Vaccine targeting HIV sites of vulnerability

项目摘要/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RV144试验 显示出针对HIV包膜V1V2区域的高效(31%，p=0.04)和高水平抗体(Abs) (Env)被发现是病毒获得量减少的唯一主要免疫相关因素。中国疫苗的研究 此后，受到SIV或SIV挑战的猴子总结了这些发现。为了改进RV144疫苗， 我们设计了针对V1V2的候选疫苗，并确定了免疫原性最强的候选疫苗：V1V2/A244-2J9C， 将CRF_01的V1V2蛋白与A244菌株A244拼接，构建成细菌三聚体蛋白支架2J9C。带着我们的 独特的疫苗策略，以使用新的重组亚单位免疫原靶向V1V2为中心，我们已经 证明了在血浆和阴道分泌物中诱导以V1V2为重点的抗体反应的能力 并从Env上其他更具免疫优势的部位转移。诱导的抗体表现出与 来自多个分支的菌株，在上次增强后的持久性为1-2年，以及抗病毒功能，包括抗体- 依赖细胞吞噬作用(ADCP)、抗体依赖细胞毒性(ADCC)和补体 激活-用完整的gp120免疫不容易实现的活动。V1V2/A244-2J9C DNA已 也被证明是一个有效的启动，集中抗体反应在特定的V2区域，占 减少了RV144的感染。总而言之，这些数据提供了支持我们铅疫苗有效性的证据 候选人和疫苗接种方法。我们的V1V2脚手架设计已经获得了美国专利， 作为受让人之一。 本申请建议在cGMP(目前良好)下生产铅免疫原V1V2/A244-2J9C 生产实践)作为DNA质粒和蛋白质，并测试其优化递送，以便为 迈向人类第一阶段临床试验的道路。为了实现这一目标，提出了四个具体目标。目标1是 构建表达V1V2/A244-2J9C的DNA表达载体。在《目标2》中，我们 将测试编码cGMP级V1V2/A244-2J9C的DNA在瞬时转染的293T中产生蛋白质 细胞和对兔的免疫原性。体外蛋白质分析将包括表达效率、质量、 寡聚、糖基化、稳定性和与一组单克隆抗体(MAbs)的反应性。目标3是 利用该质粒生产HEK293T GNTI-/-细胞库和中试批次的V1V2/A244-2J9C蛋白 最后，Aim 4将测试V1V2/A244-2J9C蛋白的纯化方法并进行体外分析 用V1V2/A244-2J9C DNA和蛋白进行兔体内免疫原性试验。 CGMP的生产将由韦斯曼生物制造公司与加州大学合作完成 由卡尔·A·罗斯博士和布莱恩·M·达蒂罗博士监督。在体外免疫原分析中， 蛋白质提纯、兔子接种和免疫评估将在Dr。 Catarina Hioe(PI，James J.Peters VA医疗中心，JJP VAMC)与Susan Zolla-Pazner博士合作 (MSSM西奈山医学院) V1V2/A244-2J9C DNA和蛋白质免疫原将是此类免疫原中第一个进入临床的 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疫苗计划。一种艾滋病毒疫苗是 对于保护在国内外处于危险中的退伍军人来说，这是无价的。这种疫苗可以作为一种 针对其他疾病的疫苗原型，如COVID19，其中针对特定表位的集中抗体反应 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.