RESULTS: Immunofluorescence microscopy with dengue specific antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody clearly showed that recombinant protein Co1-scEDIII-AGA was localized on the cell surface of the respective clones in comparison with scEDIII-Co1 and Mock cells with no fluorescence. Oral dosage applications of surface displayed Co1-scEDIII-AGA stimulated a systemic humoral immune response in the form of dengue-specific serum IgG, as well as a mucosal immune response in the form of secretory immunoglobulin A (sIgA). Antigen-specific B cell responses in isolated lymphoid cells from the spleen and Peyer\'s patches further supported an elevated mucosal immune response. In addition, surface displayed Co1-scEDIII-AGA feeding elicited strong immune responses in comparison with scEDIII-Co1 and Mock following intraperitoneal booster with purified scEDIII antigen.
CONCLUSIONS: Surface displayed preparations of Co1-scEDIII-AGA induced strong immunogenicity compared with non-displayed scEDIII-Co1. Prior studies have supported the neutralization potential of scEDIII constructs against all four serotypes. Thus, the oral administration of genetically engineered yeast whole cells displaying biologically active Co1-scEDIII fusion protein without any further processing shows prospective as a potent oral vaccine candidate against dengue viral infection.
结果:使用登革热特异性抗体和异硫氰酸荧光素缀合的抗小鼠IgG抗体的免疫荧光显微镜清楚地表明,与没有荧光的scEDIII-Co1和Mock细胞相比,重组蛋白Co1-scEDIII-AGA位于相应克隆的细胞表面。表面显示的Co1-scEDIII-AGA的口服剂量应用以登革热特异性血清IgG的形式刺激了全身性体液免疫反应,以及分泌性免疫球蛋白A(sIgA)形式的粘膜免疫反应。来自脾脏和Peyer斑块的分离淋巴样细胞中的抗原特异性B细胞反应进一步支持了升高的粘膜免疫反应。此外,与使用纯化的scEDIII抗原腹膜内加强后的scEDIII-Co1和Mock相比,表面显示的Co1-scEDIII-AGA喂养引起强烈的免疫反应。
结论:与未展示的scEDIII-Co1相比,表面展示的Co1-scEDIII-AGA制剂诱导强免疫原性。先前的研究已经支持scEDIII构建体对所有四种血清型的中和潜力。因此,口服展示生物活性Co1-scEDIII融合蛋白的基因工程酵母全细胞,无需任何进一步加工,显示出作为抗登革病毒感染的有效口服候选疫苗的前景.