HIV-1 VARIANTS WITHIN PBMC SUBPOPULATIONS IN INFANTS

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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.