SECRETORY IMMUNOGLOBULIN A AS A MUCOSAL VACCINE DELIVERY SYSTEM

Abstract: A secretory IgA antibody (sIgA) comprising a polymeric IgA antibody bound to a secretory component (SC) which SC comprises a (poly)peptide insert corresponding to a protective epitope of an antigen; a vaccine comprising such sIgA antibody; a DNA construct comprising a DNA sequence coding for a SC and a DNA insert coding for the protective epitope of an antigen; a method for producing said sIgA antibody; and a method for active immunization of a mammal which comprises administering to said mammal an effective amount of a vaccine comprising said sIgA.

Description: Secretory Immunoglobulin A as a Mucosal Vaccine Delivery System Field of the Invention The present invention relates to a novel secretory immunoglobulin A in which the secretory component is of chimeric nature, novel and comprises an immunogenic epitope which is foreign to the parent secretory component, said novel chimeric secretory component, methods of producing said secretory immunoglobulin A and said secretory component, the use of these compounds in a novel vaccine delivery system for the vaccination of a mammal, and pharmaceutical compositions comprising said secretory immunoglobulin A Background of the Invention The protection of the mucosal surfaces of the digestive, respiratory and urogenital tracts is in part mediated by secretory immunoglobulin A (slgA, Fig 1) This antibody consists of an IgA dimer associated with J chain, which is acquired during the process of polymerization in plasma cells just before secretion, and secretory component (SC) SC is derived from the polymeric immunoglobulin receptor (PIR, Fig. 2), which binds and transports polymeric immunoglobulins across mucosal and glandular epithelia Proteolytic cleavage of the extracellular portion of the receptor releases SC together with the bound IgA into mucosal secretions (Neutra et al., 1994) SC belongs to the immunoglobulin superfamily of proteins, and consists of a series of five Ig-like domains corresponding to the portion of the poly-Ig receptor facing the outside (Mostov et al , 1984, Banting et al , 1989, Schaerer et al , 1990; Krajci et al , 1991 ) In addition to the protection of mucosal surfaces by crosslinking pathogens and promoting their clearance by peristalsis or mucociliary movement, slgA in the intestinal lumen also selectively adheres to M cells (these cells transport antigens across the epithelium overlying organized lymphoid mucosal tissue). This was first observed in suckling rabbits as a local accumulation of milk slgA on M cells of Peyer's patches (Roy and Varvayanis, 1987) Subsequently, monoclonal mouse IgA, polyclonal rat IgA, and polyclonal IgG antibodies, radiolabeled or coupled to colloidal gold, were found to bind specifically to rabbit or mouse M cells and to compete with each other for binding sites CONFIRMATION COPT (Weltzin et al., 1989). Selective slgA binding and transport by M cells may play a role in the regulation of the mucosal immune response, but that role has not yet been defined. Thus, while slgA would generally prevent the contact of antigens with mucosal surfaces, it could also promote re-uptake of small amounts of antigen by M cells. Up to now it was not known whether oral application of slgA carrying a foreign epitope ("antigenized" slgA) could elicit an immunogenic response against the inserted epitope and whether recombinant chimeric SC-IgA complexes can serve as mucosal vaccine delivery systems. Obj ect of the Invention It is an object of the invention to take advantage of the fact that orally administered slgAs are efficiently transported by M cells in Peyer's patches of the small intestine and in other mucosa-associated lymphoid tissue (MALT) of the large intestine, the oral and nasal cavities, and the airways and the possibility that slgA itself could serve as a vaccine delivery vector to target foreign epitopes into MALT. However, this would require that the foreign epitope be inserted without affecting the molecular folding of SC or the assembly and the function of slgA. Also, the site of insertion must be surface-exposed, and the inserted epitope must be immunogenic. The object of this invention is to solve these problems and to generate slgA comprising a foreign epitope in the secretory component which foreign epitope does not affect the molecular folding of SC or the assembly and the function of slgA, which foreign epitope is inserted in a surface-exposed site of the SC, and which inserted foreign epitope is immunogenic. Summary of the Invention Secretory IgA was already known to bind to M cells and to be transported into the underlying organized mucosal lymphoid tissue (Weltzin et al., 1989). To act as a vaccine carrier able to deliver foreign epitopes, slgA was processed and presented to the cells of the immune system. Surprisingly it was found to trigger a systemic and a mucosal response. To show that orally administered slgA from rabbit whey is taken up and processed in mouse Peyer's patches, lymphoblasts in these latter are recovered from immunized animals, fused with myeloma partners to generate hybridoma cells (Weltzin et al., 1989) secreting immunoglobulins specific for rabbit SC and slgA. In the particular example, 10 hybridoma I clones out of 60 react with SC by ELISA out of which four recognize free and bound SC in rabbit whey on Western blots One clone produces polymeric IgM, two clones secrete IgGl, and one clone secretes IgAd IgA<i from clone 36 4 recognizes both free and IgA- bound SC blotted on nitrocellulose membrane and im unoprecipitate free SC when immobilized on Sepharose beads This indicates that mucosally administered slgA is able to elicit both a mucosal and systemic immune response against SC. The present invention demonstrates that oral immunization with "antigenized" slgA elicits both a systemic and mucosal response against the inserted epitope and that recombinant chimeric SC-IgA complexes can serve as mucosal vaccine delivery systems Detailed Description of the Invention The invention concerns a secretory IgA antibody comprising a polymeric IgA antibody bound to a secretory component which comprises a (poly)peptide insert corresponding to a protective epitope of an antigen The invention concerns further a vaccine comprising an effective amount of a secretory IgA comprising a polymeric IgA antibody bound to a secretory component which comprises a (poly)peptide insert corresponding to a protective epitope of an antigen The invention concerns further a chimeric secretory component which comprises as a (poly)peptide insert a protective epitope of an antigen The invention concerns further a secretory component as described hereinbefore and hereinafter wherein the (poly)peptide insert is a protective epitope of a pathogenic microorganism The invention concerns further a secretory component as described hereinbefore and hereinafter, wherein the (poly)peptide insert is an amino acid sequence from a bacterial, viral, parasitic or mammalian antigen which is recognized by an immune effector, i e an antibody, antigen presenting cell or lymphocyte The invention concerns further a DNA construct comprising a DNA sequence coding for a secretory component and a DNA insert coding for the protective epitope of an antigen The invention concerns further a method for producing a secretory IgA antibody comprising a polymeric IgA antibody bound to a secretory component, which comprises a (poly)peptide insert corresponding to a protective epitope of an antigen, wherein said secretory component is combined with said polymeric IgA antibody The invention concerns further a method for active immunization of a mammal which comprises administering to said mammal an effective amount of a vaccine comprising a secretory IgA comprising a polymeric IgA antibody bound to a secretory component which comprises a (poly)peptide insert corresponding to a protective epitope of an antigen Brief Description of the Figures Sequence identification numbers (SEQ ED NO) are abbreviated in the Figures as IdNo Fig 1 Schematic representation of secretory immunoglobulin A The protein is made of two immunoglobulin A monomers covalently linked through the J chain, together with secretory component wrapped around the surface of the molecule Fig 2 Schematic representation of the polymeric immunoglobulin receptor (PIR) -NH2 The amino terminus of PIR -COOH The carboxyl terminus of PER Spheres numbered I to V the five IgG-like extracellular domains of the receptor, with some important disulfide bridges and glycosylation sites drawn Secretory component (SC) comprises the N-terminus amino acid down to the cleavage site Fig 3 Scheme decribing the sites of epitope replacement/insertion within secretory component (SC) A Rabbit SC B Mouse SC The sites are expressed as amino acid numbers, with respect to the initiation methionine given number 1 Empty box Signal sequence Filled box Sites of insertion/replacement The carboxyl terminus permits to fuse fragments coding for up to 100 amino acids Fig 4 Scheme (not drawn on scale) describing the construction of plasmid pSC-11(A/M) Fig 5 Scheme (not drawn on scale) describing the construction of plasmids pSC(IpaB), pHGSl-SC, pHGSl-SC(IpaB), pHGSl-SCόxHis, pHGSl-SC(IρaB)6xHis, pLKneo- PER(IpaB) Fig 6 Purification of SC(IpaB)6xHis by Ni2 + -chelate affinity chromatography L Load, FT. Flow through, W Wash, E40 Elution with 40 mM imidazole, E80 Elution with 80 L i mM imidazole Panel A The purified protein as appearing after silver staining Panel B The purified protein as appearing after immunodetection (Western blot) Fig 7 Association of PER(wt) and PER(IpaB) with IgAd Panel A Cartoon representation of dot blot reassociation assay (DORA) Further description of the procedure is available in Rindisbacher et al (1995) Panel B Both the wild-type and chimeric SC protein produced in rW-infected CV- 1 cells bind to IgAtf with comparable affinity No interaction occurs when IgAm is used as a partner in the reconstitution reaction Anti-α chain monoclonal antibodies to the heavy chain of IgA Anti-SC pAb 982 to the rabbit secretory component Detection was performed using secondary antibodies labeled with HRP, and enhanced chemiluminescence Fig 8 Oral immunogenicity in BALB/c mice of antigenized slgA Panel A Binding specificity of the mouse sera to the indicated antigen (noted below each panel) measured by ELISA Groups of mice are 1, mice immunized with IgAd- SC(wt), 2, mice immunized with IgAd-SC(IpaB), 3, mice given PBS (negative control) Results are mean ± standard deviation of two separate triplicate experiments involving groups of three mice, and are expressed as absorbance values at 492 nm Panel B Binding specificity of the mouse sera to the specific IpaB peptide and control FLAG peptide, measured by ELISA Groups of mice are 1, mice immunized with IgAd-SC(wt), 2, mice immunized with IgAd-SC(IpaB), 3, mice given PBS (negative control) Results are mean ± standard deviation of two separate triplicate experiments involving groups of three mice, and are expressed as absorbance values at 492 nm Panel C Binding specificity of saliva samples recovered from mice in groups 1, 2 and 3 Fig 9 Construction of plasmid pBSmSC from plasmid pBSIIKS+mpIgR and a double- stranded oligonucleotide to create a novel carboxyl terminus corresponding to the putative carboxyl terminus of mSC The stop codon is underlined, half restriction sites are italicized and the EcoRl site is in bold face ζ Fig 10 Construction of plasmid pCB6mpIgR(-5') Deletion of the 5'-untranslated region in the eukaryotic expression vector pCB6 by PCR using primers SEQ ED NO 14 and SEQ ED NO 15 Fig 11 Construction of plasmids pCB6mSC(-5') Replacement of 3'-engineered mSC sequences derived from plasmid pBSmSC in plasmid pCB6mpIgR(5-) used as an expression vector Abreviations B Bglll, E EcoRl, K Kpήl, N Notl, S Smal Fig 12 Schematic representation (not drawn to scale) of the two steps involved in recombinant PCR Number 1 corresponds to oligonucleotide SEQ ED NO 18, number2 to oligonucleotide SEQ ED NO 17, number 3 to oligonucleotide number SEQ ID NO 16, and 4 to oligonucleotide SEQ ED NO 19 Nucleotides coding for the FLAG sequence are represented as a black box Fig 13 A table showing comparative results of the biochemical properties of mSC mutant proteins secreted by COS-7 cells transfected with expression vector pCBόmSC- FLAGDII( 153-160) and pCB6mSC-UreB όxHis Short description of sequences used according to Sequence Listing SEQ ID NO 1 Oligonucleotide for PCR, noncoding strand, insertion of an AccI site at position 418, hybridizing sequence 429-394 in pSC-11, used to generate pSC-11(A/M) SEQ ED NO 2 Ohgonucleotide for PCR, coding strand, hybridizing sequence 314-337 in pSC-11, used to generate pSC-11(A/M) SEQ ED NO 3. Ohgonucleotide for PCR, coding strand, insertion of a Mrol site at position 453, hybridizing sequence 448-480 in pSC-11, used to generate pSC-11(A/M) SEQ ED NO 4 Ohgonucleotide for PCR, noncoding strand, hybridizing sequence 905-881 in pSC-11, used to generate pSC-11(A/M) t SEQ ED NO 5 Ohgonucleotide coding for the Shigella invasin B (IpaB) epitope KDRTLIEQK (coding strand) with a 5' AccI half site for insertion into pSC-l l(A/M) and generation of pSC(IpaB) SEQ ED NO 6 Ohgonucleotide coding for the Shigella invasin B (IpaB) epitope KDRTLIEQK (noncoding strand) with a 3' Mrol half site for insertion into pSC-11(A M) and generation of pSC(IpaB) SEQ ED NO 7 Oligonucleotide coding for a six histidine tag (coding strand) for insertion into pHGSl-SC and generation of pHGSl-SC6xHis SEQ ED No 8 Oligonucleotide coding for a six histidine tag (coding strand) for insertion into pHGSl-SC and generation of pHGSl-SCόxHis SEQ ED NO 9 Upstream primer for PCR analysis SEQ ED NO 10 Downstream primer for PCR analysis SEQ ED NO 1 1 Amino acid sequence of Shigella invasin B epitope SEQ ED NO 12 Oligonucleotide coding for the carboxyl terminus of mSC (coding strand) for insertion into pBSETKS+mpIgR and generation of pBSmSC SEQ ED NO 13 Oligonucleotide coding for the carboxyl terminus of mSC (noncoding strand) for insertion into pBSIEKS+mpIgR and generation of pBSmSC SEQ ED NO 14 Primer for producing plasmid pCB6mpIgR(-5) SEQ ED NO 15 Primer for producing plasmid pCB6mpIgR(-5) SEQ ID NO 16 Inside primer comprising the FLAG coding sequence SEQ ED NO 17 Inside primer comprising the FLAG noncoding sequence SEQ ED NO 18 5' outside primer (coding strand) to permit amplification of a fragment comprising Kpnl and Nhel restriction sites SEQ ED NO 19 3' outside primer (noncoding strand) to permit amplification of a fragment comprising Kpnl and Nhel restriction sites SEQ ED NO 20 5' primer (coding strand) for PCR amplification of H. pylori urease carrying a 5' Xmal site SEQ ED NO 21 3' primer (noncoding strand) for PCR amplification of H. pylori urease carrying a 5' Xbal site SEQ ID NO 22 Ohgonucleotide (coding strand) for insertion of a 6xΗis tag into plasmid pCB6mSC-UreB SEQ ED NO 23 Ohgonucleotide (noncoding strand) for insertion of a 6xΗis tag into plasmid pCB6mSC-UreB The terms used in this description and the claims are further defined as follows A secretory IgA antibody (slgA) according to the invention is a novel antibody comprising a polymeric IgA antibody bound to a secretory component which comprises a (poly)peptide insert corresponding to a protective epitope of an antigen, and which is ableto trigger an immune response to the protective epitope in a mammal to which an effective amount has been administered It is able to be transported across mucosal and glandular epithelia Polymeric IgA antibodies are known in the art or can be prepared according to known procedures Such antibodies are usually dimeric or polymeric Representative examples are in particular monoclonal antibodies from hybridoma cell lines, e.g from ZAC3 hybridoma (antx-Shigella flexnen hpopolysaccharide), 2D6 hybridoma (anti- Vibrio cholerae hpopolysaccharide), IgA71 hybridoma (anti-He licobacter pylon urease), IgAC5 (anti- Shigella βexne hpopolysaccharide), Sal4 hybridoma (anti-Salmonella typhimurium), IgA380 hybridoma (anti-Sendai virus hemagglutimn-neuraminidase), IgA 59 hybridoma (anti-influenza virus hemagglutinin), ΗNK-20 hybridoma (anti-respiratory syncytial virus) A secretory component (SC) comprised by a secretory IgA antibody according to the invention, comprising a peptide insert corresponding to a protective epitope of an antigen, also called a chimeric SC, and which is able to trigger an immune response to the protective epitope, is a novel chimeric SC It is obtainable by recombinant DNA techniques in that a DNA sequence coding for an epitope which is foreign to the parent SC is inserted at a selected position of the parent SC so that the finally expressed foreign chimeric epitope in the secretory component does not affect the molecular folding of the novel SC, the assembly with polymeric IgA and the function of the novel slgA as a vaccine delivery system The parent SC can be any SC, in particular one already known in the art, and which may be readily available, such as for example the rabbit, the mouse, the rat and the human SC In the present examples, we selected rabbit SC for