The role of steady state broadly neutralizing antibody tissue levels in preventing distal site SHIV replication

项目摘要 尽管在HIV-1疫苗开发方面做出了巨大努力，但仍然缺乏能够引起广泛中和的疫苗 抗体（bNAb）足以阻止HIV感染。由于成功的被动输注bNAb， SHIV挑战恒河猴阻断全身感染，去年秋天启动了一项新的试验， 风险个体接受bNAb，VRC 01的IV输注。然而，仍有许多问题， 这些特殊的抗体是如何提供杀菌免疫的在Barouch实验室最近的一项研究中， 显示在给药前接受约1天广泛中和抗体PGT 121 IV输注的恒河猴， 用SHIV-SF 162 P3阴道内攻击的小鼠在攻击后1-3天具有远端位点病毒积累。在 此外，他们还发现病毒DNA与病毒RNA的比例较高，这意味着在病毒感染后， 尽管在1天前已经给予了广泛中和抗体，但这些远端部位仍然存在。这说明 曾经被认为是杀菌免疫的东西不能保护FRT和远端的早期感染， 尽管在10天内清除了病毒。这个模型提供了一个很好的机会来了解如何 抗体清除FRT和远端部位急性感染后的病毒。在使用cy 5的初步数据中- 标记的VRC 01 IV注射到恒河猴中，我发现抗体需要大约1周才能 在解剖学上分布以在组织中达到稳态水平，而血浆水平更早达到峰值。 利用这种新开发的标记抗体平台，我在目标1中提出用cy 5标记PGT 121， 比较阴道内给药前-7天IV输注（稳态）和-1天IV输注（非稳态） 在恒河猴中用SHIVSF 162 P3攻击并测量雌性生殖道（FRT）和远端 攻毒后48小时和1周的病毒部位蓄积。在目标2中，我将测量抗体水平 在这些远端组织以及FRT中，并将其与病毒蓄积进行比较。第二部分 为了达到这个目的，我们将研究具有病毒积累的组织的转录组学特征， 以确定病毒传播的潜在途径。 最后，在目标3中，我将利用Hope实验室的一个新平台，称为巫妖报告系统， 可以识别早期传播的感染灶。我将用SIV病毒共同挑战恒河猴 假型巫妖和SHIVSF 162 P3，以阐明稳态抗体分布的作用 对感染病灶的数量以及病毒在整个 整个FRT。在目标3的第二部分中，我们将缩小这些感染灶的范围并描述潜在的特征 免疫清除机制，如ADCC，由cy 5标记的PGT 121鉴定。突出的目标 在本申请中概述的方法将提供对稳态抗体组织分布的作用的重要见解， bNAbs提供的杀菌保护。

Project Summary Despite great efforts in HIV-1 vaccine development, a vaccine is still lacking that can elicit broadly neutralizing antibodies (bNAb) sufficient enough to block HIV infection. Due to the success of passive infusion of bNAbs in SHIV challenged rhesus macaques to block systemic infection, a new trial was launched this past fall where at risk individuals received an IV infusion of the bNAb, VRC01. However, there are still many questions as to how these specialized antibodies can provide sterilizing immunity. In a recent study by the Barouch lab, it was shown that rhesus macaques that got a -1 day IV infusion of the broadly neutralizing antibody PGT121, prior to intravaginal challenge with SHIV-SF162P3, had distal site virus accumulation 1-3 days after challenge. In addition, they found a higher ratio of viral DNA to viral RNA, which implies that there was active replication at these distal sites despite having been given a broadly neutralizing antibody 1 day before. This shows that what was once thought of as sterilizing immunity does not protect from early infection in the FRT and at distal sites, despite clearing the virus at 10 days. This model provides an excellent opportunity to understand how antibodies clear the virus following acute infection in the FRT and at distal sites. In preliminary data using cy5- labeled VRC01 IV injected into rhesus macaques I have found that it takes antibodies about 1 week to anatomically distribute to achieve steady state levels in the tissue, whereas plasma levels peak much earlier. Utilizing this newly developed labeled antibody platform, I propose in Aim 1 to label PGT121 with cy5 and compare -7 day IV infusion (steady state) and -1 day IV infusion (non-steady state) prior to intravaginal challenge with SHIVSF162P3 in the rhesus macaque and measure female reproductive tract (FRT) and distal site accumulation of virus at 48 hours and 1 week after challenge. In Aim 2, I will measure the antibody levels at these distal tissues as well as in the FRT and compare this with virus accumulation. In the second part of this aim, we will look at the transcriptomic profile of tissues that have virus accumulation compared to those that do not, with and with antibody present in an effort to identify a potential path of virus dissemination. Finally, in Aim 3 I will utilize a novel platform from the Hope lab known as the LICh reporter system, which allows identification of early transmitted foci of infection. I will co-challenge rhesus macaques with SIV pseudotyped LICh and SHIVSF162P3 and in order to elucidate the role of steady state antibody distribution has on the number of foci of infection as well as the extent to which the virus disseminates throughout the entire FRT. In the second part of Aim 3, we will narrow in on these foci of infection and characterize potential immune clearance mechanisms, such as ADCC, identified by the cy5-labeled PGT121. The highlighted aims outlined in this application will provide crucial insight into the role steady state antibody tissue distribution has on the sterilizing protection afforded by bNAbs.