WO1999013092A9 - Enhancement of mucosal antibody responses by interleukin-6 - Google Patents

Abstract

The invention provides a transgenic mouse overexpressing human interleukin-6 and methods of enhancing mucosal IgA responses in which interleukin-6 is delivered to the mucosal immune system. These methods are useful in preventing infectious diseases.

Description

ENHANCEMENT OF MUCOSAL ANTIBODY RESPONSES BY INTERLEUKIN-6

This invention was made with government support under grant number DE- 10979 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

The field of the invention is mucosal immune responses.

Background of the invention Most bacterial and viral pathogens first encounter their host at mucosal surfaces. It is generally believed that vaccination protocols that stimulate immunity at the site of entry of a pathogen result in protective immunity. Current efforts at developing mucosal vaccines have, however, been largely unsuccessful. This is because the magnitude of mucosal responses is usually small . The mucosal immune system is believed to function to a large degree independently of the systemic immune system. Systemic immunization generally fails to induce a mucosal immune response. It is believed that early stages in the maturation of B cells that secrete mucosal antibodies occur in the inductive sites, e.g., the

Peyer's patches in the gut, and that precursor plasma cells migrate to effector sites, e.g. salivary gland, intestinal lamina propia and vaginal epithelium, where they mature to IgA-secreting cells. Summary of the Invention

The invention is based on the discovery that a transcriptional regulatory element (TRE) within the 5' untranscribed region of a mouse major urinary protein (MUP V) gene directs expression of operatively linked coding segments to the submandibular gland (SMG) , also sometimes referred to as the submaxillary gland. As such this TRE can be useful in directing expression of genes encoding medically useful polypeptides to the saliva of relevant subjects. There are 2 extremely homologous genes (Mup-1.5a and Mup-1.5b) for MUP V in the mouse, only one of which (Mup-1.5a) is expressed. However, as elaborated in Example 1 below, the TRE in both is functional and specific. Thus for convenience of description, the two genes will be referred to as a single gene, i.e., Mup-1.5a/b.

The invention features an isolated DNA molecule containing a control segment that includes a TRE operatively linked to a coding segment encoding a polypeptide that enhances an antibody response and in which the TRE directs expression of the coding segment to a mucosa, e.g., the SMG. The control segment can be a sequence within a Mup-1.5a/b gene 5' untranscribed region, a sequence included within the Mup-1.5a/b gene between an EcoRI restriction enzyme site about 3.46 kilobases 5' of the Mup-1.5a/b gene transcription initiation site and the Mup-1.5a/b gene transcription initiation site, or a combination of (a) a sequence included within the Mup-1.5a/b gene between the same EcoRI restriction enzyme site of the Mup-1.5a/b gene and a SstI restriction enzyme site about 1.85 kilobases 5' of the Mup-1.5a/b gene transcription initiation site and (b) a sequence of 600 bases immediately 5' of the transcription intiation site of the Mup-1.5a/b gene. The polypeptide encoded by the coding segment can be a cytokine that can enhance an immune response (e.g., an antibody response) such as interleukin-6 (IL-6) (e.g., human IL-6) , interleukin-2 (IL-2) , interleukin-4 (IL-4) , interleukin-5 (IL-5) , interleukin-10 (IL-10) , interleukin-12 (IL-12) , or interleukin-13 (IL-13).

The invention also encompasses a transgenic (tg) non-human animal whose germ cells and somatic cells harbor a transgene which contains a control segment that includes a TRE and a coding segment encoding a polypeptide that enhances an antibody response, and in which the TRE is operatively linked to the coding segment and directs expression of it to a mucosa of the tg animal. This expression results in an enhanced antibody response in the mucosa, e.g., the SMG. The control segments of the transgenes are the same as those of the above-described DNA molecules. The polypeptide encoded by the coding segment can be a cytokine such as IL-6, e.g., human IL-6, IL-2, IL-4, IL-5, IL-10, IL-12, or IL- 13. The tg animal can be a rodent, e.g., a mouse, a bovine animal, a pig, a goat, or a sheep and enhanced antibody response can be an IgA antibody response.

Another embodiment of the invention is a method for enhancing a mucosal antibody response in a subject. The method involves delivering IL-6 to a mucosa of the subject and administering an antigen to the subject. The antigen is preferably a non-replicating antigen and delivery of the IL-6 to the mucosa results in an antibody response to the antigen in the mucosa that is greater than an antibody response in the same mucosa of a subject to which the IL- 6 was not delivered but the antigen was administered. The mucosa can be oral mucosa, intestinal mucosa, nasal mucosa, pulmonary mucosa, tracheal mucosa, or vaginal mucosa. Administration of the antigen can be to the same mucosa to which the IL-6 was delivered or a second mucosa, e.g., nasal mucosa, oral mucosa, pulmonary mucosa, tracheal mucosa, intestinal mucosa or vaginal mucosa. Delivery of IL-6 can be by administration of the IL-6 polypeptide itself to the mucosa or by delivering to a cell of the mucosa a DNA molecule containing an IL-6 coding segment operatively linked to a TRE and in which the TRE directs expression of the coding segment to the mucosa. The subject in which the antibody response is enhanced can be a mammal, e.g., a bovine mammal, a pig, a sheep, or a human. The method can include administering an adjuvant to the subject and the enhanced antibody response can be an IgA antibody response. The antigen can be a viral antigen, a bacterial antigen, or a protozoan antigen.

The invention also includes a method for enhancing a mucosal antibody response in a non-human animal in which the IL-6 with which the mucosa of the animal is contacted is encoded by a transgene harbored by the animal. The transgene contains an IL-6 coding segment operably linked to a TRE that directs expression of the IL-6 coding sequence to the mucosa. Moreover, both the germ cells and somatic cells of said animal contain the transgene.

As used herein, an "isolated DNA molecule" is a DNA molecule that is separated from the 5' and 3' coding sequences with which it is immediately contiguous in the naturally occurring genome of an organism. Isolated DNA molecules include DNA molecules which are not naturally occurring, e.g., DNA molecules created by recombinant DNA techniques. DNA molecules include cDNA, genomic DNA, and synthetic DNA (e.g., chemically synthesized) DNA. Where single-stranded, the DNA molecule may be a sense strand or an antisense strand.

As used herein, "control segment" refers to the region of a transgene or DNA molecule that contains a TRE which directs expression of an operatively linked coding segment to a mucosa tissue, e.g., the SMG. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.

Other features and advantages of the invention, e.g., prevention of oral, enteric, and pulmonary diseases, will be apparent from the following detailed description, from the drawings and from the claims.

Brief Description of the Drawings Fig. 1 is a diagram showing a partial restriction map of the DNA construct used to derive human IL-6 overexpressing tg mice.

Fig. 2 is a photograph of an ethidium bromide stained agarose electrophoretic gel of RT-PCR samples showing specific expression of the human IL-6 transgene in the SMG of tg mice.

