Among recently infected individuals observed before treatment, higher CD4 T-cell counts have been observed in those bearing HIV-1 of lower viral pol replication capacity (polRC) [1,2]. In the present study, we sought to determine whether pretreatment low polRC predicted higher CD4 cell gains during combination antiretroviral therapy (ART) [3–5].
Patients were recruited during early HIV-1 infection at five north American and seven Australian sites that participate in the Acute Infection and Early Disease Research Programme (AIEDRP). An individual was determined to be eligible for this study based on previously published criteria . Patients chosen for study had achieved an HIV-1-RNA level less than 500 copies/μl at least once after initiating ART. All participants provided written informed consent.
Viral polRC was assessed via a modification of the Phenosense phenotypic drug susceptibility assay (Monogram Biosciences, formerly ViroLogic, Inc., South San Francisco, California, USA) [6,7]. The current polRC assay is calibrated and standardized differently from previously reports, so the results are not directly comparable with previous reports . The polRC of the reference virus (set at 100%) is comparable to the average value of a large population of wild-type viruses (n = 9621) (N. Parkin, Monogram Biosciences, personal communication).
All statistical analyses were performed using the SAS system for Windows version 9.2 (SAS Institute, Cary, North Carolina, USA). In the longitudinal analysis of patients receiving ART, patients were right censored at the time they stopped ART, initiated IL-2 or were lost to follow-up. Mixed effects modeling [8,9] was used to assess the relationship of baseline and longitudinal values on longitudinal outcome variables.
We selected 243 treatment-naive individuals who met the eligibility criteria for this analysis and had specimens available for testing. Of these, 127 were recruited in New York City, 74 were recruited in San Diego or Los Angeles, 32 individuals were recruited from seven Australian sites, six from Montreal, three from Denver, and one was recruited in Boston.
At the date a first ART regimen was started, the median CD4 T-cell count was 478 cells/μl [interquartile range (IQR) 353, 659, n = 214], the HIV-1-RNA level was 4.94 log10 copies/ml (IQR 3.89, 5.73, N = 231), and polRC was 93% of control (IQR 64, 116, N = 243). ART comprised two nucleoside reverse transcriptase inhibitors, at least one protease inhibitor or at least one non-nucleoside reverse transcriptase inhibitor.
We examined the relationship between polRC and the change in CD4 T-cell count during therapy in 243 individuals from all study sites. Individuals carrying a virus of higher polRC (greater than 93% of control) experienced slower increases in CD4 T-cell counts during therapy (−0.08 fewer cells/μl per day, P = 0.002) after adjustment for HIV-1-RNA levels (−49 cells/μl lower per each 1 log10 higher HIV-1 RNA; P < 0.0001) and baseline CD4 T-cell count (−0.6 cells/μl lower per each additional CD4 T cell at therapy baseline; P < 0.0001).
When modeled as a continuous variable, higher polRC was associated with lower CD4 T-cell gain during treatment (−0.0007 fewer cells/μl per day per polRC unit; P = 0.01). This result was independent of the virological response to therapy and pretherapy CD4 T-cell count. The association between lower polRC and greater CD4 T-cell count gains during therapy remained when data from patients with evidence of phenotypic drug resistance were excluded.
The difference in CD4 T-cell count and recovery occurred after 12 months of treatment (Fig. 1) and was not caused by differential periods of follow-up. When the analysis was limited to individuals remaining in the cohort for greater than 18 months we observed that these individuals had pretreatment baseline CD4 T-cell counts (median 455 CD4 T cells/μl), and HIV-1-RNA levels (median 5.23 log10 copies/ml) comparable to the entire cohort. The polRC scores tended to be lower (median 80% of control). When restricted to those with greater than either 12 or 18 months of follow-up, polRC remained a significant and independent predictor of CD4 T-cell gains during ART.
We observed that recently infected adults with lower polRC viral infections experienced greater CD4 T-cell gains during antiretroviral treatment. The advantage in CD4 T-cell recovery was observed after 12 months of continuous treatment on ART, and was independent of virological responses to therapy. PolRC was not associated with plasma RNA levels before and during therapy, which is consistent with previous reports . We employed standard clinical assays to measure the HIV-1-RNA load, which do not measure small differences in residual viral replication during therapy. Differences in very low viral replication may affect long-term CD4 T-cell recovery after 12 months of therapy [10–12].
The basis for the relationship between viral replication capacity and the late recovery of CD4 T-cell count during ART is not clear, but may relate to the level or virulence of HIV-1 infection. Stoddart and colleagues  used the severe combined immunodeficiency–human mouse model to assess the level of viral replication in human thymic tissue, by protease inhibitor resistant variants. A virus bearing a drug-resistant protease gene was not able to infect human thymic tissue in this model, nor was it able to deplete thymic explant cultures of thymocytes (CD4 or CD8 T-cell precursors). Therefore, individuals with low polRC infections may have had less injury to the thymus, which could allow for the greater repopulation of CD4 T cells in the peripheral blood during therapy.
Our study is subject to several limitations, notably a limited sample size and the limited length of clinical follow-up. In addition, we included only individuals with acute and recent infection, whereas the majority of patients currently considering therapy have longer-term infections. The clinical utility of the polRC assay in guiding the timing of initiation or change in the treatment regimen is not known and is currently being evaluated in other populations.
The authors would like to thank the patients who participated in individual AIEDRP network site studies. They wish to thank Kimberly Schafer, Bryna Block, Florin Vaida, and Shelly Sun of the AIEDRP Statistical and Coordinating Center, and Ken Wood of Frontier Sciences, for their assistance with preparation of the manuscript, data management and derivation of the final analysis data set. They also wish to thank Michael Bates of Monogram Biosciences for assistance with the manuscript, and Jon Kaldor of the University of New South Wales for comments on the manuscript. They acknowledge Monogram Biosciences (formerly Virologic, Inc.) for providing pol replication capacity, and Phenosense drug susceptibility assays.
Sponsorship: J.D.B. is supported by K01 AI066917 (Barbour) NIH/NIAID. This work was supported by AI41531 (Levy/Hecht), MO-RR00102, AI47033 and AI41534 (Markowitz), AI36214 (Richman), AI43638 and M01-RR00425 (Daar), AI55356 and AI54907 (Campbell), AI43271 (Routy for the Investigators of the Montreal PHI Study/FRSQ), AI52403 (Walker/Rosenberg), and the AIEDRP network (5 U01 AI043638).
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