The quantitative viral outgrowth assay (QVOA) provides a precise minimal estimate of the reservoir of resting CD4 + T-cell infection (resting cell infection [RCI]). However, the variability of RCI over time during antiretroviral therapy (ART), relevant to assess potential effects of latency-reversing agents or other interventions, has not been fully described. We performed QVOA on resting CD4 + T cells obtained via leukapheresis from 37 human immunodeficiency virus (HIV)-infected patients receiving stable suppressive ART for a period of 6 years. Patients who started ART during acute (n = 17) or chronic (n = 20) HIV infection were studied once HIV RNA levels were <50 copies/mL for ≥6 months. Using random effects analysis of 160 RCI measurements, we found that RCI declined significantly over time (P < .001), with an estimated mean half-life of 3.6 years (95% confidence interval, 2.3-8.1 years), remarkably consistent with findings of prior studies. There was no evidence of more rapid decay in acute versus chronic HIV infection (P = .99) for patients suppressed ≥6 months. RCI was reliably estimated with longitudinal measurements generally showing <2-fold variation from the previous measure. When QVOA is performed in this format, RCI decreases of >6-fold were rare. We suggest that a 6-fold decline is a relevant threshold to reliably identify effects of antilatency interventions on RCI.

HIV type 1 (HIV-1) persists within resting CD4 + T cells despite antiretroviral therapy (ART). To better understand the kinetics by which resting cell infection (RCI) is established, we developed a mathematical model that accurately predicts (r = 0.65, P = 2.5 × 10 −4 ) the initial frequency of RCI measured about 1 year postinfection, based on the time of ART initiation and the dynamic changes in viremia and CD4 + T cells. In the largest cohort of patients treated during acute seronegative HIV infection (AHI) in whom RCI has been stringently quantified, we found that early ART reduced the generation of latently infected cells. Although RCI declined after the first year of ART in most acutely infected patients, there was a striking absence of decline when initial RCI frequency was less than 0.5 per million. Notably, low-level viremia was observed more frequently as RCI increased. Together these observations suggest that (i) the degree of RCI is directly related to the availability of CD4 + T cells susceptible to HIV, whether viremia is controlled by the immune response and/or ART; and (ii) that two pools of infected resting CD4 + T cells exist, namely, less stable cells, observable in patients in whom viremia is not well controlled in early infection, and extremely stable cells that are established despite early ART. These findings reinforce and extend the concept that new approaches will be needed to eradicate HIV infection, and, in particular, highlight the need to target the extremely small but universal, long-lived latent reservoir.HIV latency | viral kinetics

Central memory (T CM ) CD4؉ T cells are the principal reservoir of latent HIV-1 infection that persists despite durable, successful antiretroviral therapy (ART). In a study that measured HIV DNA in 17 patients and replication-competent HIV in 4 patients, pools of resting and activated transitional memory (T TM ) CD4؉ T cells were found to be a reservoir for HIV infection. As defective viruses account for the majority of integrated HIV DNA and do not reflect the actual frequency of latent, replication-competent proviral infection, we assessed the specific contribution of resting T TM cells to latent HIV infection. We measured the frequency of replication-competent HIV in purified resting memory cell subpopulations by a limiting-dilution, quantitative viral outgrowth assay (QVOA). HIV was routinely detected within the resting central memory compartment but was infrequently detected within the resting T TM compartment. These observations suggest that prolonged ART may limit persistent latent infection in the T TM compartment. Our results confirm the importance of latent infection within the T CM compartment and again focus attention on these cells as the most important latent viral reservoir. While proliferation may drive expansion of detectable viral genomes in cells, the frequency of replication-competent HIV must be carefully assessed. Latent infection appears to wane within the transitional memory compartment in patients who have sustained successful viral suppression via ART or were treated very early in infection. IMPORTANCE Antiretroviral therapy (ART) has led to a significant decrease in morbidity and mortality among HIV-infected patients. However, HIV integrates into the genome of CD4 ؉ T cells, generating pools of long-lived cells that are reservoirs of latent HIV. Two main subsets of CD4؉ T cells, central memory and transitional memory cells, were reported to be major reservoirs of HIV infection. However, this study primarily measured the HIV DNA content, which also includes defective proviruses that would not be able to replicate and initiate new rounds of infection. By analyzing the replication-competent virus in both cell subsets, we showed that transitional memory cells may not be a durable reservoir in patients on successful ART.

BackgroundAcute HIV infection (AHI) is a critical phase of infection when irreparable damage to the immune system occurs and subjects are very infectious. We studied subjects with AHI prospectively to develop better treatment and public health interventions.MethodsCross-sectional screening was employed to detect HIV RNA positive, antibody negative subjects. Date of HIV acquisition was estimated from clinical history and correlated with sequence diversity assessed by single genome amplification (SGA). Twenty-two cytokines/chemokines were measured from enrollment through week 24.ResultsThirty-seven AHI subjects were studied. In 7 participants with limited exposure windows, the median exposure to HIV occurred 14 days before symptom onset. Lack of viral sequence diversification confirmed the short duration of infection. Transmission dates estimated by SGA/sequencing using molecular clock models correlated with transmission dates estimated by symptom onset in individuals infected with single HIV variants (mean of 28 versus 33 days). Only 10 of 22 cytokines/chemokines were significantly elevated among AHI participants at enrollment compared to uninfected controls, and only 4 participants remained seronegative at enrollment.DiscussionThe results emphasize the difficulty in recruiting subjects early in AHI. Viral sequence diversity proved accurate in estimating time of infection. Regardless of aggressive screening, peak viremia and inflammation occurred before enrollment and potential intervention. Given the personal and public health importance, improved AHI detection is urgently needed.

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