AU2002256565B2 - Antigen targeting - Google Patents

Description

WO 02/096949 PCT/AU02/00661 Antigen Targeting FIELD OF THE INVENTION The present invention to compositions and methods for raising an immune response in animals. In particular the compositions and methods of the present invention are useful in raising mucosal and systemic immunity.

BACKGROUND OF THE INVENTION As the preferred site of entry or colonization for many pathogens, mucosal surfaces of the body play an important role in defence against numerous infections1.

However, induction of mucosal immunity, other than by live oral vaccines, has been problematic. PhysiochenTical barriers at mucosal surfaces prevent adequate amounts of intact antigen reaching underlying mucosal lymphoid tissue and antigen localization in lymphoid tissues is critical for immune induction 2. The small amount of antigen that does reach these lymphoid sites is largely ignored in a system set up to maintain non-reactivity or tolerance to a heavy burden of food and other benign antigens encountered daily.

Effective delivery of vaccine antigens to Gut Associated Lymphoid Tissue (GALT) has long been recognised as the primary hurdle for mucosal vaccine development. Strategies using the oral route impose a host of obstacles including mucus barriers, degradative gastric acid and alimentary enzymes To overcome this, co-delivery of antigen with adjuvants such as cholera toxin has been employed but the clinical application is limited due to the toxicity of such adjuvants. Direct injection of antigen into mucosal lymphoid tissue has also been used 7, but such practices would be unlikely to be accepted by vaccinees.

The present inventors postulated that delivering antigens via the blood targeted to mucosal lymphoid tissues may bypass these obstacles. The present inventors tested targeting of antigens to the Mucosal Addressin Cellular Adhesion Molecule-1 (MAdCAM-1), a receptor present in circulatory vessels in the Gut Associated Lymphoid Tissue (GALT) and found that such antigen targeting induced a rapid mucosal IgA response in the gut and augmented (1000 fold) the systemic response to antigen.

WO 02/096949 PCT/AU02/00661 SUMMARY OF THE INVENTION Accordingly in a first aspect the present invention consists in a method of raising an immune response in an animal, the method comprising administering to the animal a composition comprising a carrier and an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a second aspect the present invention consists in a targeted antigen comprising an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a third aspect the present invention consists in an antigenic composition, the composition comprising a carrier and an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a fourth aspect the present invention consists in a method of raising an immune response in an animal, the method comprising administering to the animal a composition comprising a carrier and a DNA molecule, the DNA molecule encoding an antigen and a targeting moiety which binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a fifth aspect the present invention consists in a DNA molecule, the DNA molecule encoding an antigen and a targeting moiety which binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a sixth aspect the present invention consists in an antigenic composition, the composition comprising a carrier and a DNA molecule, the DNA molecule encoding an antigen and a targeting moiety which binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a preferred embodiment of the present invention the targeting moiety binds to Mucosal Addressin Cellular Adhesion Molecule-1.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Targeting mucosal inductive sites via the blood, a, Scheme of antigen targeting to MAdCAM-1. Rat IgG2a anti-MAdCAM-1 antibodies were used to target sites of MAdCAM-1 expression in mesenteric lymph nodes (MLN) and Peyer's patches (PP) of the GALT. Targeting these specialized lymphoid sites via the blood WO 02/096949 PCT/AU02/00661 3 route bypasses physiochemical barriers associated with mucosal antigen delivery (via the oral route). b, MAdCAM-1 targeted antigen preferentially localizes to mucosal inductive sites in-vivo. Proteins were radioiodinated and injected intravenously mice per group) to quantify the amount accumulated in mucosal versus peripheral lymphoid sites. Means and standard deviations are shown. Binding of anti- MAdCAM-1 antibody MECA-367 was enhanced in MLN and PP compared with the isotype control (p *0.013 and **0.002 respectively; Student t-test). No such enhancement was found in peripheral lymphoid sites such as the spleen and inguinal lymph nodes (ILN).

Figure 2. MAdCAM-1 antigen targeting induces mucosal and augments systemic immune response. Mice (5 per group) were immunized intravenously with 100g of either anti-MAdCAM-1 antibody MECA-367 or isotype control GL117 in saline. Rat IgG2a specific antibody responses for faecal IgA a, serum IgA b, and serum IgG c, were measured by ELISA at 2,4, and 8 weeks; Mean and standard deviation of antibody titres (log) are shown. Faecal IgA responses (representing mucosal responses) were detected only when the antigen was targeted to MAdCAM-1 a.

Moreover, such targeting greatly augmented the systemic IgA and IgG response b c.

Proteins were either heat aggregated (70°C 1Smins) or cleared of aggregates by ultacentrifugation (5 X 10 5 g, 20min) to investigate their effect on the mucosal antibody response d. Mice (5 per group) were immunized intravenously with 100g of either aggregate free anti-MAdCAM-1 antibody MECA-89, aggregate free isotype control GL117, or heat aggregated isotype control GL117. Aggregation of protein had no effect on the mucosal antibody response. Moreover, targeting MAdCAM-1 with another rat IgG2a antibody MECA-89, resulted in similar enhancement in faecal antibody to that seen with MECA-367.

Figure 3. Mucosalimmune response elicited by M4dCAM-1 targeting is local. a b, Mice (3 per group) were immunized intravenously with 100g of either MECA-367 or the isotype control GL117. Five and 11 days after, mesenteric lymph node (MLN), Peyer's patches (PP) and lamina propria lymphocytes (LPL) were harvested and assayed for rat IgG2a specific IgA antibody secreting cells (ASC) by ELISPOT; Mean and standard deviation (spots/ 106) cell are shown. MAdCAM-1 targeted immunization induced antigen specific B-cell responses in MLN, PP and LPL. c, Antigen specific IgA is secreted by gastrointestinal explants after MAdCAM-1 targeting. Mice (3 per group) were immunized intravenously with 100g of either WO 02/096949 PCT/AU02/00661 4 MECA-367 or the isotype control GL117. Peyer's patches (PP) and intestinal segments (IS) were taken at 10 days and cultured in-vitro for 6 days. Antigen specific IgA in the culture supernatant was measured by ELISA; Mean and standard deviation of the optical density are shown. MAdCAM-1 targeted immunization induced a mucosal antibody response that could be detected in both PP and intestinal segment cultures.

Figure 4. MAdCAM-1 targeting enhances mucosal and systemic cytokine responses. Mice (3 per group) were immunized intravenously with 100g of either MECA-367 or isotype control GL117 and boosted intraduodenally at 2 weeks. After 3 days, spleens and MLN cells were harvested and cultured for 72hours in GL117. Cytokines IL-2, and IFN-y were measured in culture supernatant by ELISA; Mean and standard deviation of cytokine levels are shown. MAdCAM-1 targeted immunization resulted in enhanced levels of IL-2 and IFN-y from both spleen and MLN cell cultures.

Figure 5. Enhancement by MAdCAM- targeting is also effective by the intramuscular route. Mice (5 per group) were immunized intramuscularly with 100g of either anti-MAdCAM-1 (MECA-367) or isotype control (GL117) in 0.2ml of saline (O.lml into each quadriceps). Rat IgG2a specific antibody responses for faecal IgA, serum IgA and serum IgG were measured by ELISA at 2 weeks; Means SD are shown.

Figure 6. Enhancement by MAdCAM-1 targeting is specific for the targeted antigen and can be shown for anotherantigen. Mice (5 per group) were immunized intravenously with 100g of either MECA-367 or isotype control GL117 plus 500g of ovalbumin (OVA) in 0.3ml of saline. OVA specific antibody responses for faecal IgA, serum IgA and serum IgG were measured by ELISA at 2 weeks. Mice were immunized intravenously with 60g of either Fluorescein isothiocyanate (FITC) conjugated anti-MAdCAM-1 antibody (MECA-89) or isotype control antibody (GL117) in 0.2ml of saline. FITC specific antibody responses for faecal IgA, serum IgA and serum IgG were measured by ELISA at 2 weeks. Means SD are shown.

Figure 7. MAdCAM-1 targeting enhances T-cell cytokine and proliferative responses. Mice were immunized intravenously with Ig of either MECA-89 or isotype control GL117 in 0.lml of saline on days 0,2,4,7,9,12 and boosted WO 02/096949 PCT/AU02/00661 intraperitoneally at day 18 with 100g of GL117 in CFA. Ten days after, spleens and MLN cells were harvested. Antigen induced proliferation of splenic and MLN (b) T-cells was determined in a standard 5 day 3H-thymidine uptake protocol. Mean stimulation index SEM shown. Antigen induced cytokine responses were evaluated by culturing splenocytes in the presence of rat IgG2a (GL117, Cytokine levels in the supernatant were measured by sandwich ELISA; Mean SD shown.

