli. The non- limitmg examples, provided below, include a variety of compositions employing mucosal binding 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 least one antigen from a pathogen that enters its host through the mucosal membranes. This invention contemplates that the immunogen derived from the this pathogen can be selected from a variety of immunogens which are known to stimulate immune responses in a mammal susceptible to the disease caused by that pathogen. It is contemplated that the antigen includes at least one antibody stimulating determinant, preferably from a surface protem of the disease-causing pathogen. It is also contemplated that where the antigen is a polypeptide, it will contain at least one consecutive region of ammo acids, preferably from an immunoaccessible domain of the surface polypeptide of the pathogen, in addition, the 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 ammo acid sequences, multiple copies of these sequences or am o acid sequences that stimulate T-Cells or those that assist in the generation of an antibody response through the activation of T-Helper cells or other T-Cell populations Further, it is contemplated that the antibody stimulating determinant can be repeated in tandem or separated by suitable linking sequences, or the like, to further stimulate the antibody response.
Thus, the first step for selecting an antigen contemplated in this invention is to identify a non-viral pathogen capable of infecting a mammal via entry through a mucosal membrane. Next, one can optionally determine whether those mammals who are infected with the pathogen develop an antibody response to the pathogen. This can be determined experimentally or based on the literature related to the particular pathogen. 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 contacting the serum sample with cell lysates containing pathogen, or alternatively with intact pathogen, and detecting binding of serum antibody to the sample Assays useful for detecting serum antibody binding include enzyme-linked immunosorbent assays (ELISA), immunoblots such as Western blots, or the like. Such assays are well known in the art and are detailed in a variety of methodology texts including Harlow, et al. (Antibodies- A Laboratory Manual. Cold Spring Harbor Press, 1988). Optionally, those skilled in the art 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 concentration method well recognized in the art This sample is then tested for the presence of pathogen-specific immunoglobulin, in general, or slgA or IgG, specifically, using standard immunoassays well known in the art. Alternatively, those skilled in the art can determine which protein or proteins stimulate a neutralizing 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 using any number of strategies well known in the art. As one example of a method for mapping antibody 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 Today 5:397400, 1989 respectively).
These screening regimes permit the identification of determinants capable of stimulating the immune system and are also well known in the literature for a variety of pathogens, including chlamydia. Methods are also well known in the scientific literature for selecting linear antigemc determinants that stimulate antibody production from B-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. U Exp. Med. 172.203-212, 1990). To identify epitopes that stimulate antibody production in C. trachomatis see Zhong, et al. (Infect, and Imm. 59.1141-1147, 1991 ). Finally, it is contemplated that peptide mapping strategies, 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. (J Immunol 438:575-581 , 1987).
All of these steps permit one skilled in the art to identify candidate polypeptide sequences that can be linked to a mucosal binding polypeptide. These steps provide one strategy for identifying 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 to a mucosal binding polypeptide. These strategies are detailed in the examples below (see Examples 4 and 6).
As a first step for producing the composition of this invention, the mucosal binding protein is linked 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 specific 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. (Bio Technology 1 1.1574-1578,
1993) and chemically coupled to an immunogen derived from the major outer membrane protein of C trachomatis. In one preferred embodiment employing chemical coupling, the polypeptide derived from the major outer membrane protein is the VDIV peptide (SEQ ID NO: 1 ) corresponding to a portion of the fourth variable domain of the major outer membrane protein of C. trachomatis. In another preferred embodiment, the polypeptide is a linear chimeπc polypeptide that comprises the A8 domain and the VDIV domain from the major outer membrane protein of C. trachomatis (SEQ ID NO:2). Both the A8 and the VDIV domain as well as the chimenc peptide are described by Su, et al. [Vaccine 11:1159-1166, 1993).
As another embodiment of this invention, and as an example of alternative linking strategies, a polypeptide from a protein derived from a non-viral pathogen causing a sexually transmitted disease is expressed as a recombmant protein with a subunit or fragment of a mucosal binding protein. As one example of the recombinant expression of this complex, the mucosal binding protein is a portion of the cholera toxin and the antigemc determinant is derived from C. trachomatis.
It is contemplated that there are a variety of construct designs that could be used to express protein having a portion of the cholera toxin and a polypeptide from a protein derived from a pathogen capable of infecting its host through mucosal membranes. For example, the antigemc determinant or determinants from the pathogen could be placed at the ammo or carboxy terminus of the B subunit, at the am o or carboxy terminus of the A subunit, or at the ammo 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 antigemc 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 subuntt of the heat labile enterotoxin of £ coli without undue experimentation. 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-LCTBt*c2 has an identical restriction pattern to pML LCTBtac but incorporates the pUC19 origin of replication (see Lebens, et al. Biotechnology, supra). Thus, the use of this plasmid is also contemplated within the scope of this invention. In one construct detailed in Example 2, a chtmeπc protein is produced that includes a polypeptide linked at its carboxy terminus to the ammo terminus of the B subunit (CTB) from cholera toxin. As another example to indicate that this construct is similarly useful for producing stimulating antibody to other antigens, the HIV epitope IQRGPGRAFV is incorporated into the ammo terminus of the B subunit.
In another example of this embodiment, detailed in Example 3, the antigen from the pathogen is incorporated onto the ammo terminus of a polypeptide which is a fusion protein of a fragment of the A subunit of cholera toxin fused to CTB and expressed in either V. cholera or £ coli. In one aspect of this embodiment, the antigemc peptide is the VDIV sequence from the major outer membrane protein of C. trachomatis and in another preferred embodiment the antigemc peptide is the A8 sequence from the major outer membrane protein. In a third aspect of this embodiment, the peptide is a chimenc protein containing both the antigemc peptide sequences of A8 and VDIV separated by a linker (see Figure 6). It is also contemplated that this same linker introduced into, for example, pML- LCTB/1J, would facilitate the construction of multiple copies of either epitope Alternatively, the linker, or other linkers, could be used to insert the A8 and VDIV sequences into other constructs or the linker could facilitate the transfer of inserted sequences between vectors. It is urther contemplated within the scope of this embodiment that the order of the antigemc sequences can be switched and that multiple determinants of A8 or VDIV can be linked either in tandem or within the recombinant construct. The literature describes a variety of antigemc peptide sequences for C. trachomatis that are serovar specific, subgroup specific or are broadly conserved among serovars. Thus, it is also contemplated within the scope of these embodiments that other antigemc peptides from the major outer membrane protein or from other proteins of C. trachomatis would also be useful in stimulating antibody production along the mucosal surfaces.
Job ng, et al. (Infect, and Imm. 60:49154924, 1992) have produced fusion proteins in which entire bacterial proteins were linked via the ammo terminus to the A subunit fragment, CTA2. Whereas Job ng, et al. demonstrated that chimenc toxin could be produced from a cholera toxin construct, both portions of the chimera were bacterial protein and the signal sequences for chimenc expression were derived from the inserted bacterial protein. The present invention demonstrates that antigemc sequences which are foreign to the bacteria can be inserted into CTA2. The CTA2 fusion proteins with foreign antigemc sequences were coexpressed with CTB from V. Cholera to give assembled products which were excreted extracellularly and were detectable in a GM1 -ELISA. The gene products produced by Job ng et al. were 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 immunoaccessible site, in one example of this strategy, it is contemplated that a short foreign polypeptide is introduced into an internal region of the cholera B subunit in place of CTB am o acids 56-63. In one embodiment, this peptide includes the VDIV epitope, LNPTIAG, and in another embodiment, illustrating that this strategy can be used for other peptide sequences, the peptide is the HIV neutralizing epitope IQRGPGRAFV (aa 309-318 of HIV 1 isolate HTLV-ltIB).
Six additional plasmids were constructed, encoding internal HIV::CTB hybrid proteins with ten to fourteen ammo acids from the V3 loop of gp120 genetically inserted at different positions between ammo acids 52 and 65, with deletions of different CTB (Cholera toxin B subunit) am o acids (See 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 monoclonal 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 enterotoxigemc £ coli. Plasmids were constructed encoding CTB hybrid proteins which had either an eleven ammo acid peptide form Hepatitis B Virus (HBV) pre-S(2) (SEQ ID NO: 28) or one of two peptides (SEQ ID N0:30 and SEQ ID NO: 31 , see Table 1 ) related to the heat-stable toxin (ST.) of enterotoxigemc £ coli. This work demonstrated that an internal permissive site could be used to insert peptides of several different ammo acid compositions. Epitope-specific antibodies were used in GM1 -ELISA and immunoblot assays to screen the protein hybrids for their conformational integrity, for their ability 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 products stimulated strong serum antibody responses against CTB.
