Segregation of HIV-1 Protease Genotypic Resistance within Blood Compartments Identifies the CD4+ CD45RA T Lymphocytes as a Long-Lived Viral Reservoir

Stephanie L. Rose, Sarah J. Koch, Steven M. Pomeroy, Susanna L. Lamers, John W. Sleasman, and Maureen M. Goodenow.  University of Florida 1600 SW Archer Road Box 100275 Gainesville, Florida 32610 (USA).

Background: CD4 T cells are classified based on expression of CD45RA or CD45RO. Cell phenotypes reflect differences in cell half-lives, activation, and HIV-1 susceptibility. HIV-1 is found predominantly in the CD45RO CD4 subset of T cells that have a short life-span. CD4 lymphocytes and macrophages that are long-lived also serve as targets for virus infection and replication.  During acute infection in children, HIV-1 within the CD45RA subset of CD4 T cells accounts for a significant proportion of infection in peripheral CD4 lymphocytes. Combination therapy in children can produce dramatic increases in CD4 T cells that are new targets for viral infection. Material and Methods: A longitudinal study involved children who had immunological reconstitution, but incomplete suppression of virus replication, during combination therapy containing a protease inhibitor. Enriched CD4 T cell subsets expressing CD45RO or CD45RA were selected by immunomagnetic beads.  Protease genotypes were determined following amplification of HIV-1 sequences from plasma RNA and cell-associated DNA.  Viruses from both the CD45RA and CD45RO subsets replicated in co-culture with PBMC.  Gag-pol genotypes of the replicating virus were determined using cultured DNA. Results: Following 20 weeks of antiretroviral therapy plasma sequences were related to, but were more homogeneous, than PR sequences in the cellular compartment. Homogeneous populations of resistant viruses emerged in plasma before appearing in the CD45RO cells by 8 to 12 weeks of therapy. Viruses with wild type protease alleles persisted within CD45RA cells for more than 6 months before genotypic resistance appeared.  Post-therapy protease from both cell subsets evolved from a restricted lineage suggesting viral trafficking between compartments.   Viruses harbored in both the CD45RA and CD45RO CD4 T cells were replication competent and distinguishable by gag-pol genotype. Conclusion: Drug resistant viruses emerge in temporal succession in different peripheral blood compartments that reflect diverse half-lives.  Cells producing plasma virus represent a minority of infected peripheral blood cells or are sequestered in tissues.   Viruses within CD45RA cells are a dynamic population which turn over more slowly than viral populations in plasma or CD45RO cells.  CD45RA cells provide an important reservoir for drug resistant HIV-1 in patients who achieve reconstitution of CD4 lymphocytes.