Figure 8. Enhancement by MAdCAM-1 targeting is independent of splenic antigen localisation. Splenectomy or sham operations were performed on Mice (5 per group). One week after operation mice were immunized intravenously with 100g of either anti-MAdCAM-1 (MECA-367) or isotype control (GL117) in 0.2ml of saline. Rat IgG2a specific antibody responses for faecal IgA and serum IgG were measured by ELISA at 2 weeks; Means SD of two independent experiments are shown.

Figure 9. IgG can be detected from transient transfecion of antbody constructs.

Heavy and light chain constructs from GL117, MECA-367 and MECA-89 were transfected into CHO cells using FuGENE (Roche, Mannheim, Germany) reagent according to manufacturers instructions. S/N was harvested 3 days after transfection and levels of mouse IgG2c was determined by capture ELISA. Mean O.D. 450nm are shown.

Figure 10. Genetically fused antigen can be detected from transient transfection of antibody constructs. Heavy and light chain constructs from GL117, MECA-367 and MECA-89 were transfected into CHO cells using FuGENE (Roche, Mannheim, Germany) reagent according to manufacturers instructions. S/N was harvested 3 days after transfection and levels of ovalbumin (OVA) was determined by capture ELISA. Means SD 450nm) are shown.

Figure 11. Isotype control antibody (GL117) constructs retain binding to bacterial galactosidase. Heavy and light chain constructs from GL117 and MECA-89 were transfected into CHO cells using FuGENE (Roche, Mannheim, Germany) reagent according to manufacturers instructions. S/N was harvested 3 days after transfection and antibody binding to bacterial -galactosidase determined by ELISA. Means SD 450nm) are shown.

WO 02/096949 PCT/AU02/00661 6 DETAILED DESCRIPTION In a first aspect the present invention consists in a method of raising an immune response in an animal, the method comprising administering to the animal a composition comprising a carrier and an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a second aspect the present invention consists in a targeted antigen comprising an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a third aspect the present invention consists in an antigenic composition, the composition comprising a carrier and an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a preferred embodiment the composition is administered to the animal parenterally. Routes of administration include IV, IM, IP, subcutaneous and intradermal. It is preferred that the administration is by a haematogenous route.

In a fourth aspect the present invention consists in a method of raising an immune response in an animal, the method comprising administering to the animal a composition comprising a carrier and a DNA molecule, the DNA molecule encoding an antigen and a targeting moiety which binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a fifth aspect the present invention consists in a DNA molecule, the DNA molecule encoding an antigen and a targeting moiety which binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a sixth aspect the present invention consists in an antigenic composition, the composition comprising a carrier and a DNA molecule, the DNA molecule encoding an antigen and a targeting moiety which binds to at least one receptor present in circulatory vessels in Gut Associated Lymphoid Tissue.

In a preferred embodiment of the present invention the targeting moiety binds to Mucosal Addressin Cellular Adhesion Molecule-1.

Molecules which target MAdCAM-1 are known in the art. These include anti-MAdCAM-1 antibodies and alpha 4 and beta 7 integrins. It ispresently preferred that the targeting moiety is an antibody, an antibody fragment or an antibody binding domain. Further information regarding antibody fragments such as single chain Fvs WO 02/096949 PCT/AU02/00661 7 can be found in for example, Hudson PJ Kortt AA. "High avidity scFv multimers; diabodies and triabodies". J. Immunol. Meth. 231 (1999) 177-189; Adams GP Schier R. "Generating improved single-chain Fv molecules for tumor targeting". J. Immunol.

Meth. 231 (1999) 249-260; Raag R Whitlow M. "Single-chain Fvs" FASEB J. 9 (1995) 73-80; Owens RJ Young RJ. "The genetic engineering of monoclonal antibodies" J.

Immunol. Meth. 168 (1994) 149-165.

Monoclonal antibodies directed against MAdCAM-1 are known in the art Two such antibodies, MECA-89 and MECA-367, are available from ATCC under accession nos. HB-292 and I-IB-9478 respectively.

Additional ligands that target MAdCAM-1 and vascular addressins may begenerated by using peptide display libraries such as those made in phage display technology (Burton DR. "Phage display. Immunotechnology." 1995 1:87-94; Cwirla SE, Peters EA, Barrett RW, Dower WJ. Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci U S A. 1990 87:6378-82; Scott JK, Smith GP.

"Searching for peptide ligands with an epitope library." Science. 1990 249:386-90) as well as peptide libraries displayed on other surface components e.g. on flagella molecules (Westerlund-Wikstrom B. "Peptide display on bacterial flagella: principles and applications." Int J Med Microbiol. 2000 290:223-30) or on yeast (Boder ET, Wittrup KD. "Yeast surface display for screening combinatorial polypeptide libraries." Nat Biotechnol. 1997 15:553-7).

As will be recognised by those skilled in the field of protein chemistry there are numerous methods by which the antigen may be bound to the targeting moiety.

Examples of such methods include: 1) affinity conjugation such as antigen-ligand fusions where the ligand has an affinity for the targeting antibody (examples of such ligands would be streptococcal protein G, staphylococcal protein A, peptostreptococcal protein L) or bispecific antibody to cross-link antigen to targeting moiety.

2) chemical cross-linking. There are a host of well known cross-linking methods including periodate-borohydride, carbodiimide, glutaraldehyde, photoaffinity labelling, oxirane and various succinimide esters such as maleimidobenzoyl-succinimide ester. Many of these are readily available commercially e.g. from Pierce, Rockford, IL, USA. There are many references to cross-linking techniques including Hermanson GT "Bioconjugate Techniques" Academic Press, San Diego 1996; Lee YC, Lee RT. Conjugation of glycopeptides to proteins. Methods Enzymol. 1989;179:253-7; Wong SS "Chemistry of Protein Conjugation and Cross-linking" CRC Press 1991; WO 02/096949 PCT/AU02/00661 8 Harlow E Lane D "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory, 1988; Marriott G, Ottl J. Synthesis and applications of heterobifunctional photocleavable cross-linking reagents. Methods Enzymol.

1998;291:155-75.

3) genetic fusions. These can be made as recombinant antibody-antigen fusion proteins (in bacteria, yeast, insect or mammalian systems) or used for DNA immunization with or without spacers between the antibody and antigen. There are many publications of immunoglobulin fusions to other molecules. Fusions to antigens like influenza hamagglutinin are known in the art see, for example, Deliyannis G, Boyle JS, Brady JL, Brown LE, Lew AM. "A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge." Proc Natl Acad Sci U S A. 2000 97:6676-80. Short sequences can also be inserted into the immunoglobulin molecule itself [Lunde E, Western KH, Rasmussen IB, Sandlie I, Bogen B. "Efficient delivery of T cell epitopes to APC by use of MHC class II-specific Troybodies." J Immunol. 2002 168:2154-62]. Shortened versions of antibody molecules (e.g.

Fv fragments) may also be used to make genetic fusions [Reiter Y, Pastan I.

"Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer: disulfide-stabilized Fv immunotoxins." Clin Cancer Res.

1996 2:245-52].

As will be understood whatever the method of targeting moiety-antigen fusion used, such fusions need to be able to target the receptor, such as MAdCAM-1, in vivo. It is therefore highly preferred that the binding of the fusion to red blood cells or other cells vascular endothelium of the lung) which it may encounter during its hematogenous traverse is minimal as such binding may inhibit the fusion reaching the desired sites within the GALT.

For similar reasons antigens which have a high propensity for binding to cells or tissues which the fusion may encounter on its route to the GALT should also be avoided.

Clearly the fusion process should be designed or selected so as not to interfere with the ability of the targeting moiety to bind to the receptor present in circulatory vessels in the GALT. This can be tested in vitro by determining whether the fusions bind the receptor, such as MAdCAM-1 on cryostat sections of the GALT by immunohistology or bind recombinant receptor proteins by ELISA.

The antigen used in the present invention can be any antigen against which it is desired to raise an immune response. It is preferred that the antigen is selected such WO 02/096949 PCT/AU02/00661 9 that an immune response is generated against any pathogen whose main portal of entry is the gut and those that colonise mucosal surface. This would include Salmonella, Cholera, Helicobacter pylori, rectally introduced HIV, Candida, P. gingivalis, gut parasites or gut associated toxins. Moreover, the present invention may be used to induce an immune response to gut hormones gastrin) or their receptors for gut associated cancers [Watson SA, Clarke PA, Morris TM, Caplin ME.