Data is provided in Example 4 to indicate that the CTB::foreign antigen hybrids of the present invention retain all of the important characteristics of native CTB, such as folding, pentamerization, extra-cellular secretion when produced in V. cholerae and GMI-binding. Moreover, many of the inserted peptide constructs were resistant to cleavage by V. cholerae proteases. The data in this example indicated that the CTB::foreign antigen hybrids reacted with monoclonal antibody directed against the foreign antigen, both in the denatured and non-denatured forms of the protein. This indicated that the substitution of eight amino acids from a region in the native molecule with amino acids from an unrelated protein, such as gp120, VDIV, pre-S(2), and ST, produces a construct with foreign antigen 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 be modified to improve the immunogenicity. 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 sequence and that these modifications do not limit the scope to this invention.
Once 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. Methods for detecting hybrid protein expression are provided in Examples 4 and 6. In vitro assays testing mucosal binding are useful for demonstrating the intactness of the complex. Such assays include binding assays to intact mucosal epithelium cell cultures or mucosal membrane sections. If the mucosal receptor is known, specific ELISAs or receptor/iigaπd chromatographic 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 composition that 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, rectallγ, nasally, intramuscularly, intraperitoneally or intravenously. Sera or mucosal immune responses are monitored in the experimental mammal or in clinical trial by ELISA, immunoblot, or by western blot to detect antibody to either the mucosal binding polypeptide or the foreign polypeptide. It is contemplated that antibodies in serum or antibody in mucosal 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 plug, or the like. Similarly, biopsy of mucosal tissue during the testing phases of this invention will be used to assay, by histopathology or by cell activity, for immune responses specific to the mucosal binding polypeptide. For exemplary methods for preparing biopsy tissue see Eriksson, et al. or Quiding, et al. ("Perfext: A Simple Perfusion-Extraction Procedure for Quantitative Analyses of Antibodies and Cytokines in Distinct Anatomical Compartments" Abstract presented at the 12th European Immunology
Meeting. June 14-17, 1994 and J Clin. Invest. 88(11:143-148, 1991 respectively). Strategies for detecting IgG and IgA in a mammal are known in the art. These include the measurement of antibodies in serum or mucosal secretions using ELISAs, 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 intracham 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 possible 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 one to successfully perform the intended invention.
EXAMPLE 1
Chemical Coupling of Polypeptide to a Membrane Binding Protein
In this example, Cholera toxin B subunit (CTB) was produced in a mutant strain of Vibrio cholerae deleted of the cholera toxin genes and transfected with a plasmid encoding CTB (as disclosed by Lebens, et al. supra). In this expression system, CTB was recovered as secreted protein in yields at or above 1 g/l. Bacterial cultures were centrifuged at 8000 rev per mm for 220 mm and the supernatants were adjusted to pH 4 5 with dilute HCI. After precipitation with hexametaphosphate (final concentration of 2.5 g/l) for 2 hr at 23°C followed by centrifugation at 8000 rev per mm, the pellets were dissolved with 0.1M sodium phosphate buffer, pH 8.0 and dtalyzed against 0.01 M phosphate-buffered saline (PBS), pH 7.2. The dialysate was then centrifuged at 15,000 rev per mm to remove insoluble material and the supernatant was further clarified by filtration through 0.22 μm filters (Millipore, Bedford, MA). CTB was purified by standard gel filtration chromatography through columns of Sephadex G 100 (Pharmacia, Upsala, Sweden).
The polypeptides were covalentlγ conjugated to CTB using N succιnιmιdyl(3-[2-pyrιdyl] dιthιo) propioπate (SPDP, Pharmacia) as a bifunctional coupling reagent according to the manufacturer's instructions CTB was derivatized with SPDP at a molar ratio of 1 :5 in 0.1M phosphate buffer/0.1 NaCl pH 7.5. After incubation at 23 °C for 30 mm., excess SPDP was removed by gel filtration through Sephadex G 25 columns (Pharmacia) and eluted with PBS. The absorbance of the modified protein was measured at 280 nm. To estimate the degree of substitution with 2-pyrιdyl disulphide residues, the absorbance at 343 nm of 100 μl protein solution in 400 μl was measured after incubation with 50 μ\ dithiothreitol (0.1 M) for 15 minutes (molar extinction coefficient at 343 nm - 8.08 x 103 M 'c 1). This concentration was equivalent to the concentration of 2-pyrιdyl disulphide residues in the protein. Since the 2-pyrιdyl disulphide groups contribute to the absorbance at 280 nm, a correction was applied to the calculation of protein concentration: A2M due to the protein - kM ■ (B x 5.1x103) where B is the molar concentration of pyridine-2-thione released. From these results the number of moles of 2-pyridyl disulphide substituted per mole of protein were calculated. The substitution ratio obtained with these conditions ranged between 2-3 moles of SSPY per mole of CTB pentamer. Peptides and SPDP-derivatized CTB were mixed at a ratio of 5 mol peptide/ 1 mol SSPY and incubated for
24 hr at 23°C. The resulting CTB eptide conjugates were purified by gel filtration through columns of Sephadex 6-25. Conjugates were purified over GM1 -columns of Sephadex G-25. Purified conjugates were shown to retain GM1 -binding capacity and to retain both CTB and peptide specific serological reactivities by means of a solid phase ELISA using GM1 as a capture system and enzyme-labelled antibodies to CTB or to the foreign polypeptide sequence coupled to CTB. An exemplary ELISA using GM1 is provided in Examples 4 and 6, below.
In this embodiment, three different peptides were conjugated to CTB. The first, Peptide 166 (SEQ ID N0:13, A8-VDIV), is the colinear peptide disclosed by Su, et al. (Vaccine 11:1159-1 166, 1993). It contains a single cysteine residue. Peptide 172 (SEQ ID NO: 14) has a free extra cysteine coupled to the amino end and Peptide 173 (SEQ ID NO: 15) has a free extra cysteine coupled to the carboxyl end of the A8-VDIV sequence. The CTB/A8-VDIV complexes were introduced into C57 BL/6J female mice (obtained from the Animal Care
Facility of the Department of Medical Microbiology and Immunology, University of Goteborg). Mice were 6-8 weeks of age. The immunization protocol for generating mucosal antibody is provided in Example 8.
EXAMPLE 2 Genetic Fusion of Foreign polypeptide to the amino terminus of CTB
In one embodiment, the starting plasmid was pPL-lambda obtained from Pharmacia AB. Sweden. The generation of the pMLΛ 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 promoter region was recovered by band-extraction. More of the same plasmid was digested with BamHI and Pvull and again resolved on an agarose gel. This time a 2,305 bp BamHI/Pvull 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 £ coli strain N4830-1, the Gal P2 transductant of N4830 (Pharmacia, Sweden). In the resulting plasmid, pMLΛ I, the orientation of the phage lambda DNA within the plasmid has been reversed and 1 ,689 bp of pBR322 DNA has been removed. pMLΛ 1 was digested to completion with BamHI and subsequently partially digested with Haelll. The digested DNA was religated and used to transform £ coli strain N4830-1. Transformants were screened on the basis of restriction analysis for plasmids in which the lambda N gene and tL 1 terminator had been removed. The resulting plasmid was pMLΛ2 and carried the promoter ΛP, with unique downstream restriction sites BamHI, Smal and EcoRI that could be used for cloning of recombinant genes. The powerful trpk transcription terminator (derived from the trpk cassette, Pharmacia) was introduced between the unique EcoRI and Aatll sites within pMLΛ2. At the same time, an additional unique Xhol site and a Hindlll site that could be used in cloning procedures were introduced into the plasmid (see Figure 1 ).
The unique Ndel site within the plasmid was removed 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 which generated BspEI sites on either side of the introduced EcoRI site. The resulting plasmid was digested with BspEI and religated. This plasmid was used for recombinant expression in £ coli.
