Virus and Antibody Gene Sequencing Core

项目摘要 HIV-1感染者中广泛交叉反应中和抗体（bNAb）的发展 需要反复多轮包膜（Env）介导的B细胞受体刺激，菌株特异性（自体） 中和抗体（NAb）诱发和相关病毒逃逸。在这场病毒/抗体“军备竞赛”中， 产生了特定的Env逃逸突变体，在存在于细胞中的无数其他Env变体中， 感染的个体能够选择获得中和宽度的抗体谱系。虽然 建立了病毒/抗体协同进化的原理，从数量、同一性、继承性和 启动和维持bNAb谱系成熟的特定Env变体的合作尚未被证实。 测定也不清楚在一个个体中观察到的bNAb诱导途径在多大程度上可以被 推广到其他人。为了解决这些问题，这个HIVRAD团队将测试科学前提， 携带HIV-1 Env的猴-人免疫缺陷病毒（SHIV），其接合V1 V2和V3-聚糖bNAb 人类中未突变的共同祖先（UCA）和中间祖先（IA）B细胞受体（BCR）将结合 相关的BCRs在恒河猴，导致重演病毒/抗体的共同进化和 发展NAb的广度和效力。我们已经克服了一个关键的障碍， 通过产生含有SHIV的原发性和传播的创始人（TF）Env， 在恒河猴中达到高滴度，并产生早期env演变和自体NAb逃逸的模式 与感染了编码相同Envs的HIV-1病毒株的人的病毒极其相似。 这些数据表明，B细胞对共同Env抗原的应答，至少对某些Env，可能是 确定性，并通过比较病毒/抗体协同进化的模式，在远交猕猴和 感染相同Env表达病毒的人，将有可能鉴定出 病毒/BCR接合和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技术来支持该项目 服务，这将使这个HIVRAD财团解剖机制的SHIV引起保护性体液免疫， 免疫力与生物信息学和统计学核心（核心C）以及项目#1，#2和#3密切合作。 #3，我们将（i）表征SHIV感染猕猴中随时间推移的进化env准种，以及（ii）使用 对抗体重链（VHDJH）和轻链（VLJL）进行测序的桑格和nextgen测序（NGS）方法 来自SHIV感染前后的克隆B细胞培养物和恒河猴记忆B细胞的可变区，和/或 次免疫我们的目标是了解进化中的Env准种之间的动态相互作用， NAb和bNAb谱系的成员，以鉴定可重复地启动和驱动基因突变的关键Env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.