High throughput NAPPA-proteoimmunomics and Vibrio cholerae vaccine development

描述（由申请人提供）： 需要一种系统来快速鉴定针对具有高度公共和国际卫生重要性的许多A、B和C类试剂的保护性免疫的标志物。此外，需要开发平台来快速评估针对这些病原体接种疫苗后免疫应答的广度和持续时间，将应答与发现具有保护性的应答相关联。霍乱弧菌是一种B类病原体，也是霍乱的病因，霍乱是一种导致世界范围内显著发病率和死亡率的秘书性腹泻。对霍乱保护性免疫的介质知之甚少，虽然已经开发了一些霍乱疫苗，但每种疫苗都有很大的局限性。我们建议使用以前创建的试剂（已经收集的临床标本和已经生产的霍乱弧菌NAPPA蛋白微阵列）进行高通量基于蛋白质组学的筛选，以确定针对霍乱的保护性免疫标记物，并比较与疫苗接种后诱导的保护相关的免疫应答。为此，我们建议马萨诸塞州总医院（MGH）、哈佛蛋白质组学研究所（HIP）和孟加拉国达卡国际腹泻病研究中心（ICDDR，B）的研究人员建立合作研究伙伴关系。具体目标#1。使用NAPPA筛查霍乱指数患者的家庭接触者的血清，根据霍乱保护水平对反应进行分层。纯化感兴趣的抗原，并确认免疫应答。具体目标#2使用NAPPA筛选霍乱指数患者的家庭接触者的记忆B细胞的淋巴细胞上清液中的抗体，根据对霍乱的保护水平对应答进行分层。纯化感兴趣的抗原，并确认免疫应答。具体目标#3使用NAPPA筛选接种灭活口服全细胞霍乱疫苗（WC-rBS）或口服减毒活霍乱疫苗秘鲁-15的疫苗血清，比较免疫蛋白质组学应答与霍乱保护相关应答。本项目的主要目的是鉴定和纯化霍乱弧菌免疫原，人体对该免疫原的免疫反应性与霍乱的保护作用相一致。这些免疫原随后将被评估是否包含在亚单位霍乱疫苗或加强剂中。

DESCRIPTION (provided by applicant): There is a need for systems to rapidly identify markers of protective immunity against a number of category A, B and C agents of high public and international health importance. In addition, there is a need for the development of platforms to rapidly evaluate the breadth and duration of immune responses following vaccination against such pathogens, correlating responses to those found to be protective. Vibrio cholerae is a category B agent, and the cause of cholera, a secretary diarrhea that results in significant morbidity and mortality worldwide. The mediators of protective immunity against cholera are poorly understood, and although a number of cholera vaccines have been developed, each has significant limitations. We propose to use previously created reagents (already-collected clinical specimens and already-produced V. cholerae NAPPA protein microarrays) to perform high throughput proteomics-based screening to identify markers of protective immunity against cholera, and to compare immune responses correlating with protection with those induced following vaccination. To do this, we propose a cooperative research partnership between researchers at the Massachusetts General Hospital (MGH), Harvard Institute of Proteomics (HIP), and International Centre for Diarrheal Disease Research in Dhaka, Bangladesh (ICDDR,B). Specific Aim #1. Use NAPPA to screen serum of household contacts of cholera index patients, stratifying responses by level of protection from cholera. Purify antigens of interest, and confirm immune responses. Specific Aim #2. Use NAPPA to screen antibody-in-lymphocyte supernatants of memory B cells of household contacts of cholera index patients, stratifying responses by level of protection from cholera. Purify antigens of interest, and confirm immune responses. Specific Aim #3. Use NAPPA to screen serum of vaccines who have received killed oral whole cell cholera vaccine (WC-rBS) or live oral attenuated cholera vaccine Peru-15, comparing immunoproteomic responses to those correlating with protection from cholera. The major objective of this project is to identify and purify V. cholerae immunogens against which immunoreactivity in humans corresponds with protection from cholera. These immunogens would subsequently be evaluated for inclusion in subunit cholera vaccines or boosters.