Fusions of peptides to the ammo terminus of CTB were generated by inserting synthetic oligonudeotides between unique Sad and Sspl sites in the expression vector pML-LCTBΛ7 (see Figure 3). For example, the HIV RP335 peptide was placed at the ammo terminus of CTB in pML-LCTBΛ7 between the Sa and Sspl sites using two oligonudeotides: SEQ ID N0:9 and SEQ 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 site and at the same time destroying the Sspl site. Oligonudeotides corresponding to SEQ ID N0:11 and SEQ ID N0:12 were used for this vector construction. The ctxB gene in this vector is under the control of the inducible AP, promoter and was constructed essentially as described previously (Lebens, M. et al BioTechnology 1 1 :1574-1578, 1993). The vector was chosen in order to allow the construction of genetic fusions, the products of which may be deleterious to the host cells since the inducible system only allows expression under inducing conditions. Thus, using these constructs, cultures can be maintained under conditions in which expression is not switched on and the recombinant 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 p Ltac derivative plasmids. This permitted expression of the resulting 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 overexpression system for CTB and its derivatives, the 1689bp Pvull/BamHI fragment was removed. This was achieved by digestion of a plasmid into which the ctxB gene had been inserted with Pvull and BamHI, followed by blunt end repair of the plasmid with Klenow enzyme and subsequent 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 rrn_ T1T2 transcription terminator was replaced with the trpk terminator described above
A further development of the vector was to introduce the lac\ gene upstream of the tac promoter to make the vector produce cloned gene products in an inducible manner. This was done using PCR The Ptør-based expression plasmids of this invention (see Figure 2) were amplified using SEQ ID N0 16 and SEQ ID NO: 17. The /aclq gene was obtained by PCR fragment amplification of expression plasmid pMMB66 (Furste, et al. Gene 48:1 19 131 1986) using SEQ ID NO: 18 and SEQ ID N0:19. The lac\t gene was introduced between BamHI and Bglll restriction sites so that it could be removed easily to generate a plasmid giving constitutive expression from the tac promoter (see Figure 2). In a third embodiment, the same gpl 20 peptide was fused to the N-terminus of CTB by inserting complementary synthetic oligonudeotides encoding the peptide between the Sad and Xma\ restriction sites at the junction between the leader peptide and the mature CTB in the expression plasmid pJS752-3 (ApR), a derivative of pJS162 (Sanchez, et al. Proc. Natl. Acad. Sci. USA 86: 481485, 1989). This plasmid is essentially the same as pML-LCTfltar except that the EcoRI/Hindll fragment carrying the recombinant ctxB gene is derived from pJS162. The parent plasmid is the expression vector pKK223-3 (Pharmacia). Plasmid JS752-3 carries the gene encoding CTB under tac promoter control. In plasmid pJS54 (see Example 4, below), an oligo (formed from the hybridization and ligation of oligonudeotides corresponding to SEQ ID N0:20 and SEQ ID N0:21) was inserted between unique Sacl and Smal sites in pJS752-3. The recombinant V. cholerae strain producing the N-terminal CTB::HIV hybrid protein was shown to secrete the chimeric protein into the culture medium using the GM-1 ELISA provided in Example 4. The cultured cells secreted protein reactive with mAb F58/H3-aπtr-gp 120 when analyzed in a GM1-ELISA. The GM1-ELISA was useful as a tool to quantitate the amount of expressed protein and to monitor the extent of proteolytic degradation of the added peptide. Culture conditions required to produce the constructs are provided in Example 5. In another embodiment, another construct with an amino terminus substitution was made in which the gp120 epitope was placed at the N-terminus of the CTB protein with two extra amino acids, corresponding to the two first amino acids of CTB, placed N-terminally to the gpl 20 peptide, and this extended epitope was linked directly to the complete mature CTB. The resulting plasmid, pCB2gp309-318, was expressed in V. cholerae JS1569 (strain 644). When cultured at 37°C, a protein which bound to GM1 reacted with the CTB-specHic mAb LT39 and was produced within the periplasm. Unlike native CTB, it was not actively secreted into the medium. However, the protein produced by strain 644 reacted with mAb P4/D10 increasingly with time. The fused gp120 peptide (SEQ ID NO: 29) in this protein was more resistant to proteolytic degradation than the construct lacking the N-terminal amino acids of CTB. The plasmid pCB2gp309-318 was also expressed in £ coli HB101 (strain 504) and could be purified from the periplasmic space by osmotic shock lysis of the cells followed by precipitation with 80% ammonium sulfate and extensive dialysis against PBS. This protein ran at a higher molecular weight than CTB in SDS-PAGE, indicating the formation of aggregates, but the protein reacted well with both mAb P4/D10-anti-gp120 and with mAb LT39 and CT6 against pentameric and mono eric CTB.
EXAMPLE 3 Genetic Fusion of Foreign polypeptide to the amino terminus of CTA-2
The plasmid used for the expression of CTA-2 was constructed so that the CTA-2 fusion and CTB were co-expressed to obtain assembly of a hoioprotein in vivo. The ct/A2 and ctx_ genes were obtained from PCVD30
(See Kaper, J. et al. BioTechnology 2, 345-349, 1984) as an Xbal/Hindlll fragment which was cloned into pUC19 (Yanisch-Perron, C, et al. Gene 33, 103-109, 1985). The ctxk ribosome binding site and signal peptide sequence were reintroduced by the insertion of synthetic oligonudeotides between the Xbal site and a unique EcoRI site upstream from it in the vector (see Figure 4). The resulting EcoRI/Hindlll fragment carrying the ctx genes contained unique Sad and Xbal sites between which synthetic oligonudeotides can be inserted to generate ammo terminal fusions between ctxkl 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, plasmid-denved expression of CTB could be demonstrated. Plasmids carrying the ctx genes under the control of the tac promoter were maintained in V. cholerae since the levels of CTB generated in this example were too high to be tolerated by £ coli. This construct lacks the recognition site for the proteolytic cleavage involved in the maturation of CTA.
The antigens used in this work were those from Chlamydia trachomatis strains identified by Su and C aid well (Su, H. et al. Vaccine 11, 1159-1166, 1993). These include a T-cell epitope A8 SEQ ID N0:6, situated within a 25 ammo acid peptide from serovar A MOMP and a B-cell epitope VOIV SEQ ID N0:1 , from serovar B MOMP containing 17 am o acids. Within the VDIV sequence a septapeptide sequence has been mapped as an epitope reacting with the monoclonal antibody DIII-A3. The ammo acid sequences of the two peptides are shown in Figure 5 together with the synthetic nudeotide sequences used for the generation of gene fusions with CTB. The oligonucleotide corresponding to VDIV and the oligonudeotides corresponding to A8 are identified.
The first of the fusions were made to the ammo terminus of CTA2. The synthetic oligonudeotides corresponding to the foreign antigen were cloned into the vectors. It was possible to express assembled protein complexes in which the VOIV peptide was associated with CTB and detectable in a GM1 ELISA assay using Mab DI1I-A3 as the primary antibody. In this case the assembled protein was produced in £ coli under the control of the ΛP1 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. 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 Sad site of plasmid pPJ-VDIVΛ using SEQ 10 N0:7 and SEQ ID N0:8. This allowed the addition of the A8 sequence to the am o end of the VDIV sequence by cloning of a Bgll/Xbal fragment from pPJAδλ into Bgll/Nhel digested pPJVDIV , 1. pPJAJM is a CTA2 fusion vector into which the oligo encoding the A8
T-cell epitope was inserted. EXAMPLE 4
Insertion of a Neutralizing Antigen in a Surface Exposed Internal Region of the Cholera Toxin B-Subunit
In this example, novel mtrachain CTB fusion protein were prepared with foreign peptide inserted into an internal region of CTB. The resulting chimeric protein retained important functional characteristics of the native CTB including: 1 ) pentamerization; 2) GM1 ganglioside receptor binding; and 3) resistance to proteolytic degradation during production of the protem in Vibrio cholerae. 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 inserted antigen. In one example, the foreign polypeptide was inserted at the position of an internal loop structure between /?4 and al in CTB, extending with a few residues into the σ-helix, as predicted by a comparison of the resolved crystal structure of LTB (Sixma, et al. Nature 351:371-377, 1991 ). Here, the VDIV fragment TTLNPTIAGAG is incorporated into the internal CTB site using the two restriction endonuclease recognition sites Kpnl and Mscl Oligonudeotides corresponding to SEQ ID NO: 22 and SEQ ID N0:23 are hybridized, digested with the restriction enzymes Kpnl and Mscl, purified and ligated to plasmid pCB56-64.