"Antiserum raised against an epitope of the cholecystokinin B/gastrin receptor inhibits hepatic invasion of a human colon tumor." Cancer Res. 2000 60:5902-7; Smith AM, Justin T, Michaeli D, Watson SA. "Phase I/II study of G17-DT, an anti-gastrin immunogen, in advanced colorectal cancer." Clin Cancer Res. 2000 6:4719-24].

Information regarding HIV antigens such as gpl20 and other candidates can be found in Stott J, Hu SL, Almond N. "Candidate vaccines protect macaques against primate immunodeficiency viruses." AIDS Res Hum Retroviruses. 1998 Oct;14 Suppl 3:S265-70.

Informationr regarding Helicobacterpylori antigens such as urease of Helicobacterpylori and other candidates can be found in Lee CK. "Vaccination against Helicobacterpylori in non-human primate models and humans." Scand J Immunol.

2001 May;53(5):437-42.

Further information regarding antigens in which mucosal immunity is important may be found in van Ginkel FW, Nguyen HH, McGhee JR. "Vaccines for mucosal immunity to combat emerging infectious diseases." Emerg Infect Dis. 2000 Mar-Apr;6(2):123-32; and Neutra MR, Pringault E, Kraehenbuhl JP. "Antigen sampling across epithelial barriers and induction of mucosal immune responses." Annu Rev Immunol. 1996;14:275-300.

As will be recognised the third to sixth aspects of the present invention relate to DNA vaccination.

The ability of direct injection of non-replicating plasmid DNA coding for viral proteins to elicit protective immune responses in laboratory and preclinical models has created increasing interest in DNA immunisation. A useful review of DNA vaccination is provided in Donnelly et al, Journal of Immunological Methods 176 (1994) 145-152, the disclosure of which is incorporated herein by reference.

DNA vaccination involves the direct in vivo introduction of DNA encoding an antigen into tissues of a subject for expression of the antigen by the cells of the subject's tissue. DNA vaccines are described in US 5,939,400, US 6,110,898, WO 95/20660 and WO 93/19183, the disclosures of which are hereby incorporated by reference in their entireties. The ability of directly injected DNA that encodes an WO 02/096949 PCT/AU02/00661 antigen to elicit a protective immune response has been demonstrated in numerous experimental systems (see, for example, Conry etal., Cancer Res 54:1164-1168, 1994; Cardoso etal., Immuniz Virol 225:293-299, 1996; Cox etal., J Virol 67:5664-5667, 1993; Davis etal., Hum Mol Genet 2:1847-1851, 1993; Sedegah etal., Proc Natl Acad Sci USA 91:9866-9870, 1994; Montgomery etal., DNA Cell Biol 12:777-783, 1993; Ulmer et al., Science 259:1745-1749, 1993; Wang etal., Proc Natl Acad Sci USA 90:4156-4160, 1993; Xiang etal., Virology 199:132-140, 1994; Yang etal., Vaccine 15:888-891, 1997; Ulmer et alScience 259:1745, 1993; Wolff etalBiotechniques 11:474, 1991).

To date, most DNA vaccines in mammalian systems have relied upon viral promoters derived from cytomegalovirus (CMV). These have had good efficiency in both muscle and skin inoculation in a number of mammalian species. A factor known to affect the immune response elicited by DNA immunization is the method of DNA delivery, for example, parenteral'routes can yield low rates of gene transfer and produce considerable variability of gene expression (Montgomery etal., DNA Cell Biol 12:777-783, 1993). High-velocity inoculation of plasmids, using a gene-gun, enhanced the immune responses of mice (Fynan etal., Proc Natl Acad Sci USA 90:11478-11482, 1993; Eisenbraun etal, DNA Cell Biol 12:791-797, 1993), presumably because of a greater efficiency of DNA transfection and more effective antigen presentation by dendritic cells. Vectors containing the nucleic acid-based vaccine of the invention may also be introduced into the desired host by other methods known in the art, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), or a DNA vector transporter.

As used herein the term "animal" encompasses both human and non-human animals.

As used herein the term "circulatory vessel" encompasses both blood and lymphatic vessels.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications mentioned in the specification are herein incorporated by reference.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that WO 02/096949 PCT/AU02/00661 11 any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

In order that the nature of the present invention may be more clearly understood preferred forms thereof will be described with reference to the following Examples.

The present inventors investigated whether targeting mucosal inductive sites such as the mesenteric lymph nodes (MLN) and Peyer's patches (PP) from the inside (via the blood) could be used to enhance the local mucosal immune response. The targeting strategy using the haematogenous rather than luminal route, bypasses the need for antigen to penetrate through the mucous membranes or survive the harsh conditions of the alimentary lumen. The present inventors used rat IgG2a antibodies MECA-367 MECA-89 specific for the mucosal lymphocyte homing receptor MAdCAM-1 expressed in the high endothelial venules of the MLN and PP and in the flat epithelium of the lamina propria (LP) as a model antigen. Antigen binding regions of these antibodies target them to this mucosal vascular addressin, eliciting responses that can be measured against the isotypic determinants of rat IgG2a (Fig.

la). Immunization with anti-MAdCAM-1 antibody MECA-367 resulted in preferential localization of antigen to MLN and PP in-vivo (Fig. Ib), consistent with the predominant expression of MAdCAM-1 in mucosal tissues 8,10 Methods Immunizations The three immunogens used were two rat IgG2a antibodies against mouse MAdCAM-1 (MECA-367 and MECA-89) and the control rat IgG2a (GL117 which recognizes E.coli -galactosidase). The immunogens were isolated from hybridoma culture supernatant and purified on protein G Sepharose (Amersham Pharmacia Biotech, Little Chalfont, UK) or purchased from PharMingen (San Diego, CA, USA). 6- 8 week old female CBA mice were used for all experiments.

Faecal antibody isolaion Mucosal antibody was isolated from faecal samples s Briefly, 1ml of 0.1mg/mi soybean trypsin inhibitor (Sigma Chemical Co, St Louis, MO, USA) in PBS was added per 0.lg of faeces then vortexed in a mini-beadbeater (Biospec Products, WO 02/096949 PCT/AU02/00661 12 Bartlesville, OK, USA) for 10sec at 2500rpm, debris removed by centrifugation 9000g, 4°C, for 15min, and supernatant assayed for antibody.

Radioiodination In-vivo antigen targeting was demonstrated by radiotracking 5tCi iodinated protein (specific activity of 40pCi/ Lg; total protein including cold protein Protein was radiolabeled with I125 by the chloramine T method and injected intravenously. Organs harvested at 1 hour and radioactivity (cpm) for each whole tissue or 6 Peyer's patches determined on a gamma counter.

Immunological assays ELISA: Rat IgG2a specific antibody responses from serum, faecal and culture supernatant samples were determined by Enzyme-Linked Immunosorbent Assays (ELISA). Briefly, microtitre plates (Dynatech, Chantilly, VA, USA) coated with rat IgG2a (GL117, 2pg/ml in PBS) were incubated with serially diluted sera, faecal extract, or culture supernatant in blocking buffer skimmed-milk powder in PBS) overnight at 4 0 C. Bound antibody was detected after incubation with peroxidaseconjugated antibodies to mouse IgG (donkey anti-mouse, adsorbed against rat Ig, Chemicon, Temecula, CA, USA), IgA (goat anti-mouse), IgG1, IgG2a, IgG2b, or IgG3 (rat anti-mouse) (Southern Biotechnology, Birmingham, AL, USA) diluted in blocking buffer. The substrate used was tetramethyl-benzidine (T2885, Sigma Chemical Co, St Louis, MO, USA) in 0.1M sodium acetate pH 6 and reactions stopped with sulphuric acid. IgG and IgA titres were defined as the reciprocal of the highest dilution to reach an OD 5 ,,so of 0.2 and 0.1 above background respectively.

ELISPOT: To determine the number of cell secreting antibody ELISPOT assay were performed. Briefly, 96 well sterile multiscreen filtration plates (Millipore S.A.

Yvelines, Cedex, France) coated with rat IgG2a (GL117, 20g/ml in PBS) were incubated for 16hrs at 37 0 C 10%CO 2 with dilutions of single cell lymphocyte preparations isolated from mesenteric lymph nodes, Peyer's patches, spleen or lamina propria. Lamina propria lymphocytes were isolated as previously described 9. Bound antibody was detected after incubation with peroxidase-conjugated antibodies to mouse IgA (Southern Biotechnology, Birmingham, AL, USA) diluted in blocking buffer. Number of spots representing individual antigen specific ASC were counted under a stereo microscope after development with AEC substrate (Dako Co, Carpinteria, CA, USA).

