The main objective of the project for the last three years was to develop a new generation of multivalent recombinant vaccines based on non-pathogenic Gram positive bacteria Staphylococcus xylosus and Staphylococcus carnosus. The use of these bacteria represents an original approach since the more conventional approaches use pathogenic vectors such as Salmonella, Shigella, BCG or different viruses. Both staphylococci have been widely used in starter cultures for meat ripening and represent therefore ideal candidates for live bacterial vehicles for oral delivery of subunit immunogens. The main objectives were: 1. To develop an expression system containing promoters and signal sequences well recognised in the host, as well as cell wall attachment structures. Develop a shuttle vector allowing recombinant work also in E. coli. 2. To develop different assays to monitor a proper surface exposure of foreign antigenic determinants. 3. To define antigens of micro-organisms and toxins to be expressed on the surface of the bacteria. 4. To establish tools and techniques to assess mucosal immune responses expressed in the airway mucosa after different routes of administration 5. To study the immune response against recombinant antigens in different species.
Initially a surface expression system has been developed for S. xylosus taking advantage of promoter, signal sequence and cell-wall attachment structure from staphylococcal protein A. A second surface display system for S. carnosus has been developed where the expression and secretion signal used were derived from S. hyicus lipase and for cell surface-anchoring the cell-wall attaching structures from staphylococcal protein A. A number of foreign antigenic determinants (native structure and modified ones) of bacterial (streptococci), viral (human and bovine RSV, measles and Hantavirus) and protozoan (P. falciparum malaria) origin. A general method for the quantification of chimeric antigens surface displayed on bacteria has been developed. It takes advantage of fluorescence-activated cell sorting (FACS) technology. Preliminary toxicology studies have shown that wild types and recombinant forms of S. carnosus and S. xylosus are apathogenic in normal as well as immunocompromised mice. Methods have been developed to isolate and characterise lymphoid cells from murine lungs and trachea, as well as methods to explore the immune response to a mucosal antigen such as CTB and other classical antigens. Intranasal immunisation seems to be the most efficient route of antigen delivery, as measured by the local IgA and IgG antibody responses. Studies in vivo in LOU rats showed that at least two injections or oral applications are necessary to induce stable immune responses against recombinant antigens expressed on the surface of Staphylococci. Prelimi-nary studies in baboons demonstrate that recombinant Staphylococci are immunogenic by oral, nasal and parenteral route. Finally it could be shown that mice immunised with Staphylococci expressing recombinant antigens derived from respiratory syncytial virus develop high titer anti-RSV antibodies and are protected against a subsequent virus challenge.
The two described expression systems are the first reported world-wide, in which shuttle vectors have enabled high-level display of foreign antigenic determinants on the surface of Gram-positive bacteria. We have shown that surface-display is necessary to generate antigen-specific antibodies. Completely novel techniques to easily monitor the surface display have been developed, taking advantage of a reporter molecule introduced in the hybrid receptor. We have also, demonstrated that antigenic structures containing hydrophobic sequences and thus being difficult to secret, can be engineered by site-directed mutagenesis to allow proper surface exposure. Fluorescence-activated cell sorting (FACS) has been adapted for monitoring and characterisation of heterologous expressed surface antigens and has been successfully used to determine the number of chimeric receptors per recombinant bacterial cell. Finally, safety concerns and protective immune responses to the surface-displayed antigens after oral or subcutaneous administration of live recombinant staphylococci have demonstrated that both Staphylococcus xylosus and Staphylococcus carnosus are potent bacterial delivery systems of surface-displayed subunit vaccines, and have the potential as novel vaccine vehicles for both human and veterinary use.
Funding SchemeCSC - Cost-sharing contracts
91191 Gif Sur Yvette
100 44 Stockholm