In a second example, expression vectors were constructed having HIV-1 epitopes positioned internally in- frame within CTB, the CTB expression plasmid pML LCTBtac (Apfl) was mutage zed using the polymerase chain reaction (PCR), employing a modified protocol of that disclosed by Schδdel et al. ("Hybrid hepatitis B virus core/pre-S particles expressed in attenuated Salmonellae for oral immunization." In: Brown, F.. et al. (Eds), Vaccines '91. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1991, pp. 319-325.). In the first construct, the oligonudeotides used in the PCR reactions are provided as SEQ ID N0:3 and SEQ ID N0:4. These primers incorporated a sequence encoding ten ammo acids from the central portion of the V3 loop in HIV gp120 between residue 55 and 64 in CTB Two unique restriction enzyme sites, Bsslλll and Kpnl, were also introduced, with the oligonucleotide 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 deoxynucleotides under the following reaction conditions: denaturation at 94°C for 1 m , annealing of primers at 65°C for 2 mm and elongation of DNA by Tag DNA polymerase (Boehrmger Mannheim, Indianapolis, Indiana) at 72°C for 4 m . The reaction was repeated for 30 cycles, increasing the elongation step by 2 sec with each cycle To complete all synthesized DNA strands, a final incubation step at 72°C for 10 mm was performed. After phenol/chloroform extraction, the PCR product was digested with BssrlW (Boehrmger Mannheim) according to the manufacturer's instructions, phenol/chloroform extracted again and religated using T4 DNA hgase (Pharmacia, Upsala, Sweden) at 16°C for 3 h. The ligated plasmid was electroporated into V. cholerae (strain JS1569, ctxA , ctxB') by the method of Lebens et al. (supra) The DNA sequence was confirmed by the dideoxy chain termination method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977).
The CTB region was selected for substitution because it had been reported that this region reacted with antibodies recognizing primary protein structure rather than CTB conformational epitopes (Jacob et al., EMBO J. 4:3339-3343, 1985 and Kazemi, et al., Mol. Immunol. 29:865-876, 1991). In addition, based on the crystal structure of LTB, a substitution in this region would presumably not affect the beta-sheet or alpha-helix 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 (see Sixma et al., 1991, supra). Finally, a substitution is this area would likely not interfere with the site responsible for GM1 receptor binding on the mucosal surface.
The gpl 20 peptide used for these studies was taken from ammo acids 309-318, having the sequence IQRGPGRAFV (SEQ ID N0:29), representing a portion of the V3 loop of HIV-1 isolate HTLV-IIIB The sequence numbering of gp120 is based on the Los Alamos database sequence for gp120 (Los Alamos National Laboratories, Los Alamos, New Mexico). The sequence contains a principal neutralizing B-cell determinant of HIV 1 The sequence GPGR within the peptide is conserved between several HIV-1 isolates and the peptide is of great interest m the development of a peptide-based vaccine against HIV-1 (Javaheπan et al., Proc. Natl. Acad. Sci. USA 86:6768-6772, 1989). The gpl 20 peptide replaced ammo acids 56-63 of the recombinant CTB, thus adding two ammo acids to the net number of residues in the protem. Polyacrylamide gel electrophoresis (PAGE) and immunoblot analyses of partially purified proteins from culture supernatants of V. cholerae 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 grown in 50ml modified syncase medium in 250 mi Erlenmeyer flasks shaken at 250 rpm overnight at 37 °C. These plasmids can also be grown in £ coli without adjustments in vector construction.
A number of additional plasmids were prepared which incorporated foreign antigen into the CTB sequence between positions 54 and 64 of the CTB protein. The features of the different plasmids are listed in Table 1 (see 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 SEQ ID NO: 32 and SEQ ID NO:33 as oligonudeotides to incorporate the gp120 epitope into the CTB protein. Plasmid pCB56gp309-318 used SEQ ID NO: 34 and SEQ ID NO: 35, pCB52 58gp309-318 used SEQ ID NO: 36 and 37, pCB53-64gp309-318 used SEQ ID NO: 38 and SEQ ID NO: 39, pCB53-64gp307-318 used SEQ ID NO: 40 and SEQ ID NO: 41, pCB55-64gp309-322 used SEQ ID NO: 42 and SEQ ID NO: 43, pCB55-64STdeca used SEO ID NO: 44 and SEQ ID NO: 45, pCB55-64 st, used SEQ ID NO: 46 and SEQ ID NO: 47, pCB55-64psl 33-143 used SEQ ID NO: 48 and SEQ ID NO: 49, and pCB56ps133-143 used SEQ ID NO: 50 and SEQ ID NO: 51.
Plasmids pCB53-64gp309-318 and pCB53-64gp307-318 were made by the cloning of complementary synthetic oligodeoxynucleotides (oligos) between the Kpnl and BssHll sites in pCB55-64gp309 318 with the introduction of a BamHI site in the latter. In order to construct the plasmid pCB55-64gp308 322, complementary synthetic oligos encoding ammo acids 314-322 from g l 20, carrying a BssWl site, were cloned in between the unique Λ/sΛ and Mscl sites flanking the peptide inserted between 55 and 64 in the plasmid pCB5-64gp12. A BssHlllHmdlW fragment of around 1 kb, encoding ammo acid 315-322 of the inserted epitope together with ammo acids 64 to 103 of the gene encoding CTB (ctxB) and sequence up to the terminator sequence was obtained from the intermediate plasmid and subcloned between the BssHll 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-LCTBfø, introducing nucleotides coding for am o acid 309 318 from gp120, and containing a BssHll site, either as a straight insertion at position 56, resulting in plasmid pCB56GP309 318, or with deletions of the CTB ammo acids 56-64 (pCB55-65GP309-318) or ammo acids 53-57 (pCB52-58gp309-318).
To make the ST::CTB plasmids, two pairs of synthetic oligos were synthesized which encode either a ST, related decapeptide containing a neutralizing B-cell epitope, or the whole ST, of 19 aa, with a Kpnl site at the 5' and a Mscl site at the 3'-end. A Sphl site was introduced with the oligos, which were cloned into the plasmid pCB55-64gp12, to obtain the plasmids pCB55 64STI|K, and PCB55 64ST, respectively The plasmids encoding the HBV::CTB hybrid proteins were also constructed by oiigo directed PCR mutagenesis of pML-LCTBtac, introducing DNA encoding amino acids 133-143 from pre-S(2) together with an Xhol site, either between amino acids 55 and 64 of CTB, yielding the plasmid pCB55-64ps133-143, or as a straight insertion after position 56 in the plasmid pCB56ps133-143. All hybrid genes were sequenced by the dideoxy chain termination method of Sanger et al. (1977).
