Model Vaccines Exploiting Fungal Mannosylation

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Mannoproteins (MP) from Cryptococcus neoformans elicit strong T cell-mediated immune responses. Cryptococcal MP contain serine/threonine (S/T)-rich regions which serve as sites for extensive O-linked glycosylation as well as N-X-S/T sequons that serve as N-glycosylation sites. N-linked and O-linked glycans on yeasts are terminally mannosylated whereas fully processed mammalian glycoproteins rarely have terminal mannose groups. Mannose receptors (MR) on dendritic cells (DC) bind to MP, resulting in the efficient uptake, processing and presentation of peptide fragments of MP by DC. Given these findings, we hypothesize that the capacity of mannosylation to increase immunogenicity can be exploited to make recombinant vaccines that stimulate T-cell responses. To test this hypothesis, the capacity of differentially glycosylated preparations of the model antigen, ovalbumin (OVA), to elicit MHC Class I (CD8) and class II (CD4)-restricted T cell responses will be tested in vitro and in vivo. There are 3 specific aims: Aim 1 is to generate recombinant OVA preparations in bacterial, yeast, and mammalian cell vectors. Such preps should be unglycosylated, terminally mannosylated, and glycosylated but without terminal mannose groups, respectively. Addition of S/T-rich regions and N-to-Q substitutions will permit study of the relative contribution of O- and N-linkages to immunogenicity. The OVA preparations generated in aim 1 will be utilized in aim 2 to examine the effects of OVA glycosylation patterns on antigen uptake, processing and presentation in vitro, and in aim 3 to test the efficacy of the various glycosylated recombinant proteins to induce an immune response in vivo. Thus, Class I and Class ll-restricted responses to antigens that contain identical protein cores but that qualitatively and quantitatively differ with regards to glycosylation will be compared. The role of MR on DC as endocytic receptors allowing for the efficient capture of mannosylated OVA preparations will be studied using blockers of the MR and with knockout mice. Completion of these studies should add significantly to our knowledge of how innate recognition of foreign glycosylation patterns triggers adaptive immune responses and could suggests novel strategies for increasing the immunogenicity of vaccines.