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    • Total and integrated HIV DNA were measured in triplicates in

    2018-11-09

    Total and integrated HIV DNA were measured in triplicates in PBMCs using a modified nested PCR assay for CRF01_AE and B (Vandergeeten et al., 2014), and the levels were log10 transformed prior to analysis. Differences between groups were assessed using two-tailed Student\'s t-test. Generalized Estimating Equations were used to assess factors associated with proviral burden. The model was constructed using an exchangeable correlation matrix. Analyses were performed using StataCorp 2013 (StataCorp LP, College Station, TX). Figures were generated with Prism version 6.02 for Windows (GraphPad Software, La Jolla, CA).
    Results The RV217 untreated cohort started sampling at a median (IQR) of 1 (1–7) day from the first documented HIV RNA in the study (median of 13,183 copies/ml). The RV254 treated participants enrolled at a median (IQR) of 16 (12−21) days from the history of HIV exposure. The plasma HIV RNA kinetics in Fig. 1 illustrates the peak viremia at week 2 and set-point at week 4 from enrollment in the untreated group. In contrast, viremia declined rapidly after treatment referred to in RV254, and the proportions with HIV RNA<50 copies/ml were 90% at week 24, 99% at week 48 and 97% at week 144. The frequencies of PBMCs harboring total HIV DNA are shown in Fig. 2, which illustrates 3 important points 1) In the untreated RV217 cohort, the total HIV DNA values rose rapidly in the first 2weeks after enrollment without significant changes thereafter. 2) In the RV254 treated cohort, the total HIV DNA values decreased over time reaching very low levels at week 144. 3) There was a marked divergence of total HIV DNA values between the untreated and treated cohorts that occurred early and this increased with time (Table 2). The differences were 1.3 log or 20-fold at week 2 following enrollment, and 2.5 log or 316-fold by week 144. The integrated DNA values rose rapidly and peaked at week 2 followed by a significant decline at week 4 after which there was a gradual increase in integrated HIV DNA resulting in significantly higher integrated HIV DNA at week 144 compared to week 4 (p=0.02) (Fig. 2). By week 2, the treated cohort had 1.4 log or 25-fold lower integrated HIV DNA and by week 144, the difference was 2 log or 100-fold (Table 2). When only participants at the earliest AHI stages of Fiebig I/II were included (17 in RV217 and 33 in RV254), similar findings for total and integrated HIV DNA were observed as compared to the whole group (Fig. 3). The frequency of cells harboring 2-LTR circles at baseline was higher in the RV254 group (Fig. 2 and Table 1) likely because of lower proportions of Fiebig I/II individuals in RV254 than in RV217. The baseline levels of 2-LTR circles did not differ between cohorts if only the Fiebig I/II participants were included: mean (SD) 0.7 (0.8) in RV254 vs. 0.4 (0.7) in RV217, p=0.19 (Fig. 3). The levels subsequently declined significantly after 48weeks of ART (p<0.0001), whereas, in the untreated individuals, 2-LTR circles rose quickly between week 0 and week 2 (p=0.006) and remained high throughout the follow up period (Fig. 2, Fig. 3 and Table 2). As CD4+ T cell values differ significantly over time between the untreated vs. the treated groups, we also calculated the frequencies of CD4+ T cells that harbor total and integrated HIV DNA and 2-LTR circles using information of frequencies of CD4+ T cells from complete blood count (Supplemental Table 1). This resulted in greater differences between the treated and untreated cohorts with a 1585-fold difference in total HIV DNA and 501-fold difference in integrated HIV DNA observed at week 144. Two baseline factors were strongly associated with total HIV DNA at week 144 in the multivariate analysis: Total HIV DNA (coeff 0.50, 95%CI 0.44 to 0.57, p<0.001) and ART (coeff −1.39, 95%CI −1.14 to −1.64, p<0.001). Similar relationships were observed for integrated HIV DNA at week 144: baseline integrated HIV DNA (coeff 0.57, 95%CI 0.47 to 0.68, p<0.001) and ART (coeff −1.20, 95%CI −0.91 to −1.48, p<0.001). HIV DNA outcomes did not differ for the 3- vs. 5-drug regimens. In the RV217 cohort, peak HIV RNA did not correlate with total HIV DNA at set-point (week 4) (r=0.17, p=0.55) or at week 48 (r=0.11, p=0.68).