Contribution of VH Replacement to B Cell Anti-HIV Immune Response

描述(申请人提供)：中和抗体是针对细菌或病毒感染(包括艾滋病毒)的体液免疫反应的重要效应器。然而，在自然感染中或通过常规疫苗方法很难诱导针对HIV的有效中和抗体反应。对HIV病毒基因组和包膜蛋白结构的分析表明，HIV已经进化出许多策略来避免中和抗体反应。目前，已经有几组已建立的HIV中和抗体，它们识别gp41的CD4结合部位、CD4诱导的(CD4i)共受体结合部位、gp120 V3环或膜近端外区(MPER)。对这些抗体的研究提供了关于中和抗体与艾滋病毒相互作用的结构要求的宝贵信息。然而，对于这些中和抗体是如何产生的，目前还缺乏全面的分析。CD4i和MPER抗体具有带电氨基酸的长IGH CDR3区，这是自身反应性抗体的共同特征。事实上，两种有效的HIV中和MPER抗体2F5和4E10也与心磷脂和其他自身抗原发生反应。据推测，HIV中和抗体是通过罕见的重组事件产生的，表达这种抗体的B细胞被负选择。我们最近的研究结果表明，在编码抗HIV抗体的IgH基因中，VH替换产物的频率显著增加，包括抗gp41、抗gp120、抗V3环和CD4i抗体。特别是，编码CD4i抗体的12个IgH基因中有9个可能是通过VH替换产生的。VH替换通过RAG介导的二级重组发生，涉及重排的VH基因内的隐蔽RSS和上游VH基因的23bpRSS。VH替换更新了几乎整个VH编码区，但保留了一小段核苷酸作为“足迹”，优先编码带电氨基酸来延长IgH CDR3。几乎所有CD4i抗体中的VH替换“足迹”都将带负电荷的氨基酸贡献到CDR3区域，这是结合gp120的关键。基于这些结果，我们假设VH替代对B细胞抗HIV免疫反应有贡献。为了验证这一假设，(1)我们将进行单细胞PCR分析，以确定VH替换产品在HIV患者血浆细胞中的频率并表达重组抗体，以确定这些已确定的VH替换产品是否编码抗HIV或HIV中和抗体；(2)我们将使用实验性的VH替换系统来生成限制VH1-69基因使用的VH替换产品，并确定人工生成的VH替换产品是否编码抗HIV抗体。这些研究的结果将提供有关VH替代对B细胞抗HIV免疫反应的贡献的直接信息。目前的建议是基于我们最近的发现，即很大一部分抗HIV抗体是通过VH替换重组产生的。我们将使用先进的单细胞PCR方法来检测HIV患者血浆细胞中VH替换产物的频率，并表达重组抗体，以确定VH替换产品是否编码抗HIV抗体。了解VH替换对B细胞抗HIV应答的贡献对抗击HIV感染具有重要意义。

DESCRIPTION (provided by applicant): Neutralizing antibodies are important effectors in humoral immune responses against bacterial or viral infections, including HIV. However, it is difficult to elicit effective neutralizing antibody responses against HIV in natural infections or by conventional vaccine approaches. Analyses the HIV viral genome and envelop protein structures have suggested that HIV has evolved many strategies to avoid neutralizing antibody responses. Currently, there are several groups of established monoclonal HIV neutralizing antibodies, which recognize the CD4 binding site, CD4-induced (CD4i) co-receptor binding site, gp120 V3 loop, or the membrane proximal external region (MPER) of gp41. Studies of these antibodies provided valuable information regarding the structural requirements for neutralizing antibodies to interact with HIV. However, a comprehensive analysis of how these neutralizing antibodies are generated is still lacking. The CD4i and the MPER antibodies have long IgH CDR3 regions with charged amino acids, which are common features of autoreactive antibodies. Indeed, two of the potent HIV neutralizing MPER antibodies 2F5 and 4E10 also react with cardiolipin and other self antigens. It has been speculated that HIV neutralizing antibodies are generated through rare recombination events and B cells expressing such antibodies have been negatively selected. Our recent results showed that the frequencies of VH replacement products are significantly elevated in IgH genes encoding anti-HIV antibodies, including anti-gp41, anti-gp120, anti-V3 loop, and CD4i antibodies. Particularly, nine of the 12 IgH genes encoding CD4i antibodies are potentially generated through VH replacement. VH replacement occurs through RAG-mediated secondary recombination involving a cryptic RSS within a rearranged VH gene and a 23 bp RSS from an upstream VH gene. VH replacement renews almost the entire VH coding region but retains a short stretch of nucleotides as a "footprint", which preferentially encodes charged amino acids to extend IgH CDR3. Almost all the VH replacement "footprints" in the CD4i antibodies contribute negatively charged amino acids into the CDR3 regions, which are critical for binding gp120. Based on these results, we hypothesize that VH replacement contributes to the B cell anti-HIV immune response. To test this hypothesis, (1) we will perform single cell PCR analysis to determine the frequencies of VH replacement products in the plasma cells of HIV patients and express recombinant antibodies to determine if these identified VH replacement products encode anti-HIV or HIV neutralizing antibodies; (2) we will use experimental VH replacement systems to generate VH replacement products with restricted usage of VH1-69 gene and determine if the artificially generated VH replacement products encode anti-HIV antibodies. Results from these studies will provide direct information regarding the contribution of VH replacement to B cell anti-HIV immune response. The current proposal is based on our recent finding that a large fraction of anti-HIV antibodies is generated through VH replacement recombination. We will use an advanced single cell PCR approach to determine the frequencies of VH replacement products in the plasma cells of HIV patients and express recombinant antibodies to determine if VH replacement products encode anti-HIV antibodies. Understanding the contribution of VH replacement to B cell anti-HIV response will have significant implications for combating HIV infection.