HIV-1 VARIANTS WITHIN PBMC SUBPOPULATIONS IN INFANTS

Proposes研究的一个长期目标是确定病毒学因素 参与通过母体传播的儿童HIV-1感染。少校 这项提案涉及的问题是，CD-154+是否具有病毒趋向性 单核细胞在体内是早期儿科感染的特征，如果 不同的HIV-1基因变异株优先分离单核细胞 或T淋巴细胞。提出了三个具体目标：1. 检测外周血CD14+中HIV-1的基因特征 感染婴儿的单核细胞和CD4+T细胞；2.评估 中国人外周血CD14+单核细胞中HIV-1的细胞趋化表型 感染婴儿；3.定位环境的巨噬细胞决定因素 HIV-1感染的CD14+单核细胞的序列。 HIV-1感染新生儿的PBMC将通过选择进行分级 单抗和免疫磁珠进入CD14+单核细胞和 CD4+T细胞。细胞群体的纯度将通过Flow进行评估 细胞学和分子分析。中国人DNA中HIV-1基因序列的存在 每组细胞将通过扩增包膜V1至 V5区域。CD14+单核细胞和CD4+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。结果将通过非参数分析进行分析，以确定 艾滋病毒的存在或持续存在之间是否有关系- HIV-1感染者CD14+单核细胞1基因序列与临床疾病的关系 婴儿。对非同义替换的分析可能表明，取向 对于体内单核细胞来说，可以选择性地施加压力，以获得环境的可变性。 拟议的实验将从体内HIV-1的光谱中分析env 具有不同基因类型但具有相同单核/巨噬细胞的变体 表型。纵向研究将跟踪传染性疾病的演变 可能与婴儿和儿童的疾病有关的变异，以及 定位环境中参与单核/巨噬细胞的决定因素 体内的向向性。 HIV-1的巨噬细胞嗜性变异在临床上很重要，因为 感染病毒主要是表现出巨噬细胞嗜性。 培养中的表型。微噬菌体体内病毒源的研究 表型尚未被广泛确定。在初步研究中， 婴儿感染高度浓缩的外周血CD14+单核细胞。它是 外周血CD14+单核细胞感染可能是婴儿所特有的。 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如高发 感染儿童脑病的风险。或者，感染 外周血单核细胞可能是早期HIV-1感染的一般特征 提供一种机制，使艾滋病毒-1迅速系统性地传播到组织中。 提出的研究将系统地确定 HIV-1感染后早期直接在外周血单核细胞内。一本小说 战略将整合有关感染病毒的生物和遗传数据 感染患者外周血单核细胞与临床病程的关系 孩子们。通过母体传播的新生儿HIV-1感染提供了 独一无二的机会同时检查病毒的表型和基因 感染的早期阶段。结果将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.