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种人抗v3单克隆抗体（mab），关于免疫球蛋白（Ig）可变重（VH）和可变轻（VL）链基因的使用如何决定Ab的交叉反应性和功能。这些研究是基于先前的发现，即人类抗体对不同病原体表现出优先的VH基因使用。在本应用中，我们将重点放在抗体到V3环上，这是HIV-1 gp120包膜糖蛋白的一个高变区，它可能需要与它结合的抗体具有非常不同的结构特征，以适应其序列异质性。人抗V3单克隆抗体具有中和原代分离株的能力，这表明抗V3单克隆抗体可能是HIV疫苗的重要靶点。研究Ig基因在靶向特定表位中的作用最有用的方法是分析单克隆抗体中VH和VL种系基因的使用情况。因此，在Aim 1中，我们将评估VH和VL基因对中和抗v3单克隆抗体的作用，并将其与cd4结合域、CD4i表位和gp41特异性单克隆抗体进行比较。对几种人类抗v3单克隆抗体的初步分析显示，VH5-51基因片段优先使用，这在正常曲目中很少使用。44个抗v3单克隆抗体的完整分析将用于确定偏倚基因使用的程度。在目标2中，我们将解决VH和/或VL基因使用与抗v3单克隆抗体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的VH基因可能编码单克隆抗体的假设，显示更广泛的交叉反应性和更有效的中和。在Aim 3中，我们将通过晶体学分析基因使用与单抗活性之间的关系，分析VH和/或VL中抗原相互作用残基以及编码这些单抗的种系基因的序列分析。我们假设V3单克隆抗体优先使用的种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。这些结果影响了对Ab产生的免疫遗传学的理解，这可能对HIV疫苗的开发具有重要意义。

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.