Fig. 3A is a line graph demonstrating an enhanced antigen specific IgA response that is detected in the saliva of human IL-6 tg mice immunized intragastrically with the antigen (key hole limpet he ocyanin-KLH) and adjuvant (cholera toxin B subunit CT-B) . Fig. 3B is a line graph demonstrating an enhanced adjuvant specific IgA response that is detected in the saliva of human IL-6 tg mice immunized intragastrically with the antigen (KLH) and adjuvant (CT-B) .

Fig. 4A is a line graph demonstrating an enhanced antigen specific IgA response that is detected in the gastric lavage of human IL-6 tg mice immunized intragastrically with the antigen (KLH) and adjuvant (CT- B) .

Fig. 4B is a line graph demonstrating an enhanced adjuvant specific IgA response that is detected in the gastric lavage of human IL-6 tg mice immunized intragastrically with the antigen (KLH) and adjuvant (CT- B) .

Fig. 5A is a line graph demonstrating an enhanced antigen specific IgA response that is detected in the serum of human IL-6 tg mice immunized intragastrically with the antigen (KLH) and adjuvant (CT-B) .

Fig. 5B is a line graph demonstrating an enhanced adjuvant specific IgA response that is detected in the serum of human IL-6 tg mice immunized intragastrically with the antigen (KLH) and adjuvant (CT-B) .

Fig. 6A is a bar graph demonstrating an enhanced antigen specific IgA producing cell response that is detected in lymphocytes isolated from the SMG and the LP of human IL-6 tg mice immunized intragastrically with the antigen (KLH) and adjuvant (CT-B) .

Fig. 6B is a bar graph demonstrating an enhanced adjuvant specific IgA producing cell response that is detected in lymphocytes isolated from the SMG and the LP of human IL-6 tg mice immunized intragastrically with the antigen (KLH) and adjuvant (CT-B) .

Detailed Description A. OVERVIEW

To test the hypothesis that over-expression of IL- 6 in vivo at a mucosal effector site would bring about enhanced IgA synthesis in response to vaccination, tg mice that specifically over-express human IL-6 in the salivary glands and secrete the transgene-encoded human IL-6 in their saliva were derived. To generate these mice, a TRE derived from the SMG-specific mouse major urinary protein ge Mup-1.5 5b was employed. These tg mice uniquely over-express IL-6 at a mucosal effector site, i.e., the SMG.

Tg mice and control non-tg mice were orally immunized with keyhole limpet hemocyanin (KLH) and cholera toxin B subunit (CT-B) . It was determined, by enzyme-linked immunosorbent assay (ELISA) , that the tg mice secrete in their saliva about ten- fold more KLH- specific IgA than do control mice. The increase in the amount of salivary gland IgA appears to be due to both an increase in the number of KLH-specific IgA secreting plasma cells isolated from the submandibular gland as well and to enhanced synthesis of IgA per cell. It is believed that the IL-6 tg mice of the invention will also respond more efficiently to oral immunization with other antigens, e.g., those derived from microbial antigens, than normal mice. The increase in the antigen-specific IgA response is not limited to the SMG. In addition to the saliva, the levels of antigen-specific IgA are enhanced both in the intestinal secretions and in the bronchoalveolar washes (8-10 fold) , as well as in the serum (3 to 8- fold) . In the small intestine, the enhanced IgA response is paralleled by an increase (at least 7-fold) in the number of antigen-specific IgA secreting cells. Thus the IL-6 over-expressing tg mice secrete increased levels of antigen-specific IgA, not only in saliva, but also in intestinal and pulmonary secretions. This is probably because the tg mice swallow and inhale the IL-6 enriched saliva, and the ingested or inhaled IL-β stimulates the intestinal mucosal responses and pulmonary mucosal responses, respectively. However, the invention is not limited by a particular mechanism of action.

The tg mice of the invention will be useful in testing vaccines that aim at protecting the host from infection by boosting mucosal IgA production. Such vaccines would be useful in inducing immunity against oral pathogens associated with the development of adult periodontal disease by boosting salivary IgA. As indicated by the above findings, the tg mice of the invention will also be useful for testing vaccines against enteropathogens and pulmonary pathogens.

Efficacious vaccines can be used, in addition to mice, in multiple mammalian subjects, e.g. humans, non-human primates, cattle, swine or sheep.

A candidate vaccine could be tested for usefulness, for example, by contacting a mucosal surface of the tg mice with it, e.g, by oral or intragastric infusion. The test substance could be administered alone or with adjuvant (e.g., CT-B) . If desired, booster immunizations may be given once or several (two, three, four, eight or twelve, for example) times at various times (e.g., spaced one week apart). Vaccine-specific antibody (e.g., IgA) responses can then be measured by testing for the presence of such antibodies at various mucosal sites (e.g., in saliva or gastric and bronchoalveolar lavages) and systemically (e.g., in serum) using in vi tro assays familiar to those in the art, e.g., ELISA. The same or identically treated tg mice can be tested for resistance to a mucosally acquired infection from which the test vaccine was derived (e.g., influenza virus) . The test tg mice could be exposed mucosally (e.g., intranasally in the case of influenza virus) to different doses of the infectious agent. Mice can be observed for pathologic symptoms familiar to those in the art, e.g, malaise, lack of appetite, morbidity and mortality. Alternatively, they may euthanized at various time points and their tissues (e.g., lung, liver, spleen, kidney or intestine) may be assayed for relative levels of the pathogen by methods known to artisans of ordinary skill (e.g., viral plaque forming assays). The data obtained with the test tg mice can be compared to those obtained with a control group of tg mice, e.g., tg mice that were exposed to the diluent in which the test vaccine was dissolved or suspended (e.g. physiological saline) or adjuvant without vaccine if adjuvant was used for immunization. Enhanced ability of the test tg mice to produce specific antibody and increased resistance of the test tg mice to infection, relative to the control tg mice, would indicate that the test vaccine is an effective vaccine. Animals of a variety of species in addition to mice that express IL-6 transgenes similar to those described will be better protected from mucosally derived infections than normal, non-tg mammals. Since the IL-6 transgene as expressed resulted in enhanced IgA responses in the intestine and lungs, tg lines of these mammals will have enhanced resistance to infections by not only oral but also enteric and respiratory infections. Such pathogens may be viruses, bacteria or protozoans. It is likely that such animals will also show enhanced IgA- mediated immunity at other mucosal sites, e.g., vaginal or nasal and thus would show similar enhanced immunity at these sites.

Tg mammals can also be created in which the IL-6 gene is placed under control of a promoter that results in expression in other mucosal sites, e.g., the sucrase- isomaltose gene which specifically directs expression of genes to intestinal epithelial cells [Markowitz et al . (1995) Am.J. Physiol . 269(6 pt.l) : G925; Tung et al . (1997) Am. J. Physiol. 273 1 pt.l) : 883]. In such tg animals, an IL-6 enriched microenvironment will be generated both at a mucosal inductive site, the Peyer's patches (PP) , and at a mucosal effector site, the intestinal lamina propria (LP) . Since PP are a source of IgA-precursor cells that can colonize the salivary, lachrymal and mammary glands, the intestinal LP as well as the cervical glands of the uterus, stimulation of IgA responses in the microenvironment of PP will have a profound effect on IgA secreted by many mucosal sites.

