摘要目的 比较利用重组登革病毒(DENV)包膜蛋白疫苗进行新型序贯免疫与传统混合免疫策略在诱导抗病毒免疫应答上的优劣.方法 通过杆状病毒表达系统分别表达4种血清型登革病毒EDⅢ蛋白作为免疫原,用SDS-PAGE分析其纯度,Western blot分析其特异性.将雌性BALB/c小鼠随机分为阴性对照组、混合免疫组和序贯免疫组,分别在第0、2、4、6周通过皮下注射接种PBS或疫苗,在末次免疫后2周通过ELISPOT检测Th2细胞免疫应答;利用空斑减少中和试验(PRNT)测定血清中和能力.结果 与PBS阴性对照组相比,序贯免疫和混合免疫在EDⅢ蛋白刺激下,均能检测到分泌IL-4的Th2细胞反应,且序贯免疫产生的T细胞反应强于混合免疫;在肽段刺激下,仅序贯免疫诱导出分泌IL-4的Th2细胞反应;混合免疫和序贯免疫均产生针对DENV-1、DENV-2和DENV-3的中和抗体,但混合免疫产生的中和抗体滴度高于序贯免疫诱导产生的中和抗体滴度;2种免疫策略都未产生针对DENV-4的中和抗体.结论 序贯免疫较混合免疫可诱导较强的Th2细胞反应,但B细胞反应产生的中和抗体滴度低于混合免疫策略.

abstractsObjective To investigate whether a novel sequential immunization strategy was more superior to the traditional immunization strategy in eliciting immune responses by using domainⅢof dengue envelope proteins (EDⅢs) as immunogens. Methods EDⅢ subunit proteins of four serotypes of dengue viruses (DENVs) were expressed in a baculovirus expression system. SDS-PAGE and Western blot were performed to analyze the purity and specificity of purified recombinant proteins, respectively. In order to evaluate the immunogenicity of EDⅢ-based immunization strategies, female BALB/c mice were subcutane-ously immunized with PBS,tetravalent mixture of four EDⅢrecombinant proteins,or the four EDⅢproteins sequentially for four times with two weeks interval between each immunization. Two-week after the final im-munization,splenocytes were isolated and analyzed by ELISPOT assay to evaluate T cell responses and serum samples were collected for plaque reduction neutralization test(PRNT). Results Both immunization strate-gies of sequential EDⅢproteins and tetravalent EDⅢproteins could elicit stronger antigen-specific Th2(IL-4) cell responses in immunized mice than PBS did and the former was superior to the latter. Only the se-quential immunization strategy could induce Th2 cell responses in immunized mice against peptide segments of DENV2 EDⅢ. Tetravalent EDⅢ proteins performed better than the sequential immunization strategy in inducing higher levels of neutralizing antibodies against DENV-1,DENV-2 and DENV-3,while both immu-nization strategies failed to generate neutralizing antibodies against DENV-4. Conclusion Sequential immu-nization with DENV EDⅢ proteins induced stronger T cell responses, but weaker neutralizing antibody re-sponses against DENV than tetravalent EDⅢ proteins did.