WO 02/096949 PCT/AU02/00661 13 Gastrointestinal explant culture: Gastrointestinal explant cultures were performed using described methods 19,20. Briefly, Peyer's patches were removed and the remaining small intestines were stripped of epithelium with 5mM EDTA, washed and cut into 3mm 2 pieces. 20 halved Peyer's patches pieces or 20 intestinal segments were cultured on gelfoam (Amersham Pharmacia Biotech, Little Chalfont, UK) in of RPMI with 10% foetal calf serum at 37 0 C 10% CO, for 6 days and culture supernatant used for analysis.

Cell culture and Cytokine production: Lymphocytes were cultured for 72 hours at 5 X 106 cells/ml in 2ml in the presence of rat IgG2a (GL117, Cytokine levels in the supernatant were evaluated by sandwich ELISA. Recombinant cytokines as standards, coating antibody and biotinylated antibody were obtained from PharMingen (San Diego, CA, USA).

Cloning: Antigen binding domains (variable regions) of the heavy and light chains of anti-MAdCAM-1 antibodies (MECA-367 and MECA-89) and isotype control antibody (GL117) were RT-PCRed from RNA isolated from the corresponding hybirdoma, using methods previously described Gilliland et al 1996 (Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments. Tissue Antigens 47, 1-20). Variable domain of the light chains were cloned into an expression vector containing rat light chain constant region (Zhan, Martin, R. Sutherland, R. Brady, J. and Lew, A. M. (2000). Local production of anti-CD4 antibody by transgenic allogeneic grafts affords partial protection, Transplantation 70, 947-54). Variable antigen binding domains of the heavy chains were cloned into expression vectors containing mouse IgG2c constant regions as previously described (Zhan, Martin, R. Sutherland, R. Brady, J.

and Lew, A. M. (2000). Local production of anti-CD4 antibody by transgenic allogeneic grafts affords partial protection, Transplantation 70, 947-54; Martin, R. M., Brady, J. and Lew, A. M. (1998). The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice, J Immunol Methods 212, 187-92.) Antigens (OVA, helicobacter Urease B, helicobacter catalase, rotavirus VP7, cholera toxin B, mutant cholera toxin B) modified to contain Mlu-I Xba-I cloning sites for antigen substitution, were fused to the CH3 domain of the Fc of mIgG2c heavy chain as previously described (Deliyannis, Boyle, J. Brady, J. Brown, L. and Lew, A. M. (2000). A fusion DNA vaccine that targets antigen-presenting cells increases WO 02/096949 PCT/AU02/00661 14 protection from viral challenge, Proc Nati Acad Sci U S A 97, 6676-80.), using a 17 amino acid spacer.

The sequences of these constructs are set out in the Sequence Listing as follows: SEQ. ID. NO. 1 SEQ. ID. NO. 2 SEQ. ID. NO. 3 SEQ. ID. NO. 4 SEQ. ID. NO. 5 SEQ. ID. NO. 6 SEQ. ID. NO. 7 SEQ. ID. NO. 8 SEQ. ID. NO. 9 SEQ. ID. NO. 10 SEQ. ID. NO. 11 SEQ. ID. NO. 12 GL117 light chain GL117-mIgG2c-ext-OVA MECA-367 light chain MECA-367-mIgG2c-ext-OVA MECA-89 light chain MECA-89-mIgG2c-ext-OVA Cholera toxin B double mutant (dm) Cholera toxin B Helicobacterpylori catalase Helicobacter felis urease B Helicobacterpylori urease B Rotavirus VP7 Heavy and light chain constructs from GL117 and MECA-367 were transfected into CHO cells using FuGENE (Roche, Mannheim, Germany) reagent according to manufacturers instructions. Supernatant was harvested 3 days after transfection and antibody binding to mouse MAdCAM-1 was tested on frozen sections of Peyer's patches and mesenteric lymph nodes by immunofluorescence. S/N from anti-MAdCAM-1 construct (MECA-367) but not from isotype control (GL117) showed binding to mucosal high endothelial venules. This demonstrated that anti-MAdCAM-1 antibody constructs (MECA-367) retain binding to mouse MAdCAM-1.

RESULTS

MAdCAM-1 antigen targeting elicits a mucosal response and augments systemic response (Fig. As expected, mice immunized with non-targeted isotype control did not develop a faecal antibody response (Fig. 2a). In contrast, MAdCAM-1 antigen targeting induced an antigen specific faecal IgA antibody response that peaked at 2 weeks and remained detectable at 8 weeks (Fig. 2a). It should be noted that total faecal IgA immunoglobulin was not altered by targeting. In the systemic compartment, antibody responses were also augmented with MAdCAM-1 targeting.

WO 02/096949 PCT/AU02/00661 Following similar kinetics to the faecal antibody response, MAdCAM-1 antigen targeting induced a serum IgA antibody response whereas non-targeted isotype control immunization did not (Fig. 2b). The serum IgG antibody response with MAdCAM-1 targeting was enhanced 1000-fold above that without targeting (Fig. 2c).

The serum IgG response was predominantly of the IgG1 isotype and could be further elevated, along with the mucosal antibody response, by intraperitoneal boosting with targeted or non-targeted antigen (data not shown). Similarly augmented responses were obtained through targeting of another anti-MAdCAM-1 antibody (MECA-89) that recognizes an epitope from a different extracellular domain of MAdCAM-1 0 (Fig 2d). As proteins may be more immunogenic when they are aggregated, we wanted to show that the enhanced effect of MECA antibodies was not due to an increased amount of aggregation within these samples. Mucosal IgA antibody elicited by MAdCAM-1 targeting was independent of protein aggregation as faecal IgA antibody responses could be detected after immunization with aggregate free anti-MAdCAM-1 antibody; moreover, heat aggregated isotype control did not induce faecal IgA antibody response (Fig 2d). Likewise, serum IgG responses from aggregate free anti- MAdCAM-1 antibody remained 3 log higher than untreated isotype controls (data not shown).

Gut IgA is made locally in humans but can be translocated from the blood in rodents u We therefore wanted to determine whether IgA antibody in the faecal samples was of local origin. A substantial increase in antigen specific IgA antibody secreting cells was found in MLN, PP, and LP lymphocyte preparations (Fig. 3a&b).

IgA antibody secreting cells could be detected in the PP and LP as early as 5 days after primary immunization (Fig. 3a) indicating that B-cells were stimulated in these sites.

The number of IgA antibody secreting cells increased at day 11 in all three important sites of the GALT (Fig. 3b). Antibody secreting cells were not detected in the spleen at or 11 days after immunization (Fig 3a&b) suggesting that the primary source of serum antibodies were derived from the GALT and not the spleen. For further confirmation of local GALT antibody production, supernatants from gastrointestinal explant cultures were tested for antibody by ELISA. Antigen specific IgA could be detected in culture supernatants of PP and intestinal segments from MAdCAM-1 targeted, but not from the non-targeted immunizations (Fig. 3c). Thus, MAdCAM-1 antigen targeting elicits local mucosal B-cell responses in the GALT.

The presence of antigen specific IgA antibody secreting cells in the PP and LP only 5 days after immunization (Fig. 3a) suggests a strong role for the intestinal sites in the early induction of the mucosal antibody response to MAdCAM-1 targeted antigen.

WO 02/096949 PCT/AU02/00661 16 The concentration of antibody secreting cells in the MLN was unremarkable until day 11 (Fig 3a&b). It is possible that B-cell responses detected in the MLN at day 11 resulted from B-cell stimulation at this site. However, we favour the proposal that they are derived from cells trafficking from the intestine to MLN, given the delay in the MLN response and the much higher concentration of specific B cells in the two intestinal sites. T-cell responses were also measured. Enhanced antigen specific secretion of IL-2 and IFN-y could be detected from MAdCAM-1 targeted immunized mice (Fig. As these were detected only after boosting it remains moot whether this represents direct T-cell activation at this site or the result of lymphocyte trafficking.

Overall, these data indicate that augmented antigen specific antibody responses in both mucosal and systemic lymphoid compartments induced by MAdCAM-1 antigen targeting, is associated with an enhanced T-cell cytokine response (Fig. 4).

MAdCAM-1 expression is predominant in the GALT 9. However, there is physiological expression at other sites. Follicular dendritic cells (FDC) expressing MAdCAM-1 12 are found in secondary lymphoid organs and are important in antigen presentation and costimulation for B-cells and the maintenance of memory It was possible therefore, that augmented responses attained with MAdCAM-1 antigen targeting resulted from effective antigen localization to FDC. Adult mice also express MAdCAM-1 on the sinus lining cells of the spleen 1. However, we could not detect any preferential localization of MAdCAM-1 targeted antigen in the spleen (Fig. Ib).

