Heterologous polysaccharide synthesis in attenuated Salmonella

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DESCRIPTION (provided by applicant): Polysaccharide (PS) constitutes one of major classes of bio-macromolecules covering the surface of the bacterial pathogens, representing a major virulent determinant and immunogen. Several PS vaccines (including conjugated PS) have been developed to prevent the pathogen infections; these PS vaccines suffer from low yields, non-specific chemical conjugation, high costs, and delivery by injection. Recombinant attenuated Salmonella (RAS) are capable of delivering heterologous antigens, including PS, from a variety of pathogens, generating a range of immune responses including serum antibodies, mucosal IgA, and a panoply of cell-mediated immune responses at local and distal sites. These RAS are designed and constructed to deliver protein or DNA antigens. In this work, we undertake the construction of attenuated Salmonella strains designed specifically for delivery of bacterial PS antigens, including O-antigen polysaccharides (O-PS) from Gram-negative pathogens and capsular polysaccharides (C-PS) from Gram-positive pathogens. In our strains, these complex carbohydrates will be covalently linked to the Salmonella cell surface. Using our novel regulated delayed gene expression technology, all native Salmonella surface carbohydrates will be present at the time of immunization to facilitate Salmonella-host interactions necessary for optimal immunogenicity, but will be down-regulated in vivo, after colonization of host lymphatic tissues. Conversely, synthesis of heterologous carbohydrates will not occur until after the vaccine strain has colonized host tissues. Down-regulation of Salmonella carbohydrates will permit optimal expression and presentation of heterologous carbohydrates without interference or steric blocking by Salmonella surface structures. Linking the target polysaccharide antigens to the Salmonella cell surface provides the strong adjuvant effects of Salmonella LPS. In addition, these "biologically conjugated polysaccharide vaccines" will allow us to take advantage of the known ability of traditional, chemically conjugated polysaccharide antigens to stimulate both T-cell dependent and T-cell independent immune responses to the target antigen.