epitope insertion because 1 ) the cDNA for the SC portion of plgR was available (Schaerer et al , 1990), 2) the SC was shown not to perturb the binding of the slgA to the antigen (LuUau et al , 1996), 3) the SC and the IgAd can combine to form a slgA in vitro (Rindisbacher et al , 1995), and 4) a battery of monoclonal and polyclonal antibodies to the SC is available to map the possible effects of epitope substitution on protein structure (Table 1 see below) Our results first show a so far not identified function of domain I in the process of SC secretion, which nonetheless did not preclude either overexpression, or IgA binding of chimeric SC Foreign protective epitopes likewise can be any one retaining its protective, i e antigenic and immunogenic function after insertion They are for example selected from antigenic surface proteins of pathogenic fungi, bacteria, virus, parasites, or toxins, e g from intestinal, respiratory or genital bacterial pathogens, such as Shigella, Yersmia, Vibrio cholerae, Salmonella, Streptococcus pneumomae, Neissenae, or Chlamydia, from intestinal, respiratory or genital viruses, such as rotavirus, coronavirus, influenza virus, respiratory syncytial virus, herpes viruses, human papilloma virus, or human immunodeficiency virus, from intestinal, respiratory, genital toxins, such as pertussis toxin, diphteria toxin, cholera toxin, botulinal toxin, heat labile or stable toxins, or from intestinal, respiratory or genital parasites, such as shistosomia or giardia They have for example a length of about 9 up to 21 or more, preferably about 8 or 9 amino acids, and are preferentially hydrophilic for insertion on the surface of the carrier protein Such epitopes are for example the Shigella invasin epitope of IpaB of the sequence KDRTLIEQK, SEQ ) ED NO 11, or the immunogenic epitopes of the third variable (V3) loop of HIV- 1 envelope protein gp 120 in the region of between amino acid Nos 309 to 318 (Javaherian et al , 1989), an Epstein-Barr virus nuclear antigens in the region of between amino acid Nos 64 to 83 (antigen 2, Schmidt et al., 1991), Nos 339 to 347 (antigen 3, Burrows et al., 1990a), Nos. 416 to 424 (antigen 4, deCampos-Lima et al., 1994), or Nos. 290 to 299 (antigen 6, Burrows et al., 1990b), an influenza A virus hemagglutinin epitope in the region of between amino acid Nos 110 to 120 (Zaghouani et al., 1992), an influenza virus nucleoprotein epitope in the region of between amino acid Nos. 147 to 161 (Taylor et al., 1987), a bacteriophage 1 cl repressor protein in the region of between amino acid Nos 12 to 26 (Guillet et al, 1986), a diphteria toxin variant CRM197 in the region of between amino acid Nos 366 to 383 (Bixler et al., 1989), a tetanus toxin P30 epitope in the region of between amino acid Nos 947 to 967 (Panina-Bordignon et al , 1989), a mouse mammary tumor virus gp52 epitope in the region of between amino acid Nos. 143 to 153 (Dion et al , 1990), and in particular immunogenic epitopes of Helicobacter pylori, such as the urease B subunit (Labigne et al , 1991) or fragments thereof, e g in the region of between amino acid Nos 220 to 569, or Nos. 220 to 344, and the heat-shock protein GroES (Suerbaum et al , 1994) or fragments thereof It is also possible to insert mimotopes For instance, bacterial hpopolysaccharide epitopes can be converted into peptide mimotopes using monoclonal antibody screening of peptide phage libraries (Hoess et al, 1993). Foreign epitopes are preferably inserted in a position of the parent SC where they do not inadequately interfere with the natural folding of the SC, where they do not inhibit the binding to the polymeric, especially dimeric IgA in vitro, and where they still retain their antigenic and immunogenic activity The protective epitope may completely or partially replace a SC domain or be fused to the carboxy or amino terminus of the SC For example such position is a surface-exposed site of the loop connecting the E and F β strand of domain I of rabbit SC. In particular the eight amino acid sequence corresponding to position 102-109 in the protein (Fig 3) can be replaced by the nine amino acid foreign epitope derived from Shigella IpaB, SEQ ED NO 11. Further prefered insertion sites are situated in domain II and III, in particular at the positions of between amino acid Nos. 153 to 160, Nos. 194 to 201, Nos. 233 to 234, Nos. 272 to 277, Nos. 299 to 306, and the carboxy terminus, respectively, in the mouse SC sequence (Fig. 3). The foreign epitope may be inserted in an operable position of the SC amino acid sequence, provided that the overall structure can tolerate such a replacement. It is of high probability that, due to the o very conserved sequence between SC genes, a site mapped in one species will work in others A demonstration thereof is provided by Examples 8 and 9 The invention concerns further a vaccine comprising a secretory IgA comprising a polymeric IgA antibody bound to a secretory component which comprises a peptide insert corresponding to a protective epitope of an antigen The vaccine of the present invention is in the form of a pharmaceutical composition of conventional nature A pharmaceutical preparation according to the present invention for the prevention or treatment of an infection of mucosal lymphoid tissue in a mammal, including humans, comprises an effective amount of a secretory IgA antibody (slgA) according to the invention, eventually purified according to conventional methods, and a conventional pharmaceutical carrier The pharmaceutical preparation can be used nasally, genitally or rectally and in particular orally, in liquid form, or completely dried in the form of a powder, which can be pressed into tablets, filled into capsules, or applied as a dry powder in the form of a spray, e g nasal spray Suppositories or ointment are useful for rectal or vaginal application Conventional production methods are applied making use of conventional pharmaceutical carriers The method of protecting a mammal, including humans, against an infectious organism by active or passive immunization according to the present invention comprises administering of effective amounts of the pathogen-specific slgA carrying the foreign chimeric epitope The pharmaceutical vaccine is administered nasally, genitally, rectally or preferably orally for those pathogens that infect mucosal surfaces of the respiratory, urogenital or digestive tracts Pharmaceutical preparations in acid stable capsules can be used in case of intestinal infections Pharmaceutical preparations in form of nose drops or sprays are used when nasal administration is required, e g in the case of respiratory infections Suppositories or ointment are applied in case of rectal or vaginal infections The pharmaceutical preparations have to be administered according to the judgement of the physician in effective amounts depending on the concentration of the slgA and the route of administration so that a protective antigenic, immunogenic or curative effect is obtained The amounts and method of administration are to be selected further depending upon the age and weight of the patient, the nature and severity of the infection as well as the general condition of the patient In general it is sufficient to administer the slgA in amounts of about 1 to 50 milligrams, e g. orally or nasally, per patient either once or several, e g four times within one or two months I! The invention concerns further a secretory component (SC) of an IgA which comprises as a (poly)peptide insert a protective epitope of an antigen as defined hereinbefore, in particular wherein the peptide insert is an amino acid sequence from a bacterial, viral or parasitic antigen or a toxin antigen which is recognized by an antibody Such SCs are prepared according to conventional recombinant DNA techniques, whereby a DNA insert coding for the epitope is prepared, e g. by chemical synthesis, inserted in proper reading frame into the DNA coding for the SC in a position defined hereinbefore to produce a chimeric DNA coding for the SC and the foreign epitope The chimeric DNA is inserted into a proper expression vector which is then introduced into a proper mammalian cell where it is converted under suitable conditions into chimeric, glycosylated SC protein The expressed chimeric SC comprising the foreign antigenic and immunogenic protective epitope(s) is recovered and purified according to conventional methods, e g by Ni 2+ -chelate affinity chromatography (Rindisbacher et al , 1995) The invention concerns further a chimeric DNA construct comprising a DNA sequence coding for a secretory component