BACKGROUND Quantification of integrated proviral HIV DNA by repetitive-sampling Alu-HIV PCR is a candidate virological tool to monitor the HIV reservoir in patients. However, the experimental procedures and data analysis of the assay are complex and hinder its widespread use. Here, we provide an improved and simplified data analysis method by adopting binomial and Poisson statistics. METHODS A modified analysis method on the basis of Poisson statistics was used to analyze the binomial data of positive and negative reactions from a 42-replicate Alu-HIV PCR by use of dilutions of an integration standard and on samples of 57 HIV-infected patients. Results were compared with the quantitative output of the previously described Alu-HIV PCR method. RESULTS Poisson-based quantification of the Alu-HIV PCR was linearly correlated with the standard dilution series, indicating that absolute quantification with the Poisson method is a valid alternative for data analysis of repetitive-sampling Alu-HIV PCR data. Quantitative outputs of patient samples assessed by the Poisson method correlated with the previously described Alu-HIV PCR analysis, indicating that this method is a valid alternative for quantifying integrated HIV DNA. CONCLUSIONS Poisson-based analysis of the Alu-HIV PCR data enables absolute quantification without the need of a standard dilution curve. Implementation of the CI estimation permits improved qualitative analysis of the data and provides a statistical basis for the required minimal number of technical replicates.

Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy.

The dynamics of HIV reservoir accumulation off antiretroviral therapy (ART) is underexplored. Levels of integrated HIV DNA in peripheral blood mononuclear cells (PBMCs) were longitudinally monitored before and after antiviral therapy. HIV integration increased over time in both elite controllers (ECs; n ‫؍‬ 8) and noncontrollers (NCs; n ‫؍‬ 6) before ART, whereas integration remained stable in patients on ART (n ‫؍‬ 4). The median annual fold change was higher in NCs than in ECs and negatively correlated with CD4/CD8 T-cell ratio. Cytotoxic T lymphocyte (CTL) function as assessed by infected CD4 T-cell elimination (ICE) and granzyme B activity did not significantly change over time in ECs, suggesting that the gradual increase in integrated HIV DNA observed in ECs was not a result of progressive loss of immune-mediated control. Also, acutely infected (n ‫؍‬ 7) but not chronically infected (n ‫؍‬ 6) patients exhibited a significant drop in integrated HIV DNA 12 months after ART initiation. In conclusion, in the absence of ART, integrated HIV accumulates over time both in NCs and in ECs, at variable individual rates. Starting ART early in infection leads to a greater drop in integrated HIV DNA than does initiating treatment after years of infection. The increase in integrated HIV DNA over time suggests that early treatment may be of benefit in limiting HIV reservoirs. IMPORTANCEThe establishment of a latent reservoir represents a barrier to cure among HIV-infected individuals. The dynamics of HIV reservoir accumulation over time in patients before antiviral therapy is underexplored, in large part because it is difficult to accurately and reproducibly measure the size of HIV reservoir in this setting. In our study, we compared the dynamics of integrated HIV DNA over time in ECs and NCs before and after ART was initiated. We found that integrated HIV DNA levels progressively increase over time in the absence of ART, but with a higher, albeit variable, rate in NCs compared to ECs. In addition, integrated HIV DNA declines more dramatically when ART is initiated in acute rather than chronic HIV infection, suggesting important differences between acute and chronic infection. Our study highlights the role of HIV replication and CTL control in reservoir accumulation in sanctuary sites and why ART appears to be more effective in acute infection.

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Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy.