MOLECULAR ANALYSIS & MODELING OF HIV-1 TRANSMISSION, CONTAINMENT AND ESCAPE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项研究涉及全球卫生倡议的重大挑战#6：了解哪些免疫反应提供(针对艾滋病毒-1的)保护性免疫。识别全长传播的HIV-1基因组可以通过直接分析那些实际导致生产性临床感染的病毒，在HIV-1的发病机制、杀微生物剂和疫苗研究中发挥作用。我们已经在12例急性感染者(9例B分支和3例C分支)中表明，通过单基因组扩增和血浆病毒粒子RNA测序可以推断传播/创始病毒的完整HIV-1基因组。这使得可以对传播的/创始病毒进行分子克隆和生物学分析，并对宿主选择压力的组合在进化中的病毒准物种上留下的遗传印记进行全面的全基因组评估。 传播的病毒编码完整的规范基因(gag-polvif-vpr-tat-rev-vPU-env-nef)，并在原代人类CD4(+)T淋巴细胞中有效复制，但在单核细胞来源的巨噬细胞中复制较少。传播的病毒是嗜性的CD4和CCR5，并显示包膜桥联片层和可变环3的共受体结合面的隐藏。在感染后2个月，3个受试者的传播/创始病毒几乎完全被两到五个高度选择的基因组位点的不同病毒所取代，到12-20个月，病毒在17-34个离散位置出现集中突变。 这些发现揭示了与HIV-1粘膜传播有关的病毒特性，以及一组有限的快速进化的适应性突变，主要但不完全是由早期的细胞毒性T细胞反应驱动的。 我们最近描述了两种来自赞比亚受试者的自体单抗6.4C和13.6A，每种抗体都识别创始人Env上的V1V2依赖靶点。利用这些单抗，我们鉴定了V1V2结构域中的两个突变，它们参与了单一抗体水平的逃逸。我们扩大了这些研究的范围，以测试糖基化的改变是否对NAB耐药是必要的，并进一步确定单抗的表位。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This study addresses Grand Challenge #6 of the Global Health Initiative: Learn which immunological responses provide protective immunity (against HIV-1). Identification of full-length transmitted HIV-1 genomes could be instrumental in HIV-1 pathogenesis, microbicide, and vaccine research by enabling direct analysis of those viruses actually responsible for productive clinical infection. We have shown in 12 acutely infected subjects (9 clade B and 3 clade C) that complete HIV-1 genomes of transmitted/founder viruses can be inferred by single genome amplification and sequencing of plasma virion RNA. This allowed for molecular cloning and biological analysis of transmitted/founder viruses and comprehensive genome-wide assessment of the genetic imprint left on the evolving virus quasispecies by a composite of host selection pressures. Transmitted viruses encoded intact canonical genes (gag-pol-vif-vpr-tat-rev-vpu-env-nef) and replicated efficiently in primary human CD4(+) T lymphocytes but much less so in monocyte-derived macrophages. Transmitted viruses were CD4 and CCR5 tropic and demonstrated concealment of coreceptor binding surfaces of the envelope bridging sheet and variable loop 3. At 2 mo after infection, transmitted/founder viruses in three subjects were nearly completely replaced by viruses differing at two to five highly selected genomic loci; by 12-20 mo, viruses exhibited concentrated mutations at 17-34 discrete locations. These findings reveal viral properties associated with mucosal HIV-1 transmission and a limited set of rapidly evolving adaptive mutations driven primarily, but not exclusively, by early cytotoxic T cell responses. We have recently described two autologous monoclonal antibodies, 6.4C and 13.6A, generated from a Zambian subject, that each recognize a V1V2-dependent target on the founder Env. Using these Mabs, we identified two mutations in the V1V2 domain that were involved in escape at the single antibody level. We have extended these studies to test whether a change in glycosylation is necessary for Nab resistance and to further define epitopes of the Mabs.