United States Patent:  6,110,898

Assignee:  University of Maryland, Baltimore (Baltimore, MD)

Filed:  May 23, 1997

The invention consists of a method for inducing production of a mucosal immune response in a host by administration of an antigen-encoding polynucleotide preparation, comprising DNA or RNA encoding an antigenic epitope to a mucosal inductor site in the mucosal tissue of the host. Naked DNA may be administered directly to mucosa, for instance in saline drops, or in a recombinant gene expression vector. Preferably, the recombinant gene expression vectors are not capable of replication or dessimination. The invention also includes the use of live viral vaccines wherein the viruses include immunostimulatory polynucleotides of the invention. According to a preferred method of the invention, a target protein antigen is administered through its expression by a recombinant gene expression vector.

SUMMARY OF THE INVENTION

In one aspect, the invention comprises a method for transfection of polynucleotides which express immunogenic proteins into cells of mucosal tissue located at mucosal inductor sites so as to result in a mucosal immune response to the encoded antigens wherein secretory IgA antibodies are produced to the encoded antigens. In another aspect, the invention comprises a method for inducing a protective mucosal response against mucosal-contracted pathogens, such as respiratory, intestinal and vaginal viruses by administration to a vertebrate of a polynucleotide vaccine.

An antigen-encoding polynucleotide preparation, comprising recombinant expression vectors, such as chimeric viral vectors, for use in genetic immunization via mucosal administration are also provided herein. The antigen-encoding polynucleotide preparation of the invention can include immunostimulatory polynucleotides which help to elicit a vigorous mucosal humoral immune response.

As used with respect to the invention, the term "antigen-encoding polynucleotide preparation" refers to plasmids or cosmids which encode a peptide or protein of interest (e.g., antigens, cytokines, chemokines, heat-shock proteins, and other immunomodulatory agents). The vector may contain a gene encoding an adjuvant, such as the cholera toxin B subunit, to enhance the immunostimulatory effect of the vaccine. In addition to cholera toxin, other natural compounds with mucosal adjuvant properties are streptococcal antigen, and the heat labile toxin (LT) of E. coli.

The present invention exploits the discovery that gene expression delivery systems directed to mucosal inductor sites adjacent to or containing "mucosal-associated lymphoid aggregates" (MALT) in the mucosal tissue can elicit antigen expression therein with resulting mucosal immune responses, including secretion of sIgA. Such mucosal inductor sites are found in the tonsils and nasal lymphoid tissue, (the Waldeyer's ring of oropharyngeal lymphoid tissue in the nasal, palatinal and lingual tonsils)), in bronchus-associated lymphoid tissue (BALT), and in gut-associated lymphoid tissue (Peyer's patches). The effector sites from which sIgA is secreted may be located in anatomically remote glands, such as the lacrimal, salivary and mammary glands, as well as in the nasal mucosa, upper respiratory tract, small intestine, large intestine and genital tract. A unique cooperation between the mucosal B-cell system and the secretory component (SC) expressed basolaterally on glandular epithelial cells accounts for this phenomenon. The B cells responsible for local sIg production are initially stimulated in MALT cells, from which they migrate as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory site.

The present invention exploits this integrated system by administering polynucleotide vaccines so as to contact cells located at mucosal inductor sites, especially those in the gut, genital, and upper and lower respiratory system, and thereby advantageously promote production of sIgA to titer levels that are protective against diseases caused by pathogens, especially respiratory, genital, and enteric viruses, which initially enter the body through the mucosa.

The antigen-encoding polynucleotide preparation may also encode polypeptides of interest, such as additional antigens or adjuvants and cytokines. The preferred vectors for use in the method of this invention are viral recombinant gene expression vectors and non-viral recombinant gene expression vectors associated with delivery vehicles (e.g., liposomes or colloidal particles) into which polynucleotides of the invention have been inserted. In addition, using the same techniques by which polynucleotides of the invention are incorporated into viral gene expression vectors, the polynucleotides may be incorporated into live viral vaccines to augment the immune response to viral antigens.

For purposes of monitoring gene expression or testing immune response, these vectors may be modified to include known reporter or antigen genes. For example, the pRSV lac-Z DNA vector described in Norton, et al., Mol. Cell. Biol., 5:281, (1985), may produce .beta.-galactosidase with protein expression. .beta.-Galactosidase has also been used as a test antigen expressed from recombinant SFV vectors. Luciferase and chloramphenicol acetyl transferase ("CAT"; see, e.g., Gorman, et al., supra, re construction of a pRSV-CAT plasmid) may also be used. Convenient plasmid propagation may be obtained in E. coli (see, e.g., Molecular Cloning: A Laboratory Manual, supra.)

For use as a tolerizing vaccine, a mixture of polynucleotides or separately coadministered group of polynucleotides may include a gene operatively encoding for an immunosuppressive cytokine (such as TGF.beta.) and a separate gene operatively encoding for a relevant histocompatibility protein. This approach could be adapted for use in inducing tolerance to foreign antigens (including alloantigens) as well as self-antigens.

In the preferred embodiment, the invention comprises a method for immunizing a host against antigen using gene expression vectors of the invention. According to a preferred method of the invention, such expression vectors are introduced into mucosal tissues of the host having a relatively high concentration of mucosal inductor tissue therein. Introduction of the antigen-encoding polynucleotide preparation into the host may be by any suitable means known in the art.

Claim 1 of 38 Claims

1. A method for inducing a mucosal immune response in a host comprising locally administering to said host an antigen-encoding polynucleotide preparation, whereby administration of said polynucleotide preparation is specifically targeted to mucosal inductor sites.

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