This lack of preferential localization and the lack of antibody secreting cells in the spleen (Fig 3a&b) would indicate that the localization to the spleen or FDC alone was not important for the augmented responses. We therefore argue that localization to the endothelia of the GALT is the key mechanism for the augmented responses. For the same reasons outlined above, it is likely that the enhancement of systemic antibody responses (Fig. 2b&c) resulted primarily from antigen targeting to the GALT and not the spleen. This is further supported by the fact that an increase in serum IgA parallels that of faecal IgA (Fig. 2a&b) and that systemic antibody can result from mucosal responses 6,1s16. This is not surprising as the GALT comprises the majority of secondary lymphoid tissue in the body.

Localization of antigen to lymphoid sites is a powerful way of generating immune responses 1, w 7. We found that antigen delivered to a mucosal vascular addressin in the lymphoid tissue of the gut using the blood route would elicit strong mucosal responses. The blood route avoids the need for antigen to penetrate through mucous membranes or survive the harsh conditions throughout the alimentary tract.

WO 02/096949 PCT/AU02/00661 17 It will be appreciated by persons skilled in the art that numerous variations and/or modifications maybe made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 02/096949 PCT/AU02/00661 18 References 1. Czerkinsky, C. et al. Mucosal immunity and tolerance: relevance to vaccine development. ImmunolRev170, 197-222. (1999).

2. Zinkernagel, R. M. et al. Antigen localisation regulates immune responses in a dose- and time- dependent fashion: a geographical view of immune reactivity.

ImmunolRev156, 199-209 (1997).

3. Ermak, T. H. Giannasca, P. J. Microparticle targeting to M cells. Adv Drug DelivRev34, 261-283 (1998).

4. Frey, A. Neutra, M. R. Targeting of mucosal vaccines to Peyer's patch M cells. BehringInst itt, 376-389 (1997).

Elson, C. O. Ealding, W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J Immunol 132, 2736-2741 (1984).

6. Kawabata, Terao, Fujiwara, Nakagawa, I. Hamada, S. Targeted salivary gland immunization with plasmid DNA elicits specific salivary immunoglobulin A and G antibodies and serum immunoglobulin G antibodies in mice. InfectImmun 67, 5863-5868 (1999).

7. Lehner, T. et al. Protective mucosal immunity elicited by targeted iliac lymph node immunization with a subunit SIV envelope and core vaccine in macaques. NatMed2, 767-775 (1996).

8. Briskin, M. McEvoy, L. M. Butcher, E. C. MAdCAM-1 has homology to immunoglobulin and mucin-like adhesion receptors and to IgA1. Nature 363, 461-464 (1993).

9. Briskin, M. et al. Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue. Am JPatholl51, 97-110 (1997).

Streeter, P. Berg, E. Rouse, B. Bargatze, R. F. Butcher, E. C. A tissuespecific endothelial cell molecule involved in lymphocyte homing. Nature 331, 41-46 (1988).

11. Delacroix, D. L. et al. The liver in the IgA secretory immune system. Dogs, but not rats and rabbits, are suitable models for human studies. Hepatology3, 980- 988 (1983).

12. Szabo, M. Butcher, E. C. McEvoy, L. M. Specialization of mucosal follicular dendritic cells revealed by mucosal addressin-cell adhesion molecule- 1 display. JImmuno1158, 5584-5588 (1997).

WO 02/096949 PCT/AU02/00661 19 13. Tew, J. G. et al. Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells. ImmunolRevl56, 39-52 (1997).

14. Kraal, Schornagel, Streeter, P. Holzmann, B. Butcher, E. C.

Expression of the mucosal vascular addressin, MAdCAM-1, on sinus-lining cells in the spleen. AmJPatholl47, 763-771 (1995).

Harokopakis, Childers, N. Michalek, S. Zhang, S. S. Tomasi, M.

Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens. JmmunolMethods 185, 31-42 (1995).

16. Sato, Y. et al. Injection of plasmid DNA into the gastric mucosa induces mucosal and systemic immunity. Celllmmunoll99, 58-63 (2000).

17. Boyle, J. Brady, J. L. Lew, A. M. Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction. Nature 392, 408-411 (1998).

18. Bromander, A. Ekman, Kopf, Nedrud, J. G. Lycke, N. Y. IL-6deficient mice exhibit normal mucosal IgA responses to local immunizations and Helicobacter felis infection. Jlmmunol 156, 4290-4297 (1996).

19. Kramer, D. R. Cebra, J. J. Early appearance of "natural" mucosal IgA responses and germinal centers in suckling mice developing in the absence of maternal antibodies. fJmmunoll54, 2051-2062 (1995).

20. Losonsky, G. Fantry, G. Reymann, M. Lim, Y. Validation of a gastrointestinal explant system for measurement of mucosal antibody production. Clin Diagn Lab Immunol6, 803-807 (1999).

WO 02/096949 WO 02/96949PCT/AU02/00661 SEQUENCE LISTING <110> <120> <130> <150> <151> <160> <170> <210> <211> <212> <213> <400> The Council of the Queensland Institute of Medical Research Antigen Targeting 13162563 PR5 241 2001-05-25 12 Patentln version 3.1 1 G99

DNA

Rattus sp.

I

atggctccag atccagatga aactgcaaag gaagcgccaa ttcagtiggea gatgttgcca aagctggaac gaacagttag gacatcagtg gttactgatc aaggctgact tcacccgtcg ttcaactttt cccagtctcc caagtcagaa aagtcctgat gtggatctgg catattactg tgaaacgggc caactggagg tcaagtggaa aggacagcaa atgaaagtca tcaagagctt agggcttttg ctgctctgcc ttcectcctg tctgcatctg tattaataag aacttagact atattatace gacaatttgc tacagattac acactcacca ctatcagtat aacagtgggc tgatgctgca ccaactgtat tgcctcagtc gtgtgcctca gattgatggc actgaacgac agacagcacg tacagcatga taacctctat acctgtgagg caacaggaat gagtgttag tcccagccat tgggagacag ggtatcagca aaacgggctt tcagcagcct ccacgtttgg ctatcttccc tgaacaactt gagatggtgt gcagcaccct ttgttcataa.

gagatgtgac agtcectctc aaagcttgga ctcatcaagg gcagcctgaa acctgggacc accatccacg ctatcccaga cctggacagt ctcgttgacc gacatcatcc <210> 2 <211> 2333 <212> DNA <213> RattuS sp.

