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

描述（由申请人提供）：HIV-1疫苗开发的主要挑战之一是激发针对HIV Env的广泛中和抗体（bNAbs）。最近从hiv感染者中分离出的几种bNAbs表明，人类B细胞库可以产生针对保守Env区域的bNAbs。然而，关于如何在慢性HIV-1感染期间引起广泛的反应，以及如何将这些反应与Env疫苗引起的更有限的反应进行比较，仍然存在巨大的知识差距。为了填补这一信息空白，我们建议定义和改进针对HIV-1 Env复合体中功能最保守和可接近的元件的中和抗体的激发，即CD4结合位点（CD4bs），这对于病毒与受体CD4结合至关重要。之前，我们开发了一种多色Env表位特异性B细胞分选和RT-PCR策略来利用hiv感染者的记忆B细胞区室，从而分离出CD4bs bNAb， VRC01，它可以中和90%的循环原代病毒。有趣的是，我们发现VRC01和CD4i抗体亚群可以相互增强对gp120的结合亲和力，这表明在自然感染过程中，CD4i单克隆抗体可能与VRC01样bNAbs的进化有关。我们采用这种表位特异性记忆B细胞分选策略来扩展我们的研究，以深入了解Env B细胞的反应，总结如下。(1)研究接种env疫苗的非人灵长类动物（NHPs）所引起的中和抗体反应。我们将对env特异性B细胞进行记忆性B细胞FACS分选、克隆分析和IgG基因深度测序。(2)在选定的hiv感染者中确定纵向进化的中和抗体反应。我们将通过分析一些高度选定的hiv -1感染者的CD4bs bNAbs及其“互补”抗体（包括CD4i抗体）来研究与bNAb反应相关的因素。(3)通过检测Env-CD4i MAb复合物作为免疫原是否能促进CD4bs bNAbs的激发，进一步推进免疫原设计；ii)通过AAV载体平台长期暴露Env抗原可促进B细胞亲和成熟。我们将在小动物身上测试这些假设。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理解和开发一种安全和保护性的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.