Isolation and Deep Sequencing of Monoclonal Antiobodies Against HIV

一些HIV感染者会产生针对HIV的强有力的抗体反应，但目前尚不清楚这种情况发生的频率，也不清楚它是如何发生的。我们对HIV+血清进行了系统性评价，以发现具有广泛中和活性的血清，了解中和的广度如何发生，并从此类患者的细胞中分离抗体。 我们观察到约20%的患者具有广泛中和抗体。在该组中，我们选择了具有最广泛抗体应答的患者，并收集PBMC以分离单克隆抗体。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PCR以分离IgG基因。然后将基因克隆并表达为完整IgG。迄今为止，这些方法已经产生了几种新型抗HIV单克隆抗体，包括VRC 01和VRC 02，可以中和90%的HIV-1毒株。我们还进行了454次深度测序，以研究这些和其他个体中mAb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.