Furthermore, delivery of the IL-6 polypeptide to the mucosae of the above recited mammals together with or separate from an antigenic vaccine can result in enhanced IgA mediated immunity to viruses, bacteria, or protozoans at these sites. Delivery can be by administration of the IL-6 polypeptide itself or by delivery of a polynucleotide (e.g., an expression vector) encoding it to the above recited sites. These methods of the invention will be applicable to the above species as well as humans .

B.METHODS OF IMMUNIZATION The invention features methods for protecting subjects from infection by microorganisms such as viruses as well as intracellular bacteria and protozoans. The methods involve induction of neutralizing antibody responses specific for antigens produced by these microorganisms. Such antibodies can be IgM, IgG, IgA or IgE antibodies. They will preferably be of the IgA class. This is achieved by delivery of IL-6 and antigen to a mucosal surface, e.g., oral, intestinal, vaginal, nasopharyngeal or tracheal mucosae. Relevant microorganisms include but are not restricted to those that enter their hosts via mucosal barriers, e.g. HIV-1, influenza virus, enteric viruses such as rotaviruses, hepatitis A virus, papilloma virus, feline immunodeficiency virus, feline leukemia virus, simian immunodeficiency virus, listeria bacteria and mycobacteria such as M. tuberculosis and M. leprae, Helicobacter pylori, and parasites, e.g., Toxoplasma gondii . Since the responses elicited by mucosal immunization occur in the systemic as well as the mucosal immune system, the methodologies may also be applied to protection from infection by pathogens that enter their hosts via non-mucosal (e.g., HIV-1 in some scenarios such as a "stick" by a contaminated syringe needle, rabies virus and malarial protozoans) as well as mucosal routes. In light of the above consideration, immunization via mucosae can also be used for immunotherapy of infections.

Polypeptide agents to be used in the invention include IL-6, other cytokines (see infra) , certain antigens (see infra) , and adjuvants. The adjuvants are, in some instances, co-administered at the mucosal tissue site with the antigens. These adjuvants include, but are not limited to, cholera toxin (CT) , E. coli heat labile toxin (LT) , mutant CT (MCT) [Yamamoto et al . (1997) J. Exp. Med. 185 :1203] and mutant E. coli heat labile toxin (MLT) (Di Tommaso et al . (1996) Infect. Immunity 64_.: 974] . MCT and MLT contain point mutations that substantially diminish toxicity without substantially compromising adjuvant activity relative to that of the parent molecules. Polypeptides and peptides may also include variants of those described above. These variants may contain different amino acids (preferably conservative changes) from the parental molecules but retain the biological activity of the parental molecules, e.g., the ability to enhance antigen-specific antibody responses subsequent to mucosal immunization. Similarly, fragments of polypeptides shorter than full-length polypeptides can also be utilized, provided that they also retain the above-recited biological activity. Such variants and fragments can be synthesized by standard means, and are readily tested in assays known to those in the art .

Polypeptides and peptides of the invention also include those described above but modified for in vivo use by addition at either or both the amino- and carboxy- terminal ends, of a blocking agent in order to facilitate survival of the relevant polypeptide or peptide in vivo . This can be useful in those situations in which the termini tend to be degraded ("nibbled") by proteases prior to cellular or ER uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxy terminal residues of the polypeptide or peptide to be administered. This can be done either chemically during the synthesis of the peptide or by recombinant DNA technology (see Section B.2 infra) . Alternatively, blocking agents such as pyroglutamic acid or other molecules known to those in the art may be attached to the amino and/or carboxy terminal residues, or the amino group at the amino terminus or carboxyl group at the carboxy terminus replaced with a different moiety. Likewise, the polypeptides or peptides can be covalently or noncovalently coupled to pharmaceutically acceptable "carrier" proteins prior to administration. B.l In vivo methods to deliver IL-6 to mucosae of a subject

In methods of the invention that induce mucosal antibody responses in a subject (i.e., a mammal), IL-6 is delivered to the effector (e.g., SMG, or LP) and, preferably, also the inductive (e.g., PP) mucosal immune system. Delivery involves either the derivation of tg lines of the relevant mammal over-expressing IL-6 in a mucosal tissue or administering to a mammal either the IL-6 polypeptide itself or a polynucleotide (e.g., an expression vector) encoding the IL-6. Examples of appropriate mammals include but are not restricted to humans, non-human primates, horses, cattle, swine, sheep, dogs, cats, mice, rats, guinea pigs, hamsters, rabbits and goats.

B.1.1 Transgenic (tg) lines

The invention features non-human tg animals that harbor relevant transgenes in both their somatic cells and their germ cells. The transgenes within the tg animals contain at least one coding segment that encodes a polypeptide that enhances antibody responses. Such a polypeptide can be a non-cytokine molecule such as CD40 ligand or a cytokine . CD40 ligand is a molecule expressed by activated T cells that interacts with CD40 on B cells. This molecular interaction serves to enhance B cell antibody producing responses. The cytokine can be any cytokine that enhances antibody responses and is preferably IL-6, e.g., human IL-6. The coding sequence is operatively linked to a TRE (e.g., a promoter or an enhancer) that directs expression of the coding sequence to the mucosa of the animal. Mucosa-specific TRE include, without limitation, the enhancer of the Mup- 1.5a/b gene (see (Example 1) which directs expression to the SMG and the TRE of the sucrase-isomaltase gene which directs expression to intestinal epithelial cells. The TRE will preferably be that of the Mup-1.5a/b gene. In one embodiment, the transgene can include the whole Mup- 1.5a/b gene, including regulatory and structural genes, as illustrated in Fig. 1. Alternatively, the control segment can be a subregion of 5' untransribed region of the Mup-1.5a/b gene that includes the enhancer elements and promoter. The 5' untranscribed region segment utilized can be the whole 5' untranscribed region or it can include a sequence from within a stretch of DNA bounded by, at its 5' end, an EcoRI restriction enzyme site about 3.46 kilobases 5' of the Mupl .5a/b gene transcription initiation site and, at its 3' end, by the Mup-1.5a/b transcription initiation site. Alternatively, the 5' untransribed region segment can include a combination of (a) a sequence from a stretch of DNA bounded by, at its 5' end, the EcoRI restriction enzyme site mentioned above and, at its 3' end, a SstI restriction enzyme site about 1.85 kilobases 5' of the Mup-1.5a/b transcription initiation site and (b) a sequence of 600 bases immediately 5' of the transcription initiation site of Mup-1.5a/b gene. Methods of establishing the minimal length of the TRE required for function (e.g., direction of expression of the coding sequence to the mucosae such the SMG) are known to those in the art .

