Reverse genetics to develop a second generation Rift Valley fever vaccine

描述(申请人提供)：裂谷热病毒(RVFV)属于布尼亚病毒科白喉病毒属，是最重要的新兴病毒之一。它被列为NIAID A类病原体。RVFV通过蚊子传播，在人和牲畜中都会导致严重的疾病。一部分受感染的人会患上出血热、脑炎或视网膜血管炎；受感染的反刍动物的后代往往会在子宫中死亡。RVFV在撒哈拉以南非洲国家流行，但由于气候变化、航空运输和/或生物恐怖主义，其他国家正在为可能引入RVFV做准备。唯一真正有效的对策是接种疫苗。RVFV基因组为三段负链，由S、M和L三个片段组成。基因组编码4个主要结构蛋白(N、Gn、GC和L)，2个非结构蛋白(Nss和Nsm)和一个78kD的蛋白，其功能尚不清楚。在化学诱变剂存在下，野生型ZH548株在人二倍体MRC-5细胞中连续传代12代，形成候选减毒活疫苗MP-12。我们在小鼠模型中的初步数据表明，MP-12被部分衰减的M-片段和L-片段的联合作用所减弱。目前的MP-12疫苗对人类的使用构成了重大风险，因为病毒的减毒还没有完全完成，而且M节段或L节段的逆转可能会增加MP-12的毒力。因此，有必要对MP-12的衰减机制进行表征，以进一步提高其安全性。我的长期目标是建立针对高毒力负链RNA病毒的有效对策，特别强调接种疫苗。中心假设是，通过反向遗传学将突变引入S或M节段，同时保持免疫原性，可以进一步改进当前候选MP-12疫苗的安全性。总体目标是确定MP-12基因组中现有的衰减突变的特征，并通过将进一步的衰减突变加入到S或M片段中来提高MP-12的安全性。具体目标1：鉴定和鉴定MP-12的衰减性突变；特异性目标2：在不降低MP-12免疫原性的情况下减弱MP-12的S片段；特异性目标3：通过修饰Gn或GC的胞质结构域来减弱MP-12的M片段。这项拟议的研究将利用反向遗传学用于疫苗开发的优势，并开发出具有高度免疫原性和非常安全的下一代减毒活疫苗候选疫苗。 公共卫生相关性：裂谷热病毒MP-12毒株是一种减毒活疫苗候选毒株，有可能通过几个突变增加毒力。我们将鉴定和表征现有MP-12疫苗的减毒突变，并进一步生产减毒但具有免疫原性的第二代MP-12疫苗。

DESCRIPTION (provided by applicant): Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is one of the most important emerging viruses. It is listed as an NIAID category A pathogen. RVFV is transmitted by mosquitoes and causes severe disease in both humans and livestock. A proportion of infected humans develop hemorrhagic fever, encephalitis or retinal vasculitis; the offspring of infected ruminants often die in utero. RVFV is endemic in sub-Saharan African countries, but other countries are preparing for potential introductions of RVFV due to climate change, air transport, and/or bioterrorism. The only truly effective countermeasure is vaccination. RVFV has a tripartite negative-stranded RNA genome composed of the S-, M- and L-segments. The genome encodes 4 major structural proteins (N, Gn, Gc and L), 2 nonstructural proteins (NSs and NSm) and a 78-kD protein whose function is poorly characterized. A candidate live-attenuated vaccine, MP-12, was developed by 12 serial passages of the wild-type ZH548 strain in human diploid MRC-5 cells in the presence of a chemical mutagen. Our preliminary data in the mouse model suggest that MP-12 is attenuated by the combined effect of partially attenuated M- and L-segments. The current MP-12 vaccine poses a significant risk for use in humans because attenuation of the virus is not complete, and reversion of either the M- or L- segment could potentially increase the virulence of MP-12. Therefore, it is essential to characterize the mechanism of MP-12 attenuation to further improve its safety. My long term goal is to establish effective countermeasures against highly virulent negative-stranded RNA viruses, with special emphasis on vaccination. The central hypothesis is that the current candidate MP-12 vaccine can be further improved for safety by introducing mutations into either the S- or M-segment by reverse genetics while retaining immunogenicity. The overall objective is to characterize existing attenuation mutations in the MP-12 genome, and improve the safety of MP-12 by incorporating further attenuation mutations into the S- or M-segment. The three specific aims are proposed as follows: Specific Aim 1: To identify and characterize attenuation mutations of MP-12, Specific Aim 2: To attenuate the MP-12 S-segment without reducing the immunogenicity of MP-12, and Specific Aim 3: To attenuate the MP-12 M-segment by modifying the cytoplasmic domains of Gn or Gc. The proposed study will harness the advantage of using of reverse genetics for vaccine development, and develop a next generation of live-attenuated RVFV vaccine candidates that are highly immunogenic and very safe. PUBLIC HEALTH RELEVANCE: Rift Valley fever virus MP-12 strain, a live-attenuated vaccine candidate, has a potential risk to increase the virulence by a few mutations. We will identify and characterize the attenuation mutations of existing MP-12 vaccine and generate further attenuated but immunogenic second generation MP-12 vaccines.