Heterologous polysaccharide synthesis in attenuated Salmonella

描述(申请人提供)：多糖(PS)是覆盖在细菌病原体表面的主要生物大分子之一，代表主要的毒力决定因素和免疫原。已开发出几种PS疫苗(包括结合PS)来预防病原体感染；这些PS疫苗存在产量低、非特异性化学结合、成本高和注射给药等缺点。重组减毒沙门氏菌(RAS)能够携带包括PS在内的多种病原体的异源抗原，产生包括血清抗体、粘膜免疫球蛋白A在内的多种免疫应答，并在局部和远端产生一系列细胞免疫应答。这些RA的设计和构造是为了运送蛋白质或DNA抗原。在这项工作中，我们承担了专为运送细菌PS抗原而设计的减毒沙门氏菌菌株的构建，包括来自革兰氏阴性病原体的O-抗原多糖(O-PS)和来自革兰氏阳性病原体的荚膜多糖(C-PS)。在我们的菌株中，这些复杂的碳水化合物将共价连接到沙门氏菌细胞表面。使用我们新的调节延迟基因表达技术，所有天然沙门氏菌表面碳水化合物将在免疫时存在，以促进沙门氏菌与宿主之间的相互作用，这是最佳免疫原性所必需的，但在宿主淋巴组织定植后，在体内将下调。相反，异源碳水化合物的合成只有在疫苗株在宿主组织中定植后才会发生。沙门氏菌碳水化合物的下调将允许异源碳水化合物的最佳表达和呈递，而不会受到沙门氏菌表面结构的干扰或空间位阻。将目标多糖抗原连接到沙门氏菌细胞表面提供了沙门氏菌脂多糖的强大佐剂作用。此外，这些“生物偶联多糖疫苗”将使我们能够利用传统的、化学偶联的多糖抗原的已知能力来刺激T细胞依赖和T细胞非依赖的免疫反应。

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.