of an IgA and a DNA insert coding for the protective epitope of an antigen Such construct is in particular an expression vector able to express the chimeric SC in an eukaryote, in particular in a mammalian, including a human, cell Representative constructs are coding sequences inserted into expression vectors including pCB6, pCB7, pcDNA3, pCI, pSI, recombinant vaccinia viruses, and recombinant baculoviruses The invention concerns further a method for producing a secretory IgA antibody comprising a polymeric IgA antibody bound to a chimeric secretory component, which comprises a peptide insert corresponding to a protective epitope of an antigen, wherein said secretory component is combined with said polymeric IgA antibody The polymeric IgA and the chimeric SC are in particular such as defined hereinbefore. The combination of the two components, in particular of dimeric IgA and chimeric SC to give the slgA is preferably carried out in vitro as described by Rindisbacher et al (1995) Briefly, 1 microgram of purified recombinant SC in PBS buffer and 5 micrograms of purified dimeric IgA in PBS buffer are mixed together in 100 microliters (final volume) of PBS buffer, and allowed to reassociate at ambient temperature for 16 hours. The invention concerns further a method for active immunization of a mammal which comprises administering to said mammal of an effective amount of a vaccine comprising a secretory IgA comprising a polymeric IgA antibody bound to a secretory n component which comprises a (poly)peptide insert corresponding to a protective epitope of an antigen. Abbreviations Throughout the entire description the following abbreviations are used: cDNA complementary DNA CV-1 African green monkey kidney cells (ATCC CCL70) ELISA enzyme linked immunosorbent assay FPLC fast protein liquid chromatography HRP horse radish peroxidase IgAp IgA polymer IgAd dimeric IgA IgAd-SC(IpaB) dimeric IgA with secretory component contaning IpaB IgAι-SC(wt) dimeric IgA with wild type secretory component IgA m monomeric IgA IpaB Shigella invasin protein antigen B M cells cells transporting antigens across epithelium overlaying organized lymphoid tissue MALT mucosa-associated lymphoid tissue pl lK late promoter of the 1 IK envelope protein of vaccinia virus pAb polyclonal antibody PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline, pH 7.2 PCR polymerase chain reaction pHGSl-SC plasmid Fig. 5 pHGSl-SCόxHis plasmid Fig. 5 pEBI-SC(IpaB) plasmid Fig. 5 pSC-l l(A/M) plasmid Fig. 4 pSC-11 plasmid Fig. 4, (Schaerer et al, 1990) PIR polymeric immunoglobulin receptor PER(IpaB) PER comprising the IpaB epitope PIR(wt) PER wild type pLKneo-PER(IpaB) plasmid Fig. 5 pLKneo-PER plasmid Fig. 5 rW recombinant vaccinia virus SC secretory component SC(IpaB) secretory component comprising the IpaB epitope SC-IgA complex of secretory component and IgA SDS sodium dodecylsulfate slgA secretory immunoglobulin A TKL thymidine kinase, left arm of w TK gene TKR thymidine kinase, right arm of w TK gene Tris-HCl tris(hydroxymethyl)aminomethane with hydrogene chloride w vaccinia virus wHGSl-SC(IpaB)6xHis recombinant vaccinia virus used for the expression of SC(IpaB)6xHis wHGSl-SCόxHis recombinant vaccinia virus used for the expression of SCόxHis wt wild type ZAC3 hybridoma cell hne secreting IgA antibodies Monoclonal antibodies (mAb) and polyclonal antisera (pAb): The monoclonal antibodies (mAb) and polyclonal antisera (pAb) used in the examples and their properties are listed in Table 1. Table 1 ' ■ ( Abbreviations pAb: polyclonal antiserum; mAb. monoclonal antibody; EP immunoprecipitation, ELISA enzyme linked immunosorbent assay. The following examples serve to further illustrate the invention without restricting it In the examples the following materials and general methods were used Example 1: Construction of chimerized SC cDNA plasmid pSC(IpaB). A rabbit PIR cDNA fragment (allele t61) is recovered from plasmid pSC-11 (Fig 4, Schaerer et al , 1990) by Hindlll/Saul digestion The 1082 bp fragment serves as a template for two PCRs aiming at the insertion of two unique restriction sites for subsequent sequence replacement within domain I of the rabbit SC clone PCR conditions are as follows 94°C for one minute, 58°C for 1 minute, and 72°C for 2 minutes, repeated for 30 cycles using a thermocycler from Cetus Corporation In the first reaction, an AccI site is created at position 418 using oligonucleotides SEQ ID NO 1 (hybridizing sequence 429-394) and SEQ ED NO 2 (hybridizing sequence 314-337) In order to facilitate subsequent ligation of PCR products, the 5' primer comprises the unique NotI site at position 324, and the 3' primer carries a 5' end Bglll site In the second reaction, a Mrol site is inserted at position 453 using oligonucleotides SEQ ED NO 3 (hybridizing sequence 448-480) and SEQ ED NO 4 (hybridizing sequence 905-881) To allow subsequent cloning steps, a Bglll site is added at the 5' end of the 5' primer, and the 3' primer is selected because it contains the unique BstEII site at position 888 The 128 bp and 469 bp PCR products digested with Bglll are hgated together, then digested with NotI and BstEII and finally inserted into pSC-11 treated with the same two enzymes To confirm the correct nature of the recombinant DN PCR-derived inserts are sequenced by the method of Sanger et al. (1977) Several of the genes involved in invasion of cultured cells by Shigella flexnen, an enteropathogen that causes dysentery in humans, have been identified and designated as ipa for "invasion plasmid antigen" (Maurelli et al., 1985; Baudry et al, 1987). Two proteins /5 ~ encoded by ipa genes, IpaB and IpaC, are exposed on the bacterial surface, and might explain why antibodies to these proteins are seen in infected animals or recovering patients (Phalipon et al , 1992) For insertion into SC, we selected the immunodominant linear B cell epitope consisting of residues Lys-Asp-Arg-Thr-Leu-Ele-Glu-Gln-Lys (KDRTLIEQK) of IpaB (Barzu et al., 1993) The nine amino acid sequence is chosen in order to change the length of the original loop by only a single amino acid Ohgonucleotides coding for the Shigella invasin B (IpaB) epitope (KDRTLIEQK) are synthesized with a 5' AccI half site on the coding strand (SEQ ED NO 5) and a 3' Mrol half site on the non coding strand (SEQ ED NO 6) Following annealing, the hybrid is introduced into the newly created AccI and Mrol sites of plasmid pSC-l l(A/M) (Fig 4), The resulting construct is referred to as pSC(IpaB) (Fig 5) Example 2: Construction of expression and insertion vectors pHGSl-SC6xHis and pHGSl-SC(IpaB)6xHis. Epitope insertion is performed by inserting the fragment NotI (324)/SalI (1834) recovered from pSC(IpaB) into pLKneo-PER (Hirt et al , 1992) digested with the same two enzymes The resulting construct is called pLKneo-PER(lpaB) (Fig 5) Rabbit SC cDNA is engineered to contain the coding sequences for amino acids 1 to 571 fused to a C-terminus histidine hexamer tag The DNA fragment is cloned into W insertion plasmid pHGSl (Bertholet et al , 1985), which directs homologous recombination into the TK locus of the viral genome and contains regulatory sequences ensuring high expression levels in eukaryotic cells The insertion plasmids to generate rWs are constructed as follows Plasmid pSC- 11 is digested with Ncol at position 122 and Sail at position 1834 (putative C-terminus, Mostov et al , 1984). The Ncol and Sail sites are filled in using Mungbean exonuclease, and the fragment is hgated into the blunt-ended EcoRI site of pHGSl (Bertholet et al, 1985) containing the strong pi IK late promoter inserted in the body of the viral TK gene. This results in the in frame fusion of the 1 IK ATG to the second codon of the SC coding region Addition of a C-terminal six histidine tag is carried out by annealing of ohgonucleotides SEQ ED NO 7 and SEQ ED NO 8 followed by insertion into the Kpnl site located 3 bp upstream of the Sail site of pHGSl-SC. The sequence encodes 6 histidine residues, a io translational termination codon, as well as Xhol and Bglll sites to determine the orientation The resulting plasmid is designated pHGSl-SCόxHis (Fig. 5). Plasmid pHGSl-SC(IpaB)6xHis (Fig. 5) encoding antigenized SC is obtained by cleaving plasmid pHGSl-SC6xHis with NotI and BstEII and replacing the fragment by the corresponding mutated fragment coming from plasmid pSC(IpaB) (Fig. 5). Example 3: Generation of vaccinia virus recombinant vectors wHGSl-SC6xHis and wHGSl-SC(IpaB)6xHis. Vaccinia virus recombinant vectors wHGSl-SC6xHis and wHGSl-SC(IpaB)6xHis are generated by homologous recombination into the thymidine kinase (TK) gene of constructs pHGSl-SCόxHis and pHGSl-SC(IpaB)6xHis as described (Rindisbacher et al., 1995) Integration of the SC gene construct in the viral genome is checked by PCR after each round of plaque purification using TK upstream primer SEQ ED NO 9 and TK downstream primer SEQ ED NO No 10. Example 4: Large scale production and purification of recombinant rabbit SC in CV- 1 cells. Stationary phase CV-1 cells (ATCC CCL 70) at a density of 2 x 10 7 cells/175 cm 2 T-flask are washed with PBS, and infected with vaccinia virus recombinant vectors wHGSl-SC6xHis and wHGSl-SC(IpaB)6xHis expressing SCόxHis or SC(IpaB)6xHis, respectively, at a multiplicity of infection (M.O.I.) of 2. Infected cells are cultured in Dulbecco's modified Eagle's medium in the absence of serum and antibiotics for 24 hours. The culture supernatant is recovered by centrifugation at 120 x g for 15 min The production of recombinant wild type and chimeric SC is determined by immunodetection (Rindisbacher et al, 1995). Purification of secreted recombinant SC proteins is performed by Ni 2+ -chelate affinity chromatography according to the procedure given in Rindisbacher et al. (1995). The cell culture medium containing SCόxHis or SC(IpaB)6xHis is brought to pH 7.8, and subsequently loaded onto a column of Ni2 + -nitrilotriacetic acid agarose (Qiagen) equihbrated in 20 mM phosphate sodium, 500 mM NaCl, pH 7.8. All purification steps are performed at 4°C with prechilled buffers. One milliliter settled beads is used per 5 milligrams protein. Washing steps with 5 column volumes is carried out using equilibration buffer at pH 7.8 containing 8 mM imidazole. Elution is performed with equilibration buffer 1} at pH 7 8 containing 40 mM and 80 mM imidazole Fractions containing the purified protein are identified by SDS-PAGE with silver staining and immunodetection Buffer exchange against PBS is carried out using Centriprep-50 units (Amicon), and the material is kept at 4°C until further required For silver staining of recombinant SC, a selection of Ni 2+ -chelate column fractions are separated by SDS-PAGE, and then treated according to the procedure of Morrissey (1981) For Western blot analysis of recombinat SC, a selection of Ni 2+ -chelate column fractions are separated by SDS-PAGE prior to transfer onto blotting membranes, and detected with the goat antiserum pAb 982 at a dilution of 1 500 Binding of goat antiserum pAb 982 is then detected using HRP-conjugated anti-goat IgG antibodies at a dilution of 1 3,000, and the chemio-luminescence detection reagent (Amersham) The results are shown in Figure 6 A and B Example 5: Assay for binding rSC and IgAs. The interaction between recombinant SC and IgA is determined by dot blot reassociation assay (DORA) as described in Rindisbacher et al (1995) Murine IgAd and IgAm are obtained from hybridoma ZAC3 (Lullau et al , 1996) The results are shown in Figure 7 A and B Example 6: Reconstitution of IgAd-SC(IpaB). 350 micrograms of SC(IpaB) or SC(wt) are associated in vitro with 1 75 milligrams of IgAd purified from ZAC3 hybridoma (Lullau et al , 1996) at room temperature in PBS Reconstituted IgAd-SC complexes are purified by FPLC (fast protein hquid chromatography system from Pharmacia) onto a 140 cm x 1.6 cm Superdex 200 (Pharmacia) column (Lullau et al , 1996) equihbrated and eluted with PBS Fractions containing the reconstituted slgA are pooled and concentrated by filtration using Centriprep-50 units (Amicon) equilibrated in PBS IgAd-SC antibodies are sterilized by passage through a 0 2 micrometer pore size Acrodisc filter (Gelman Sciences) and stored at 4°C until use / θ Example 7: Mucosal immunization with reconstituted IgAd-SC(IpaB). Immunization! of mice with a mixture of 100 micrograms of in vitro reconstituted IgAd-SC(IpaB) antibodies in PBS together with 10 micrograms of cholera toxin (Calbiochem) as an adjuvant in a final volume of 200 microliters of PBS demonstrates that chimerized reconstituted slgA can serve as a mucosal delivery system Germ-free BALB/c mice are administered the antibodies by intragastric intubation via PE10 polyethylene tubing connected to an 1 milliliter syringe Two groups of three mice are challenged four times at day 1, 10, 44 and 51, and blood and sahva are collected 10 days after the last immunization Biological fluids are kept frozen until use Controls consist of immunization with 100 micrograms of IgAd-SC(wt)/cholera toxin, and PBS buffer Immune and control sera are collected and tested by enzyme linked immunosorbent assay (ELISA) Shigella lysates containing or lacking the IpaB antigen are applied to 96-well plates (Dynatech) to serve as the capture reagent for the antibodies in the sera to be tested Wells are blocked with PBS- 0 05% Tween-20 (Bio-Rad) for 30 minutes, and dilutions of the IpaB-specific antisera and control sera are added Following incubation for 2 hours, the wells are washed with PBS- 0 05% Tween-20, and binding of antibodies is measured by detection with a secondary antibody coupled to HRP Read-out values correspond to the absorbance at 492 nanometers of the orthophenyl diamine substrate converted by HRP in each individual wells High titers of antibodies to SC are obtained in sera from mice challenged with either IgAd-SC(IpaB) or IgAd-SC(wt) (Fig 8 left panel), thereby confirming the immunogenicity of SC when delivered into gastric mucosa A strong specific response against the IpaB-containing lysate can be detected by incubation of the sera of the mice immunized with IgAd-SC(IpaB) (Fig 8 middle panel) This indicates that serum antibodies in mice immunized with IgAd- SC(IpaB) can recognize the IpaB epitope in its native environment Background levels of similar low intensity are obtained with the sera of animals immunized with IgAd-SC(wt) or PBS alone (Fig. 8A) No signal is obtained when a lysate lacking IpaB is coated (Fig 8 right panel) Sera from mice immunized with IgAd-SC(IpaB) also bind to the free IpaB peptide, but not to the unrelated FLAG (EBI-Kodak) peptide (Fig 8B) Sahva samples of two mice challenged with IgAd-SC(IpaB) exhibits a reproducible, albeit weak binding to Shigella lysates containing IpaB (Fig 8C). Antibodies against SC are detected in all sahva samples of ammals immunized with IgAd-SC(IpaB) or IgAd-SC(wt) (Fig 8C) Data in Fig 8A-C thus demonstrate that gastric mucosa delivery of antigenized slgA can serve as a vaccine system capable to elicit both a systemic and a mucosal antibody response '5 The results are shown in Fig 8 A-C Example 8: Construction of expression vectors for m SC-FLAG Enzymes used for cloning were purchased from Boehringer Mannheim and Life Technologies, Inc. Deprotected synthetic oligodeoxynucleotides were obtained from Microsynth GmbH (Balgach, Switzerland) The 3095-bp long cDNA encoding mouse plgR (Piskurich et al , 1995) was cloned into the EcoRI site of Bluescript π KS+ (Stratagene), producing plasmid pBSIΕKS+mpIgR Because the authentic carboxyl terminus of mouse SC has not yet been precisely mapped, we chosed, based on comparison with human SC to stop the translation of the protein after Arg . The 3' region coding for the transmembrane domain and the cytoplasmic tail was deleted using the strategy depicted in Fig 9 The replacing double stranded ohgonucleotide (codmg strand SΕQ ΕD NO 12 5^C^GCCTTTTGCCAACGAAAGATAGAAT_ICGC-3', non-coding strand SΕQ ID NO 13 5^GGCCC G\A7TCTATCTTTCGTTGGCAAAAGGCCC-3') coding for the amino acids Pro to Arg contain half of a 5' Smal site (italic), a stop codon (bold), an ΕcoRI site (underhned), and half of a 3' NotI site to facilitate its insertion The resulting plasmid was named pBSmSC The 5 '-untranslated region (UTR) of the mouse plgR cDNA in pCB6 was deleted according to the strategy described in Fig 10 using primers SΕQ ΕD NO 14 5'-CCAGATCTCACAAGCAATGAGGCTCTACTTG-3' and SΕQ D NO 15 5'-CCGGT ACCCAGGCCAC ACTTG-3 ' The resulting plasmid was named pCB6mpIgR(-5') Plasmid pCB6 is an eukaryotic expression vector bearing a cytomegalovirus (CMV) promoter, a polyadenylation signal, and a SV40 ongin of replication (Brewer and Roth, 1991) To allow expression of mSC- FLAG constructs, the KpnI-ΕcoRI fragment of the constructs in Fig 9 was introduced into pCB6mpIgR(-5') as shown in Fig 11, resulting in the production of plasmid pCB6mSC(- 5') We used recombinant PCR (Higuchi, 1990) to introduce into pCB6mSC(-5') one copy of the FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) coding sequence at sites emphasized in Figure 3. As an example, the construction of mSC-FLAGDΕI(153-160) is described Inside primers contained the FLAG coding sequence (bold) as overlapping region SEQ ED NO 16 (coding strand) 5 -GACTACAAGGACGACGATGACAAGAAGAAATCCCTGTGTAAGAAGAC-3 ' and SEQ ED NO 17 (noncoding strand) 5 -CTΥGTCATCGTCGTCCTTGTAGTCGCATTCAATGGTCACATTTCTGCC-3' Outside primers 2o SEQ ED NO 18 (coding strand) 5'-GGGAGCTAC AAGTGTGGCCTGG-3 ', and SEQ ED NO 19 (noncoding strand). 