Heterologous polysaccharide synthesis in attenuated Salmonella

描述（由申请人提供）：多糖（PS）是覆盖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簇和免疫原。已经开发了几种PS疫苗（包括缀合的PS）来预防病原体感染;这些PS疫苗具有低产量、非特异性化学缀合、高成本和通过注射递送的缺点。重组减毒沙门氏菌（RAS）能够递送来自多种病原体的异源抗原，包括PS，在局部和远端位点产生一系列免疫应答，包括血清抗体、粘膜伊加和一系列细胞介导的免疫应答。这些RAS被设计和构建为递送蛋白质或DNA抗原。在这项工作中，我们进行了减毒沙门氏菌菌株的建设，专门设计用于交付细菌PS抗原，包括O-抗原多糖（O-PS）从革兰氏阴性病原体和荚膜多糖（C-PS）从革兰氏阳性病原体。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。使用我们的新型调控延迟基因表达技术，所有天然沙门氏菌表面碳水化合物将在免疫时存在，以促进最佳免疫原性所必需的沙门氏菌-宿主相互作用，但在宿主淋巴组织定殖后，将在体内下调。相反，在疫苗株定殖宿主组织之前，不会合成异源碳水化合物。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，而不受沙门氏菌表面结构的干扰或空间阻断。将目标多糖抗原连接到沙门氏菌细胞表面提供沙门氏菌LPS的强佐剂作用。此外，这些“生物缀合的多糖疫苗”将允许我们利用传统的化学缀合的多糖抗原的已知能力来刺激对靶抗原的T细胞依赖性和T细胞非依赖性免疫应答。

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.