Neutralization of Eastern equine encephalitis virus by human monoclonal antibodies

项目摘要/摘要 这个项目的总体目标是解决人类利用的中和的分子和结构基础。 抗东部马脑炎病毒(EEEV)的单抗。北美血统 乙型脑炎病毒(NA-EEEV)是脑炎甲型病毒中毒力最强的，其中70%为人类，90%为人类 马的死亡率。此外，在那些在感染中幸存下来的人中，高达80%的人显示出神经症状 恶化。美国平均每年报告8例人类感染病例。然而，最近的涨势 在以人为食的蚊子物种中检测到NA-EEEV引起了人们对东部大爆发的担忧 我们。此外，NA-EEEV被认为是NIH B类优先病原体和USDA/CDC选择剂 它作为生物恐怖主义毒剂的潜在威胁。没有批准的抗病毒药物或获得许可的人类 疫苗可用于NA-EEEV。对甲型病毒的抗体反应已被证明是一种重要的 免疫反应的一部分，在授予保护性免疫和帮助清除和恢复 感染。然而，人类中抗体作用的基本分子和结构机制 NA-EEEV的定义仍然不明确。为了填补这一知识空白，本研究的目标是确定 人源单抗中和NA-EEEV的机制。这项研究的首要假设是 人中和单抗的主要作用方式是稳定病毒颗粒，抑制病毒融合 宿主细胞。为了验证这一假设，我将从自然感染NA-EEEV的幸存者中分离出人类单抗，以 BSL-2嵌合病毒Sindbis/EEEV(SINV/NA-EEEV)和重组NA-EEEV结构蛋白。这就做 鉴定分离的人源单抗的特异性反应性和中和效力 SINV/NA-EEEV。在中和NA-EEEV的人源单抗中，我将确定步骤(S) 复制周期单抗通过体外机制分析中和病毒。识别中和 抗原决定簇，我将使用表位映射、丙氨酸扫描突变和结构生物学 技巧。人NA-EEEV单抗识别的中和抗原决定簇的测定 将告知单抗如何与NA-EEEV相互作用。这一项目取得的成果将填补在以下方面的重大空白 了解人类对NA-EEEV的抗体反应，将确定潜在的保护相关因素，以及 可能有助于设计有效的治疗方法和疫苗来对抗这种临床相关的甲型病毒。这个 建议书概述了一项全面的团契培训计划，将为我在 病毒免疫学领域。为了完成这一计划和研究，我们组建了一个协作团队 由主要研究人员和在免疫学、病毒学、 和结构生物学领域为本研究提供支持和指导。

PROJECT SUMMARY/ABSTRACT The overall goal of this project addresses the molecular and structural basis of neutralization utilized by human monoclonal antibodies (mAbs) against Eastern equine encephalitis virus (EEEV). The North American lineage of EEEV (NA-EEEV) is the most virulent of the encephalitic alphaviruses with up to 70% human and 90% equine mortality rate. In addition, of those that survive infection, up to 80% show signs of neurological deterioration. On average, eight human cases a year in the US are reported. However, the recent rise in detection of NA-EEEV in human-feeding mosquito species raises concern for a large outbreak in the eastern US. Moreover, NA-EEEV is considered a NIH Category B priority pathogen and USDA/CDC Select Agent due to its potential threat as a bioterrorism agent. There are no approved antiviral drugs or licensed human vaccines available for NA-EEEV. The antibody response to alphaviruses has been shown to be an important part of the immune response in conferring protective immunity and aiding in the clearance and recovery from infection. However, the fundamental molecular and structural mechanisms of action of antibodies in humans to NA-EEEV remain poorly defined. To fill this gap in knowledge, the objective of this study is to determine the mechanisms by which human mAbs neutralize NA-EEEV. The overarching hypothesis of this study is that the principle mode of action of human neutralizing mAbs is to stabilize the virus particle and inhibit viral fusion to host cells. To test this hypothesis, I will isolate human mAbs from naturally infected survivors of NA-EEEV to the BSL-2 chimeric virus, Sindbis/EEEV (SINV/NA-EEEV) and recombinant NA-EEEV structural proteins. I will characterize the panel of human mAbs isolated for specific reactivity and neutralization potency against SINV/NA-EEEV. Of the human mAbs that neutralize NA-EEEV, I then will determine the step(s) in the replication cycle the mAbs neutralize the virus through in vitro mechanistic assays. To identify neutralizing antigenic determinants, I will use epitope mapping, alanine scanning mutagenesis, and structural biology techniques. Determination of the neutralizing antigenic determinants recognized by human NA-EEEV mAbs will inform how mAbs interact with NA-EEEV. The results obtained from this project will fill major gaps in knowledge about the human antibody response to NA-EEEV, will identify potential correlates of protection, and may facilitate the design of efficient therapeutics and vaccines against this clinically relevant alphavirus. The proposal outlines a comprehensive fellowship training plan that will prepare me for a high-impact career in the field of viral immunology. To accomplish this program and research, we have assembled a collaborative team consisting of principal investigators and staff scientists with exceptional expertise in the immunology, virology, and structural biology fields to provide support and guidance for this study.