VACCINE DEVELOPMENT FOR ALPHAVIRUSES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 包括委内瑞拉(VEEV)、东部(EEEV)和西部马脑炎病毒(WeEV)在内的甲型病毒是高度发展的生物战和恐怖主义(BWT)病原体和重要的自然出现的人畜共患病病毒。目前对这些病毒的生物防御是不够的，平民或军事人员暴露可能会造成广泛的发病率和死亡率。人们迫切需要获得许可的有效疫苗、关键的第一道防线和生物防御研究的重要工具。我们将重点放在活病毒疫苗上，以利用它们高水平的抗原呈递、诱导体液免疫和细胞介导免疫的能力，以及接触复制病毒所具有的更快、更持久的抗体水平。在我们的甲型病毒疫苗开发项目的拟议继续中，我们将完善和进一步验证一套针对VEEV、EEEV和WeEV的候选疫苗的安全性和有效性。将在嵌合疫苗基因组中测试额外的衰减策略，以利用我们最近确定的一些甲型病毒的衣壳蛋白抑制宿主细胞转录的优势，这是毒力的一个重要机制。作为进一步的安全措施，将开发一种新的遗传策略，以完全削弱嵌合疫苗病毒在蚊子细胞中的复制能力，无论是在体外还是在体内。最后，为了进一步评估这些嵌合体用于人类免疫的可能性，食蟹猴将用两种不同的Sindbis/East马脑炎候选疫苗进行免疫，以进行安全性、免疫原性和减毒效果的初步评估。除了保护平民和军事人群免受预期的甲型病毒BWT事件的影响，以及保护从事关键BWT和公共卫生研究的实验室人员，我们将开发的方法还可以被用来快速有效地开发针对新识别的新出现的甲型病毒或工程甲型病毒武器的新疫苗。在新世界的许多地方，它们也将有助于保护自然暴露于这些人畜共患病病原体的危险人群。 到目前为止，我们已经完成了使用食蟹猴(猕猴)的EEEV候选疫苗的有效性研究。动物接种辛德比斯病毒北美(Sind-NAEEEvv)或南美(Sind-SAEEEvv)变异疫苗或生理盐水假免疫。取血测定抗体产生(α-EEEV-Ig G)。以目标致死剂量(吸入1.0E+07 PFU)用气雾剂对动物进行攻击，感染来自北美的EEE病毒(FL-9393)。接受SIND-NAEEEvv变种的动物大多得到保护(5/6存活率；83%)，而接种南美变种(Sind-SAEEEvv)或假接种(生理盐水)的动物因暴露而死亡(分别为1/6；16%和0/4；0%)。对挑战后的动物进行的遥测监测显示，成功接种疫苗的动物核心温度的正常日变化几乎没有受到干扰，心率和心电图也没有异常。相反，受影响的动物则经历了核心温度的显著变化(体温过高)和心电信号的中断。对组织，特别是大脑的组织病理学分析表明，病毒入侵和严重的脑炎。我们正在食蟹猴身上重复这个实验，以确认Sind-EEEvv在这个模型中的有效性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Alphaviruses including Venezuelan (VEEV), eastern (EEEV) and western equine encephalitis viruses (WEEV) are highly developed agents of biological warfare and terrorism (BWT) and important, naturally emerging zoonotic viruses. Current biodefense against these viruses is inadequate, and widespread morbidity and mortality could be inflicted upon exposure of civilians or military personnel. Effective, licensed vaccines, critical first lines of defense and important tools for biodefense research, are badly needed. We have focused on live virus vaccines to take advantage of their high levels of antigen presentation, their ability to induce both humoral and cell-mediated immunity, and the faster and longer lived antibody levels characteristic of exposure to replicating viruses. In the proposed continuation of our alphavirus vaccine development project, we will refine and further validate the safety and efficacy of a set of vaccine candidates against VEEV, EEEV and WEEV. Additional attenuation strategies will be tested in the chimeric vaccine genomes to take advantage of our recent determination that the capsid proteins of some alphaviruses inhibit host cell transcription, an important mechanism of virulence. As a further safety measure, a novel genetic strategy will be developed to completely debilitate the ability of the chimeric vaccine viruses to replicate in mosquito cells, both in vitro and in vivo. Finally, to further evaluate the potential of these chimeras for vaccination of humans, Cynomolgus macaques will be immunized with 2 different Sindbis/Eastern equine encephalitis vaccine candidates in a preliminary assessment of safety, immunogenicity and attenuation. In addition to protection of civilian and military populations from an anticipated alphavirus BWT event, and protection of laboratory personnel doing critical BWT and public health research, the methods we will develop can be exploited to rapidly and efficiently develop new vaccines against newly recognized, emerging alphaviruses or an engineered alphavirus weapon. They will also be useful for protecting populations at risk of natural exposure to these zoonotic agents in many parts in the New World. Thus far, we have completed an efficacy study using the candidate EEEv vaccines using cynomolgus macaques (Macaca fascicularis). Animals were primed with a Sindbis virus vectored North American (SIND-NAEEEvv) or South American (SIND-SAEEEvv) variant vaccine or sham immunized with saline. Animals were bled and antibody production (alpha-EEEv IgG) was determined. The animals were challenged with a North American origin EEE virus (FL-9393) by aerosol at a target lethal dose (1.0E+07 PFU inhaled). Animals receiving SIND-NAEEEvv variant were mostly protected (5/6 survival; 83%) whereas animals vaccinated with the South American variant (SIND-SAEEEvv) or sham-vaccinated (saline) died as a result of exposure (1/6; 16% and 0/4; 0% survival respectively). Telemetric monitoring of the animals post challenge showed the animals successfully vaccinated experienced minimal to no disruption in normal diurnal variation in core temperature or abnormalities in heart rate and ECG. The affected animals, alternatively, experienced significant changes (hyperthermia) in core temperature and disruption in ECG signal. Histopathological analysis of tissues, specifically the brain, indicated viral invasion and significant encephalitis. We are repeating this experiment in cynomolgus macaques to confirm the efficacy of SIND-EEEvv in this model.