<400> 2 atggctgtcc tggtgctgtt gctctgectg gtgacattttc ceagctgtgt cctgtcccag WO 02/096949 WO 02/96949PCT/AU02/00661 21 gtgcagctga aagagtcagg acctggtctg gtgcagccct cacagaccct gtctctcacc 120 tgcactgtct ctgggttctc actaattagc tatcatgtaa cctgggttcg ccagcctcct 180 ggaaagagtc tggtgtggat gggaacaata tggactggtg gaggtagaaa ttataattcg 240 gctgaacaat cccgactgag catcagccgg gacacctcca agagccaagt tttcttaaaa 300 atgaacagtc tgcaacctga agacacaggc acttactact gtgccagaca tcgagggggg 360 tataactacg gctttgatta ctggggccaa ggagtcatgg tcacagtctc ctcagctgaa 420 acaacagccc catctgtcta tccactggct cctggaactg ctctcaaaag taactccatg 480 gtgactctgg gatgcctggt caagggctat ttccctgagc cagtcaccgt gacctggaac 540 tctggagccc tgtccagtgg tgtgcdcdcc ttcccagctc tcctgcagtc tggcctctac 600 accctcagca gctcagtgac tgtaacctcg aacacctggc ccagccagac catcacctgc 660 aatgtggccc acccggcaag cagcaccaaa gtggacaaga aaattgagcc cagagtgccc 720 ataacacaga acccctgtcc tccactcaaa gagtgtcccc catgcgcagc tccagacctc 780 ttgggtggac catccgtctt catcttccct ccaaagatca aggatgtzact catgatctcc 840 ctgagcccca tggtcacatg tgtggtggtg gatgtgagcg aggatgaccc agacgtccag 900 atcagctggt ttgtgaacaa cgtggaagta cacacagctc agacacaaac ccatagagag 960 gattacaaca gtactctccg qgtggtcagt gccctcccca tccagcacca ggactggatg 1020 agtggcaagg agttcaaatg caaggtcaac aacagagccc tcccatcccc catcgagaae. 1080 accatctcaa aacccagagg gccagtaaga gctccacagg tatatgtctt gcctccacca 1140 gcagaagaga tgactaagaa agagttcagt ctgacctgca tgatcacagg cttcttacct 1200 gccgaaattg ctgtggactg gaccagcaat gggcgtacag agcaaaacta caagaacacc 1260 gcaacagtcc tggactctga tggttcttac ttcatgtaca gcaagctcag agtacaaaag 1320 agcacttggg aaagaggaag tcttttcgcc tgctcagtgg tccacgaggt gctgcacaat 1380 caccttacga ctaagaccat ctcccggtct ctgggtccgg agctgcaact ggaggagagc 1440 lzgtgcggagg cqcagqacgg ggagctcgac acgcgtgagc tcatcaattc ctgggtagaa 1500 agtcagacaa atggaattat cagaaatgtc cttcagccaa gctccgtgga ttctcaaact 1560 gcaatggttc tggttaatgc cattgtcttc aaaggactgt gggagaaagc atttaaggat 1620 gaagacacac aagcaatgcc tttcagagtg actgagcaag aaagcaaacc tgtgcagatg 1680 atgtaccaga ttggtttatt tagagtggca tcaatggctt ctgagaaaat gaagatcctg 1740 gagcttccat ttgccagtgg gacaatgagc atgttggtgc tgttgcctga tgaagtctca 18300 ggccttgagc agcttgagag tataatcaac tttgaaaaac tgactgaatg gaccagttct 1860 aatgttatgg aagagaggaa gatcaaagtg tacttacctc gcatgaagat ggaggaaaaa 1920 tacaacctca catctgtctt aatggctatg ggcattactg acgtgtttag ctcttcagcc 1980 aatctgtctg gcatctcctc agcagagagc ctgaagatat ctcaagctgt ccatgcagca 2040 catgcagaaa tcaatgaagc aggcagagag gtggtagggt cagcagaggc tggagtggat 2100 gctgcaagcg Lctctgaaga atttagggct gaccatccat tcctcttctg tatcaagcac 2160 atcgcaacca acgccgttct cttctttggc agatgtgttt ccccttaaaa agaagaaagc 2220 WO 02/096949 WO 02/96949PCT/AU02/00661 22 tgaaaaactc tgtcccttcc aacaagaccc agagcactgt agtatcaggg gtaaaatgaa 2280 aagtatgtta tctgctgcat ccagacttca taaaagctgg agcttaatct aga 2333 <210> 3 <211> 699 <212> DNA <213> Rattus sp.

<400> 3 atggctccag ttcaactttt atccagatga cccagtctcc agctgcaaaa caagtcagaa gaagctccca aactcctgat ttcagtggca gtggatctgg gatgttgcca catattactg aaactggaat tgagtcgggc gaacagttaa catctggagg gacatcagtg tcaagtggaa gttactgatc~ aggacagcaa aaggctgact atgaaagtca tcacccgtcg tcaagagctt agggcttttg ctgctctgc ttcagtcctg tctgcatctg taetaataag aacttagact atattttaca aacaatttgc tacagattac acactcacca ctatcagtat aacagtgggc tgatgctgca ccaactgtat tgccacagtc gtgtgcttcg gattgatggc actgaacgac agacagcacg tacagcatga taacctc tat acctgtgagg caacaggaat gagtgttag tcccagccat tgggagacag ggtatcagca aaacgggcat tcagcagcct ccacgtttgg ccatcttccc tgaacgactt gagatggtgt gcagcaccct ttgttcataa gagatgtgac agtcactctc aaagcttgga cccatcaagg gcagcctgaa agctgggacc accatccatg ctatcecaga cctggacagt ctcgttgacc gacatcatcc <210> 4 <211> 2348 <212> DNA <213> Rattus sp.

4 atggctgtcc tggtgctgtt gtgcagctga aggagtcagg tgcactgtct ctgggttctc ggaaagggtc tggagtggat gctctcaaat cccgattgag ttgaacagtc tgcaaactga gctctgcctg gtgacatttc acctggtctg gtgcggccct aataaccagt aacggtgtaa gggagcaata tggagtggtg catcagcagg gacacctcca agacacagcc atttacttct caagc tgtgc cacagaccct gctgggttcg gaagtagaga agagccaagt gtaccagatc cctgtcccag gtccctcacc ccagcctccg ttataattca tttcttaaac ggattatcat WO 02/096949 WO 02/96949PCT/AU02/00661 gatggtacct gtctcctcag aaaagtaact accgtgacct cagtctggcc cagaccatca gagcccagag gcagctccag gtac'tcatga gacccagacg caaacccata caccaggact tcccccatcg gtcttgcctc acaggcttct aactacaaga ctcagagtac gaggtgctgc caactggagg aattcctggg gtggattctc aaagcattta aaacctgtgc aaaatgaaga cctgatgaag gaatggacca aagatggagg tttagctctt gctgtccatg gaggctggag ttctgtatca taaaaagaag caggggtaaa aatctaga ccctatatta ctgaaacaac ccatggtgac ggaactctgg tctacaccct cctgcaatgt tgcccataac acctcttggg tetccctgag tccagatcag gagaggatta ggatgagtgg agaaaaccat caccagcaga tac!ctgccga acaccgcaac aaaagagcac acaatcacat agagctgtgc tagaaagtca aaactgcaat aggatgaaga agatgatgta tcctggagct tctcaggcct gttctaatgt aaaaatacaa cagccaatct cagcacatgc tggatgctgc agcacatcgc aaagctgaaa atgaaaagta ctatgttatg agccccatct cctgggatgc agccctgtcc cagcagctca ggcccacccg acagaacccc tggaccatcc ccccatggtc ctggtttgtg caacagtact caaggagt to ctcaaaaccc agagatgact aattgctgtg agtcctggac ttgggaaaga tacgactaag ggaggcgeag gacaaatgga ggttctggtt cacacaagca ccagattggt tccatttgcc tgagcagctt tatggaagag cctcacatct gtctggcatc agaaatcaat aagcgtctct aaccaacgcc aactctgtcc tgttatctgc gatgcctggg gtctatccac ctggtcaagg agtggtgtgc gtgactgtaa gcaagcagca tgtcctccac gtcttcatct acatgtgtgg aacaacgtgg ctccgggtgg aaa tgcaagg agagggccag aagaaagagt gactggacca tctgatggtt ggaagtcttt accatctcc gacggggagc attatcagaa aatgccattg atgcctttca ttatttagag agtgggacaa gagagtataa aggaagatca gtcttaatgg tcctcagcag gaagcaggca gaagaattta gttctcttct cttccaacaa tgcatccaga gtcaaggagc ttcagtcact tggctcctgg gctatttccc acacottoc cctcgaacac ccaaagtgga tcaaagagtg tccctccaaa tggtggatgt aagtacacac tcagtgccct tcaacaacag taagagctcc tcagtctgac gcaatgggcg cttacttcat tcgcctgctc ggtctctggg tcgacacgcg atgtcc ttca aactgctctc tgagccagtc agctctcctg ctggcccagc caagaaaatt tcccccatgc gatcaaggat gagogaggat agetoagaca ccccatccag agccctccca acaggtatat ctgcatgatc tacagagcaa gtacagcaag agtggtccac tccggagctg tgagctcatc gccaagctcc 420 480 540 600 660 72 0 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2348 tcttcaaagg actgtgggag gagtgactga gcaagaaagc tggcatcaat ggcttctgag tgagcatgtt ggtgctgttg tcaactttga aaaactgact aagtgtactt acctcgcatg ctatgggcat tactgacgtg agagcctgaa gatatctcaa gagaggtggt agggtcagca gggctgacca tccattcctc ttggcagatg tgtttcccct gacccagagc actgtagtat cttcataaaa gctggagctt WO 02/096949 WO 02/96949PCT/AU02/00661 <210> <2121> 699 <212> DNA <213> Rattus sp.

