Reverse genetics to develop a second generation Rift Valley fever vaccine

描述（由申请人提供）：裂谷热病毒（RVFV）属于布尼亚病毒科白蛉病毒属，是最重要的新兴病毒之一。它被列为NIAID的A类病原体。裂谷热病毒由蚊子传播，在人类和牲畜中引起严重疾病。一部分感染者出现出血热、脑炎或视网膜血管炎；受感染的反刍动物的后代通常在子宫内死亡。裂谷热病毒在撒哈拉以南非洲国家流行，但其他国家正在为气候变化、航空运输和/或生物恐怖主义可能导致的裂谷热病毒传入做准备。唯一真正有效的对策是接种疫苗。裂谷热病毒具有由S-、M-和l -片段组成的三段式负链RNA基因组。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）和一个功能不明确的78-kD蛋白。在化学诱变剂存在的情况下，将野生型ZH548菌株在人二倍体MRC-5细胞中连续传代12次，研制出候选减毒活疫苗MP-12。我们在小鼠模型中的初步数据表明，MP-12是由部分减弱的M段和l段共同作用而减弱的。目前的MP-12疫苗对人类使用具有重大风险，因为病毒的衰减不完全，M或L段的逆转可能会增加MP-12的毒力。因此，为了进一步提高MP-12的安全性，有必要对其衰减机理进行表征。我的长期目标是建立针对高毒力负链RNA病毒的有效对策，特别强调疫苗接种。中心假设是，目前的候选MP-12疫苗可以在保持免疫原性的同时，通过反向遗传将突变引入S段或m段，从而进一步提高安全性。总体目标是表征MP-12基因组中现有的衰减突变，并通过将进一步的衰减突变纳入S或m段来提高MP-12的安全性。提出了三个特异性目的：特异性目的1：鉴定和表征MP-12的衰减突变，特异性目的2：在不降低MP-12免疫原性的情况下减弱MP-12的s段，特异性目的3：通过修饰Gn或Gc的细胞质结构域来减弱MP-12的m段。拟议的研究将利用反向遗传学用于疫苗开发的优势，并开发具有高度免疫原性和非常安全的下一代RVFV减毒活疫苗候选疫苗。

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.