Table f. Properties of the different plasmids and the corresponding hybrid proteins
Recombin¬ Unique restnction ant CTB aa Aa in inserted Aa sequence of enzyme sites
Plasmid V. Cholerae deleted epitope inserted epitope introduced with the strain insert number'
pCB55-64gp309-318° 407 56-63 gpl 20 309-318' IQRGPGRAFV BssHll. Kpm pCB55-65gp309-318 408 56-64 gpl 20 309-318 IQRGPGRAFV BsMl, Kpm pCB56gp309-318 439 gpl 20 309-318 IQRGPGRAFV BssHW pCB52-5βgp309-318 440 53-57 gpl 20 309-318 IQRGPGRAFV Bssrlll pCB53-64gp309-318 460 54-63 gpl 20 309-318 IQRGPGRAFV aøαHII. Kpm pCB53-64gp307-318 586 54-63 gpl 20 307-318 IRIQRGPGRAFV BssWll. Kpm, BarmW pCB55-64gp309-322 550 56-63 gpl 20 309-322 IQRGPGRAFVTIGK BssWl, Kprή pCB2gp309-318 644 (504*) gpl 20 309-318 IQRGPGRAFV BssHW pJS54 285 gpl 20 309-317 IQRGPGRAFPGYAHG* Xmai pCB55-64STdeca 551 56-63 ST decapephde CAELCCNPAC Spm pCB55-64st, 557 56-63 ST 1-19 NSSNYCCELCCNPACT SplA GCY pCB55-64ps133-143 395 56-63 pre-S(2) 133-143 OPRVRGLYFPA XheA
pCB56ps133-143 549 pre SI2| 133-143 OPRVRGLYFPA Xhol
a) The maternal strain is V. cholerae JS1569 b) The nomenclature used for the plasmids is as follows: CB indicates that the maternal protein is CTB; the first number indicates the position in CTB of the inserted peptide la straight insertion without deletions is noted with the aa which the insert is placed after the two numbers (eg. 55-64) indicate that the aa between the numbers are deleted); the next two letter describes the origin of the inserted aa, gp, for gpl 20, ps for pre-S(2) and ST for STa, and the last numbers or letters indicated the aa inserted c) Numbering of gpl 20 aa accordingl to the Los Alamos database d! Strain number 504 is an £ coli HB101 strain carrying the plasmid pCB2gp309-318 e) Am o acids in bold constitutes the intervening linker sequence EXAMPLE 5
Bacterial Growth Conditions for
Hybrid Polypeptide Production
The recombinant V. cholerae strains producing the hybrid protein polypeptides were cultured in a modified Syncase medium (Lebens et al., Biotechnology 11:1574-1578, 1993) with 100 μg/ml Ampicillin at 37° C with shaking. Samples were taken at 3, 6, 8, 13 and 24 h after inoculation and both the cells and the culture supernatant were analyzed in a GM1 -ELISA (see Example 4 above and 6 below) with the mAb LT39-anti-CTB and F58/H3-anti-gp120. The major part of the produced hybrid proteins were found in the supernatant. The amount of CTB produced was calculated using a standard curve with purified recombinant CTB. The titers of F58/H3 binding were defined as the interpolated dilution giving an Ats0 of 0.4 above background.
Cultures of £ coli were grown in the same way using L-broth rather than syncase medium. EXAMPLE 6
Screening for Expression of the Hybrid Proteins:
Characterization of the different internal hybrid proteins
The internal HIV::CTB, HBV::CTB and ST::CTB hybrid proteins were synthesized in the V. cholerae strain JS1569 (ctxA, ctx,) which was transformed with the different plasmids by electroporation. The resulting recombinant bacterial strains were cultured at 37° C in the presence of 100 μg/ml Ampicillin. Hybrid protein was found in the culture medium which could be precipitated by acidification in the presence of hexametaphosphate (Lebens et al., BioTechniques 1993). Precipitates were redissolved and dialyzed extensively against PBS and then analyzed in SDS-PAGE and immunoblot. Polyacrγlamide Gel Analysis and Immunoblottiπg
1 -3 μg of purified recombinant CTB and hybrid proteins were partially purified from culture supernatants by precipitation with sodium hexametaphosphate, as described by Lebens et al. (Biotechniques, 1993). Samples were prepared in Laemmii sample buffer with 5' mercaptoethanol and were loaded both boiled and unboiled onto 15-17% polyacrylamide 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 eiectrophoresis was performed at a constant voltage of 200 V for approximately 1 h. Gels were either stained with Coomassie Brilliant Blue or electrophoretically transferred to nitrocellulose membranes for immunoblot analyses. After blocking with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS), the membranes were incubated with either the two CTB specific mAb LT39 and CT6, reacting with pentameric and monomeric CTB respectively, mAb P4/D10 against gpl 20, mAb ST1:3 against
ST, or mAb 5520 against pre-S(2). Horse-radish peroxidase labelled goat-anti-mouse IgG (Jackson) was used as secondary antibody and the membranes were developed with the chromogenic substrate 4-chioro- 1 - naphthoi (Bio-Rad). The CTB::HIV fusion protein migrated like native CTB in standard Coomassie-blue stained PAGE in both pentameric and monomeric forms. The inserted heterologous gp120 epitope in the hybrid protein was detected in standard immunoblots using mAb F5B/H3, directed against the V3 loop of gp120. The gpl 20 epitope was recognized both when the hybrid protein was present in the assembled pentameric form of the protein and after dissociation into monomers. This also indicated that there was a significant retention of three-dimensional structure in the assembled peπtamers.
The internal CTB::HIV hybrid protein showed an increasing titer with mAb F58/H3 proportional to the amount of protein produced and secreted. The peptide was resistant to proteolytic degradation when placed internally in CTB. The additional HIV epitope - containing plasmids producing HIV::CTB hybrid proteins, pCB55
65gp309-318, pCB52 58gp309 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 the polypeptide chains. The structural differences between the proteins which were produced in detectable levels and the proteins which were not produced at detectable levels were minimal. The ST::CTB hybrid proteins, encoded by the plasmids pCB55-64ST(K1 (strain 551 ) 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 am o acids 55 and 64 of CTB. Comparative expression levels were 15-30 μgfml for pCB55-64STiK1 (strain 551) and pCB55-64ST, (strain 557) as compared with 5-15 μg/ml for the HBV::CTB and the HIV::CTB chimeras encoded by the plasmids pCB55-64gp309 318, pCB53-64gp309 318 and pCB53-64gp307-318. Thus, the ammo acid composition of the foreign insert is also important. The ST sequences include several cysteines which may form internal disulfide bridges that potentially stabilize the structure of the inserted peptide and of the entire chimenc protein.
The HIV::CTB 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 μg/ml after 24 hours). When analyzed in SDS-PAGE and immunoblot assays, the protein migrated as a sharp band of the size of pentameric CTB when analyzed unboiled. However, the mam part of the protein fell apart when boiled in sample buffer containing SDS and ?-mercaptoethanol. The HBV::CTB hybrid protein from strain 549, with a peptide from pre-S{2) inserted at the same position, behaved in exactly the same way. Resistance to proteolytic cleavage is probably dependent on the conformation and accessibility of the peptide in each individual hybrid protein.
It is worth noting that proteins can maintain a conformation which resembles CTB enough to migrate as CTB pentamers on a polyacryla ide gel, bind to GMI gang osides and be recognized by mAb LT39-antι-CTB, 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 unduly susceptible to cleavage Analysis of the hybrid protein in GM1 -ELISA Assays
Cholera toxin protein was detected using a GM1 -ELISA that has been disclosed in the art (see Sanchez et al., FEBS Lett. 241 :110-114, 1988 and Svennerholm et al., J. Clin. Microbial. 24:585-590, 1986). Microtiter wells were coated overnight at room temperature with 0.3 nmol GM1-ganglιoside (Sigma, St. Louis, Missouri) in 100 μl PBS and blocked with 0.1 % BSA in PBS (PBS-BSA) for 30 mm at 37°C.
After three washings with PBS, samples were diluted in PBS-BSA and incubated for one hour. Recombinant CTB was also used at concentrations starting at 0.5 μg/ml. This and all subsequent incubations were performed at ambient temperature. After subsequent washings with 0.05% Tween-20 in PBS (PBS-T), either mAb LT39-antι-CTB, or foreign epitope specific antibody such as mAb F57/H3-antι-V3 loop of gp120, TB mAb P4/D10-antι-gp120, ST1:3-antι STa or 5520-antι-pre-S(2), were added in PBS with
0.1 % BSA and 0.05% Tween-20 (PBS-BSA-T) and incubated for one hour. Horse-radish peroxidase labelled goat-anti-mouse-lgG (Jackson Laboratories) in PBS-BSA-T was added as second antibody to the wells after washings with PBS T, and after one hour the plates were washed again and then developed with the chromogenic substrate orthophenylenediamme (OPD) in citrate buffer with 0.012% H202. The absorbance was measured at 450 nm after 10-20 mm. The amounts of each hybrid protein added in the first well of the microtiter plate was adjusted to 10 μg as estimated from Coomassie stained potyacrylamide gels with recombmant CTB as a standard and were also confirmed by the reaction with mAb LT39-antι-CTB. Titers were defined as the interpolated serum dilution giving a A450 of 0.2 above background.