The invention features tg lines of non-human animals, e.g., rodents (e.g., mice, hamsters, and rats) rabbits, non-human primates, bovine animals (cows and bulls), sheep, goats, pigs, horses, dogs, cats, and birds (e.g., chickens, ducks, and turkeys). Methods of deriving tg lines of animals are well known in the art [e.g. Hogan et al . , (1986) Manipulating the mouse embryo: a laboratory manual, Cold Spring Harbor Press, N.Y.] . An example of a method for the production of a tg mouse line is provided infra in Example 1. A protocol for the production of a tg pig can be found in White and Yannoutsos, Current Topics in Complement Research: 64th Forum in Immunology, pp. 88-94; US Patent No. 5,523,226; US Patent No. 5,573,933; PCT Application WO93/25071; and PCT Application WO95/04744. A protocol for the production of a tg mouse can be found in US Patent No. 5,530,177. A protocol for the production of a tg rat can be found in Bader and Ganten, Clinical and Experimental Pharmacology and Physiology, Supp . 3:S81-S87, 1996. A protocol for the production of a tg cow can be found in Transgenic Animal Technology, A Handbook, 1994, ed. , Carl A. Pinkert, Academic Press, Inc. A protocol for the production of a tg sheep can be found in Transgenic Animal Technology, A Handbook, 1994, ed. , Carl A.

Pinkert, Academic Press, Inc. Furthermore Deboer et al . [U.S. Patent No. 5,741,957], using methods analogous to those developed in mice in their laboratory, generated tg lines of cattle expressing human lactoferrin in their milk, thereby establishing the feasibility of generating tg lines of large animals using methods similar to those used for producing tg lines of small animals (e.g., mice). In addition, Cibelli et al . [(1998) Science, 280 : 1256] have developed an efficient method of generating tg animals that is particularly useful for large animals (e.g., cattle). In this method non- quiescent fetal fibroblast cells are used to produce cloned tg animals. In brief, a transgene of interest is inserted into a fibroblast cell of the relevant species. The transgenic fibroblast cell is then fused to a mature oocyte from which the nucleus has been removed. Nuclear transplant techniques known to those in the art could also be used for this step. The resulting embryo is implanted into the uterus of a recipient female animal . Methods for deriving tg birds (e.g., chickens) have also been developed [PCT Application WO 97/47739] . All patents and other references cited herein are hereby incorporated by reference .

A tg animal of the invention can be one that specifically overexpresses an IL-6 gene in a mucosal tissue, e.g., the SMG, intestinal epithelium or nasal, tracheal, pulmonary, or oral mucosa. One tg mouse strain of the invention specifically over-expresses a human IL-6 tg in the submandibular gland (SMG) and thus secretes IL- 6 in saliva. The IL-6 gene may be derived from different mammalian species, e.g, human, mouse, cattle, swine or sheep. The tg animals of the invention may also mucosally over-express other transgene encoded cytokines that enhance immune responses, e.g., granulocyte- macrophage colony-stimulating factor (GM-CSF) , interleukin-2 (IL-2) , interleukin-4 (IL-4) , interleukin-5 (IL-5) , interleukin-7 (IL-7) , interleukin-13 (IL-13) , gamma-interferon, or tumor necrosis factor-α; (TNF-o.) . Tg animals may harbor one or multiple cytokine transgenes (e.g., two, three, four or five). Thus, for example, a tg animal may harbor both an IL-6 and an IL-4 coding sequence. Where the tg animals contain more than one exogenous coding sequence encoding a polypeptide that enhances an immune response, one or more of the coding sequences may be introduced by interbreeding of parents transgenic for different coding sequences. In tg animals expressing more than one tg coding sequence, at least one of the coding sequences will be under the control of a TRE directing expression to a mucosa. The tg animals can be immunized by the same methods described infra .

B . 1 . 2 Administration of soluble IL-6

IL-6 may be delivered to the mucosal immune system of a mammal using techniques substantially the same as those described infra for delivery to human subjects. IL-6 may be delivered to the mucosal immune system of a human in its unmodified state, dissolved in an appropriate physiological solution, e.g. physiological saline. Alternatively, it may be modified as detailed in supra in order to prevent extracellular or intracellular degradation. It may also be delivered encapsulated in liposomes [Gabizon et al . (1990) Cancer Res. 50 :6371; Ranade (1989) J. Clin. Pharmacol. 29:685] or an appropriate biodegradable polymeric microparticle (also referred to as a "microsphere" , "nanosphere" ,

"nanoparticle, or "microcapsule" ) . Naturally, it is desirable that the IL-6 be selectively targeted to the mucosae. This can be achieved by contacting the soluble antigens directly with the relevant mucosal surface, e.g. by oral, rectal, vaginal, nasal, or tracheal infusion or implantation .

Soluble IL-6 can be co-administered mucosally with antigen. It can also be given mucosally but at a different mucosal site than that of antigen administration. Alternatively, soluble IL-6 can be injected systemically (e.g., intravenously or subcutaneously) at the same time, or a different time, as mucosal antigen administration. Antigens include, but are not restricted to: a) the microbes themselves; b) related microbes; c) attenuated or heat-killed forms of a) or b) ; d) polypeptides, carbohydrates or lipids derived from a) or b) ; or e) peptide fragments derived from microbial polypeptides. Thus the antigens can be non-replicating antigens. Non-replicating antigens are antigens that are substantially incapable of reproducing themselves, either intra- or extracellularly . Thus, non-replicating antigens include, without limitation, heat, ultra-violet light, or ionizing radiation attenuated microbes, genetically modified non-replicative variants of microbes, polypeptides, carbohydrates, or lipids obtained from microbes, and active fragments of such polypeptides, carbohydrates, or lipids. Polypeptide or polypeptide fragment antigens may also be administered using polynucleotides encoding them by methods similar to those for administering IL-6 (see infra) . Antigens may be administered mucosally either alone or together with the adjuvants described supra . Antigens and their administration are well known to those in the art.

Examples of other cytokines that may be used, singly or in combination with IL-6, include, without limitation, GM-CSF, IL-2, IL-4, IL-5, IL-7, IL-13, gamma- interferon or TNF-α.. Methods of delivery would be as for IL-6. Administration may be single or multiple (two, three, four, five, six, eight or twelve administrations, for example) . Where multiple, they may be spaced from one day to one year apart .

It is well known in the medical and veterinary arts that dosages for any one subject depend on many factors, as well as the particular compound to be administered, the time and route of administration and other drugs being administered concurrently. Dosages for the antigens of the invention will vary, but, where soluble, can be approximately 0.01 mg to 100 mg per administration. Dosages for the mucosal adjuvants will be approximately 0.001 mg to 100 mg per administration. Dosages for IL-6 will be approximately 1 μg/kg to 500 μg/kg. Methods of determining optimal doses are well known to pharmacologists, physicians or veterinarians of ordinary skill. Routes will be, as recited supra, i.e., mucosal, e.g., oral, rectal, vaginal, nasal, or tracheal.

