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

最近使用具有跨分支中和活性的选定患者血清的研究表明，这种特异性 至少三分之一的中和活性仍未确定。这些结果表明有必要 确定新的表位，以指导开发有前景的疫苗的努力。我们提出了一种创新的 使用未使用的选定技术组合生产人源单抗的方法 由任何其他组一起使用，即，使用与病毒样颗粒一起选择的单个Ig G记忆B细胞 (VLP)，从中高效地产生重组单抗(MAbs) 分子技术。另一项创新包括更有效地分选和选择仅特定于B细胞 用于三聚体包膜(Env)蛋白。B细胞将来自感染了不同HIV-1病毒的捐赠者 其血浆抗体交叉中和第二层病毒的亚型。我们假设这些被选中的志愿者 为天然三聚体HIV-1上存在的新的未知表位产生中和抗体 包膜和对这些表位的反应性将使用VLP进行检测。具体目标1.生产 来自单个B细胞的重组单抗。血液样本将来自两个久负盛名的 受感染的对象。艾滋病毒/艾滋病疫苗免疫中心将提供三份PBMC样本 (CHAVI)和10个PBMC样本将从喀麦隆受试者身上获得，他们的血清已被证明 调解跨分支中和活性。单抗将从所选的单个环境特异性B细胞中产生 用GFP标记的VLP表达三聚体Env蛋白。免疫球蛋白可变区基因将被扩增 利用RT-PCR，将其克隆到表达载体中，并将其用于293T细胞的转染 MAB生产。总共将对10-15个受试者的PBMC样本进行研究，产生300-450个单抗。 特定目的2.新的多种功能活性(中和、ADCC和ADCVI)的表征 单抗。纯化的单抗将在功能分析中进行中和、ADCC和/或ADCVI活性测试。 单抗的中和活性将在我们的实验室中筛选，以对抗伪病毒和初级分离株 选定的单抗将由合作者针对伪型病毒的标准小组进行测试。新单抗 结合两个或三个抑制功能(中和、ADCC和/或ADCVI)将优先用于表位 仅交叉中和单抗和非中和单抗与Fc&R介导的单抗的映射 活动。特异性目的3.新单抗的表位定位使用VLP选择特定于环境的B 细胞将产生针对各种已知表位和存在于环境中的表位的单抗 三聚体。将使用各种测绘技术，包括免疫化学和病毒分析以及 晶体分析。将特别关注mAbs到第四系和新定义的mAbs的映射 表位和中介双(或三)功能。具有高效、交叉中和和的单抗表位 不同的活性将作为未来免疫原设计的模板，以诱导保护性抗体。

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.