<400> atggctccag atccagatga aac tgcaaag gaagctccca ttcagtggca gatgtagcca dagctggagt gaacagttag gacatcagtg gttactgatc aaggctgact tcacccgtcg ttcaactctt cccagtctcc caagtcagaa aactcctgat gtggatctgg catatttctg tgaaacgggc caactggagg tcaagtggaa aggacagcaa atgaaagtca tcaagagctt agggctgctg ttcattcctg tattaacaag atataatcca tactgatttc ccttcagcat tgatgctgca tgcctcagtc gattgatggc agacagcacg taacctctat caacaggaat ctgctctggc tctgcatctg tacttagact aacagtttgc acacttacca aacagtgggt ccaactgtat gtgtgcctca actgaacgac tacagcatga acctgtgagg gagtgttag tcccagccat tgggagacag ggtatcagca aaacaggaat tcdgcagccL ggacgttcgg ctatcttccc tgaacaactt gagatggtgb gcagcaccct ttgttcataa gagatgtgac agtcactatc aaagcttggt cccatcaagg gcagcctgaa tggaggcacc accatccacg ctatcccaga cctggacagt c tcgt tgacc gacatcatec <210> 6 <211> 23d2 <212> DNA <213> Rattus sp.

<400> 6 atggattggg cagatccagt tcctgcaagg ccaggaaagg ggtgatgact ttgcagatca ttctatgatt tcagctgaaa aactccatgg acctggaact tgtggaactt tggtacagtc cttctgggta gcttgaagtg tccaaggacg gcaacctcaa cctactggta caacagcccc tgactctggg ctggagccct gctatttctg tggacctgaa taccttcaca gatgggctgg gtttgtcctc aaatgaggac C tt tgac ttc atctgtctat atgcctggtc gtccagtggt atggcagttg c tgaagaagc gaccatgcaa atcaacacct tctttggaag acggctacat tggggcccag ccactggctc aagggctatt gtgcacacct cccaaacagg ctggagagtc tgcactgggt atactgggaa cctctgccag atttctgtgc gaaccatggt ctggaactgc tccctgagcc tcccagctct tgcccaagca agtgaagatc gaaacaggct gccaacatat cactgcaaat aagatcttat caccgtgtcc tctcaaaagt agtcaccgtg cctgcagtct WO 02/096949 WO 02/96949PCT/AU02/00661 ggcctctaca ccctcagcag ctcagtgact gtaacctcga acacctggcc cagccagace 660 atcacctgca atgtggccca cccggcaagc agcaccaaag tggacaagaa aattgagccc 720 agagtgccca taacacagaa cccctgtcct ccactcaaag agtgtccccc atgcgcagct 780 ccagacctct tgggtggacc atccgtcttc atcttccctc~ caaagatcaa ggatgtactc 840 atgatctccc tgagccccat ggtcacatgt gtggtggtgg atgtgagcga ggatgaccca 900 gacgtccaga tcagctggtt tgtgaacaac gtggaagtac acacagctca gacacaaacc 960 catagagagg attacaacag tactctccgg gtggtcagtg ccctccccat ccagcaccag 1020 gactggatga gtggcaagga gttcaaatgc aaggtcaaca acagagccct cccatccccc 1080 atcgagaaaa ccatctcaaa acccagaggg ccagtaagag ctccacaggt atatgtcttg 1140 cctccaccag cagaagagat gactaagaaa gagttcagtc tgacctgcat gatcacaggc 1200 ttctacctg ccgaaattgc tgtggactgg accagcdatg ggcgtacaga gcaactac 1260 aagaacaccg caacagtcct ggactctgat ggttcttact tcatgtacag caagctcaga 1320 gtacaaaaga gcacttggga aagaggaagt cttttcgcct gctcagtggt ccacgaggtg 1380 ctgcacaatc accttacgac taagaccatc tcccggtctc tgggtccgga gctgcaactg 1440 gaggagagct gtgcggaggc gcaggacggg gagctcgaca cgcgtgagcz catcaattcc 1500 tgggtagaaa gtcagacaaa tggaattatc agaaatgtcc ttcagccaag ctccgtggat 1560 tctcaaactg caatggttct ggttaatgcc attgtcttca aaggactgtg ggagaaagca 1620 tttaaggatg aagacacaca agcaatgcct ttcagagtga ctgagcaaga aagcaaacct 1680 gtgcagatga tgtaccagat tggtttattt agagtggcat caatggcttc tgagaaaatg 1740 aagatcctgg agcttccatt tgccagtggg acaatgagca tgttggtgct gttgcctgat 1800 gaagtctcag gccttgagaa gcttgagagt ataatcaact ttgaaaaact gactgaatgg 1860 acagttbta atgttatgga agagaggaag atcaaagtgt acttacctcg catgaagatg 1920 gaggaaaaat acaacctcac atctgtctta atggctatgg gcattactga cgtgtttagc 1980 tcttcagcca atctgtctgg catctcctca gcagagagcc tgaagatatc tcaagctgtc 2040 catgcagcac atgcagaaat caatgaagca ggcagagagg tggtagggtc agcagaggct 2100 ggagtggatg ctgcaagcgt ctctgaagaa tttagggctg accatccatt cct cttctgt 2160 atcaagcaca tcgcaaccaa cgccgttctc ttctttggca gatgtgtttc cccttaaaaa 2220 gaagaaagct gaaaaactct gtcccttcca acaagaccca gagcactgta gtatcagggg 2280 taaaatgaaa agtatgttat ctgctgcatc cagacttcat aaaagctgga gcttaatcta 2340 3 0 ga 2342 <210> 7 <211> 324 <212> DNA WO 02/096949 WO 02/96949PCT/AU02/00661 <213> Vibrio cholerae <400> 7 acgcgtaccc acgctaaatg atcattactt gattocacaaa gaagctaaag attagtatgg cgcagaatat ataagatatt ttaagaatgg aaaaagcgat tcgaaaagtt camattaatc tactgatttg tgtgcagaat accacaacac acaaatacat ttcgtataca gaatctctag ctggaaaaag agagatggct tgcaactttt caagtagaag taccaggtag tcaacatata tgddaggatg aaggataccc tgaggattgc atatcttact atgtgtatgg aataataaaa cgcctcatgc gattgccgca taga <210> 8 <211> 324 <212> DNA <213> Vibrio cholerae <400> 8 acgcgtaccc cgcagaatat acgctaaatg ataagatatt atcattactt ttaagaatgg gattcacaaa aaaaagtgat gaagctaaag tcgaaaagtt attagtatgg caaattaatc tactgatttg tgtgcagaat accacaacac acaaatacat ttcgtataca gaatctctag ctgagaaaag agagatggct tgcaactttt caagtagaag taccaggtag teaacatata tgaaaggatg aaggataccc tgaggattgc atatcttact atgtgtatgg aataataaaa cgcctcatgc gattgccgca taga <210> 9 <211> 417 <212> DNA <213> H-elicobacter pylori <400> 9 acgcgtatgc aaaacgggta ttacggctct ttacaaaact atacgcctag ctcattgcct ggctataaag aagataagag tgcaagggat cctaagttca acttagctca tattgagaaa gagtttgaag tgtggaattg ggattacaga gctgaggata gcgattacta cacccaacca ggtgattact accgctcatt gccagctgat gaaaaagaaa ggttgcatga cactattgga gagtctttag CtCatgttaC ccatdaggaa attgtggata aacaattgga gcatzttcaag WO 02/096949 WO 02/96949PCT/AU02/00661 27 aaagctgacc ccaaatacgc tgagggagtt aaaaaagctc ttgaaaaaca ccaaaaaatg 360 atgaaagaca tgcatggaaa agacatgcac cacacgaaaa agaaaaagta atctaga 417 <210> <211> 1719 <212> DNA <213> Helicobacter felis <400> cgcgccccat gaaaaagatt tcacgaag aatatgtttc tatgtatggt cccactaccg gggatcgtgt tagactcggc gacactgatt tgatcttaga agtggagcat gattgcacca 120 cttatggtga agagatcaaa tttgggggcg gtaaaactat ccgtgatggg atgagtcaaa 180 ccaatagccc tagctcttat gaattagatt tggtgctcac taacgccctc attgtggact 240 atacgggcat ttacaaagcc gacattggga ttaaagacgg caagattgca ggcattggca 300 aggcaggcaa taaggacatg caagatggcg tagataataa tctttgcgta ggtcctgcta 360 cagaggcttt ggcagctgag ggcttgattg taaccgctgg tggeatcgat acgcatattc 420 actttatctc tccccaacaa atccctactg cttttgccag cggggttaca accatgattg 480 gaggaggcac aggacctgcg gatggcacga atgcgeccac catcactccc ggacgcgcta 540 atctaaaaag tatgttgcgt geageecgaag aata ccat gaatctaggc tttttggcta 600 aggggaatgt gtcttacgaa ccctctttac gcgatcagat tgaagcaggg gcgattggtt 660 ttaaaatcca cgaagactgg ggaagcacac ctgcagctat tcaccactgc ctcaatgtcg 720 ccgatgaata cgatgtgcaa gtggctatcc acaccgatac ccttaacgag gcgggctgtg 780 tagaacac cctagaggcg attgccgggc gcaccatcca taccttccac actgaagggg 840 ctgggggtgg acacgctcca gatgttatca aaatggcagg ggaatttaac attctacccg 900 cctctactaa cccgaccatt cctttcacca aaaacactga agccgagcac atggacatgt 960 taatggtgtg ccaccacttg gataaaagta tcaaggaaga tgtgcagttt gccgattcga 1020 ggattcgccc ccaaactatc gcggctgaag accaactcca tgacatgggg atcttttcta 1080 tcaccagctc cgactctcag gctatgggac gcgtaggcga ggtgatcaca cgcacttggc 1140 agacagcaga caaaaacaaa aaagagtttg ggcgcttgaa agaggaaaaa ggcgataacg 1200 acaacttccg catcaaacgc tacatctcta aatacaccat caaccccgcg atcgcgcatg 1260 ggatttctga ctatgtgggc tctgtggaag tgggcaaata cgccgacctc gtgctttgga 1320 gtccggcttt ctttggcatt aagcccaata tgattattaa gggcggattt attgcgctct 1380 ctcaaatggg cgatgccaat gcgtctattc ccacccctca gcccgtctat taccgtgaaa 1440 tgtttggaca ccatgggaaa aacaaattcg acaccaatat cactttcgtg tcccaagcgg 1500 cttacaaggc agggatcaaa gaagaactag ggctagatcg cgtggtattg ccagtgaaaa 1560 WO 02/096949 WO 02/96949PCT/ATJO2/00661 28 actgtcgcaa tatcactaaa aaggacctca aattcaacga tgtgaccgca catattgatg tcaaccctga aacctataag gtgaaagtgg atggcaaaga ggtaacctct aaagcagcag atgaattgag cctagcgcaa ctttataatt tgttCtaga <210> Li <211> 1717 <212> DNA <213> Hellcobacter pylori <400> 11 1620 1680 1719 acgcgtatga gataaagtga tatggcgaag aacaacccta accggtattt ggcggtaaca gaagccttag ttcatttcac ggcggaactg ttaaaatgga ggtaacgctt aaaattcacg gacaaatacg gaagacacta ggcggcggac tccactaacc atggtgtgcc atccgccctc accagttctg acagctgaca aacttcagga actagcgagt ccagcattct caaatgggcg ttcgctcatc aaaagattag gattgggcga agcttaaatt gcaaagaaga ataaagcgga aagacatgca ccggtgaagg cccaacaaat gtcctgctga tgctcagagc ctaacgatgc aagactgggg atgtgcaagt tggctgctat acgctcctga ecaccatoc accacttgga aaaccattgc actctcaagc aaaacaagaa tcaaacgcta atgtcggttc ttggcgtgaa atgcgaacgc gtggtaaagc cagaaaagaal tacagacttg cggtggcggt actggatcta tattggtatt agatggcgtt tttgatcgta cctacagct tggcactaac ggctgaagaa gagcttagc caccactcct cgctatccac tgctggacgc tattattaa tttcaccgtg taaaagcatt ggctgaagac gatgggccgt agaatttggc cttgtctaaa tgtagaagtgj acctaacatg ttctatccct taaatacgat tatgtttcta atcgctgaag aaaaccctaa atcatcacta aaagatggca aaaaacaatc actgctggtg tttgcaagcg gcgactacta tattctatga gatcaaattg tctgcaatca acagacactt actatgcaca gtagccggtg aatacagaag aaagaagatg actttgcatg gtgggtgaag cgcttgaaag tacaccatta ggcaaagtag atcatcaaag acccctcaac gcaaacatca tgtatggccc tactacaggt tagaacatga ctacaccatt gagaaggcat gagccaatct acgctttaat cgtggattac aaatcgctgg cattggtaaa ttagcgtggg tcctgctact gtattgacac acacatccac gtgtaacaac catgattggt tcactccagg atttaggttt aagccggtgc atcatgcgtt tgaatgaagc ctttccacac aacacaacat cagagcacat ttcagttcgc acutggggat ttatcactag aagaaaaagg acccagcgat ctgacttggt gtgggttcat cggtttatta cttttgtgtc tagaagaaat cttggctaaa gattggcttt agatgttgcg cggttgtgta tgaaggcgct to ttcccgc t ggacatgctt tgattcaagg tttctcaatc aacttggcaa cgataacgac cgctcatggg attgtggagt tgcattaagc cagagaaatg tcaagcggct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 02/096949 WO 02/96949PCT/AU02/00661 29 tatgacaaag gcattaaaga agaattagga cttgaaagac aagtgttgcc ggtaaaaaat tgcagaaaca tcaccaaaaa agacatgcaa ttcaacgaca ctaccgctca cattgaagtc aatcctgaaa cttaccatgt gttcgtggat ggcaaagaag taacttctaa accagctaat aaagtgagct tggcgcaact ctttagcatt ttctaga <210> 12 <211> 853 <212> DNA <213> Rotavirus VP7 <400> 12 1560 1620 1680 17 17 acgcgtggca caatcggaaa gagataaacg ccaacagggt caactatact atgtcggagc ctgtactact actattaaag gctgcgactt ggcgtaaatc ctcggtccac actgcggatc tggtggcaag aagcgatctc ttacagctct ttaaccttcc catttctgac ataactcatg cagtctattt gtgattacaa ttgcggactt atcagcaaac tatgtccact ttgaagaaat acaaacttga gggaaaacgt ctacaactgc tgttctatac gatcactgaa aga aattactgga ttctacccta gaaagacacg taaagaatac cgttgtgctg gatactgaat agacgaagca taacac tcag tgcgactgcg cgttacaact agcagttata accacaaacc tgtcgttgat ctcagcgac t tcaatggaca tgcctttact ctzatcgcaac accgacatag atgaaatatg gaatggcttt aacaaatgga acgctaggaa gagaagt tag gcgacttgta caagtaggcg gagagaatga tacgtaaatc ttttattata cggcatatgc atccaaaaa tattcctgac cagcattctc acgcttcatt gcaacccaat tatctatggg tagctgtct cgataatgga cgattcgcaa gttctgacgt tgcgcattaa agataatttc gagtgtaggt aaactcaact ggcagetact gaaaggatgg agttgatccg gcaaatggat ggacatcaca ttcctcctgt tgtcgtagat ctgcaagaaa aatagacata ttggaaaaaa agcaatgtcc ataactgaag