5'-TAAAGCAGCTCTGGCTCAGGCG-3\ were designed to permit the amplification of a fragment containing Kpnl and Nhel restriction sites adapted to the substitution of wild type mSC sequences in pCB6pmSC(5-) The regions that have been amplified by PCR were sequenced (Sanger et al , 1977) The construct, while keeping the original Kozak and signal peptide sequences, lack both the 5' and 3' untranslated regions See Fig 12 and short description thereto. Example 9: Preparation of pCB6mSC-UreB:6xHis. In this case, a H. pylori urease B fragment is cloned at the carboxyl terminus of mSC. To get rid off the 5'-UTR, plasmid pBSEEKS+mpIgR was digested with Kpnl, purified on agarose gel, and ligated on itself, producing plasmid pBSIEKS+mpIgR(-5') The urease fragment was prepared by PCR amplification using primers SEQ ED NO 20 5'-CGGCCCGGGCACGAAGACTGGGGCACC-3' (coding strand, Xmal site shown in italic) and SEQ ED NO 21 5'-GC7C7 G^CCGAAAATGCTAAAGAGTTGCGC-3' (noncoding strand, Xbal site shown in italic) and inserted into the Xmal and Xbal sites of pBSIEKS+mpIgR(-5'), creating plasmid pBS EKS+mSC-UreB Plasmid pBSIEKS+mSC- UreB was digested with Kpnl and Xbal to produce a fragment containing mSC and the urease fragment, and introduced into the same sites of plasmid pCB6mSC(-5') to obtain plasmid pCB6mSC-UreB Finally, plasmid pCB6mSC-UreB was cut at the unique Xbal site downstream of the urease insert, and a 6xHis tag was inserted in frame using a double- stranded ohgonucleotide comprising the sequences SEQ ED NO 22 5'-CTAGACCATCACCATCACCATCA-3' (coding strand) and SEQ ED NO 23. 5'-CTAGTGATGGTGATGGTGATGGT-3' (noncoding strand) The final construct was called pCB6mSC-UreB 6xHis. Example 12: Culture and transfection of COS-7 cells COS-7 cells (ATCC CRL 1651) were cultured under 5% CO2 at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 4 5 g/l glucose, 20 mM HEPES, and 10 mg/ml gentamycin (all from Life Technologies, Inc.) About 10 7 cells in 0 4 ml of PBS were transfected by electroporation (400 V, 250 mF) for about 10 ms in 0 4-cm cuvettes (Bio-Rad) with 25 mg of the pCBόmSC constructs The DNA was prepared using the MaxiPrep kit of Qiagen, and used without further purification Cells (~2 x 10 7 cells in 20 ml medium) were seeded into 15-cm dishes, and the medium was aι replaced the next morning Cultures were maintained for 4-7 more days, and supernatants were harvested Example 13: Protocol for the capture ELISA of mouse SC The wells of Nunc MaxiSorp ELISA plates were coated with 50 μl of affinity purified IgG to native mSC (2 μg/ml in 50 mM sodium carbonate/bicarbonate, pH 9 6) Wells were blocked with 0 2 ml of PBS buffer containing 5% (w/v) nonfat dry milk and 0 05% (w/v) Tween-20 (Bio Rad) Samples containing mSC proteins serially diluted into 50 μl of PBS and incubated for 1 h at room temperature Purified mSC from mouse milk (0 to 200 ng/ml) was used as standard After washing with PBS containing 0 05% Tween-20, bound mSC was detected using biotinylated IgG to native mSC (40 μg/ml) HRP-coupled Extr Avidin (Sigma, 1 1,000) was developed with 1,2-phenylenediamine as a chromogene The reaction was stopped with one volume of 2 M H2SO4, and plates were read at 492 nm using 620 nm as reference wavelength The apparent Mr was determined by chromatography on 8% polyacrylamide gel (Acrylamide 29 , Bisacrylamide 1) in 1% sodium dodecylsulfate along with molecular weight markers (Bio Rad), according to the method of Laemmli (1970) Reduction was perfomed in the presence of 100 mM (final concentration) of dithiothreitol (Fluka) Example 14: Protocol for measurement of binding to dimeric IgA Binding of mSC mutants to immobilized IgA The wells of Nunc MaxiSorp ELISA plates were coated with 50 μl of either purified ZAC3 IgAd (5 μg/ml) or ZAC3 IgA (2 5 μg/ml) dissolved in PBS Control wells were coated with pooled mouse IgG (Sigma, 2 mg/ml) or were not coated Wells were blocked with 0 2 ml of Tris-buffered sahne (TBS, 25 mM Tris-HCl, 137 mM NaCl, 2 7 mM KCl, pH 7 5) containing 5% (w/v) nonfat dry milk and 0 05% (w/v) Tween-20 (Bio Rad) mSC proteins at various concentrations in 0 1 ml of PBS were incubated for 1 hr at room temperature Saturation of the signal occured with 5 ng/ml of mutant mSC protein After washing with TBS containing 0 05% Tween-20, either rabbit IgG to native mSC (10 μg/ml) or mouse anti-FLAG monoclonal antibody M2 (10 μg/ml, Eastman-Kodak) was apphed for 1 h at room temperature HRP-coupled secondary antibodies (1 1,000 dilution) were developed with 1,2-phenylenediamine as chromogene Reactions were stopped with one volume of 2 M H2SO4, and plates were read at 492 nm using 620 nm as reference wavelength Comparative results are shown in the table of Fig 13 The binding to dimeric IgA is higher Also, very importantly, the secretion of the recombinant mutant mSC is better References Banting, G , Brake, B , Braghetta, P , Luzio, J P , and Stanley, K K (1989) FEBS Lett 254, 177-183 Barzu, S , Nato, F , Rouyre, S , Mazie, J C , Sansonetti, P J , and Phalipon, A (1993) Infect Immun 61, 3825-3831 Baudry B , Maurelh A T , Clerc P , Sadoff J C , and Sansonetti, P J (1987) J Gen Microbiol 133, 3403-3413 Bertholet, C , Drillien, R , and Wittek, R (1985) Proc Natl Acad Sci U S A 82, 2096- 2100 Bixler, G S , Eby, R , Dermody, K , Woods, R M , Seid, R C , and Pillai, S , (1989) Lmmunobiol Proteins Peptides 5, 175-180 Brewer, C B , and Roth, M G (1991) J. Cell Biol. 114, 413-421 Burrows, S R , Sculley, T B , Misko, I S , Schmidt, C , and Moss, D j (1990a) J Exp Med 171, 345-349 Burrows, S R , Misko, I S , Sculley, T B , Schmidt, C , and Moss, D J (1990b) j Virol 64, 3974-3976 Corthesy, B , Kaufmann, M , Phalipon, A , Peitsch, M , Neutra, M R , and Kraehenbuhl, j - P (1996) j Biol Chem 271, 33670-33677 Dion, A , Knittel, J , and Morneweck, S (1990) Virology 179, 474-477 deCampos-Lima, P O , Levitsky, V , Brooks, J , Lee, S P , Hu, L F , Rickinson, A B , and Masucci, M G (1994) j Exp Med 179, 1297-1305 Guillet, J -G , Lai, M -Z , Briner, T J , Smith, J A , and Gefter, M L (1986) Nature 324, 260-262 Hanly, C W , Cook, L , Kingzette, M , Cox, W , Frutiger, S , Hughes, G J , and Jaton, j -C (1987) J Immunol 139, 1597-1601 Higuchi, R (1990) in PCR Protocols: A Guide to Methods and Applications (Ennis, M A , Gelfand, D H , Sninsky, J J , and White, T J , eds) pp 177-183, Academic Press Inc , New York Hirt, R , Hughes, Poulain-Godefroy, O , Billotte, J , Kraehenbuhl, j -P , and Fasel, N (1992) Gene 111, 199-206 Hoess, R H (1993) Curr Opin Structural Biology 3, 572-579 l Javaherian, K , Langlois, A.J., McDanal, C, Ross, K.L , Eckler, L.I , Jellis, C.L , Profy, A T , Rusche, J.R , Bolognesi, D P., Putney, S.D., and Matthews, T J (1989) Proc Natl Acad Sci U S A 86, 6768-6772 Kuhn, L.C., Kocher, H.P., Hanly, W C , Cook, L., Jaton, J -C, and Kraehenbuhl, J -P (1983) J. Biol Chem 258, 6653-6659 Krajci, P., Grzeschik, K. H., Geurts van Kessel, A. H M., Olaisen, B., and Brandtzaeg, P (1991) Hum Genet. 87, 642-648 Laemmli, U.K (1970) Nature 227, 680-685 Labigne, A F., Cussac, V., and Courcoux, P. (1991) J. Bacteriol 173, 1920-1931 LuUau, E., Heyse, S , Vogel, H , Marison, I , von Stockar, U., Kraehenbuhl, J.-P , and Corthesy, B (1996) J Biol Chem 271, 16300-16309 Maurelh, A T , Baudry, B , d'Hauteville, H , Hale, T L., and Sansonetti, P J (1985) Infect Immun 49, 164-171 Morrissey, J H (1981) Anal Biochem 117, 307-310 Mostov, K E , Friedlander, M , and Blobel, G (1984) Nature 308, 37-43 Neutra, M R , Michetti, P , and Kraehenbuhl, J.-P (1994) In Physiology of the Gastrointestinal Tract (Johnson, L. R , ed), p 685, Raven Press, New York Panina-Bordignon, P , Tan, A , Teπriijtelen, A , Demotz, S., Corradin, G , and Lanzavecchia, A (1989) Eur J Immunol 19, 2237-2242 Phalipon, A , Arondel, J., Nato, F , Rouyre, S , Mazie, J C , and Sansonetti, P J (1992) Infect Immun 60, 1919-1926 Piskurich, J F , Blanchard M H , Youngman K R , France, J A , and Kaetzel, C S (1995) J Immunol. 154, 1735-1747 Rindisbacher, L , Cottet, S , Wittek, R , Kraehenbuhl, J -P., and Corthesy, B (1995) J Biol Chem 270, 14220-14228 Roy, M J., and Varvayanis, M (1987) CeU Tissue Res 248, 645-651 Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc Natl. Acad Sci U.S.A 74, 5463- 5467 Schaerer, E., Verrey, F., Racine, L., Talichet, C , Rheinhardt, M., and Kraehenbuhl, J -P (1990) J. Cell Biol. 110, 987-998 Schmidt, C , Burrows, S.R , ScuUey, T.B., Moss, D.J., and Misko, I.S. (1991) Proc Natl Acad Sci. U.S.A 88, 9478-9482 Solari, R , Kuhn, L.C., and Kraehenbuhl, J.