Neutralization of Eastern equine encephalitis virus by human monoclonal antibodies

人单克隆抗体中和东方马脑炎病毒

• 批准号: 9757522
• 负责人: Lauren Williamson
• 金额: $ 2.97万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9757522
• 关键词: Address; Alanine; Alphavirus; American; Antibodies; Antibody Repertoire; Antibody Response; Antibody Specificity; Antigens; Antiviral Agents; Binding; Biological Assay; Bioterrorism; Capsid Proteins; Category B pathogen; Cells; Centers for Disease Control and Prevention (U.S.); Complex; Culicidae; Detection; Deterioration; Disease Outbreaks; Eastern Equine Encephalitis Virus; Electron Microscopy; Epitope Mapping; Epitopes; Equus caballus; Fellowship; Glycoproteins; Goals; Heterodimerization; Human; Human papillomavirus 16 E1 protein; Hybridomas; Immune; Immune response; Immune system; Immunity; Immunology; In Vitro; Infection; Knowledge; Laboratories; Libraries; Membrane; Molecular Conformation; Molecular Structure; Monoclonal Antibodies; Mus; Mutagenesis; Neurologic Signs; Neutralization Tests; Plaque Assay; Play; Prevention; Principal Investigator; Quantitative Reverse Transcriptase PCR; Recombinants; Recovery; Reporting; Research; Resolution; Role; Scanning; Scientist; Structural Protein; Structure; Surface; Survivors; Techniques; Technology; Testing; Therapeutic; Training; Transmembrane Domain; United States National Institutes of Health; Vaccines; Viral; Virion; Virulent; Virus; Virus Diseases; Virus Replication; X-Ray Crystallography; career; clinically relevant; clinically significant; crosslink; design; experience; feeding; human monoclonal antibodies; human mortality; insight; mortality; murine antibody; neutralizing antibody; neutralizing monoclonal antibodies; particle; prevent; programs; protein structure; prototype; receptor; stoichiometry; structural biology; virology

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.

项目总结/摘要 该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）的单克隆抗体（mAb）。北美血统 EEEV的（NA-EEEV）是脑炎甲病毒中毒性最强的，高达70%的人和90%的人 马的死亡率此外，在感染后存活的人中，高达80%的人表现出神经系统疾病的迹象。 恶化美国平均每年报告8例人类病例。然而，最近， 在人类喂养的蚊子物种中检测到NA-EEEV引起了对东部大规模爆发的关注 我们此外，NA-EEEV被认为是NIH B类优先病原体和USDA/CDC选择剂， 它作为生物恐怖分子的潜在威胁目前还没有批准的抗病毒药物或许可的人类 可用于NA-EEEV的疫苗。对甲病毒的抗体反应已被证明是一个重要的 免疫反应的一部分，赋予保护性免疫力，并帮助清除和恢复， 感染然而，抗体在人体中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，本研究的目的是确定 人mAb中和NA-EEEV的机制。这项研究的首要假设是， 人中和mAb的主要作用模式是稳定病毒颗粒并抑制病毒融合， 宿主细胞为了验证这一假设，我将从自然感染的NA-EEEV幸存者中分离人单克隆抗体， BSL-2嵌合病毒、Sindbis/EEEV（SINV/NA-EEEV）和重组NA-EEEV结构蛋白。我会 表征分离的人mAb组的特异性反应性和中和效价， SINV/NA-EEEV。在中和NA-EEEV的人mAb中，然后我将确定制备中的步骤。 在复制周期中，mAb通过体外机制测定中和病毒。识别中和 抗原决定簇，我将使用表位作图，丙氨酸扫描诱变，和结构生物学 技术.人NA-EEEV mAb识别的中和抗原决定簇的测定 将告知mAb如何与NA-EEEV相互作用。该项目取得的成果将填补以下方面的主要空白： 关于NA-EEEV的人抗体应答的知识，将确定保护的潜在相关性， 可以促进针对这种临床相关甲病毒的有效治疗剂和疫苗的设计。的 建议概述了一个全面的奖学金培训计划，这将使我准备在一个高影响力的职业生涯， 病毒免疫学领域。为了完成这项计划和研究，我们组建了一个合作团队， 由在免疫学，病毒学， 和结构生物学领域的研究成果，为本研究提供支持和指导。