Claims (7)

1. 3. A method according to claim 1 or claim 2 wherein the composition is administered by the haematogenous route.
2. 4. A method according to any one of claims 1 to 3 wherein the antigen is from Salmonella, Cholera, Helicobactcr pylori, HTV Candida, P. gingivals, gut parasites, gut associated toxins, gut hormones, gut honnrmone receptors or gut associated cancers. A method according to any one of claims 1 to 4 wherein the antigen is bound to the targeting moiety by affinity conjugation, chemical cross-linking or genetic fusions.
3. 6. A targeted antigen comprising an antigen bound to a targeting moiety wherein the targeting moiety is an antibody, an antibody fragment or an antibody binding domain and the targeting moiety binds to Mucosal Addressin Cellular Adhesion Molecule-I present in circulatory vessels in Gut Associated Lymphoid Tissue.
4. 7. A targeted antigen according to claim 6 wherein the antibody is MECA-89 or MECA-367.
5. 8. A targeted antigen according to claim 6 or claim 7 wherein the antigen is from Salmonella, Cholera, Helicobactcr pylori, HIV, Candida, P. gingivalis, gut parasites, gut associated toxins, gut hormones, gut hormone receptors or gut associated cancers. 202436522_ I COMS ID No: SBMI-07874468 Received by IP Australia: Time 12:40 Date 2007-06-22 03 9679 3111 Blake Dawson WaldrOn 12:43:11 22-06-2007 9/11 O o 31
6. 9. A targeted antigen according to any one of claims 6 to 8 wherein the antigen is bound to c, the targeting moiety by affinity conjugation, chemical cross-linking or genetic fusions. An antigehic composition comprising a carrier and a targeted antigen according to any one of claims 6 to 9. INO NO 11. A DNA molecule encoding the targeted antigen according to any one of claims 6 to 9. O O 10 12. An antigenic composition comprising a carrier and a DNA molecule according to claim 11.
7. 13. The use of a targeted antigen according to any one of claims 6 to 9 in the manufacture of a medicament for the raising of an immune response in an animal. 202436522 1 COMS ID No: SBMI-07874468 Received by IP Australia: Time 12:40 Date 2007-06-22