Abundant HIV-infected cells in blood and tissues are rapidly cleared upon ART initiation during acute HIV infection

Abstract

The timing and location of the establishment of the viral reservoir during acute HIV infection remain unclear. Using longitudinal blood and tissue samples obtained from HIV-infected individuals at the earliest stage of infection, we demonstrate that frequencies of infected cells reach maximal values in gut-associated lymphoid tissue and lymph nodes as early as Fiebig stage II, before seroconversion. Both tissues displayed higher frequencies of infected cells than blood until Fiebig stage III, after which infected cells were equally distributed in all compartments examined. Initiation of antiretroviral therapy (ART) at Fiebig stages I to III led to a profound decrease in the frequency of infected cells to nearly undetectable level in all compartments. The rare infected cells that persisted were preferentially found in the lymphoid tissues. Initiation of ART at later stages (Fiebig stages IV/V and chronic infection) induced only a modest reduction in the frequency of infected cells. Quantification of HIV DNA in memory CD4⁺ T cell subsets confirmed the unstable nature of most of the infected cells at Fiebig stages I to III and the emergence of persistently infected cells during the transition to Fiebig stage IV. Our results indicate that although a large pool of cells is infected during acute HIV infection, most of these early targets are rapidly cleared upon ART initiation. Therefore, infected cells present after peak viremia have a greater ability to persist.

Fig. 1.. Rapid establishment of a large pool of infected cells in blood and tissues during acute HIV infection.

A, Frequency of peripheral blood mononuclear cells (PBMCs) harboring integrated HIV DNA according to the Fiebig stage before antiretroviral therapy (ART) initiation (Fiebig I n=14, Fiebig II n=31, Fiebig III n=40, Fiebig IV/V n=12 and chronic n=37). B, Frequency of lymph node mononuclear cells (LNMCs) harboring integrated HIV DNA according to the Fiebig stage before ART initiation (Fiebig I n=7, Fiebig II n=7, Fiebig III n=10, Fiebig IV/V n=6 and chronic n=3). C, Frequency of colon cells harboring integrated HIV DNA according to the Fiebig stage before ART initiation (Fiebig I n=3, Fiebig II n=12, Fiebig III n=31, Fiebig IV/V n=10 and chronic n=7). In A-C, each symbol represents an individual sample. Undetectable samples are plotted as “zero” and are represented as open circles. Columns represent the median values with interquartile range. Values were transformed in log₁₀([copies/10⁶ cells]+1). Nonparametric Mann Whitney U tests were performed to compare Fiebig I to all other stages and chronic to all other stages: ns p>0.05, * p≤0.05, ** p≤0.009, *** p≤0.0009, **** p≤0.0001. D, Percentages of lymph node, sigmoid colon biopsies and blood matched samples with detectable and undetectable integrated HIV DNA in acutely infected individuals. Chi-Square tests were performed to compare blood to colon, blood to lymph nodes and colon to lymph nodes E, F, Correlations between plasma viremia and frequencies of cells harboring integrated HIV DNA in lymph nodes and sigmoid colon biopsies, respectively. Values were transformed in log₁₀([copies/10⁶ cells]+1) and the nonparametric Spearman’s tests were used to calculate p- and r values. G, Ratio between total and integrated HIV DNA values in lymph nodes, sigmoid colon biopsies and blood during acute infection. The ratio between total HIV DNA and integrated HIV DNA was quantified in each sample and represented by individual symbols. Samples with undetectable integrated HIV DNA (denominator = 0) were excluded from the analysis. Columns represent the median values with interquartile range. A one-way ANOVA test was performed to compare the ratio between the 3 compartments: ns p>0.05, * p≤0.05, ** p≤0.009, *** p≤0.0009, **** p≤0.0001.

Fig. 2.. ART initiation during acute infection leads to profound decreases in the frequency of infected cells in blood and tissues.