B.1.3 Administration of IL-6 utilizing polynucleotides encoding IL-6

Delivery of IL-6 can be by administration of a polynucleotide (e.g., an expression vector) encoding IL-6 to a mucosal cell. An expression vector is composed of or contains a nucleic acid in which a polynucleotide sequence encoding IL-6 and/or another cytokine or other cytokines (as recited above for vectors used in the production of tg mice) of the invention is operatively linked to a promoter or enhancer-promoter combination. A promoter is a TRE composed of a region of a DNA molecule typically within 100 nucleotide pairs in front (upstream of) of the point at which transcription starts. Another TRE is an enhancer. An enhancer provides specificity in terms of time, location and expression level. Unlike a promoter, an enhancer can function when located at variable distances from the transcription site, provided a promoter is present. An enhancer can also be located downstream of the transcription initiation site. A coding segment of an expression vector is operatively linked to a transcription terminating region. To bring a coding segment under control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the peptide or polypeptide between one and about fifty nucleotides downstream (3') of the promoter. Examples of particular promoters are provided infra . Expression vectors and methods for their construction are known to those familiar with the art.

Suitable vectors include plasmids, and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses and adeno-associated viruses, among others.

This "genetic immunization" approach requires delivery of a genetic construct directly into the mucosa of a subject, preferably targeting it to the cells or tissue of interest (e.g., cells in the SMG, PP or LP) . This can be achieved by administering it directly to the relevant mucosa (e.g., orally in the case of the SMG or rectally in the case of PP or LP) .

Tissue specific targeting may also be achieved by the use of a molecular conjugate composed of a plasmid or other vector attached to poly-L-lysine by electrostatic or covalent forces. Poly-L-lysine binds to a ligand that can bind to a receptor on target cells [Cristiano et al . (1995) J. Mol. Med 73:479]. Similarly, cell specific antibodies (e.g., B-cell specific antibodies such anti- CD19 or anti-CD20) can be bound to vectors and thereby target them to the relevant cells of the mucosal immune system. A TRE inducing relatively tissue or cell- specific expression can be used to achieve a further level of targeting. Appropriate tissue-specific TREs include, for example, the TRE regulating expression of the SMG acinar cell -specific major urinary protein ge Mup-1.5a/b or the intestinal epithelial cell-specific sucrase-isomaltase gene TRE. Vectors can also be delivered by incorporation into liposomes. TRE containing segments used for such vectors include those described above for transgenes harbored by the tg animals of the invention. As indicated above, coding segments can encode a wide range of cytokines or other molecules (e.g., CD40 ligand) that increase antibody responses. The vectors can also contain more than one (e.g., 2,3,4, or 6) coding segments. In such vectors, at least one of the coding segments is under the control of a mucosa expression directing TRE.

DNA may be administered in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a human, e.g., physiological saline. A therapeutically effective amount is an amount of the DNA of the invention which is capable of producing a clinically desirable result in a treated subject. As is well known in the medical arts, the dosage for any one subject depends upon many factors, including the subject's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for administration of DNA is from approximately 10⁶ to 10¹² copies of the DNA molecule. This dose can be repeatedly administered, as needed. Routes of administration will be the mucosal routes recited for soluble IL-6 supra in Section B.1.2. The antigens and mucosal adjuvants can also be administered in the same combinations by the same routes and at the same dosages recited in Section B.1.2. It is anticipated that delivery of a polypeptide that enhances an antibody reponse (e.g., CD40 ligand or the cytokines listed above) , either by administration of the polypeptide itself or by administration of a polynucleotide (e.g., a DNA molecule) encoding the polypeptide, will result in at least a 1.5-fold, preferably at least a 3 -fold, more preferably at least a 6-fold, and most preferably at least a 12 -fold increase in a mucosal immune response (e.g., a mucosal IgA response) .

B.2 Sources of peptides and polypeptides agents

Peptides and polypeptides used in the methods of the invention may be obtained by a variety of means . Smaller peptides (less than 100 amino acids long) may be conveniently synthesized by standard chemical methods familiar to those skilled in the art [e.g see Creighton (1983) Proteins: Structures and Molecular Principles, W.H. Freeman and Co., N.Y.] . Larger peptides (longer than 100 amino acids) can be produced by a number of methods including recombinant DNA technology (see infra) . Some polypeptides (e.g., IL-6, CT, LT and some polypeptide antigens) may be purchased from commercial sources .

Polypeptides such as IL-6, CT, LT or certain polypeptide antigens may be purified from biological sources by methods well-known to those skilled in the art (Protein Purification, Principles and Practice, second edition (1987) Scopes, Springer Verlag, N.Y.]. Polypeptides such as IL-6, CT, MCT, LT, MLT or certain polypeptide antigens may also be produced in their naturally occurring, truncated, chimeric, or fusion protein forms by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vi tro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al . (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.,; and Ausubel et al . , eds. (1989) , Current Protocols in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., N.Y. Alternatively, RNA encoding the proteins may be chemically synthesized. See, for example, the techniques described in Oligonucleotide Synthesis, (1984) Gait, M.J. ed. , IRL Press, Oxford, which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to express the nucleotide sequences. Where the peptide or polypeptide is soluble, it can be recovered from: (a) the culture, i.e., from the host cell in cases where the peptide or polypeptide is not secreted; or (b) from the culture medium in cases where the peptide or polypeptide is secreted by the cells. The expression systems also encompass engineered host cells that express the polypeptide in si tu, i.e., anchored in the cell membrane. Purification or enrichment of the polypeptide from such an expression system can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. Alternatively, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the protein, but also to assess biological activity.

The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (for example, E. coli and B . subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the nucleotide sequences; yeast transformed with recombinant yeast expression vectors; insect cells infected with recombinant viral expression vectors (baculovirus) ; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors; or mammalian cells (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses . In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the gene product being expressed. For example, when a large quantity of such a protein is to be produced, e.g. for in vivo immunization, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 [Ruther et al . (1983) EMBO J. 2_.: 1791], in which the coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors [Inouye & Inouye (1985) Nucleic Acids Res. 13_.: 3101; Van Heeke & Schuster (1989) J. Biol. Chem. 264 : 5503] ; and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) . In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e . g. , the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vi tro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the gene product in infected hosts [e.g. , See Logan & Shenk (1984) Proc. Natl. Acad. Sci. USA 81 :3655] . Specific initiation signals may also be required for efficient translation of inserted nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. [Bittner et al . (1987) Methods in Enzymol . 153 :516] . In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications ( e . g. , glycosylation) and processing { e . g. , cleavage) of protein products may be important for the function of the protein. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. Mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and WI38.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the sequences described above may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements ( e . g. , promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the gene product. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the gene product .

A fusion protein may be readily purified by utilizing an antibody or a ligand that specifically binds to the fusion protein being expressed. For example, a system described by Janknecht et al . [(1991) Proc. Natl. Acad. Sci. USA 8_8-'⁸9⁷²] allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. If desired, the histidine tag can be selectively cleaved with an appropriate enzyme .

The following examples are meant to illustrate the invention and not to limit it.

