Virus and Antibody Gene Sequencing Core

项目总结 HIV-1感染者广谱交叉反应中和抗体的研究进展 需要反复循环包膜(Env)介导的B细胞受体刺激，菌株特异性(自体) 中和抗体(NAB)的激发和相关病毒的逃逸。在这场病毒/抗体“军备竞赛”中， 产生了特定的环境逃逸突变体，在存在于 受感染的个体，能够选择获得中和广度的抗体谱系。尽管 病毒/抗体协同进化的原理已经建立，数量、身份、继承和 启动和维持bNAb谱系成熟的特定环境变异体的合作尚未得到 下定决心。在一个个体中观察到的bNAb诱导途径在多大程度上也不清楚。 推广到其他人。为了解决这些问题，HIVRAD团队将测试以下科学假设 猴-人类免疫缺陷病毒(SHIV)携带HIV-1包膜蛋白，结合V1V2和V3-糖蛋白bNAb 人类未突变的共同祖先(UCA)和中间祖先(IA)B细胞受体(BCR)将结合 猕猴中相关的BCR，导致了病毒/抗体共同进化和 NAB广度和效力的发展。我们已经克服了实现这些目标的一个关键障碍 目的通过生成包含SHIV的初级和传播型方正(Tf)环境，复制 到猕猴体内的高滴度，以及早期环境进化和自体NAB逃逸的产生模式 这些病毒与人类感染HIV-1病毒株编码相同的env病毒的基因惊人地相似。 这些数据表明，B细胞对共同的env抗原的反应可能，至少对某些env来说是 通过比较近交系猕猴和近交系猕猴中病毒/抗体协同进化的模式 人类感染相同的环境表达病毒，将有可能确定共同的途径 病毒/bcr接触和bNAb开发，可应用于疫苗设计。病毒和 抗体基因测序核心(核心B)将通过提供最先进的测序来支持这一项目 将允许这个HIVRAD财团剖析SHIV引发保护性体液的机制的服务 豁免权。与生物信息学和统计核心(核心C)以及项目1、2和#密切合作 #3，我们将(I)描述受新城疫病毒感染的猕猴随着时间的推移而进化的环境拟物种，以及(Ii)使用 Sanger和NextGen测序(NGS)方法测定抗体重链和轻链 SHIV感染前后克隆性B细胞培养和恒河猴记忆B细胞的可变区 免疫接种。其目标是了解进化中的环境准物种和 NAB和bNAb谱系成员识别可重复启动和驱动的关键环境逃逸突变 BNab成熟。

PROJECT SUMMARY The development of broadly cross-reactive neutralizing antibodies (bNAbs) in HIV-1 infected individuals requires iterative rounds of envelope (Env)-mediated B cell receptor stimulation, strain specific (autologous) neutralizing antibody (NAb) elicitation, and associated virus escape. In this virus/antibody “arms race”, particular Env escape mutants are generated which, among the myriads of other Env variants present in infected individuals, are able to select for antibody lineages that acquire neutralization breadth. Although the principle of virus/antibody co-evolution has been established, the number, identity, succession, and cooperation of the particular Env variants that initiate and sustain bNAb lineage maturation have not been determined. It is also unclear to what extent the pathways of bNAb induction observed in one individual can be generalized to others. To address these questions, this HIVRAD team will test the scientific premise that simian-human immunodeficiency viruses (SHIVs) bearing HIV-1 Envs that engage V1V2 and V3-glycan bNAb unmutated common ancestor (UCA) and intermediate ancestor (IA) B cell receptors (BCR) in humans will bind related BCRs in rhesus macaques, leading to a recapitulation of virus/antibody coevolution and the development of NAb breadth and potency. We have already overcome a critical hurdle toward these objectives by generating primary and transmitted founder (TF) Env containing SHIVs, which replicate to high titers in rhesus macaques and yield patterns of early env evolution and autologous NAb escape that are astonishingly similar to those of humans infected with HIV-1 strains encoding the same Envs. These data suggest that B cell responses to a common Env antigen may, at least for some Envs, be deterministic and that by comparing the patterns of virus/antibody coevolution in outbred macaques and humans infected with the same Env expressing virus, it will be possible to identify common pathways of virus/BCR engagement and bNAb development that can be applied to vaccine design. The Virus and Antibody Gene Sequencing Core (Core B) will support this project by providing state-of-the-art sequencing services that will allow this HIVRAD consortium to dissect the mechanisms of SHIV elicited protective humoral immunity. Working closely with the Bioinformatics and Statistics Core (Core C) as well as Projects #1, #2 and #3, we will (i) characterize the evolving env quasispecies in SHIV infected macaques over time, and (ii) use Sanger and nextgen sequencing (NGS) methods to sequence antibody heavy (VHDJH) and light (VLJL) chain variable regions from clonal B cell cultures and rhesus memory B cells before and after SHIV infection and/or immunization. The goal is to understand the dynamic interactions between the evolving Env quasispecies and members of NAb and bNAb lineages to identify key Env escape mutations that reproducibly initiate and drive bNAb maturation.