Title:  Vaccine delivery system

United States Patent:  6,180,111

Assignee:  University of Maryland (College Park, MD)

Filed:  May 19, 1998

The invention relates to a hyperblebbing strain of Neisseria gonorrhoeae which produces large amounts of blebs useful for production of blebosomes containing antigens for use as a vaccine delivery vehicle or as a diagnostic reagent. The invention also relates to a method for producing high levels of a desired protein in purified form using the hyperblebbing strain of N. gonorrhoeae, and to a vaccine delivery systems containing the blebosomes expressing the desired antigen.

SUMMARY OF THE INVENTION

The present invention is directed to a mutant of N. gonorrhoeae strain WR302 that is defective in an unknown growth parameter. This defect causes the gonococcus to grow abnormally and slough off its outer membrane at a very high frequency or hyperbleb. This strain produces many more outer membrane vesicles (blebs) than other strains and the blebs are readily visualized by electron microscopy.

The present invention is also directed to the production of other N. gonorrhoeae strains with the hyperblebbing phenotype by using a non-selective spot transformation technique. This technique allows for easy identification of transformants of N. gonorrhoeae in the absence of selective pressure. The technique comprises the steps of mixing a limiting number of cells with an excess amount of DNA, spotting the mix onto the surface of an agar plate, incubating the mix, and re-streaking and selecting the transformed cells. The hyperblebbing strains are altered in their expression of lipooligosaccharide (LOS). The transformants can be identified phenotypically based on their acquisition of new monoclonal antibody reactivity and the diffusion of blebs away from the colony.

By using the spot-transformation technique described above on strains of N. gonorrhoeae with different mutations and/or already possessing genes for different antigens, hyperblebbing strains of N. gonorrhoeae expressing antigens for different diseases can be produced. Blebosomes collected from these hyperblebbing strains can be used to produce a vaccine for immunization against these diseases. Vaccines produced in this way have an advantage over whole cell vaccines because the antigens are present in the absence of other cellular components. In addition, the antigens are assembled in a natural biological membrane allowing the antigen to form a native conformation, more closely mimicking what is encountered in the natural organism.

These blebosomes can be used in diagnostic assays wherein the presence of antibodies against disease can be detected in samples from a patient suspected of having the disease.

Blebosomes can also be used as a delivery system for other biologically made molecules, such as chemotherapeutic agents for use in chemotherapy, or immune enhancers/suppressors or antibiotics.

Further, the blebosomes can be used to expedite the purification of membrane proteins from the gonococcus (either natural or engineered). Blebosomes are significantly enriched for outer membrane proteins of the gonococcus since most of the cells cytoplasmic components are absent. Isolation of blebosomes then represents a significant purification of those gonococcal membrane proteins. Similarly, blebosomes can be enriched for proteins which have been engineered to be expressed in the blebosome, for example a receptor protein, and would offer a significant improvement in aiding in the production and purification of such proteins.

Therefore, it is an object of the present invention to provide a hyperblebbing strain of Neisseria gonorrhoeae which produces large amounts of blebs useful for the production of blebosomes containing specific antigens for use as a vaccine delivery vehicle or as a diagnostic agent.

It is another object of the present invention to provide a method for the production of hyperblebbing strains of N. gonorrhoeae.

It is another object of the present invention to provide a method for the production of large amounts of a desired protein in purified form, said method comprising introducing the desired gene into a hyperblebbing strain of N. gonorrhoeae such that the protein is expressed in blebosomes, and isolating said blebosomes.

It is yet another object of the present invention to provide a vaccine delivery system comprising the blebosomes expressing an antigen specific for said disease which would provide immunity against said disease.

Claim 1 of 19 Claims

What is claimed as new and is desired to be secured by Letters Patent of the United States is:

1. A vaccine for providing in a subject immunity against a disease, said vaccine comprising blebosomes wherein a heterologous immunogenic polypeptide specific for said disease is expressed on the surface of said blebosomes, and

wherein said heterologous immunooenic polypeptide is present in a pharmacologically effective dose in a pharmaceutically acceptable excipient.

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