Vaccine targeting HIV sites of vulnerability

项目概要/摘要 目前还没有有效的艾滋病毒疫苗。在2b/3期疫苗试验中，只有泰国RV 144试验 显示出有效性（31%，p=0.04），以及针对HIV包膜V1 V2结构域的高水平抗体（Ab） (Env)被发现是减少病毒获得的唯一主要免疫相关性。疫苗研究 SIV或SHIV攻击的猴子已经概括了这些发现。为了改进RV 144疫苗， 我们设计了靶向V1 V2的候选疫苗并鉴定了最具免疫原性的：V1 V2/A244- 2 J 9 C， 将CRF_01.AE菌株A244的V1 V2剪接到细菌三聚体蛋白支架中的蛋白，2 J 9 C。与我们 独特的疫苗策略集中于使用新型重组亚单位免疫原靶向V1 V2，我们已经 证明了在血浆和阴道分泌物中诱导Ab应答的能力，其集中于V1 V2 并从Env上的其他免疫显性位点转移。所引发的Ab显示与以下的交叉反应性： 来自多个进化枝的菌株，最后一次加强后1-2年的持久性，以及抗病毒功能，包括Ab- 依赖性细胞吞噬作用（ADCP）、Ab依赖性细胞毒性（ADCC）和补体 活化-用完整的GP 120免疫不容易获得的活性。V1 V2/A244- 2 J 9 C DNA具有 也被证明是一种有效的引物，将Ab反应集中在特定的V2区域， 减少RV 144的感染。总之，这些数据提供了支持我们的先导疫苗有效性的证据 候选人和疫苗接种方法。我们的V1 V2支架设计已获得美国专利，VA 作为受让人之一。 本申请提出在cGMP（现行良好）下生产先导免疫原V1 V2/A244- 2 J 9 C 生产实践）作为DNA质粒和蛋白质，并测试其优化的交付，以铺平道路 人类I期临床试验的进展为实现这一目标，提出了四个具体目标。目标1： 生成E的主库。用V1 V2/A244- 2 J 9 C表达质粒转化大肠杆菌。在目标2中， 将在瞬时转染的293 T细胞中检测cGMP级V1 V2/A244- 2 J 9 C编码DNA的蛋白质产生 细胞和兔的免疫原性。体外蛋白质分析将包括表达效率、质量， 在一个实施方案中，所述方法包括寡聚化、糖基化、稳定性和与一组单克隆抗体（mAb）的反应性。目标3是 使用质粒生产HEK 293 T GnTi-/-细胞的主库和V1 V2/A244- 2 J 9 C蛋白的中试批次 目标1最后，目的4将测试V1 V2/A244- 2 J 9 C蛋白的纯化方法并进行体外分析 然后用V1 V2/A244- 2 J 9 C DNA和蛋白进行兔体内免疫原性试验。 cGMP的生产将由Waisman BioManufacturing公司完成，该公司与University of 威斯康星-麦迪逊，在卡尔A。作者声明：Brian M.达蒂洛体外免疫原分析， 蛋白纯化、兔接种和免疫评估将在Dr. 卡塔里纳Hioe（PI，James J. Peters VA Medical Center，JJP VAMC）与Susan Zolla-Pazner博士合作 （西奈山医学院，MSSM）。 V1 V2/A244- 2 J 9 C DNA和蛋白质免疫原将是第一个走向临床的同类免疫原。 审判预防艾滋病毒感染和/或疾病的有效疫苗是国家战略的重要组成部分， 卫生与公众服务部和退伍军人事务部的疫苗计划。艾滋病毒疫苗是 保护在国内外处于危险之中的退伍军人是非常宝贵的。这种疫苗也可以作为一种 针对其他疾病的疫苗原型，如COVID 19，其中针对特定表位的集中抗体反应 这是保护所必需的。

Project Summary/Abstract Effective HIV vaccines are not yet available. Of the phase 2b/3 vaccine trials, only the Thai RV144 trial showed efficacy (31%, p=0.04), and high levels of antibodies (Abs) against the V1V2 domain of HIV envelope (Env) were found to be the only primary immune correlate of reduced virus acquisition. Studies of vaccines in SIV or SHIV-challenged monkeys have since recapitulated these findings. To improve upon the RV144 vaccine, we have designed V1V2-targeted vaccine candidates and identified the most immunogenic: V1V2/A244-2J9C, a protein with V1V2 of CRF_01.AE strain A244 spliced into a bacterial trimeric protein scaffold, 2J9C. With our unique vaccine strategy centered on targeting V1V2 using novel recombinant subunit immunogens, we have demonstrated the capacity to induce an Ab response in plasma and vaginal secretion that was focused on V1V2 and diverted from other more immunodominant sites on Env. The elicited Abs displayed cross-reactivity with strains from multiple clades, durability of 1-2 years after the last boost, and antiviral functions including Ab- dependent cellular phagocytosis (ADCP), Ab-dependent cellular cytotoxicity (ADCC), and complement activation—activities that are not readily achieved by immunization with intact gp120. V1V2/A244-2J9C DNA has also been shown as an effective prime, focusing the Ab response on the specific V2 region that accounted for reduced infection in RV144. Altogether these data provide evidence supporting the validation of our lead vaccine candidate and vaccination approach. A US patent has been issued for our V1V2-scaffold designs, with the VA as one of the assignees. This application proposes to produce the lead immunogen V1V2/A244-2J9C under cGMP (current Good Manufacturing Practice) as both DNA plasmid and protein, and test its optimized delivery in order to pave the way toward a human phase I clinical trial. To accomplish this goal, four specific aims are proposed. Aim 1 is to generate a master bank of E. coli transformed with the V1V2/A244-2J9C-expressing DNA plasmid. In Aim 2 we will test the cGMP-grade V1V2/A244-2J9C-encoding DNA for protein production in transiently transfected 293T cells and for immunogenicity in rabbits. In vitro protein analysis will include expression efficiency, mass, oligomerization, glycosylation, stability, and reactivity with a panel of monoclonals Abs (mAbs). Aim 3 is to produce a master bank of HEK293T GnTi-/- cells and a pilot batch of V1V2/A244-2J9C protein using the plasmid from Aim 1. Finally, Aim 4 will test purification methods and conduct in vitro analysis for V1V2/A244-2J9C protein from Aim 3 and then perform in vivo rabbit immunogenicity testing with V1V2/A244-2J9C DNA and protein. The cGMP production will be done by Waisman BioManufacturing, associated with the University of Wisconsin-Madison, under the supervision of Drs. Carl A. Ross and Brian M. Dattilo. In vitro immunogen analysis, protein purification, rabbit vaccination, and immune assessment will be performed in the laboratory of Dr. Catarina Hioe (PI, James J. Peters VA Medical Center, JJP VAMC) in collaboration with Dr. Susan Zolla-Pazner (Mount Sinai School of Medicine, MSSM). The V1V2/A244-2J9C DNA and protein immunogens will be the first of their kind to move toward clinical trials. An effective vaccine to prevent HIV infection and/or disease is an essential portion of the Strategic National Vaccine Plan of the Departments of Health and Human Services and Veterans Affairs. An HIV vaccine is invaluable to protect Veterans who are at risk at home and abroad. This vaccine could as well serve as a prototype for vaccines against other diseases, like COVID19, where a focused Ab response to specific epitopes is requisite for protection.