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Objective HIV-1 replication and microbial translocation occur concomitant with systemic immune activation. This study delineates mechanisms of immune activation and CD4 T cell decline in pediatric HIV-1 infection. Design Cross-sectional and longitudinal cellular and soluble plasma markers for inflammation were evaluated in 14 healthy and 33 perinatally HIV-1-infected pediatric subjects prior to and over 96 weeks of protease-inhibitor-containing combination antiretroviral treatment [ART]. All HIV-1-infected subjects reconstituted CD4 T cells either with suppression of viremia or rebound of drug-resistant virus. Methods Systemic immune activation was determined by polychromatic flow cytometry of blood lymphocytes and ELISA for plasma soluble CD27 [sCD27], soluble CD14 [sCD14], and tumor necrosis factor [TNF]. Microbial translocation was evaluated by limulus amebocyte lysate assay to detect bacterial lipopolysaccharide [LPS] and ELISA for anti-endotoxin core antigen IgM antibodies. Immune activation markers were compared to viral load, CD4% and LPS by regression models. Comparisons between healthy and HIV-1 infected or between different viral outcome groups were performed by non-parametric rank sum. Results Microbial translocation was detected in healthy infants but resolved with age (P<0.05). LPS and sCD14 levels were elevated in all HIV-1 infected subjects (P<0.05 and P<0.0001, respectively) and persisted even if CD4 T cells were fully reconstituted, virus optimally suppressed, and lymphocyte activation resolved by ART. Children with CD4 T cell reconstitution but viral rebound following ART continued to display high levels of sCD27. Conclusions Microbial translocation in pediatric HIV-1-infection is associated with persistent monocyte/macrophage activation independent of viral replication or T cell activation.

Next generation sequencing (NGS) is superseding Sanger technology for analysing intra-host viral populations, in terms of genome length and resolution. We introduce two new empirical validation data sets and test the available viral population assembly software. Two intra-host viral population ‘quasispecies’ samples (type-1 human immunodeficiency and hepatitis C virus) were Sanger-sequenced, and plasmid clone mixtures at controlled proportions were shotgun-sequenced using Roche's 454 sequencing platform. The performance of different assemblers was compared in terms of phylogenetic clustering and recombination with the Sanger clones. Phylogenetic clustering showed that all assemblers captured a proportion of the most divergent lineages, but none were able to provide a high precision/recall tradeoff. Estimated variant frequencies mildly correlated with the original. Given the limitations of currently available algorithms identified by our empirical validation, the development and exploitation of additional data sets is needed, in order to establish an efficient framework for viral population reconstruction using NGS.

BackgroundDeep sequencing provides the basis for analysis of biodiversity of taxonomically similar organisms in an environment. While extensively applied to microbiome studies, population genetics studies of viruses are limited. To define the scope of HIV-1 population biodiversity within infected individuals, a suite of phylogenetic and population genetic algorithms was applied to HIV-1 envelope hypervariable domain 3 (Env V3) within peripheral blood mononuclear cells from a group of perinatally HIV-1 subtype B infected, therapy-naïve children.ResultsBiodiversity of HIV-1 Env V3 quasispecies ranged from about 70 to 270 unique sequence clusters across individuals. Viral population structure was organized into a limited number of clusters that included the dominant variants combined with multiple clusters of low frequency variants. Next generation viral quasispecies evolved from low frequency variants at earlier time points through multiple non-synonymous changes in lineages within the evolutionary landscape. Minor V3 variants detected as long as four years after infection co-localized in phylogenetic reconstructions with early transmitting viruses or with subsequent plasma virus circulating two years later.ConclusionsDeep sequencing defines HIV-1 population complexity and structure, reveals the ebb and flow of dominant and rare viral variants in the host ecosystem, and identifies an evolutionary record of low-frequency cell-associated viral V3 variants that persist for years. Bioinformatics pipeline developed for HIV-1 can be applied for biodiversity studies of virome populations in human, animal, or plant ecosystems.

HIV infection strikingly shifts the proportion of naive and late activated CD45RA(+)CD8(+) T cells. Homeostasis within this T-cell population reflects TREC levels and the extent of T-cell receptor Vbeta perturbations.