Isolation and Deep Sequencing of Monoclonal Antiobodies Against HIV

一些感染艾滋病毒的患者对艾滋病毒产生了强大的抗体反应，但目前尚不清楚这种情况发生的频率，也不清楚这种情况是如何发生的。我们已经对HIV+血清进行了系统的评估，以找到那些具有广泛中和活性的血清，了解中和的广度是如何发生的，并从这些患者的细胞中分离抗体。 我们观察到大约20%的人有广泛的中和抗体。在这组患者中，我们选择了抗体反应最广泛的患者，并收集了PBMC以分离单抗。我们的方法包括分离HIV-1特异性的记忆B细胞，并进行单细胞聚合酶链式反应来分离免疫球蛋白基因。然后，这些基因被克隆并以完整的IgG形式表达。到目前为止，这些方法已经产生了几种新的抗HIV单抗，包括VRC01和VRC02，它们可以中和90%的HIV-1毒株。我们还进行了454次深度测序，以研究这些个体和其他个体中单抗的克隆关系和谱系。

Some HIV infected patients develop potent antibody responses against HIV, but it is not clear how often this happens, nor how it occurs. We have undertaken a systemic evaluation of HIV+ sera to find those with broad neutralizing activity, understand how breadth of neutralization occurs and to isolate antibodies from the cells of such patients. We have observed that about 20% have broadly neutralizing antibodies. Among this group, we selected those patients with the broadest antibody response and have collected PBMC in order to isolate monoclonal antibodies. Our methodology includes isolating memory B-cells that are HIV-1 specific, and performing single cell PCR to isolate IgG genes. The genes are then cloned and expressed as full IgGs. To date, these approaches have yielded several novel anti-HIV monoclonal antibodies including VRC01 and VRC02 that neutralize 90% of HIV-1 strains. We have also performed 454 deep sequencing to study the clonal relationships and lineage of mAbs in these and other individuals.