HIV-1 VARIANTS WITHIN PBMC SUBPOPULATIONS IN INFANTS

这项研究的一个长期目标是确定病毒学因素 通过母亲传播的儿童HIV-1感染。 主要 本提案中提出的问题是CD-154+的病毒嗜性 体内单核细胞是早期儿科感染的特征，如果 HIV-1的不同遗传变异体优先向单核细胞分离 或T淋巴细胞。 提出了三个具体目标：1。 到 确定外周血CD 14+中HIV-1的遗传特征 来自感染婴儿的单核细胞和CD 4 + T细胞; 2. 评估 HIV-1在外周血CD 14+单核细胞群中的亲细胞表型 受感染的婴儿;以及3. 定位env的巨噬细胞决定簇 来自HIV-1感染的CD 14+单核细胞的序列。 来自HIV-1感染新生儿的PBMC将通过选择使用 单克隆抗体和免疫磁珠进入CD 14+单核细胞， CD 4 + T细胞。 将通过流式细胞仪评估细胞群的纯度。 细胞计数和分子分析。 DNA中HIV-1序列的存在 每个细胞群将通过扩增包膜V1进行分析， V5区域。 CD 14+单核细胞和CD 4 + T细胞中的病毒基因型将 通过对克隆的V1-V5的V1、V2和V3结构域进行测序来确定 产品. 将通过非参数分析对结果进行分析，以确定 艾滋病毒的存在或持续存在之间是否存在关系- HIV-1感染者CD 14+单核细胞中CD 14 - 1序列与临床疾病 婴儿。 对非同义替换的分析可能表明， 对于单核细胞在体内可以是一个选择性的压力为env的变异性。 拟议的实验将从体内HIV-1的光谱中分析env 具有不同基因型但相同单核细胞/巨噬细胞的变体 表型 纵向研究将跟踪传染性疾病的演变， 可能与婴儿和儿童疾病相关的变异，以及 将决定因子定位在env中， 体内向性 HIV-1的嗜巨噬细胞变体在临床上很重要，因为 感染病毒主要是那些显示嗜巨噬细胞的病毒， 培养物中的表型。 体内病毒来源与微噬菌体 表型尚未广泛确定。 在初步研究中， 高度富集的外周CD 14+单核细胞在婴儿中被感染。 是 外周血CD 14+单核细胞的感染可能是婴儿独有的。 新生儿单核细胞对HIV-1感染的易感性可能有助于 儿童艾滋病独特的临床表现等高发 感染儿童的脑病。 或者，感染 外周单核细胞可能是早期HIV-1感染的一般特征， 为HIV-1向组织的快速系统性传播提供机制。 拟议的研究将系统地确定 HIV-1在感染后早期直接进入外周血单核细胞。 一种新型 战略将整合有关病毒感染的生物学和遗传学数据， 外周血单核细胞与临床病程的关系 孩子 新生儿经母体感染HIV-1提供了一个 独特的机会来检查病毒的表型和基因型， 感染的最早阶段。 研究结果对设计具有重要意义 阻断HIV-1感染的策略。

A long-term goal of the proposes research is to define virological factors involved in pediatric HIV-1 infection by maternal transmission. The major questions addressed in this proposal ar whether virus tropism for CD-154+ monocytes in vivo is a feature of early pediatric infection and if different genetic variants of HIV-1 segregate preferentially to monocytes or to T lymphocytes. There are three specific aims proposed: 1. To determine the genetic characteristics of HIV-1 in peripheral CD14+ monocytes and CD4+ T cell from infected infants; 2. To assess the cytotropic phenotype of HIV-1 in peripheral CD14+ monocyte populations from infected infants; and 3. To localize the macrophage determinants of env sequences from HIV-1 infected CD14+ monocytes. PBMC from HIV-1 infected neonates will be fractionated by selection using monoclonal antibodies and immunomagnetic beads into CD14+ monocytes and CD4+ T cells. Purity of the cell populations will be assessed by flow cytometry and molecular analyses. Presence of HIV-1 sequences in DNA from each population of cells will be assayed by amplification of envelope V1 to V5 regions. Genotypes of viruses in CD14+ monocytes and CD4+ T cells will be determined by sequencing V1, V2, and V3 domains of cloned V1-V5 products. Results will be analyzed by nonparametric analysis to determine whether there is a relationship between the presence or persistence of HIV- 1 sequences in CD14+ monocytes and clinical disease in HIV-1 infected infants. Analysis of nonsynonymous substitutions may indicate that tropism for monocytes in vivo can be a selective pressure for env variability. Proposed experiments will analyze env from a spectrum of in vivo HIV-1 variants with different genotypes but the same monocyte/macrophage phenotype. Longitudinal studies will follow the evolution of infectious variant that could be related to disease in infants and children, and localize the determinants in env that contribute to monocyte/macrophage tropism in vivo. Macrophage tropic variants of HIV-1 are clinically important because infecting viruses are predominately ones that display a macrophage tropic phenotype in culture. The source in vivo of viruses with a microphage phenotype has not been determined extensively. In preliminary studies, highly enriched peripheral CD14+ monocytes are infected in infants. It is possible that infection of peripheral CD14+ monocytes is unique to infants. Susceptibility of neonatal monocytes to HIV-1 infection may contribute to unique clinical manifestation of pediatric AIDS such as the high incidence of encephalopathy in infected children. Alternatively, infection of peripheral monocytes may be a general feature of early HIV-1 infection and provide a mechanism for rapid systemic dissemination of HIV-1 to tissues. The research proposed will determine systematically the characteristics of HIV-1 directly within peripheral monocytes early after infection. A novel strategy will integrate biological and genetic data about viruses infecting peripheral monocytes with the clinical course of disease in infected children. Neonatal HIV-1 infection by maternal transmission provides a unique opportunity to examine both phenotype and genotype of viruses at the earliest stages in infection. Results will be significant for designing strategies to block infection by HIV-1.