Analysis of the internal gp120 epitope hybrid protein in a GM1 -ELISA indicated that GM1 binding was retained together with affinity for the CTB pentamer-specif ic mAb, LT39. Under the same conditions, the CTB::HIV protein also reacted with the anti-gp 120 mAb F58/H3, indicating that the gp120 epitope was exposed on the surface of the molecule. The ELISA assays for GM1 expression used the mAb LT39 and included a standard curve with recombinant CTB starting at 0.5 μg/ml, and reactivity with mAb P4/D10, which was defined as the interpolated serum dilution giving a Ati0 of 0.4 above background. Antigenic properties of the g 120 epitope in the HIV::CTB proteins
The ten am o acid-peptide from gp 20 (aa 309-318) yielded a relatively strong signal when placed N-termιπally in CTB (see Example 3), or between aa 55 and 64, indicating that the epitope was surface exposed in these two proteins (from strain 504 and 407) When increasing the length of the inserted epitope to twelve aa (as in strain 586), the reaction of the epitope with antibody was weaker. Either the epitope was less well exposed on the surface or it adopted a conformation which the mAb did not recognize.
The HIV::CTB hybrid proteins were also analyzed in immunoblot with the same antι-gp120-mAb. When run in the unboiled pentameric form, the inserted gpl 20 epitope was only 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 monomeric form. This could imply that the epitope was exposed in the monomers form, but became more buried or distorted when pentamers were formed. At the same time, the reaction in GM1-ELISA assays indicated that after binding to GM1, the pentameric forms of some of the proteins exposed the inserted HIV epitope enough to be recognized by the antι-gp120 monoclonal antibody. It is possible 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 analyzed in immunoblot with monoclonal P4/D10. The weak monomeric band of around 11 kD, which presumably is still uncieaved, reacts with the mAb, but the majority of the protein, which was degraded, showed no antt-gp 120 reactivity.
Antigenic properties of the ST and pre-S(2) epitopes in the ST::CTB and HBV::CTB hybrid proteins
When analyzed in GM1 -ELISA, the ST, epitopes inserted between ammo acids 55 and 64 in CTB strains 551 and 557 were detected with the ST-specific neutralizing mAb ST1:3 (see Svennerholm et al., J Clin. 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., Res. Microbiol. 47:971-979, 1990). In immunoblot assays, the mtrachain ST, peptides were only detected in the monomeric protein form. This is consistent with what was seen 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 5520-antι-pre-S(2) (see Milich et al., J. Immunol. 137: 2703-2710, 1986) either in GM1-ELISA or in the unboiled pentameric form in immunoblot assays, but were readily observed in the boiled monomeric form. These results indicated that when foreign antigen was inserted into internally in CTB within the
56-64 am o acid region, Ala-64 should be positioned C-termιnally to the insert to get production of the protein. This observation is consistent with other scientists who have reported that Ala-64 is important for the stability of pentameric CTB. Likewise, the N-terminal position of Pro-53 was important to the insert since when deleted, as in pCB52-58gp309-318, 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 less than the level of reactivity of the CTB epitopes Since the mtrachain fusion protein was resistant to proteolytic cleavage during production in V. cholerae. degradation of the foreign peptide epitope is probably not the explanation for the reduced immunogemcitγ of the foreign epitope in the hybrid protein as compared with level of CTB reactivity. It is more likely that the surface density of the inserted foreign epitope is low compared to the several different, strong, mainly conformational CTB epitopes. Thus, this invention also contemplates that multiple 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 also affect stability. Thus, those skilled in the art should also contemplate varying the length of the insert in those substitutions where stability is potentially a problem. The ELISA screening methods in combination with polyacrγlamide gel electrophoresis staining and immunoblot analysis serve to guide those skilled in the art to prepare appropriate combinations and to screen these combinations for expressed protein. Importantly, the results provided here indicated that CTB can be modified without loss of synthesis of the desired protein.
EXAMPLE 7
Immunization Protocol for Optimizing
Serum Antibody Levels Female C57B1/6 mice were immunized intraperitoneally (i.p.) with three doses of 10 μg of the
CTB::HIV hybrid protein partially purified from JS1569 supernatant. The first dose was given with Freund's complete adjuvant (DIFCO Laboratories, Detroit, Michigan) and the subsequent doses with Freund's incomplete adjuvant. As negative controls, mice were immunized using the same scheme with a CTB hybrid protein with an irrelevant foreign epitope or with Freund's adjuvant alone. Serum samples were collected before the first dose and seven days after the second and third dose. Sera were analyzed in ELISA, using either CTB (bound to GM1 in GM1 -coated microwells using the methods of Example 6) or recombinant gpl 20 (Bolmstedt et al., J. Gen. Virol. 73:3099-3105, 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 majority of the mice tested. The mice receiving only Freund's adjuvant gave no antibody responses against either CTB or gp120.
EXAMPLE 8 Immunization Protocol for Producing Vaginal Immune response
Female mice were given 10 mg of progesterone subcutaneously 10 and 3 days before the first immunization and were then treated once a week with progesterone. Groups of mice were immunized either four times intravaginally (at 1-2 week intervals between each dose) or with three intraperitoneal doses followed by an intravaginal dose. Each iπtravaginal immunization dose consisted of ca. 0.5 mg CTB-peptide conjugate (see Example 1). Each dose was estimated to contain approximately 0.4 mg CTB and 0.1 mg A8-VDIV peptide and additionally included 5 meg cholera toxin as extra adjuvant and each intraperitoneal dose contained one third this amount. EXAMPLE 9
Immunization to produce
Mucosal Antibody
The CTB fusion proteins are isolated from bacterial culture supernatants using the expression system described by Lebens, et al. (BioTechnology, supra). The fusion proteins are isolated by GM1 affinity purification as disclosed by Tayot, et al. (Europ. J. Biochem. 113. 249-258) and dialyzed against PBS. The specific protein concentrations of the samples used for immunization are determined by ELISA using a CTB standard.
In an experimental model, male monkeys are immunized with 40-250 μg 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 Freund 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 specific antibodies against CTB and the foreign antigen by ELISA. In addition, the ability of the sera to neutralize the pathogen was assayed in the infant mouse test. Serum from immunized monkeys in a dilution of 1/5 was mixed with an equal volume of pathogen and introduced into the mouths of infant mice. Mice were monitored for the presence of the disease over time. For human immunization it is contemplated that the chimenc 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 mg for repeated immunizations. 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 3-10 ml for rectal or vaginal immunization. It is contemplated that the oral immunization is likely to be given together in a pharmaceutical acceptable buffer containing about 25-200 ml liquid containing about 2 grams of sodium bicarbonate or equivalent acid buffering agent.
EXAMPLE 10 Experimental Screening for IgA and IgG Mucosal Antibody
Serum, mesenteπc lymphnodes, spleen, cervix, vagina, small intestine, colon and rectum were collected from mice sacrificed (and perfused with heparin containing buffer) one week after the last immunization dose with the preparation of Example 1 using the immunization protocol of Example 8. The organs were frozen and then extracted with 2% (WfV) of saponm using the PERFEXT method (Quidmg, et al. J. Clin. Invest. 88(11:143-148, 1991 ). The sapon extracts were tested for IgA and IgG anti-CTB and anti-VDIV antibodies by ELISA. ELISA plates were coated with GM1 (0.3 nmol/ml) or A8 VDIV peptide (1 μg/ml) respectively. In this example, immunized mice were of the C57/B1 strain and were 8-10 weeks old at the onset of immunizations. The immunization protocol was 1 mg medroxyprogesterone acetate (Depoprovera; UpJohn Company, Kaiamazoo, Ml) subcutaneously 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.
