Development of recombinant VSV vaccines for emerging bunyaviruses

项目摘要/摘要 新出现的病毒感染仍然是对人类健康的全球威胁。布尼亚病毒是核糖核酸的最大目 包括许多与临床相关的人类病原体的病毒，如拉萨病毒、裂谷热和各种 引起病毒性出血热(VHF)的汉坦病毒。SFTSV，或伴有血小板减少的高烧 综合征病毒，是一种新出现的由壁虱传播的布尼亚病毒，已在东亚暴发(中国，日本， 韩国、越南)，病死率高达30%。现在已经在一个大型的 地理环境和SFTSV已经在许多野生和家养动物物种中发现，这突出了一种 人畜共患病蔓延到人类的风险。尽管人类通常是死胡同的宿主，人与人之间 已记录通过血液和粘膜分泌物进行传播。此外，SFTSV还具有 分段基因组，增加基因重组和突变的能力。由于这些功能，在其2017年 《研究与发展蓝图》世界卫生组织将SFTSV确定为可能导致 未来可能会出现严重的疫情。正如我们所了解到的，从最近人畜共患疫情的经验来看，防备 是最重要的。我们建议继续开发一种重组水泡性口炎 猪瘟病毒(RVSV)疫苗。RVSV是一种获得批准的具有免疫原性的疫苗接种平台 而且事实证明是可以忍受的。我们课题组的初步工作表明，rVSV-SFTSV可以诱导保护 在小鼠中，在免疫低下的动物和注射CNS时是可以耐受的，并能产生交叉保护 对一种相关的本亚病毒的反应。此外，我们观察到SFTSV特异性T细胞和抗体的升高 对SFTSV尖峰蛋白Gn和Gc的反应。在第一阶段，我们建议改善 我们的载体将提高病毒滴度，以促进疫苗的生产，并增强疫苗的免疫原性 动物。在目标1中，我们将重点关注rVSV的反向工程突变，这将改善SFTSV Gn/Gc 加入到颗粒中，从而提高病毒的复制和体外产量。我们将描述这第二个 使用分子和细胞分析对病毒复制、GnGc表达进行评分的第二代(Gen2)疫苗 并将GnGc掺入VSV病毒粒子中。在目标2中，我们将评估我们的Gen2疫苗的能力 在致命的SFTSV挑战研究中保护动物。我们假设GnGc表达增加和 与复制Gen2载体相比，复制Gen2载体将改善接种动物的免疫反应 Gen1载体。我们将评估免疫保护的相关性，如增强中和和交叉保护 与Gen1 rVSV-SFTSV疫苗接种的动物相比，抗体以及强大的T细胞反应。如果成功， 我们的第一阶段将为第二阶段奠定基础，以推进制造并严格研究保护 我们的疫苗在更先进的动物模型中的能力。

PROJECT SUMMARY/ABSTRACT Emerging viral infections remain a global threat to human health. Bunyaviruses are the largest order of RNA viruses that includes many clinically relevant human pathogens such as Lassa, Rift Valley Fever and various hantaviruses which cause viral hemorrhagic fever (VHF). SFTSV, or Severe fever with thrombocytopenia syndrome virus, is an emerging tick-borne bunyavirus that has caused outbreaks in Eastern Asia (China, Japan, Korea, Vietnam) with up to a 30% case fatality rate. The host tick vector has now been discovered over a large geographical setting and SFTSV has been found in numerous wild and domestic animal species highlighting a risk for zoonotic spillover into humans. Though humans are usually dead-end hosts, human to human transmission has been documented through blood and mucosal secretions. Furthermore, SFTSV has a segmented genome which increases ability to reassort genes and mutate. Due to these features, in its 2017 “Research and Development Blueprint” the WHO identified SFTSV as one of 11 pathogens likely to cause a sever outbreak in the future. As we have learned, from experience with recent zoonotic outbreaks, preparedness is of the utmost importance. We are proposing to continue development of a recombinant vesicular stomatitis virus (rVSV)-based vaccine for SFTSV. rVSV is an approved vaccination platform that is immunogenically potent and proven tolerable. Preliminary work from our group has demonstrated that rVSV-SFTSV can elicit protection in mice, is tolerable in immunocompromised animals and upon CNS injection, and can generate cross protecting responses to a related bunyavirus. Furthermore, we observe elevated SFTSV-specific T cell and antibody responses against both SFTSV spike proteins, Gn and Gc. In this Phase I, we propose to improve the design of our vector which will increase viral titers to facilitate vaccine manufacturing and increase immunogenicity in animals. In aim 1, we will focus on reverse engineering mutations into rVSV that will improve SFTSV Gn/Gc incorporation into particles thereby improving virus replication and yield in vitro. We will characterize these 2nd generation (Gen2) vaccines using molecular and cell-based assays scoring for viral replication, GnGc expression in infected cells and incorporation of GnGc in VSV virions. In aim 2, we will assess the ability of our Gen2 vaccine to protect animals in lethal SFTSV challenge studies. We hypothesize that increased GnGc expression and replication of the Gen2 vectors will lead to improved immune responses in vaccinated animals compared to the Gen1 vector. We will assess immune correlates of protection such enhanced neutralizing and cross protective antibodies as well as robust T cell responses compared to Gen1 rVSV-SFTSV vaccinated animals. If successful, our Phase I would set the foundation for a Phase II to advance manufacturing and stringently study protective capacity of our vaccine in more advanced animal models.