A, Longitudinal measures of total, integrated HIV DNA and 2-LTR circles in PBMCs from acutely infected participants (n=81). Samples from chronically infected participants were used as controls (total n=16, integrated n=31, 2-LTR n=13). Measurements were performed before treatment initiation and thereafter at 2, 12, 24, 36, 48, 72 and 96 weeks of ART for acutely treated participants and at week 0, 24 and 96 for chronically treated individuals. B, Longitudinal quantifications of integrated HIV DNA in participants treated in acute vs chronic infection (Fiebig I n=11, Fiebig II n=27, Fiebig III n=34, Fiebig IV/V n=9 and chronic n=31). Nonparametric paired t tests were performed to compare baseline to 12 weeks measurements in acutely treated individuals and baseline to 48 and 96 weeks measurements in chronically treated participants. Quantifications were performed at the same time points as in A. In A and B, mean values and 95% confidence interval are represented. C, Frequency of PBMCs harboring integrated HIV DNA after >24 weeks of ART (Fiebig I n=34, Fiebig II n=59, Fiebig III n=82, Fiebig IV/V n=30 and chronic n=41). D, Frequency of LNMCs harboring integrated HIV DNA after >24 weeks of ART (Fiebig I n=6, Fiebig II n=6, Fiebig III n=10, Fiebig IV/V n=7 and chronic n=5). E, Frequency of sigmoid colon cells harboring integrated HIV DNA after >24 weeks of ART (Fiebig I n=7, Fiebig II n=11, Fiebig III n=22, Fiebig IV/V n=7 and chronic n=7). In C-E, each symbol represents an individual sample. Open circles denote measurements below the limit of detection of the assay and are plotted as zero. Columns represent medians with interquartile ranges. Values are transformed in log₁₀([copies/10⁶ cells]+1). Nonparametric Mann Whitney t tests were performed to compare Fiebig I and chronic controls to all other stages: ns p>0.05, * p≤0.05, ** p≤0.009, *** p≤0.0009, **** p≤0.0001. F, Quantity of integrated HIV DNA in acutely infected and treated individuals was compared to chronic controls (black bars). Columns represent median values. Nonparametric Mann Whitney t tests were performed to compare acutely infected participants from different Fiebig stages to chronic controls: ns p>0.05, * p≤0.05, ** p≤0.009, *** p≤0.0009, **** p≤0.0001. G, Average fold decrease in the frequencies of cells harboring integrated HIV DNA in blood, sigmoid colon biopsies and lymph nodes according to the Fiebig stage after >24 weeks of ART. Columns represent mean values. H, Average fold decrease in the frequency of circulating CD4+ T cells producing tat/rev RNA after PMA/ionomycin stimulation according to the stage of infection. Columns represent mean values. In G and H the limit of detection was used to calculate the ratio for undetectable samples.

Fig. 3.. HIV reservoir in CD4+ T cells subsets before and after ART initiation in acute infection.

A-E Frequency of sorted naïve, T_CM, T_TM and T_EM harboring integrated HIV DNA before and after >24 weeks of ART according to the Fiebig stage. A, Fiebig I baseline n= 7, ART n=6 B, Fiebig II baseline n= 8, ART n=6 C, Fiebig III baseline n=8, ART n=16 D, Fiebig IV/V baseline n=6, ART n=6 E, Chronic baseline n=6, ART n=8. Nonparametric Mann Whitney t tests were performed to compare infection frequencies before and after ART in each CD4+ T cell subset according to the Fiebig stage: ns p>0.05, * p≤0.05, ** p≤0.009, *** p≤0.0009, **** p≤0.0001. Each point represents an individual sample. Open circles denote measurements below the limit of detection of the assay and are plotted as zero. Columns represent medians with interquartile range. Values were transformed in log₁₀([copies/10⁶ cells]+1). F, Average fold decrease in the frequencies of cells harboring integrated HIV DNA in T_CM, T_TM and T_EM cells according to Fiebig stage. Columns represent mean values. Limits of detection were used for the undetectable samples. G, Frequencies of naïve, T_CM, T_TM and T_EM cells harboring integrated HIV DNA after >24 weeks of ART in acutely treated individuals compared to chronically treated individuals. Columns represent median values. Open symbols denote measurements below the limit of detection of the assay and are plotted as zero. Values were transformed in log₁₀([copies/10⁶ cells]+1) and a one-way ANOVA was performed to compare the different CD4+ T cells subsets individuals treated in acute and chronic infection: ns p>0.05, * p≤0.05, ** p≤0.009, *** p≤0.0009, **** p≤0.0001.