TABLE 2. IMMUNE RESPONSES IN THE FEMALE GENITAL TRACT AND SERUM OF MICE IMMUNIZED BY VARIOUS ROUTES WITH A CHEMICALLY PREPARED CONJUGATE BETWEEN CHOLERA TOXIN B SUBUNIT AND THE A8-VDIV CO-LINEAR PEPTIDE DERIVED FROM THE MAJOR OUTER MEMBRANE PROTEIN OF CHLAMYDIA TRACHOMATIS
ELISA antibody titer to A8-V0IV
Immunization Vagina Cervix Fallopian Serum uteri tubes
IgA IgG IgA IgG IgA IgG IgA IgG
None < .5 < .5 < .5 < .5 < .5 < .5 <.5 < .5 Intravag al x 4 290 .5 920 .5 50 .5 3 43
Oral x 4 < .5 < .5 < .5 < .5 22 < .5 < .5 3
Intraperitoneal x 3 + 14 99 40 130 38 nd 2 3500 intravagmal x 1
Oral x 4 + intravagmal x 2 < .5 2 < .5 < .5 < .5 < .5 < .5 1
This table indicates that with repeated intravagmal immunization or a combination of repeated intraperitoneal priming followed by a vaginal booster immunization, the CTB-A8-VDIV conjugate could induce substantial specific genital mucosal IgA antibody formation to the Chlamydia trachomatis A8-VDIV antigen in test samples comprising the vaginal, cervical and Fallopian tube mucosae. After multiple intraperitoneal immunizations, the IgA response was also associated with a substantial IgG response in both the genital tract tissues and in serum. Immunogeπicity of the foreign sequences in the CTB hybrid proteins
To determine the ability of the internally inserted peptides to induce immune responses, groups of mice of strain C57B1/6 were immunized intraperitoneally (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 chimenc proteins, with Freund's adjuvant. All mice responded with high titered serum anti-CTB IgG-responses. As noted above, the ten ammo acid HIV epitope induced serum IgG-respoπses against gp120 when placed between ammo acids 55 and 64 of CTB. A weak response against g l 20 was also induced by the gp 20 moiety in the mice immunized with the internal HIV::CTB hybrids from strain 460 (ten gpl 20 ammo acids between 53 and 64) and 586 (twelve gpl 20 ammo acids between 53 and 64) and by the N- terminally placed gp120-epitope in the protein from strain 504. in fact, all these proteins showed immunogenic properties similar to the previously described HIV::CTB protein.
The ST decapeptide 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 i munological reactivity of the inserted peptide was modest in vitro. 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 triers of antibody than the decapeptide in the protein from strain 551.
The HBV::CTB chimeric protein from strain 395 was given either i.p. or perorally (p.o.) with cholera toxin (CT) as adjuvant to either BALB/c or C57B1/6 mice. High serum-anti-CTB titers where obtained in both p.o. and i.p. immunized mice, whereas the most significant levels of aπti-pre-S(2)-ab were induced with i.p. immunizations using CT as adjuvant. Table 3 illustrates the various immunization protocols and their results.
Table 3. Serum IgG titers after immunization with the HBV::CTB hybrid protein.
Mouse strain CT* Route" antι-CTBc anti-pre-S(2)e
2 doses 3 doses 2 doses 3 doses
C57BU6 (H-2 ) - p o 53 000* 23 400 0 0 BALB/c (H-2") - p.o 0 2 400 0 0
C57BL/6 + p o 102 400 256 400 0 0
BALB/c + p.o 4 000 38 800 0 0
C57BL 6 IP 170000 546000 0 1060
BALB/c IP 32000 153600 0 0
C57BL/6 i-P 10β 682000 8500 3300 BALB/c 'P 10β 461000 6400 3600
a) Mice were immunized with or without 5 μg (p o ) or 1 5 μg (i p ) cholera toxin (CT) as adjuvant b) The animals were given does of 30 μg (p o ) or 7 5 μg (i p ) of the HBV CTB hybπd protein from strain 395 on day 0. 14 and 24 c) Recombinant LTB bound to GM1 was used as antigen for anti-CTB-lgG determinations d) A synthetic peptide containing aa residues 133-153 from pre-S(2) (Schϋdel et al . 1990) was used as antigen for antι-preS(2)-lgG determinations e) Sera were collected on day 7 after does 2 and 3 and sera from mice from the same group (2 or 3 mice per group were pooled before analyses Mean reciprocal serum dilutions yielding an A3K>3 x the A4β2 of preimmune sera are indicated as titers While particular embodiments of the invention have been described in detail, it will be apparent to those skilled in the art that these embodiments are exemplary rather than limiting, and the true scope of the invention is that defined in the following claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: Holmgren, Jan
Lebens, Michae1
(ii) TITLE OF THE INVENTION: IMMUNOGENS FOR STIMULATING MUCOSAL
IMMUNITY
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(A) ADDRESSEE: Knobbe, Martens, Olson and Bear
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(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Kaiser, AnneMarie
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(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 619-235-8550
(B) TELEFAX: 619-235-0176
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO: 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acids
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICA : NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :
Phe Asp Val Thr Thr Leu Asn Pro Thr lie Ala Gly Ala Gly ASD Val
1 5 10 15*
Lys
(2) INFORMATION FOR SEQ ID NO:2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acids
(C) 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:2 :
Ala Leu Asn lie Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala
1 5 10 15
Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr Leu Asn
20 25 30
Pro Thr lie Ala Gly Ala Gly Asp Val Lys 35 40
(2) INFORMATION FOR SEQ ID NO:3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:3:
GGGGCGCGCT TTCGTTGCGA TCGAAAGGAT GAAGGATACC CTGAGGATTG C 51
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:4 : GGGGCGCGCC CCGGACCACG CTGGATACTA CCTGGTACCT CTACTTGAAA AGTTGC 56
(2) INFORMATION FOR SEQ ID NO: 5 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 125 base pairs (3) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: (ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 33...125 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :
AATTCGATTC TGTTAAACAA AGGGAGCATT AT ATG GTA AAG ATA ATA TTT GTG 53
Met Val Lys lie He Phe Val 1 5
TTT TTC TTA TCA TCA TTT TCA TAT GCA AAT GAT GAT AAG TTA GGA GCT 101 Phe Phe Leu Ser Ser Phe Ser Tyr Ala Asn Asp Asp Lvs Leu Gly Ala 10 15 20
CCT GAT TCT AGA GCG ATG AGT AAT 125
Pro Asp Ser Arg Ala Met Ser Asn 25 30
(2) INFORMATION FOR SEQ ID NO: 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 ammo acids
(B) TYPE: amino acids
(C) 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:6 :
Ala Leu Asn He Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala
1 5 10 15
Thr Thr Gly Tyr Leu Lys Gly Asn Ser 20 25
(2) INFORMATION FOR SEQ ID NO:7 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 7 :
CCGGCTAGCG CAGCT 15
(2) INFORMATION FOR SEQ ID NO: 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 8 :
GCGCTAGCCG GAGCT 15
(2) INFORMATION FOR SEQ ID NO: 9 :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (3) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 9:
CCTATTCAGC GTGGTCCGGG GCGCGCTTTT GTTGCTCCTC AAAAT 45
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 10: ATTTTGACCA GCAACAAAAG CGCGCCCCGG ACCACGCTGA ATAGGAGCT 49
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 11:
CCTCAAATTC AGCGTGGTCC GGGGCGCGCT TTTGTTAAC 39
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 12:
GTTAACTTTA GCCGCGCCCC GGACCACGCT GAATTTGAGG AGCT 44
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: amino acids
(C) 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: 13:
Ala Leu Asn He Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala
1 5 10 15
Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr Leu Asn
20 25 30
Pro Thr He Ala Gly Ala Gly Asp Val Lys 35 40
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 amino acids
(B) TYPE: amino acids
(C) 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: 14:
Cys Ala Leu Asn He Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly
1 5 10 15
Ala Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr Leu
20 25 30
Asn Pro Thr He Ala Gly Ala Gly Asp Val Lys 35 40
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acids
(C) 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:15:
Ala Leu Asn He Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala
1 5 10 15
Thr Thr Gly Tyr Leu Lys Gly Asn Ser Phe Asp Val Thr Thr Leu Asn
20 25 30
Pro Thr He Ala Gly Ala Gly Asp Val Lys Cys 35 40
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 16:
GGGGGGGATC CCCTCGCGCG TTTCGGTGAT GAC 33
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 17:
GGGGGAGATC TCTGAAATGA GCTGTTGACA ATTATC 36
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 18:
GGGGGAGATC TGCCAGAACC GTTATGATGT CGG 33
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 19:
GGGGGGGATC CCGAACGCCA GCAAAGACGT A 31
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 20: ATTCAGCGTG GTCCGGGTCG TGCTTTTG 28
(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 (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CCGGGAAAAC GACGACCCGG ACCACGCTGA ATAGCT 36