-P. (1985) J. Biol Chem 260, 1141-1145 2H Suerbaum, S , Thiberge, J M , Kansau, I , Ferrero, R L , and Labigne, A F (1994) Mol Microbiol 14, 959-974 Taylor, P M , Davey, J , Howland, K , Rothbard, J B , and Askonas, B A (1987) Immunogenetics 26, 267-272 Weltzin, R , Lucia- Jandris, P , Fields, B N , Kraehenbuhl, J -P , and Neutra, M R (1989) J CeU Biol 108, 1673-1685 Zaghouani, H , Krystal, M , Kuzu, H , Moran, T , Shah, H , Kuzu, Y , Schulman, J , and Bona, C (1992) J Immunol 148, 3604-3609 SEQUENCE LISTING (1) GENERAL INFORMATION (l) APPLICANT (A) NAME SWISS INSTITUTE FOR EXPERIMENTAL CANCER RESEARCH (B) STREET Ch des 155 (C) CITY Epalinges s/Lausanne (E) COUNTRY Switzerland (F) POSTAL CODE (ZIP) CH 1066 (G) TELEPHONE xx41-21-6925858 (H) TELEFAX xx41-21-6526933 (ii) TrrLE OF INVENTION Secretory Immunoglobulin A as a Mucosal Vaccine Delivery Sy stem (ui) NUMBER OF SEQUENCES 23 ) COMPUTER READABLE FORM (A) MEDIUM TYPE Floppy disk 03) COMPUTER IBM PC compatible (C) OPERATING SYSTEM PC-DOS/MS-DOS (D) SOFTWARE Patentin Release #1 0, Version #1 30 (EPO) (vi) PRIOR APPLICATION DATA (A) APPLICATION NUMBER EP 96203051 6 (B) FILING DATE 01-NOV-1996 (2) INFORMATION FOR SEQ ID NO 1 0) SEQUENCE CHARACTERISTICS (A) LENGTH 48 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (11) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = Oligonucleotide" (in) HYPOTHETICAL NO ANTI-SENSE YES 5 (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Oryctolagus cuiuculus (vii) IMMEDIATE SOURCE (B) CLONE pSC-ll(A/M) l o (vui) POSITION IN GENOME 03) MAP POSrnθN 429-394 (C) UNITS bp (ix) FEATURE (A) NAME/KEY pnmer_bιnd 1 5 03) LOCATION complement (13 48) (xi) SEQUENCE DESCRIPTION SEQ ID NO 1 GGGAAGATCT TCTAAGTATA CCACAAACTC CCC ITITI CA GGGAAGTC 48 (2) INFORMATION FOR SEQ ID NO 2 20 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 24 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear 25 (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc = Oligonucleotide" (in) HYPOTHETICAL NO (iλ ) ANTI-SENSE NO (\ ) FRAGMENT TYPE internal 30 (vi) ORIGINAL SOURCE (A) ORGANISM Oryctolagus cumculus (vii) IMMEDIATE SOURCE 03) CLONE pSC-l l(A M) (vui) POSΓΠON IN GENOME 35 03) MAP POSrπON 314-337 (C) UNITS bp (ix) FEATURE (A) NAME/KEY pπmer_bιnd 03) LOCATION complement (1 24) 40 (xi) SEQUENCE DESCRIPTION SEQ ED NO 2 2k AAGAGGAGAG CGGCCGCTGC GTGA 24 (2) INFORMATION FOR SEQ ED NO 3 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 45 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (u) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc = 'Oligonucleotide" (iii) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Oryctolagus curuculus (MI) IMMEDIATE SOURCE 03) CLONE pSC-l l(A/M) (Mil) POSΓΠON IN GENOME 03) MAP POSmON 448-480 (C) UNITS bp (ix) FEATURE (A) NAME/KEY pnmer bind 03) LOCATION complement (13 45) (M) SEQUENCE DESCRIPTION SEQ ED NO 3 GGGAAGATCT TCGACTCCGG AAGCTACAAG TGTGGCGTGG GAGTC 45 (2) INFORMATION FOR SEQ ED NO 4 0) SEQUENCE CHARACTERISTICS (A) LENGTH 25 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single φ) TOPOLOGY linear (u) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc = Ohgonucleotide" (ui) HYPOTHETICAL NO (iv) ANTI-SENSE YES (v) FRAGMENT TYPE internal (M) ORIGINAL SOURCE (A) ORGANISM Oryctolagus cuniculus (vu) IMMEDIATE SOURCE } 03) CLONE pSC-l l(A/M) ( m) POSITION IN GENOME 03) MAP POSITION 905-881 (C) UNITS bp (ix) FEATURE (A) NAME/KEY pnmer_bιnd 03) LOCATION complement (1 25) (xi) SEQUENCE DESCRIPΗON SEQ ED NO 4 GCACATTCAA AGGTCACCGA GCCCC 25 (2) INFORMATION FOR SEQ ED NO 5 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 32 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc = "oligonucleotide" (in) HYPOTHETICAL NO (iλ) ANTI-SENSE NO (\ ) FRAGMENT TYPE internal (M) ORIGINAL SOURCE (A) ORGANISM Shigella i asin B epitope KDRTLIEQK (MI) IMMEDIATE SOURCE (B) CLONE pSC(IpaB) (MΠ) POSITION IN GENOME (A) CHROMOSOME/SEGMENT Epitope KDRTLIEQK (C) UNΓΓS bp (ix) FEATURE (A) NAME/KEY mιsc_feature 03) LOCATION 5 31 (xi) SEQUENCE DESCRIPΗON SEQ ED NO 5 ATACAAAGAC AGGACATTGA TTGAGCAGAA AT 32 (2) INFORMATION FOR SEQ ED NO 6 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 34 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single φ) TOPOLOGY linear It (n) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = "ohgonucleotide" (in) HYPOTHETICAL NO (ι\) ANTI-SENSE YES (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Shigella invasm B epitope KDRTLIEQK (non coding strand) (MI) IMMEDIATE SOURCE (B) CLONE pSC(IpaB) (Mil) POSITION IN GENOME (A) CHROMOSOME/SEGMENT Epitope KDRTLIEQK (non codmg strand) (C) UNITS bp (ix) FEATURE (A) NAME/KEY misc eature (B) LOCATION 6 32 (xi) SEQUENCE DESCRIPTION SEQ ED NO 6 CCGGATTTCT GCTCAATAAC TGTCCTGTCT TTGT 34 (2) INFORMATION FOR SEQ ED NO 7 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 38 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (P) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc = "oligonucleoude" (in) HYPOTHETICAL NO (n) ANTI-SENSE NO (\) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM six histidine tag (MI) IMMEDIATE SOURCE 03) CLONE pHGSl-SCόxHis (vm) POSΓΠON EN GENOME (C) UNITS bp (ix) FEATURE (A) NAME/KEY mιsc_feature (B) LOCATION 3 20 (xi) SEQUENCE DESCRIPTION SEQ ED NO 7 CTCATCACCA TCACCATCAC TAAAGATCTC GAGTGTAC 38 % (2) INFORMATION FOR SEQ ED NO 8 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 38 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (n) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc = "ohgonucleoUde" (ui) HYPOTHETICAL NO (iv) ANTI-SENSE YES (v) FRAGMENT TYPE internal (M) ORIGINAL SOURCE (A) ORGANISM six histidine tag (MI) IMMEDIATE SOURCE 03) CLONE pHGSl-SC6xHιs (Mil) POSΓΠON IN GENOME (C) UNITS bp (ix) FEATURE (A) NAME/KEY miscjeature 03) LOCATION 15 32 (xi) SEQUENCE DESCRIPTION SEQ ED NO 8 ACTCGAGATC TTTAGTGATG GTGATGGTGA TGAGGTAC 38 (2) INFORMATION FOR SEQ ED NO 9 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 23 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (n) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc = "ohgonucleoUde" (in) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (M) ORIGINAL SOURCE (A) ORGANISM pHGS plasmid sequence (vm) POSΓΠON EN GENOME (C) UNITS bp (ix) FEATURE (A) NAME/KEY pnmer_bmd 03) LOCATION complement (1 23) (xi) SEQUENCE DESCRIPΗON SEQ ED NO 9 GCTACGCTAG TCACAATCAC CAC 23 (2) INFORMATION FOR SEQ ED NO 10 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 20 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = "ohgonucleoUde" (in) HYPOTHETICAL NO ANTI-SENSE YES (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM vaccmia MΓUS thymidine kinase gene (ix) FEATURE (A) NAME KEY pπmer_bιnd 03) LOCATION complement (1 20) (xi) SEQUENCE DESCRIPTION SEQ ED NO 10 GTCCCATCGA GTGCGGCTAC 20 (2) INFORMATION FOR SEQ ED NO 11 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 9 ammo acids (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE pepUde (ui) HYPOTHETICAL NO (iv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (vi) ORIGINAL SOURCE (A) ORGANISM Shigella invasin B epitope (vu) IMMEDIATE SOURCE (B) CLONE pHGSl-SC(IpaB)6xHιs (vui) POSITION IN GENOME (A) CHROMOSOME/SEGMENT Shigella mvasm B epitope (ix) FEATURE 3>i (A) NAME KEY PepUde (B) LOCATION 1 9 (xi) SEQUENCE DESCRIPTION SEQ ED NO 11 Lys Asp Arg Thr Leu He Glu Gin Lys 1 5 (2) INFORMATION FOR SEQ ED NO 12 0) SEQUENCE CHARACTERISTICS (A) LENGTH 31 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single 09) TOPOLOGY lmear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc="Ohgonucleotιde"" (in) HYPOTHETICAL NO ANTI-SENSE NO (Λ ) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE pBSmSC (Mil) POSITION IN GENOME 03) MAP POSITION 607-614 (C) UNITS bp (xi) SEQUENCE DESCRIPΗON SEQ ED NO 12 GGGCCTTTTG CCAACGAAAG ATAGAATTCG C 31 (2) INFORMATION FOR SEQ ED NO 13 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 35 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single OD) TOPOLOGY lmear (u) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON desc/="OhgonucleoUde"" (ui) HYPOTHETICAL NO (iv) ANTI-SENSE YES (v) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE pBSmSC (Mil) POSΓΠON IN GENOME 03) MAP POSITION 607-614 1 (C) UNITS bp (ix) FEATURE (A) NAME/KEY mιsc_feature 03) LOCATION complement (1 35) (xi) SEQUENCE DESCRIPΗON SEQ ED NO 13 GGCCGCGAAT TCTATCTTTC GTTGGCAAAA GGCCC 35 (2) INFORMATION FOR SEQ ED NO 14 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 