Example 1. Construction of a human IL-6 cDNA/Mup-1.5a/b fusion gene and derivation of tg mouse lines harboring it

The entire coding region of human IL-6 cDNA, including the signal peptide, was excised from a cloning vector by NlalV restriction enzyme digestion and sandwiched into a unique Sma I site in exon 1 of the pH construct which includes about 3.46 kilobases of 5' flanking sequences of the submandibular gland-specific gene Mup-1.5a/b [Son et al . (1991). Mol. Cell. Biol. i :4244-4254 , incorporated herein by reference in its entirety] . There are 2 Mup-1.5 genes in the BALB/c mouse genome, Mup-1.5a and Mup-1.5b, only one of which (Mup- 1.5a) is expressed. The genes are extremely homologous, with only 1 nucleotide difference in the coding sequences and 1 nucleotide difference in the 3' untranslated region. In the introns the 2 genes differ at 2 positions. There is a T→C transition in intron II and a deletion of (GT)₃ in a GT-rich region of intron III. The Mup-1.5 gene utilized for preliminary experiments [Son et al . , supra] and the tg animals of the invention was actually the non-expressed gene, i.e., Mup-1.5b. However, as is clear from both the preliminary experiments and those below, the TRE of the Mup-1.5b gene acts as it does in the expressed Mup-1.5a gene., i.e., it directs expression to the SMG. It seems therefore that any differences in the untranscribed regions between the 2 genes do not effect the specificity of expression.

This contention was supported by sequence analysis of the 1.6 kilobase 5' untranscribed region established to contain the appropriate TRE(s) [Son et al ., supra] . In light of these considerations the relevant TREs of the 2 genes are interchangeable and thus, for ease of description, the genes are referred to as a single gene, i.e., Mup-1.5a/b. The TRE within Mup-1.5a/b was (a) broadly mapped to a region of the gene between an EcoRI restriction enzyme site about 3.46 kilobases 5' of the transcription initiation site and the transcription initiation site and (b) more narrowly mapped to the above mentioned 1.6 kilobase region between the same EcoRI restriction enzyme site and a SstI site about 1.85 kilobases 5' of the transcription initiation site. In the latter case, in addition to the l.b kilobase region, the construct contained about 600 nucleotides of DNA immediately 5' of the transcription initiation site of the Mupl .5a/b gene. This 600 nucleotide fragment included promoter sequences. The construct used to derive the tg mice is shown in Fig.l. The tg lines were derived by the Rockefeller University Transgenic Mouse Facility. The construct which was liberated from its cloning vector with Sail and HindiII restriction enzymes was microinjected into FVB/N fertilized mouse eggs. Viable embryos derived from the injected eggs were reimplanted into the oviducts of pseudopregnant foster mothers. Tg offspring were identified by PCR or Southern blot analysis of genomic DNA isolated from the tail ends of the mice. Expression was tested for by RT-PCR or Northern blot analysis of RNA isolated from various tissues of the mice. Approximately 50% of tg mice showed SMG-specific expression of the IL-6 construct. These lines were chosen for further study. Over-expression of IL-6 by the salivary glands of the tg mice does not cause any observable pathologies. The tg mice are healthy, having normal life spans and normal reproductive capacities.

Example 2. SMG-specific expression of human IL-6 transgene in tg mice lμg of DNase I treated RNA extracted from the indicated tissues (Fig. 2) of a tg mouse was reverse transcribed (RT) in 10 μl total volume, lμl of the RT reaction was amplified in 25 μl of total volume using primers specific for human IL-6 [5': 5'- ACCTCTTCAGAACGAATTGACAAA-3' (SEQ ID NO:l) and 3': 5'-

AGCTGCGCAGAATGAGATGAGTTGT-3' (SEQ ID NO : 2 ) ] or for beta- actin [5': 5 ' -TGGAATCCTGTGGCATCCATGAAAC-3 ' (SEQ ID NO: 3) and 3' : 5 ' -TAAAACGCAGCTCAGTAACATCCG-3 ' (SEQ ID NO: 4)] using a GeneAmp-RNA PCR kit (Perkin Elmer) . The RT-PCR samples were subjected to ethidium bromide agarose gel electrophoresis. To show that DNA was not amplified, reverse transcriptase was omitted from the RT reaction (-RT lane) . The tg line from which the RNA was isolated expresses the human IL-6 transgene in the SMG only (Fig. 2) . As in the indicated tissues, transgene expression was also not seen in bone marrow.

Example 3. Human IL-6 activity in saliva and antigen specific IgA secretion into saliva in tg mice Saliva (50-100μl) was collected from adult female tg mice (16-3, 16-4, 16-38, 16-29, 16-60) and adult female non-tg mice (16-1, 16-31) following subcutaneous injection of a the saliva inducer pilocarpine hydrochloride dissolved in PBS (60 μg/lOg of body weight) . The presence of human IL-6 in the saliva was assessed by bioassay with IL-6 dependent B9 hybridoma cells. B9 cells were seeded in 96-well plates and cultured, 5,000 per well, in lOOμl of culture medium containing serial dilutions of saliva, saliva plus monoclonal anti-human IL-6 antibody shown not to cross- react with murine IL-6 (5IL-6, 13μg) , and saliva plus monoclonal anti-murine IL-6 antibody (20F3, 18 μg) . Recombinant human IL-6 was employed as a standard. After 4 days of incubation, the number of viable cells was determined by 3- (4 , 5-dimethylthiazol-2-yl) -2 , 5- diphenyltetrazolium bromide (MTT) assay [Aarden et al . , Eur. J. Immun., 37:1411]. 1 μg of recombinant human IL-6 is equivalent to 88,459 U/ml , in this assay. As can be seen in Table 1, only the tg mice produced significant levels of IL-6. Table 1. Secretion of human IL-6 into the saliva of tg mice over-expressing a human IL-6 transgene

10-week-old tg female mice (B37, B39, B44, B52) and non-tg female littermates (Al, A2 , A3, A4) were immunized orally with 2.4 mg of keyhole limpet hemocyanin (KLH, Sigma) and 10 μg of cholera toxin B (CT-B, List Biological Laboratory), dissolved in 0.5 ml of PBS containing 3% sodium carbonate. Following immunization, both groups of mice were boosted three times at 10 -day intervals. Three days following the 3rd boost, saliva was collected from mice subcutaneously injected with 100 μl of pilocarpine (6mg/ml) . Saliva was cleared by centrifugation, and the samples were adjusted to equal protein concentration as determined by the BioRad Protein Assay, and stored at -70 °C. To measure the KLH-specific IgA response (Fig. 3A) , Falcon MicroTest III plates were coated with 100 μl of 100 μg/ml KLH. To measure the CT- B-specific IgA response (Fig. 3B) , the plates were coated with 3 μg/ml ganglioside GM1 (which binds CT-B) followed by 100 μl of 3 μg/ml CT-B. Each coating was done at 4°C overnight. After 5 washes with PBS and 0.05% Tween 20, plates were blocked with 100 μl of 5% dry milk for 1 hour at 37°C. 100 μl of serially diluted saliva was then added in duplicate and incubated for 1.5 hours at 37°C. After 5 washes, 100 μl of 1:500 diluted alkaline phosphatase-conjugated rabbit anti-mouse IgA (Zymed) was added, and incubated for 1 hour at 37°C. The color development was carried out by adding 100 μl of substrate buffer (48 ml diethanolamine, 24.5 mg MgCl₂ in 500 ml H₂0, pH 9.8) containing 1 mg/ml Phosphatase Substrate tablets (Sigma) . The plates were read at 405 nm using a STL Elisa Reader. The OD405 nm data points represent the average of duplicate wells. The tg mice mounted a salivary IgA response to the antigen, KLH, approximately ten-fold greater than control non-tg mice (Fig. 3A) . Both tg and non-tg mice mounted an IgA response to the antigenic adjuvant CT-B (Fig 3B) . The response to CT-B was however significantly greater in the tg mice than in the non-tg mice.

