Development of novel subbunit vaccine targeting mutiple alphaviruses

东方和西方马脑炎病毒（EEEV和WEEV;甲病毒属;披膜病毒科）是蚊媒病毒， 引起人类严重脑炎的甲病毒。目前还没有针对甲病毒的人类疫苗， 这是一种广泛传播的疾病。我们先前已经开发了描述 这些甲病毒，并已表明，阳离子脂质体-DNA复合物（CLDC）引起的保护性 WEEV攻击后小鼠先天免疫的激活。在这项提案中，基于CLDC的疫苗 将开发包括具有新型N-聚糖的E2-E1和E1糖蛋白的LANAC平台。我们 将检验用WEEV E2-E1或基于E1的LANAC疫苗免疫具有保护性的假设 对多种甲病毒的免疫力。该提案的具体目标如下： 并使用常规方法产生重组形式的WEEV包膜糖蛋白E1和E2-E1， 杆状病毒-昆虫细胞系统和具有人源化蛋白A/-糖基化的杆状病毒-昆虫细胞系统 通路目的2）评价重组WEEV囊膜糖蛋白（无脊椎动物型）的保护作用 A-聚糖或脊椎动物型A-聚糖）作为亚单位疫苗在小鼠中的应用 CLDC的。将在小鼠模型中皮下和粘膜免疫后评价保护作用 以及气溶胶、皮下和蚊子递送的病毒攻击。目的3）确定体液和 细胞免疫机制负责最有效的保护小鼠暴露后 与具有CLDC佐剂的WEEV E1和E2-E1糖蛋白疫苗相比。这些免疫反应 包括定量IgG、IgM和伊加抗体和中和抗体滴度，并绘制潜在 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 免疫的CD 1、B6、缺乏B细胞的小鼠和T细胞耗尽的小鼠中的糖蛋白。目标4）描述 WEEV E1糖蛋白在小鼠中对其他甲病毒提供交叉保护的能力。横 将在CSU评价CLDC的保护性糖蛋白对EEEV、VEEV和辛德毕斯病毒（SINV）的作用 和基孔肯雅和罗斯河病毒（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博士约翰斯顿的实验室和SERCEB。我们将评估针对多种病毒挑战的保护 途径，并评估免疫应答和保护的寿命。这个研究项目符合 在RMRCE免疫调节，佐剂和疫苗综合研究重点，并将 与RP 1.5、RP 1.6、RP 3.3交互，利用Core C和E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.