The Immunoglobulin Gene Usage for Anti-V3 Monoclonal Antibodies

描述(由申请人提供)：这些研究旨在检测44个人类抗V3单抗(MAb)，以了解免疫球蛋白(Ig)可变重链(VH)和可变轻链(VL)基因的使用如何决定抗体的交叉反应和功能。这些研究是基于先前的发现，即人类针对不同病原体的抗体显示出优先使用VH基因的情况。在本应用中，我们专注于Abs to the V3环，这是HIV-1 gp120包膜糖蛋白的一个高变区，可能需要与其结合的Abs具有非常不同的结构特征，以适应其序列异质性。人源性抗V3单抗具有中和初代分离株的能力，提示抗V3单抗可能是HIV疫苗的重要靶点。研究Ig基因在靶向特定表位中的作用的最有用的方法是分析单抗中VH和VL胚系基因的使用情况。因此，在目标1中，我们将评估VH和VL基因用于中和抗V3单抗，并将其与针对CD4结合区、CD4i表位和gp41的单抗进行比较。对几种人源性抗V3单抗的初步分析表明，VH5-51基因片段在正常谱系中很少使用。对44个抗V3单抗的完整分析将用于确定偏向基因使用的程度。在目标2中，我们将解决VH和/或VL基因使用与抗V3单抗的交叉中和活性之间是否存在相关性的问题。初步实验支持优先使用VH基因可能编码mAbs的假设，表现出更广泛的交叉反应和更有效的中和。在目标3中，我们将通过结晶学、分析VH和/或VL中的抗原相互作用残基以及编码这些mAb的胚系基因的序列分析来检验基因使用与mAb活性之间的关系。我们推测，V3单抗优先使用的胚系基因可能编码与抗原相互作用的残基，并在抗体的CDR中保持完整。这一结果对理解抗体产生的免疫遗传学有影响，这可能对HIV疫苗的开发具有关键意义。 叙述：在这项计划中，我们将研究免疫球蛋白基因对人类中和抗体HIV-1的V3区域的作用。我们的初步结果表明，有一个特定的免疫球蛋白基因片段被优先用于制造抗V3抗体，所产生的抗体对来自不同HIV-1亚型的病毒具有非常有效的中和作用。在设计具有诱导保护性抗体能力的有效HIV疫苗时，识别编码最有效的中和抗体的免疫球蛋白基因将是至关重要的。

DESCRIPTION (provided by applicant): These studies are designed to examine 44 human anti-V3 monoclonal antibodies (mAbs) with respect to how the immunoglobulin (Ig) variable heavy (VH) and variable light (VL) chain gene usage determines Ab cross-reactivity and function. The studies are based on previous findings in that human Abs against different pathogens exhibit preferential VH gene usage. In this application we focus on Abs to the V3 loop, a hypervariable region of the HIV-1 gp120 envelope glycoprotein, which may require very different structural features in the Abs that bind to it in order to accommodate its sequence heterogeneity. Human anti-V3 mAbs have the capacity to neutralize primary isolates and this suggests that Abs against V3 might constitute an important target for HIV vaccine. The most useful approach to study the role of Ig genes in targeting particular epitopes is the analysis of the VH and VL germline gene usage among mAbs. Therefore, in Aim 1 we will evaluate the VH and VL gene usage for neutralizing anti-V3 mAbs and compare this with mAbs specific to the CD4-binding domain, the CD4i epitope, and gp41. Preliminary analysis of several human anti-V3 mAbs has revealed a preferential usage of the VH5-51 gene segment that is rarely used within the normal repertoire. Complete analysis of the 44 anti-V3 mAbs will be used to determine the extent of the biased gene usage. In Aim 2, we will address the question of whether there is a correlation between VH and/or VL gene usage and cross-neutralizing activity of anti-V3 mAbs. Pilot experiments support the hypothesis that preferentially used VH genes may encode mAbs displaying broader cross-reactivity and more efficient neutralization. In Aim 3, we will examine the relationship between gene usage and mAb activity by crystallography, analyzing the antigen-interacting residues in VH and/or VL and sequence analysis of the germline genes encoding these mAbs. We hypothesize that the germline genes preferentially used by the V3 mAbs may encode residues which interact with the antigen and remain intact in the CDRs of the antibody. The results impact on understanding the immunogenetics of Ab production which could have critical implications for HIV vaccine development. NARRATIVE: In this proposal, we will study immunoglobulin gene usage for the human neutralizing antibodies against the V3 region of HIV-1. Our preliminary results suggest that there is a preferential use of one particular immunoglobulin gene segment for making anti-V3 antibodies and that the resultant antibodies are very efficient at neutralizing viruses from different HIV-1 subtypes. Identifying the immunoglobulin genes that encode the most potent neutralizing antibodies will be essential when designing an effective HIV vaccine with capability of inducing protective antibodies.