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

描述（由申请方提供）：中和抗体是针对细菌或病毒感染（包括HIV）的体液免疫应答中的重要效应物。然而，在自然感染中或通过常规疫苗方法很难引起针对HIV的有效中和抗体应答。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，有几组已建立的单克隆HIV中和抗体，其识别CD 4结合位点、CD 4诱导的（CD 4 i）共受体结合位点、gp 120 V3环或gp 41的膜近端外部区域（MPER）。对这些抗体的研究提供了关于中和抗体与HIV相互作用的结构要求的有价值的信息。然而，对这些中和抗体如何产生的全面分析仍然缺乏。CD 4 i和MPER抗体具有带电荷氨基酸的长IgH CDR 3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2F 5和4 E10也与心磷脂和其他自身抗原反应。据推测，HIV中和抗体是通过罕见的重组事件产生的，并且表达这种抗体的B细胞已被阴性选择。我们最近的研究结果表明，在编码抗HIV抗体的IgH基因中，包括抗gp 41、抗gp 120、抗V3环和CD 4 i抗体，VH置换产物的频率显著升高。特别地，编码CD 4 i抗体的12个IgH基因中的9个可能通过VH置换产生。VH置换通过RAG介导的二级重组发生，涉及重排的VH基因内的隐蔽RSS和来自上游VH基因的23 bp RSS。VH置换更新了几乎整个VH编码区，但保留了一小段核苷酸作为“足迹”，其优先编码带电荷的氨基酸以延伸IgH CDR 3。CD 4 i抗体中几乎所有的VH置换“足迹”都将带负电荷的氨基酸贡献到CDR 3区中，这对于结合gp 120至关重要。基于这些结果，我们假设VH替换有助于B细胞抗HIV免疫应答。为了验证这一假设，（1）我们将进行单细胞PCR分析以确定HIV患者的浆细胞中VH置换产物的频率，并表达重组抗体以确定这些鉴定的VH置换产物是否编码抗HIV或HIV中和抗体;（2）我们将使用实验性VH替代系统来产生具有限制使用的VH 1的VH替代产物。69基因，并确定人工产生的VH置换产物是否编码抗HIV抗体。来自这些研究的结果将提供关于VH置换对B细胞抗HIV免疫应答的贡献的直接信息。目前的提议是基于我们最近的发现，即大部分抗HIV抗体是通过VH置换重组产生的。我们将使用先进的单细胞PCR方法来确定HIV患者浆细胞中VH替代产物的频率，并表达重组抗体以确定VH替代产物是否编码抗HIV抗体。了解VH替代对B细胞抗HIV应答的贡献将对对抗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.