31 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc ="Ohgonucleotιde"" (IU) HYPOTHETICAL NO ANTI-SENSE NO (Λ ) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE pCB6mpIgR(-5') ( il) POSΓΠON EN GENOME 03) MAP POSITION 607-614 (C) UNITS bp (ix) FEATURE (A) NAME/KEY pnmer_bmd 03) LOCATION complement (1 31) (xi) SEQUENCE DESCRIPTION SEQ ED NO 14 CCAGATCTCA CAAGCAATGA GGCTCTACTT G 31 (2) INFORMATION FOR SEQ ID NO 15 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 21 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single D) TOPOLOGY linear (u) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc ="01ιgonucleoUde"" (ui) HYPOTHETICAL NO (rv) ANTI-SENSE NO (v) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE pCB6mpIgR(-5') (vm) POSΓΠON EN GENOME (B) MAP POSITION 607-614 (C) UNITS bp (ix) FEATURE (A) NAME/KEY pπmer_b d 03) LOCATION complement (1 21) (xi) SEQUENCE DESCRIPTION SEQ ED NO 15 CCGGTACCC A GGCCACACTT G 21 (2) INFORMATION FOR SEQ ID NO 16 0) SEQUENCE CHARACTERISTICS (A) LENGTH 47 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc ="OhgonucleoUde"" (ui) HYPOTHEΗCAL NO ANTI-SENSE NO (\) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE mSC-FLAGDII(153-160) (Mil) POSITION IN GENOME (B) MAP POSπiON 153-160 (C) UMTS bp (ix) FEATURE (A) NAME/KEY pπmer nnd 03) LOCATION complement (1 47) (xi) SEQUENCE DESCRIPTION SEQ ED NO 16 GACTACAAGG ACGACGATGA CAAGAAGAAA TCCCTGTGTA AGAAGAC 47 (2) INFORMATION FOR SEQ ED NO 17 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 48 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY lmear (u) MOLECULE TYPE other nucleic acid 3ι (A) DESCRIPΗON /desc ="OhgonucleoUde"" (in) HYPOTHETICAL NO (iv) ANTI-SENSE YES (v) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE mSC-FLAGDII(153-160) (vm) POSΓΠON ΓN GENOME (B) MAP POSITION 153-160 (C) UNITS bp (ix) FEATURE (A) NAME/KEY pnmer_bmd 03) LOCATION complement (1 48) (xi) SEQUENCE DESCRIPΗON SEQ ED NO 17 CTTGTCATCG TCGTCCTTGT AGTCGCATTC AATGGTCACA TTTCTGCC 48 (2) INFORMATION FOR SEQ ED NO 18 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 22 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (u) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc ="Ohgonucleoude"" (in) HYPOTHETICAL NO ) ANTI-SENSE NO (Λ ) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE mSC-FLAGDII(153-160) (Mil) POSITION IN GENOME (C) UMTS bp (ix) FEATURE (A) NAME KEY pπmer_bιnd 03) LOCATION complement (1 22) (xi) SEQUENCE DESCRIPTION SEQ ED NO 18 GGGAGCTACA AGTGTGGCCT GG 22 (2) INFORMATION FOR SEQ ED NO 19 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 22 base pairs 03) TYPE nucleic acid 3r (C) STRANDEDNESS single 09) TOPOLOGY lmear (n) MOLECULE TYPE other nucleic acid (A) DESCRIPΗON /desc ="OhgonucleoUde"" 5 (ui) HYPOTHETICAL NO (iv) ANTI-SENSE YES (v) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE mSC-FLAGDπ(153-160) l o (MU) POSΓΠON IN GENOME (C) UNITS bp (ix) FEATURE (A) NAME/KEY pnmer_bιnd 03) LOCATION complement (1 22) 15 (xi) SEQUENCE DESCRIPΗON SEQ ED NO 19 TAAAGCAGCT CTGGCTCAGG CG 22 (2) INFORMATION FOR SEQ ID NO 20 0) SEQUENCE CHARACTERISTICS 20 (A) LENGTH 27 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (n) MOLECULE TYPE other nucleic acid 25 (A) DESCRIPTION /desc = 7des="01ιgonucleotιde"" (in) HYPOTHETICAL NO ANTI-SENSE NO (\) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 30 03) CLONE pBSUKS+mSC-UreB (vui) POSITION IN GENOME (C) UNITS bp (ix) FEATURE (A) NAME KEY pnmer_bιnd 35 (B) LOCATION complement (1 27) (xi) SEQUENCE DESCRIPΗON SEQ ED NO 20 CGGCCCGGGC ACGAAGACTG GGGCACC 27 (2) INFORMATION FOR SEQ ED NO 21 40 (l) SEQUENCE CHARACTERISTICS 3t (A) LENGTH 31 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (u) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc ="OhgonucleoUde"" (in) HYPOTHETICAL NO (iv) ANTI-SENSE YES (v) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE pBSIIKS+mSC-UreB (vm) POSΓΠON IN GENOME (C) UNΠS bp (ix) FEATURE (A) NAME/KEY pnmer_bιnd 03) LOC AΗON complement (1 31) (xi) SEQUENCE DESCRIPTION SEQ ED NO 21 GCTCTAGACC GAAAATGCTA AAGAGTTGCG C 31 (2) INFORMATION FOR SEQ ED NO 22 0) SEQUENCE CHARACTERISTICS (A) LENGTH 23 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY linear (n) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc ="OhgonucleoUde"" (in) HYPOTHETICAL NO ANTI-SENSE NO (v) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE pCB6mSC-UreB 6xHιs (vm) POSΓΠON EN GENOME (C) UNITS bp (ix) FEATURE (A) NAME/KEY mιsc_feature 03) LOC AΗON complement (1 23) (xi) SEQUENCE DESCRIPTION SEQ ED NO 22 CTAGACCATC ACCATCACCA TCA 23 (2) INFORMATION FOR SEQ ED NO 23 (l) SEQUENCE CHARACTERISTICS (A) LENGTH 23 base pairs 03) TYPE nucleic acid (C) STRANDEDNESS double (D) TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc ="OhgonucleoUde"" (in) HYPOTHETICAL NO (iv) ANTI-SENSE YES (v) FRAGMENT TYPE internal (MI) IMMEDIATE SOURCE 03) CLONE pCB6mSC-UreB 6xHιs (Mil) POSITION IN GENOME (C) UNITS bp (ix) FEATURE (A) NAME/KEY mιsc_feature 03) LOCATION complement (1 23) (xi) SEQUENCE DESCRIPΗON SEQ ED NO 23 CTAGTGATGG TGATGGTGAT GGT 23

Claims: What is claimed is: 1 A secretory IgA antibody comprising a polymeric IgA antibody bound to a secretory component which comprises a (poly)peptide insert corresponding to a protective epitope of an antigen 2 A secretory antibody according to claim 1 , wherein the protective epitope is inserted in a position where it does not inadequately interfere with the natural folding of the SC, where it does not inhibit the binding to the polymeric, especially dimeric IgA in vitro, and where it still retains its antigenic and immunogenic activity 3 A secretory antibody according to claim 1 , wherein the protective epitope of an antigen is inserted in a surface exposed side of the loop connecting the E and F β strand of domain I of rabbit SC 4 A secretory antibody according to claim 1 , wherein the protective epitope of an antigen is fused to the carboxy or amino terminus of SC 5 A secretory antibody according to claim 1, wherein a SC domain is replaced by a (poly)peptide carrying a protective epitope of an antigen 6 A chimeric secretory component which comprises as a (poly)peptide insert a protective epitope of an antigen 7 A chimeric secretory component according to claim 6 wherein the (poly)peptide insert is a protective epitope of a pathogenic microorganism 8 A chimeric secretory component according to claim 6 wherein the (poly)peptide insert is an amino acid sequence from a bacterial, viral, parasitic or mammalian antigen which is recognized by an immune effector, i e antibody, antigen presenting cell or lymphocyte 10 ' A DNA construct comprising a DNA sequence coding for a secretory component and a DNA insert coding for the protective epitope of an antigen X 1() A DNA construct according to claim lϋ comprising a DNA sequence coding for a secretory component and a DNA insert coding for the protective epitope of an antigen which is inserted at a position where the finally expressed foreign chimeric epitope in the secretory component does not effect the foldimg of the novel SC, the assembly with the polymeric IgA and the function of the novel slgA as a vaccine delivery system. \1 11 A method for producing a secretory IgA antibody according to claim l, wherein the secretory component according to claim 6 is combined with a polymeric IgA antibody. 3 3 RECTIFIED SHEET (RULE 91) ISA/EP J^. A method for purifying a secretory IgA aπt:body according to claim 1 , and produced according to claim J . 14'. A mucosal vaccine comprising an effective amount of a secretory IgA antibody IS. A method for the active immunization of a mammal which comprises administering to said mammal an effective protecting amount of a vaccine according to claim hf. \€. A method for the active immunization of a mammal according to claim \%, wherein the vaccine is administered via the nasal route, eg. in form of an aerosol or nasal drops; the oral route, e. g. in form of tablets or capsules, the vaginal or rectal route, e. g. in form of a suppository or an ointment. RECTIFIED SHEET (RULE 91) ISA/EP

Inventors: CORTHESY BLAISE, KAUFMANN MURIEL, KRAEHENBUHL JEAN-PIERRE, PEITSCH MANUEL, CORTHESY, BLAISE, KAUFMANN, MURIEL, KRAEHENBUHL, JEAN-PIERRE, PEITSCH, MANUEL
Applicants: CORTHESY BLAISE, KAUFMANN MURIEL, KRAEHENBUHL JEAN PIERRE, PEITSCH MANUEL, SUISSE RECH EXPERIMENT, CORTHESY, BLAISE, INSTITUT SUISSE DE RECHERCHES EXPERIMENTALES SUR L, KAUFMANN, MURIEL, KRAEHENBUHL, JEAN-PIERRE, PEITSCH, MANUEL
Application year: 1997
Main classification: C12N15
IPC codes: A61K38, A61K39, C07K1, C07K14, C07K16, C07K19, C12N15, C12P21