The Immunoglobulin Gene Usage for Anti-V3 Monoclonal Antibodies

描述（由申请方提供）：这些研究旨在检查44种人抗V3单克隆抗体（mAb）的免疫球蛋白（IG）可变重链（VH）和可变轻链（VL）基因使用如何决定Ab交叉反应性和功能。这些研究是基于先前的发现，即针对不同病原体的人Ab表现出优先的VH基因使用。在这个应用程序中，我们专注于抗体的V3环，HIV-1 gp 120包膜糖蛋白的高变区，这可能需要非常不同的结构特征的抗体结合，以适应其序列的异质性。人源抗V3单克隆抗体具有中和原代分离株的能力，这表明抗V3单克隆抗体可能成为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mAb中VH和VL种系基因的使用。因此，在目的1中，我们将评估中和抗V3 mAb的VH和VL基因使用，并将其与特异性针对CD 4结合结构域、CD 4 i表位和gp 41的mAb进行比较。对几种人抗V3单克隆抗体的初步分析揭示了VH 5 -51基因片段的优先使用，其在正常库中很少使用。将使用44种抗V3 mAb的完整分析来确定偏倚基因使用的程度。在目的2中，我们将解决VH和/或VL基因使用与抗V3 mAb的交叉中和活性之间是否存在相关性的问题。初步实验支持这样的假设，即优先使用的VH基因可以编码显示更广泛的交叉反应性和更有效的中和的mAb。在目标3中，我们将通过晶体学、分析VH和/或VL中的抗原相互作用残基以及编码这些mAb的种系基因的序列分析来检查基因使用与mAb活性之间的关系。我们假设V3 mAb优先使用的生殖系基因可能编码与抗原相互作用并在抗体CDR中保持完整的残基。这些结果影响了对抗体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。 叙述：在这项计划中，我们将研究免疫球蛋白基因的用途，为人类中和抗体对HIV-1的V3区。我们的初步结果表明，有一个特定的免疫球蛋白基因片段的优先使用，用于制造抗V3抗体，并得到的抗体是非常有效的中和来自不同的HIV-1亚型的病毒。在设计具有诱导保护性抗体能力的有效HIV疫苗时，鉴定编码最有效中和抗体的免疫球蛋白基因将是必不可少的。

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.