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 57 base pairs (3) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 22:
GCGTAGCGGT ACCACCACTC TGAACCCAAC TATTGCTGGA GCTGGCTGGC CAGCACG 57
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:23:
CCGTGCTGGC CAGCCAGCTC CAGCAATAGT TGGGTTCAGA GTGGTGGTAC CGCTACGC 58
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(11) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CCATTTGATA TTACCACTTT AAATCCAACA ATTGCTGGTG CTGGTGATGT TAAACCCGGG 60 T 61
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (lil) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CTAGACCCGG GTTTAACATC ACCAGCACCA GCACCAGCAA TTGTTGGATT TAAAGTGGTA 60 ATATCAAATG GAGCT 75
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 76 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:26:
CCCGCTTTAA ATATTTGGGA TCGTTTTGAT GTTTTTTGTA CATTAGGTGC TACCACTGGT 60 TATAAAGGTA ATAGTT 76
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:27:
CTAGAACTAT TACCAAAATA ACCAGTGGTA GCACCTAATG TACATTTAAC ATCAAAACGA 60 TCCCAAATAT TTAAAGCGGG AGCT 84
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acids
(C) 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:28:
Asp Pro Arg Val Arg Gly Leu Tyr Phe Pro Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acids
(C) 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:29:
He Gin Arg Gly Pro Gly Arg Ala Phe Val 1 5 10
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acids
(C) 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:30:
Cys Ala Glu Leu Cys Cys Asn Pro Ala Cys 1 5 10
(2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acids
(C) 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:31:
Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr
1 5 10 15
Gly Cys Tyr
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:32:
GGGGCGCGCT TTCGTTGCGA TCGAAAGGAT 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 (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GGGGCGCGCC CCGGACCACG CTGGATACTA CCTGGTACCT CTACTTGAAA AGTTGCACCA 60 TTCTTAAAAG 70
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:34:
GGGGCGCGCT TTCGTTCATA TAGATTCACA AAAAAAAGCG AT 42
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:35:
GGGGCGCGCC CCGGACCACG CTGGATTTGA CTACCTGGTA CTTCT 45
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:36:
GGGGCGCGCT TTCGTTATAG ATTCACAAAA AAAAGCGAT 39
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:37:
GGGGCGCGCC CCGGACCACG CTGGATTACT TCTACTTGAA AAGTTGCA 48 (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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 TGGTCCGGGG 20
(2) INFORMATION FOR SEQ ID NO: 39 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:39 :
CGCGCCCCGG ACCACGCTGG ATTGGTAC 28
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (Vi) ORIGINAL SOURCE:
( i) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
CAATCCGGAT CCAGCGTGGT CCGGGG 26
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 1:
CGCGCCCCGG ACCACGCTGG ATCCGGATTG GTAC 34
(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) HYPOTHETICAL: NO (iv) ANTISENSE: NO (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE:
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CTTTTCCTAT TGTAACGAAA GCGCGCCCCG GACCACGCTG GATCAAAAAT GCA 53
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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-.43:
TTTTTGATCC AGCGTGGTCC GGGGCGCGCT TTCGTTACAA TAGGAAAA 48
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:44:
CAGGTAGTTG CGCTGAATTG TGTTGTAATC CTGCATGCG 39
(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:45:
CGCATGCAGG ATTACAACAC AATTCAGCGC AACTACCTGG TAC 43
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:46:
CAGGTAGTAA TAGCAGCAAT TACTGCTGTG AATTGTGTTG TAATCCTGCA TGCACTGGAT 60 GTTACG 66
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:47:
CGTAACATCC AGTGCATGCA GGATTACAAC ACAATTCACA GCAGTAATTG CTGCTATTAC 60 TACCTGGTAC 70
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:48:
GGGCCTCGAG TCCGAGGCCT ATACTTTCCG GCGATTGAAA GGATGAAGGA TACCCTG 57
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 49:
GGGCTCGAGG GTCACTACCT GGTACCTCTA CTTGAAAAGT TG 42
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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: 50:
GGGCTCGAGG ATCTTGACTA CCTGGTACTT CTACTTGAAA AG 42
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: 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:51:
GGGCTCGAGT TCGTGGTCTG TACTTCCCGG CTCATATAGA TTCACAAAAA AAAGCGATTG 60 AAAG 64

Claims

WHAT IS CLAIMED IS:
1. A mucosal binding 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.
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 ammo 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 ma|or outer membrane protein of chlamydia.
8. The composition of Claim 4, wherein said B-cell stimulating antigen is from the VDIV region of the ma|or outer membrane protein of chlamydia.
9. The composition of Claim 8, wherein said antigen further comprises a T-helper cell stimulating antigen from the ma|or outer membrane protein of chlamydia.
10. The composition of Claim 9, wherein said T-helper cell stimulating antigen is from the
A8 region.
1 1. 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 maior 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 rnaior 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 enterotoxigemc E. coli.
34. The composition of Claim 33, wherein said antigen is from the ST, protein of enterotoxige c £ 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 CTAI2) subunit.
41. The polynucleotide of Claim 40, wherein the nucleic acid encoding the B-cell stimulating antigen encodes a peptide which includes the ammo 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 gpl 20.
46. The polynucleotide of Claim 45, wherein said nucleic acid encoding a peptide from HIV gp120, encodes the peptide IQRGPGRAFV.
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 ammo 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 ST, protein of eπterotoxigeπic £ coli.
50. The polynucleotide of Claim 49, wherem said nucleic acid encoding the B-cell stimulating antigen encodes peptide having the am o 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.
PCT/GB1995/002708 1994-11-17 1995-11-17 Immunogens for stimulating mucosal immunity WO1996016178A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP95937948A EP0792365A1 (en) 1994-11-17 1995-11-17 Immunogens for stimulating mucosal immunity
JP8516667A JPH10509325A (en) 1994-11-17 1995-11-17 An immunogen that stimulates mucosal immunity
MX9703676A MX9703676A (en) 1994-11-17 1995-11-17 Immunogens for stimulating mucosal immunity.
AU38767/95A AU3876795A (en) 1994-11-17 1995-11-17 Immunogens for stimulating mucosal immunity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US34224194A 1994-11-17 1994-11-17
US08/342,241 1994-11-17

Publications (1)

Publication Number Publication Date
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Country Status (7)

Country Link
EP (1) EP0792365A1 (en)
JP (1) JPH10509325A (en)
CN (1) CN1173204A (en)
AU (1) AU3876795A (en)
CA (1) CA2205130A1 (en)
MX (1) MX9703676A (en)
WO (1) WO1996016178A1 (en)

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WO1999059627A2 (en) * 1998-05-15 1999-11-25 Green Allan M Verotoxin b subunit for immunization
EP1372528A2 (en) * 2001-04-05 2004-01-02 Chiron Corporation Mucosal boosting following parenteral priming
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WO1998028005A1 (en) * 1996-12-24 1998-07-02 Smithkline Beecham Biologicals S.A. Chlamydia vaccines
US8524652B2 (en) 1997-07-18 2013-09-03 Inserm Chimeric polypeptide comprising the fragment B of shiga toxin and peptides of therapeutic interest
US7488809B2 (en) 1997-07-18 2009-02-10 Insem-Transfert Chimeric polypeptide comprising the fragment B of shiga toxin and peptides of therapeutic interest
WO1999059627A2 (en) * 1998-05-15 1999-11-25 Green Allan M Verotoxin b subunit for immunization
WO1999059627A3 (en) * 1998-05-15 2000-01-20 Allan M Green Verotoxin b subunit for immunization
JP2002515456A (en) * 1998-05-15 2002-05-28 アラン エム グリーン Verotoxin B subunit for immunization
US8367071B2 (en) 1998-05-15 2013-02-05 Inserm-Transfert Verotoxin B subunit for immunization
JP4820000B2 (en) * 1998-05-15 2011-11-24 アラン エム グリーン Verotoxin B subunit for immunization
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
EP1372528A4 (en) * 2001-04-05 2005-03-16 Chiron Corp Mucosal boosting following parenteral priming
EP1372528A2 (en) * 2001-04-05 2004-01-02 Chiron Corporation Mucosal boosting following parenteral priming
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
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EP0792365A1 (en) 1997-09-03
CN1173204A (en) 1998-02-11
AU3876795A (en) 1996-06-17
JPH10509325A (en) 1998-09-14
CA2205130A1 (en) 1996-05-30
MX9703676A (en) 1998-03-31

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