Viral and Placental Determinants of HIV-1 Subtype C Mother-to-Child Transmission

摘要本研究旨在全面描述与HIV-1亚型C （hiv - 1c）母婴传播（MTCT）相关的宿主和病毒生物学。我们假设hiv - 1c MTCT是由胎盘和病毒决定因素介导的。为了解决这一假设，我们将：1)基因型和表型垂直传播包膜（env）基因，2)表征从存档的胎盘活检中分离的包膜基因，3)描述MTCT期间的胎盘环境。所有实验将使用先前从744名hiv - 1c感染的马拉维孕妇中收集的样本，这些孕妇的特征为非传播性（NT）、子宫内（ID）母婴对（MOPs）和产内（IP） MOPs。在一项初步研究中，我们在48个MOPs中枚举了HIV-1 C V1/V2环境多样性，发现婴儿的变异明显少于母亲，并证实HIV环境多样性在MTCT期间以非随机方式受到限制。特异性目的1验证了这种限制是由特定的环境基因型促进的假设，它将通过单基因组扩增克隆全长包膜基因来完成：a)比较V1/V2环长度，b)枚举假定的糖基化位点，c)克隆的环境基因对CCR5、CxCR4、CD4hi和CD4lo细胞进行假型。作为俄亥俄州立大学微生物相互作用生物学中心的一名独立研究者，我将专注于IU MTCT，并验证胎盘是独特的hiv - c隔室的假设。使用异源双工跟踪测定，我将比较来自匹配外周血、胎盘血和胎盘活检的HIV-1C环境基因（特异性目的2）。胎盘特异性变异将使用在指导阶段学习的异双工跟踪测定来表征。在Specific Aim 3中，胎盘特异性变异将针对原代胎盘滋养细胞和霍夫鲍尔细胞进行假型分型。最后，在Specific Aim 4中，先前收集的胎盘活检将分析hiv - 1c、CCR5、CD4和CXCR4的表达。本项目的结果将全面描述hiv - 1c包膜基因和有利于HIV-1亚型C MTCT的胎盘环境。

Viral and Placental Determinants of HIV-1 Subtype C Mother-to-Child Transmission ABSTRACT This proposal aims to comprehensively describe host and virus biology associated with HIV-1 subtype C (HIV-1 C) mother-to-child transmission (MTCT). We hypothesize that HIV-1 C MTCT is mediated by both placental and viral determinants. To address this hypothesis, we will: 1) genotype and phenotype vertically transmitted envelope (env) genes, 2) characterize envelope genes isolated from archived placental biopsies, 3) describe the placental environment during MTCT. All experiments will use previously collected samples from a well-characterized cohort of 744 HIV-1C-infected pregnant Malawian women characterized as nontransmitting (NT), in utero (ID) mother-offspring pairs (MOPs) and intrapartum (IP) MOPs. In a preliminary study, we enumerated HIV-1 C V1/V2 env diversity in 48 MOPs and found that infants had significantly fewer variants than their mothers and confirmed that HIV env diversity is restricted during MTCT in a non-stochastic manner. Specific Aim I tests the hypothesis that this restriction is facilitated by specific env genotypes and it will be accomplished using single-genome amplification to clone full-length envelope genes to: a) compare V1/V2 loop lengths, b) enumerate putative glycosylation sites, c) pseudotype the cloned env genes against CCR5, CxCR4, CD4hi and CD4lo cells. As an independent investigator in the Center for Microbial Interfact Biology at Ohio State, I will focus on IU MTCT and test the hypothesis that the placenta is a unique HIV1-C compartment. Using a heteroduplex tracking assay, I will compare HIV-1C env genes from matched peripheral blood, placental blood, and placental biopsies (Specific Aim 2). Placental specific variants will be characterized using the heteroduplex tracking assays learned during the mentored phase. In Specific Aim 3, placental specific variants will be psuedotyped against primary placental trophoblasts and Hofbauer cells. Finally, in Specific Aim 4, previously collected placental biopsies will be analyzed for HIV-1 C, CCR5, CD4, and CXCR4 expression. The results of this project will provide a comprehensive description of the HIV-1 C envelope genes and the placental environment conducive to HIV-1 subtype C MTCT.