Production of Cross-Neutralizing HIV-1 Antibodies from Single B Cells

最近使用具有交叉进化枝中和活性的选定患者血清的研究显示， 至少三分之一的中和活性仍然没有被表征。这些结果表明，需要 确定新的抗原决定簇，指导开发有前景的疫苗。我们提出了一个创新的 使用未使用的技术的选定组合生产人mAb的方法 任何其他团体，即，使用用病毒样颗粒选择的单个IgG+记忆B细胞 重组单克隆抗体（mAb）将使用高效的 分子技术进一步的创新包括更有效的分选和选择仅特异性的B细胞 三聚体包膜（Env）蛋白。这些B细胞将来自感染了多种HIV-1的捐赠者 血浆抗体交叉中和二级病毒的亚型。我们假设这些被选中的志愿者 产生针对天然三聚体HIV-1上存在的新的尚未鉴定的表位的中和Ab 这些表位的反应性将使用VLP检测。具体目标1.生产 来自单个B细胞的重组mAb。血液样本将来自两个已建立的队列， 被感染的对象3份PBMC样本将由HIV/AIDS疫苗免疫学中心提供 （CHAVI）和10个PBMC样品将从其血清已被显示为 介导跨进化枝中和活性。mAb将由选择的单个Env特异性B细胞产生 与表达三聚体Env蛋白的GFP标记的VLP一起。免疫球蛋白可变基因会被扩增 使用RT-PCR，克隆到表达载体中，并将所述基因用于转染293 T细胞， mAb生产。总共将研究10-15例受试者的PBMC标本;产生300-450个mAb。 具体目标2.表征新化合物的各种功能活性（中和、ADCC和ADCVI）。 单克隆抗体将在功能测定中测试纯化的mAb的中和、ADCC和/或ADCVI活性。 将在我们的实验室中针对假病毒和原代分离株筛选mAb的中和活性， 选定的单克隆抗体将由合作者针对一组标准假型病毒进行测试。新mAb 组合两种或三种抑制功能（中和、ADCC和/或ADCVI）将具有表位优先性。 作图，随后是仅交叉中和的那些mAb和与Fc R介导的免疫球蛋白结合的非中和mAb。 活动具体目标3.新单克隆抗体的表位作图。使用VLP选择Env特异性B 细胞将导致针对各种已知表位和存在于Env上的表位的mAb的产生 三聚体。将使用各种定位技术，包括免疫化学和病毒测定以及 晶体学分析映射将特别关注mAb到四元和新定义的 表位和介导双重（或三重）功能。具有强效、交叉中和和免疫抑制作用的mAb的表位 不同的活性将作为模板用于将来设计免疫原以诱导保护性Ab。

Recent studies using selected patients' sera with cross-clade neutralizing activity revealed that the specificity of at least one-third of the neutralizing activity remains uncharacterized. These results demonstrate the need to identify new epitopes which can guide efforts to develop a promising vaccine. We propose an innovative approach to produce human mAbs using a selected combination of techniques which are not being used together by any other group, i.e., the use of single IgG+ memory B cells, selected with virus-like particles (VLPs) from which recombinant monoclonal antibodies (mAbs) will be generated using highly efficient molecular techniques. A further innovation includes more efficient sorting and selection of B cells specific only for trimeric envelope (Env) proteins. The B cells will be derived from donors infected with diverse HIV-1 subtypes whose plasma Abs cross-neutralize Tier 2 viruses. We hypothesize that these selected volunteers produce neutralizing Abs to new as yet unidentified epitopes that are present on the native trimeric HIV-1 envelope and that reactivity to such epitopes will be detected using VLPs. SPECIFIC AIM 1. Production of recombinant mAbs from single B cells. The blood specimens will come from two well-established cohorts of infected subjects. Three PBMC samples will be provided by the Center for HIV/AIDS Vaccine Immunology (CHAVI) and 10 PBMCs samples will be obtained from Cameroonian subjects whose sera have been shown to mediate cross-clade neutralizing activity. The mAbs will be produced from single Env-specific B cells selected with GFP-tagged VLPs expressing trimeric Env proteins. The immunoglobulin variable genes will be amplified using RT-PCR, cloned into expression vectors, and the genes will be used for the transfection of 293T cells for mAb production. In total, PBMC specimens from 10-15 subjects will be studied; yielding 300-450 mAbs. SPECIFIC AIM 2. Characterization of various functional activities (neutralizing, ADCC and ADCVI) of new mAbs. The purified mAbs will be tested in functional assays for neutralization, ADCC and/or ADCVI activity. The neutralizing activity of mAbs will be screened against pseudoviruses and primary isolates in our lab and selected mAbs will be tested against a standard panel of pseudotyped viruses by a collaborator. New mAbs combining two or three inhibitory functions (neutralization, ADCC and/or ADCVI) will have priority for epitope mapping followed by those mAbs that cross-neutralize only and non-neutralizing mAbs with the FcγR-mediated activity. SPECIFIC AIM 3. Epitope mapping of new monoclonal Abs. Using VLPs for selection of Env-specific B cells will result in production of mAbs against various known epitopes and those which are present on Env trimers. A variety of mapping techniques will be used, including immunochemical and viral assays as well as crystallographic analysis. Mapping will be particularly focused on mAbs to quaternary and newly defined epitopes and that mediate double (or triple) functions. Epitopes of mAbs with potent, cross-neutralizing and varied activities will serve as templates for the future design of immunogens to induce protective Abs.