High-resolution definition of B cell responses to HIV Env for immunogen design

描述（由申请方提供）：HIV-1疫苗开发的主要挑战之一是激发抗HIV Env的广泛中和抗体（bNAb）。最近从HIV感染的个体中分离的几种bNAb证明了人B细胞库可以产生靶向保守Env区的bNAb。然而，关于在慢性HIV-1感染期间如何引起广泛的应答，以及这些应答如何与Env疫苗接种引起的更有限的应答进行比较，仍然存在巨大的知识差距。为了填补这一信息空白，我们建议定义和改善中和抗体对HIV-1 Env复合物中功能上最保守和最容易获得的元素的诱导，即CD 4结合位点（CD 4 bs），这对病毒与受体CD 4的结合至关重要。以前，我们开发了一种HIV Env表位特异性B细胞分选和RT-PCR策略，以利用HIV感染个体的记忆B细胞区室，这导致了CD 4 bs bNA B，VRC 01的分离，其中和>90%的循环原发病毒。有趣的是，我们发现VRC 01和CD 4 i抗体亚群可以相互增强对gp 120的结合亲和力，这表明CD 4 i单克隆抗体可能与自然感染过程中VRC 01样bNAb的进化有关。我们采用了这种表位特异性记忆B细胞分选策略来扩展我们的研究，以获得对Env B细胞应答的了解，总结如下。(1)表征接种Env的非人灵长类动物（NHP）接种疫苗引发的中和抗体应答。我们将对Env特异性B细胞进行记忆B细胞FACS分选、克隆分析和IgG基因深度测序。(2)确定选定的HIV感染者中中和抗体反应的纵向演变。我们将研究与bNAb反应的因素，从一些高度选择的HIV-1感染者，通过分析他们的CD 4 b bNAb和他们的“互补”抗体，包括CD 4 i抗体。(3)通过检查i）Env-CD 4 i MA B复合物作为免疫原是否可以改善CD 4 bs bNAb的引发; ii）通过AAV载体平台的持久Env抗原暴露是否可以改善B细胞亲和力成熟来推进免疫原设计。我们将在小动物身上验证这些假设。这项研究的总体结果将有助于我们对HIV中和抗体反应的基本了解，并有助于开发安全和保护性的HIV疫苗。

DESCRIPTION (provided by applicant): One of the major challenges of HIV-1 vaccine development is the elicitation of broadly neutralizing antibodies (bNAbs) against HIV Env. The recent isolation of several bNAbs from HIV-infected individuals demonstrates that the human B cell repertoire can generate bNAbs targeting the conserved Env region. However, there is still a tremendous knowledge gap regarding how the broad responses are elicited during chronic HIV-1 infection and, additionally, how these compare to the much more limited responses elicited by Env vaccination. To fill this information gap, we propose to define and improve the elicitation of neutralizing antibodies toward the most functionally conserved and accessible element of the HIV-1 Env complex, namely the CD4 binding site (CD4bs), which is crucial for virus engagement with receptor CD4. Previously, we developed a multicolor Env epitope-specific B cell sorting and RT-PCR strategy to exploit the memory B cell compartment of HIV-infected individuals, which led to the isolation of CD4bs bNAb, VRC01, which neutralizes >90% of circulating primary viruses. Interestingly we find that VRC01 and CD4i antibody subsets can mutually enhance binding affinity for gp120, suggesting that CD4i MAbs may be associated with the evolution of VRC01-like bNAbs during the natural infection process. We adapted this epitope-specific memory B cell sorting strategy to extend our study to gain insight of Env B cell response, which is summarized as follows. (1) To characterize the neutralizing antibody response elicited by vaccination of Env-inoculated non-human primates (NHPs). We will perform memory B cell FACS sorting, clonal analysis and IgG gene deep sequencing of Env-specific B cells. (2) To determine the longitudinally evolving neutralizing antibody response in selected HIV-infected individuals. We will investigate factors associated with bNAb response from a few highly selected HIV-1-infected individuals by analyzing their CD4bs bNAbs and their "complementary" antibodies including CD4i antibodies. (3) To advance immunogen design by examining if i) Env-CD4i MAb complex as immunogen can improve the elicitation of CD4bs bNAbs; ii) long-lasting Env antigen exposure via AAV vector platform can improve the B cell affinity maturation. We will test these hypotheses in small animals. The overall outcome of this study will contribute to our basic understanding of HIV neutralizing antibody responses and the development of a safe and protective HIV vaccine.