Development of novel subbunit vaccine targeting mutiple alphaviruses

东西方马脑炎病毒(EEEV和Weev；甲型病毒；Togaviridae)是蚊虫 引起人类严重脑炎的甲型病毒。目前还没有针对甲型病毒的人类疫苗。 广泛性疾病的案例。我们之前已经建立了小鼠模型，描述了糖尿病的发病机制 这些甲型病毒，并已表明，阳离子-脂质体-DNA复合体(CLDC)诱导保护 Weev攻击后小鼠天然免疫的激活。在这项提案中，基于CLDC的疫苗 将开发的平台(LANAC)包括具有新的N-糖链的E2-E1和E1糖蛋白。我们 将测试Weev E2-E1或基于E1的LANAC疫苗免疫可产生保护作用的假设 对多种甲型病毒具有免疫力。该提案的具体目标如下：目标1)构建 并使用常规的方法生产Weev包膜糖蛋白的重组形式E1和E2-E1 杆状病毒-昆虫细胞系统和人源化蛋白A/-糖基化的杆状病毒-昆虫细胞系统 路径。目的2)评价重组包膜Weev糖蛋白(无脊椎型)提供的保护作用 A/-多聚糖或脊椎动物A/-多聚糖)作为小鼠亚单位疫苗与 在小鼠模型中，将评估皮下和粘膜免疫后的保护作用 以及气雾剂、皮下和蚊子传播的病毒挑战。目标3)确定幽默和 对暴露后小鼠提供最有效保护的细胞免疫机制 对带有CLDC佐剂的Weev E1和E2-E1糖蛋白疫苗。这些免疫反应 包括对免疫球蛋白抗体、免疫球蛋白M和免疫球蛋白A抗体进行定量，中和抗体滴度，并潜在地定位 Weev糖蛋白的保护性T细胞表位。我们还将比较对E1和E2-E1的免疫应答 免疫CD1、B6、缺乏B细胞的小鼠和T细胞耗竭的小鼠的糖蛋白。目标4)描述 Weev E1糖蛋白在小鼠体内对其他甲型病毒提供交叉保护的能力。十字 CSU将评估带有CLDC的保护性糖蛋白对EEEV、VEEV和SINBIS病毒(SINV)的影响 和基孔肯亚和罗斯河病毒(CHIKV和RRV)在北卡罗来纳大学教堂山作为合作的一部分 罗伯特·E·约翰斯顿博士的实验室和SERCEB。我们将评估针对多个病毒挑战的保护 并评估免疫反应和保护的持久性。这项研究项目适合 在RMRCE范围内，综合研究的重点是免疫调节、佐剂和疫苗，并将 与RP1.5、RP1.6、RP3.3进行交互，并利用Core C和E的资源。

Eastern and Western equine encephalitis viruses (EEEV and WEEV; Alphavirus; Togaviridae) are mosquitoborne alphaviruses causing severe encephalitis in humans. There are no human vaccines for alphaviruses in case of widespread disease. We have previously developed mouse models describing pathogenesis of these alphaviruses and have shown that cationic-liposome-DNA complexes (CLDC) elicit protective activation of innate immunity in mice following WEEV challenge. In this proposal, CLDC-based vaccine platforms (LANAC) will be developed that include E2-E1 and E1 glycoproteins having novel N-glycans. We will test the hypothesis that immunization with WEEV E2-E1 or E1-based LANAC vaccines elicits protective immunity against multiple alphaviruses. The specific aims of the proposal are as follows: Aim 1) Construct and produce recombinant forms of the WEEV envelope glycoproteins, E1 and E2-E1 using a conventional baculovirus-insect cell system and a baculovirus-insect cell system with a humanized protein A/-glycosylation pathway. Aim 2) Evaluate protection provided by recombinant envelope WEEV glycoproteins (invertebratetype A/-glycans or vertebrate-type A/-glycans) as subunit vaccines in mice when used in combination with CLDC's. Protection will be evaluated following subcutaneous and mucosal immunization in the mouse model and aerosol-, subcutaneous-, and mosquito-delivered virus challenge. Aim 3) Identify the humoral and cellular immunological mechanisms responsible for the most efficient protection of mice following exposure to the WEEV E1 and E2-E1 glycoprotein vaccines with CLDC adjuvant. These immunological responses include quantifying IgG, IgM and IgA antibody and neutralizing antibody titers and mapping potentially protective T cell epitopes to WEEV glycoproteins. We will also compare immune responses to E1 and E2-E1 glycoproteins in immunized CD1, B6, mice which lack B-cells, and T-cell depleted mice. Aim 4) Characterize the ability of WEEV E1 glycoprotein to provide cross-protection in mice to other alphaviruses. Cross protective glycoproteins with CLDC's will be evaluated for EEEV, VEEV, and Sindbis virus (SINV) at CSU and Chikungunya and Ross River viruses (CHIKV and RRV) at UNC-Chapel Hill as part of a collaboration with Dr. Robert E. Johnston's lab and SERCEB. We will evaluate protection against multiple virus challenge routes and evaluate the longevity of immunological responses and protection. This research project fits within the RMRCE Integrated Research Focus on Immunomodulation, Adjuvants and Vaccines, and will interact with RP1.5, RP1.6, RP 3.3 and utilize the resources of Core C and E.