Tg mice from which the SMG had been surgically removed mounted salivary IgA responses to KLH and CT-B that were indistinguishable to those mounted by control non-tg mice from which the SMG had been surgically removed. This experiment showed that the increased salivary IgA responses in the tg mice were due to expression of the transgene in the SMG and were not due to ectopic effects.

Example 4. Antigen-specific IgA responses in gut lavage of tg mice

10-week-old tg female mice (B37, B39, B44, B52) and non-tg female littermates (Al , A2 , A3, A4) were immunized orally with 2.4 mg of KLH and 10 μg of CT-B, dissolved in 0.5ml of PBS containing 3% sodium carbonate. Following immunization, both groups of mice were boosted 4 times at 10-day intervals. Five days following the 4th boost, the mice were sacrificed and gut lavage was collected as described by Vajdy et al . [(1995) J. Exp. Med. 181 :41-53) . To measure the KLH-specific IgA response (Fig. 4A) , Falcon Micro Test ID plates were coated with 100 μl of 100 μg/ml KLH. To measure the CT- B-specific IgA response (Fig. 4B) , the plates were coated with 3 μg/ml ganglioside GM1 followed by lOOμl of 3 μg/ml CT-B. Each coating was done at 4°C overnight. After 5 washes with PBS, and 0.5% Tween 20, plates were blocked with lOOμl of 5% dry milk for 1 hour at 37 °C . After 5 washes, lOOμl of 1:500 diluted gut lavage was then added in duplicate and incubated for 1.5 hours at 37°C. The color development was done by adding 100 μl of substrate buffer (48 ml diethanolamine, 24.5 mg MgCl₂ in 500 ml H₂0, pH 9.8) containing lmg/ml Sigma Phosphatase Substrate tablets. The plates were read at 405 nm using a STL Elisa Reader. The OD405 nm data points represent the averages of duplicate wells. While detectable antigen- specific (Fig. 4A) and adjuvant-specific (Fig. 4B) IgA responses were seen in the non-tg mice, the tg mice mounted significantly greater responses. The response to KLH in the tg mice was 8-10 times that in control non-tg mice .

Example 5. Antigen-specific IgA responses in serum of tg mice

10-week-old tg female mice (B37, B39, B44, B52) and non-tg female littermates (Al , A2 , A3, A4) were immunized orally with 2.4 mg of KLH and 10 μg of CT-B, dissolved in 0.5ml of PBS containing 3% sodium carbonate. Following immunization, both groups of mice were boosted 4 times at 10 -day intervals. Both groups of mice were sacrificed five days following the 4th boost. To measure the KLH-specific IgA response (Fig. 5A) , Falcon MicroTest III plates were coated with 100 μl of lOOμg/ml KLH. To measure the CT-B-specific IgA response (Fig. 5B) , plates were coated with 3μg/ml ganglioside GM1 followed by lOOμl of 3 μg/ml CT-B. Each coating was done at 4°C overnight. After 5 washes with PBS, and 0.5% Tween 20, plates were blocked with lOOμl of 5% dry milk for 1 hour at 37 °C. lOOμl of serially diluted serum was then added in duplicate and incubated for 1.5 hours at 37°C. After 5 washes, 100 μl of 1:500 diluted alkaline phosphatase- conjugated rabbit anti-mouse IgA (Zymed) was added, and incubated for 1 hour at 37°C. The color development was done by adding 100 μl of substrate buffer (48 ml diethanolamine, 24.5 mg MgCl₂ in 500 ml H₂0, pH 9.8) containing lmg/ml Sigma Phosphatase Substrate tablets. The plates were read at 405 nm using a STL Elisa Reader. The OD405 nm data points represent the average of duplicate wells. The IgA responses to antigen (Fig. 5A) and to adjuvant (Fig. 5B) were significantly more potent in tg mice than in non-tg mice. The response to KLH in the tg mice was 3-8 times that in control non-tg mice.

Example 6. Antigen-specific IgA-secreting cells in tg mice

10-week-old tg female mice (B37, B39, B44, B52) and non-tg female littermates (Al , A2 , A3, A4) were immunized orally with KLH and CT-B as described in Example 3. Five days after the fourth boosting, mononuclear cells were prepared from SMG using the method of Mega et al . [In Advances in Mucosal Immunology, J. Mestecky et al . , ed. Plenum Press, New York, 1995], and from the intestinal LP, using the method of Vajdy et al . [(1995). Exp. Med. 181:411. The numbers of KLH-specific IgA secreting cells (Fig. 6A) , and CT-B-specific IgA secreting cells (Fig. 6B) were determined by ELISPOT assay. 96-well plates (Numc Immuno-Plate) were coated at 4°C overnight with lOOμg/ml of KLH or 3 μg/ml of ganglioside GM1 followed by 3 μg/ml of CT-B, and the plates were blocked with 5% dry milk. 1X10⁴ viable mononuclear cells were added to each well, in triplicate, and incubated for 3 hours at 37°C. After washes with PBS and 0.5% Tween 20, lOOμl of 1:500 diluted alkaline phosphatase-conjugated rabbit anti-mouse IgA (Zymed) was added, and the plates were further incubated for 2 hours at 37°C. Antibody-forming cells (AFC) were enumerated by addition of 75 μg/ml 5-bromo-4-chloro-3 -indolyl phosphate (BCIP) and lOOμg/ml nitro blue tetrazolium (NBT) in 100 mM Tris, pH9.5 , lOOnM NaCl, 25 mM MgCl₂. The AFCs were counted under a dissecting microscope, using 3OX magnification. The graph shows the average of four individual mice. The IgA forming cell responses to antigen (Fig. 6A) and adjuvant (Fig. 6B) were more potent in both SMG and LPL in the tg than in control non-tg mice and responses in SMG were greater in the SMG than the LPL. Since the increase in the number of KLH-specific IgA secreting cells in the SMG was about 3 -fold and the increase in salivary KLH-specific IgA was about 10-fold, (Example 3), the increase in salivary IgA antibody was probably due to both enhanced synthesis of IgA per cell as well as an increase in IgA secreting cell number.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. An isolated DNA molecule comprising: a control segment that includes a transcriptional regulatory element; and a coding segment encoding a polypeptide that enhances an antibody response, wherein said regulatory element is operatively linked to said coding segment and directs expression of said coding segment to a mucosa.

2. The DNA molecule of claim 1, wherein said regulatory element directs said expression to the submandibular gland.

3. The DNA molecule of claim 2, wherein said control segment comprises a sequence within at least one of a Mup-1.5a gene 5' untranscribed region and a Mup-1.5b gene 5' untranscribed region.

4. The DNA molecule of claim 3 , wherein said control segment comprises: a sequence included within the Mup-1.5a gene between an EcoRI restriction enzyme site about 3.46 kilobases 5' of the Mup-1.5a gene transcription initiation site and said Mup-1.5a gene transcription initiation site; or a sequence included within the Mup-1.5b gene between an EcoRI restriction enzyme site about 3.46 kilobases 5' of the Mup-1.5b gene transcription initiation site and said Mup-1.5b gene transcription initiation site.

5. The DNA molecule of claim 4, wherein said control segment comprises: a sequence included within the Mup- 1.5a gene between said EcoRI restriction enzyme site of said Mup- 1.5a gene and a SstI restriction enzyme site about 1.85 kilobases 5' of the Mup- 1.5a gene transcription initiation site and a sequence of about 600 bases immediately 5' of the Mup- 1.5a gene transcription initiation site; or a sequence included within the Mup- 1.5b gene between said EcoRI restriction enzyme site of said Mup- 1.5b gene and a SstI restriction enzyme site about 1.85 kilobases 5' of the Mup- 1.5b gene transcription initiation site and a sequence of about 600 bases immediately 5' of the Mup-1.5b gene transcription initiation site.

6. The DNA molecule of claim 1, wherein said polypeptide is a cytokine.

7. The DNA molecule of claim 6, wherein said cytokine is interleukin-6 (IL-6) .

8. The DNA molecule of claim 7, wherein said IL-6 is human IL-6.

9. The DNA molecule of claim 6, wherein said cytokine is selected from the group consisting of interleukin-2 (IL-2) , interleukin-4 (IL-4) , interleukin-5 (IL-5) , and interleukin-13 (IL-13) .

10. A transgenic non-human animal whose germ cells and somatic cells contain a transgene comprising: a control segment that includes a transcriptional regulatory element; and a coding segment encoding a polypeptide that enhances an antibody response, wherein said transcriptional regulatory element is operatively linked to said coding segment and directs expression of said coding segment to a mucosa of said transgenic animal, and wherein said expression results in an increased antibody response in said mucosa.

11. The transgenic animal of claim 10, wherein said regulatory element directs said expression to the submandibular gland.

12. The transgenic animal of claim 10, wherein said control segment comprises a sequence within at least one of a Mup-1.5a gene 5' untranscribed region and a Mup- 1.5b gene 5' untranscribed region.

13. The transgenic animal of claim 12, wherein said control segment comprises: a sequence included within the Mup- 1.5a gene between an EcoRI restriction enzyme site about 3.46 kilobases 5' of the Mup- 1.5a gene transcription initiation site and said Mup-1.5a gene transcription initiation site; or a sequence included within the Mup- 1.5b gene between an EcoRI restriction enzyme site about 3.46 kilobases 5' of the Mup-1.5b gene transcription initiation site and said Mup-1.5b gene transcription initiation site.

14. The transgenic animal of claim 13, wherein said control segment comprises: a sequence included within the Mup- 1.5a gene between said EcoRI restriction enzyme site of said Mup- 1.5a gene and a SstI restriction enzyme site about 1.85 kilobases 5' of the Mup- 1.5a gene transcription initiation site and a sequence of about 600 bases immediately 5' of the Mup- 1.5a gene transcription initiation site; or a sequence included within the Mup-1.5b gene between said EcoRI restriction enzyme site of said Mup- 1.5b gene and a SstI restriction enzyme site about 1.85 kilobases 5' of the Mup- 1.5b gene transcription initiation site and a sequence of about 600 bases immediately 5' of the Mup- 1.5b gene transcription initiation site.

15. The transgenic animal of claim 12, wherein said polypeptide is a cytokine.

16. The transgenic animal of claim 15, wherein said cytokine is IL-6.

17. The transgenic animal of claim 16, wherein the IL-6 is human IL-6.

18. The transgenic animal of claim 15, wherein said cytokine is selected from the group consisting of IL-2, IL-4, IL-5, and IL-13.

19. The transgenic animal of claim 10, wherein said animal is a rodent .

20. The transgenic animal of claim 19, wherein said rodent is a mouse.

21. The transgenic animal of claim 10, wherein said animal is a bovine animal.

22. The transgenic animal of claim 10, wherein said animal is selected from the group consisting of a pig, a goat, and a sheep.

23. The transgenic animal of claim 10, wherein said antibody response is an IgA antibody response.

24. A method for enhancing a mucosal antibody response in a subject, the method comprising: delivering IL-6 to a mucosa of the subject, and administering an antigen to the subject, said antigen being a non-replicating antigen, wherein said delivering results in an antibody response to said antigen in said mucosa that is greater than an antibody response in the mucosa of a subject to which the IL-6 was not delivered but the antigen was administered.

25. The method of claim 24, wherein the mucosa is oral mucosa.

26. The method of claim 24, wherein the mucosa is intestinal mucosa.

27. The method of claim 24, wherein the mucosa is selected from the group consisting of nasal mucosa, pulmonary mucosa, tracheal mucosa, and vaginal mucosa.

28. The method of claim 24, wherein said administering is by delivery of said antigen to said mucosa or a second mucosa selected from the group consisting of nasal mucosa, oral mucosa, pulmonary mucosa, tracheal mucosa, intestinal mucosa and vaginal mucosa .

29. The method of claim 24, wherein delivering of IL-6 is by administering the IL-6 polypeptide to the mucosa .

30. The method of claim 24, wherein delivering is by delivering to a cell of the mucosa a DNA molecule comprising an IL-6 coding segment and a transcriptional regulatory element, wherein said transcriptional regulatory element is operatively linked to said coding segment and directs expression of said coding segment to said mucosa.

31. The method of claim 24, wherein the subject is a mammal .

32. The method of claim 31, wherein the mammal is a bovine mammal .

33. The method of claim 31, wherein the mammal is a pig.

34. The method of claim 31, wherein the mammal is a sheep .

35. The method of claim 31, wherein the mammal is a human.

36. The method of claim 24, further comprising administering an adjuvant to the subject.

37. The method of claim 24, wherein said antibody response is an IgA antibody response.

38. The method of claim 24, wherein the antigen is a viral antigen.

39. The method of claim 24, wherein the antigen is a bacterial antigen.

40. The method of claim 24, wherein the antigen is a protozoan antigen.

41. A method for enhancing a mucosal antibody response in a non-human animal, the method comprising contacting a mucosa of the subject with soluble IL-6, said soluble IL-6 being encoded by a transgene comprising an IL-6 coding segment operably linked to a transcriptional regulatory element that directs expression of the IL-6 coding sequence to said mucosa, wherein the germ cells and somatic cells of said animal contain said transgene.