The Effects of Human Immunodeficiency Virus Type 1 (HIV-1) Antigen-Expanded Specific T-Cell Therapy and Vorinostat on Persistent HIV-1 Infection in People With HIV on Antiretroviral Therapy.

BACKGROUND: The histone deacetylase inhibitor vorinostat (VOR) can reverse human immunodeficiency virus type 1 (HIV-1) latency in vivo and allow T cells to clear infected cells in vitro. HIV-specific T cells (HXTCs) can be expanded ex vivo and have been safely administered to people with HIV (PWH) on antiretroviral therapy. METHODS: Six PWH received infusions of 2 × 107 HXTCs/m² with VOR 400 mg, and 3 PWH received infusions of 10 × 107 HXTCs/m² with VOR. The frequency of persistent HIV by multiple assays including quantitative viral outgrowth assay (QVOA) of resting CD4+ T cells was measured before and after study therapy. RESULTS: VOR and HXTCs were safe, and biomarkers of serial VOR effect were detected, but enhanced antiviral activity in circulating cells was not evident. After 2 × 107 HXTCs/m² with VOR, 1 of 6 PWH exhibited a decrease in QVOA, and all 3 PWH exhibited such declines after 10 × 107 HXTCs/m² and VOR. However, most declines did not exceed the 6-fold threshold needed to definitively attribute decline to the study intervention. CONCLUSIONS: These modest effects provide support for the strategy of HIV latency reversal and reservoir clearance, but more effective interventions are needed to yield the profound depletion of persistent HIV likely to yield clinical benefit. Clinical Trials Registration. NCT03212989.

CD3 downregulation identifies high-avidity human CD8 T cells.

CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.

Stable Latent HIV Infection and Low-level Viremia Despite Treatment With the Broadly Neutralizing Antibody VRC07-523LS and the Latency Reversal Agent Vorinostat.

We tested the combination of a broadly neutralizing HIV antibody with the latency reversal agent vorinostat (VOR). Eight participants received 2 month-long cycles of VRC07-523LS with VOR. Low-level viremia, resting CD4+ T-cell-associated HIV RNA (rca-RNA) was measured, and intact proviral DNA assay (IPDA) and quantitative viral outgrowth assay (QVOA) were performed at baseline and posttreatment. In 3 participants, IPDA and QVOA declines were accompanied by significant declines of rca-RNA. However, no IPDA or QVOA declines clearly exceeded assay variance or natural decay. Increased resistance to VRC07-523LS was not observed. This combination therapy did not reduce viremia or the HIV reservoir. Clinical Trials Registration. NCT03803605.

Sequence Evaluation and Comparative Analysis of Novel Assays for Intact Proviral HIV-1 DNA.

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

Longitudinal Dynamics of Intact HIV Proviral DNA and Outgrowth Virus Frequencies in a Cohort of Individuals Receiving Antiretroviral Therapy.

BACKGROUND: The replication-competent human immunodeficiency virus (HIV) reservoir is the major barrier to cure. The quantitative viral outgrowth assay (QVOA), the gold-standard method to quantify replication-competent HIV, is resource intensive, which limits its application in large clinical trials. The intact proviral DNA assay (IPDA) requires minimal cell input relative to QVOA and quantifies both defective and intact proviral HIV DNA, the latter potentially serving as a surrogate marker for replication-competent provirus. However, there are limited cross-sectional and longitudinal data on the relationship between IPDA and QVOA measurements. METHODS: QVOA and IPDA measurements were performed on 156 resting CD4 T-cell (rCD4) samples from 83 antiretroviral therapy-suppressed HIV-positive participants. Longitudinal QVOA and IPDA measurements were performed on rCD4 from 29 of these participants. RESULTS: Frequencies of intact, defective, and total proviruses were positively associated with frequencies of replication-competent HIV. Longitudinally, decreases in intact proviral frequencies were strikingly similar to that of replication-competent virus in most participants. In contrast, defective proviral DNA frequencies appeared relatively stable over time in most individuals. CONCLUSIONS: Changes in frequencies of IPDA-derived intact proviral DNA and replication-competent HIV measured by QVOA are similar. IPDA is a promising high-throughput approach to estimate changes in the frequency of the replication-competent reservoir.

Impact of Biological Sex on Immune Activation and Frequency of the Latent HIV Reservoir During Suppressive Antiretroviral Therapy.

BACKGROUND: Persistent HIV infection of long-lived resting CD4 T cells, despite antiretroviral therapy (ART), remains a barrier to HIV cure. Women have a more robust type 1 interferon response during HIV infection relative to men, contributing to lower initial plasma viremia. As lower viremia during acute infection is associated with reduced frequency of latent HIV infection, we hypothesized that women on ART would have a lower frequency of latent HIV compared to men. METHODS: ART-suppressed, HIV seropositive women (n = 22) were matched 1:1 to 22 of 39 ART-suppressed men. We also compared the 22 women to all 39 men, adjusting for age and race as covariates. We measured the frequency of latent HIV using the quantitative viral outgrowth assay, the intact proviral DNA assay, and total HIV gag DNA. We also performed activation/exhaustion immunophenotyping on peripheral blood mononuclear cells and quantified interferon-stimulated gene (ISG) expression in CD4 T cells. RESULTS: We did not observe evident sex differences in the frequency of persistent HIV in resting CD4 T cells. Immunophenotyping and CD4 T-cell ISG expression analysis revealed marginal differences across the sexes. CONCLUSIONS: Differences in HIV reservoir frequency and immune activation appear to be small across sexes during long-term suppressive therapy.

HIV-Specific, Ex Vivo Expanded T Cell Therapy: Feasibility, Safety, and Efficacy in ART-Suppressed HIV-Infected Individuals.

Adoptive T cell therapy has had dramatic successes in the treatment of virus-related malignancies and infections following hematopoietic stem cell transplantation. We adapted this method to produce ex vivo expanded HIV-specific T cells (HXTCs), with the long-term goal of using HXTCs as part of strategies to clear persistent HIV infection. In this phase 1 proof-of-concept study (NCT02208167), we administered HXTCs to antiretroviral therapy (ART)-suppressed, HIV-infected participants. Participants received two infusions of 2 × 107 cells/m2 HXTCs at a 2-week interval. Leukapheresis was performed at baseline and 12 weeks post-infusion to measure the frequency of resting cell infection by the quantitative viral outgrowth assay (QVOA). Overall, participants tolerated HXTCs, with only grade 1 adverse events (AEs) related to HXTCs. Two of six participants exhibited a detectable increase in CD8 T cell-mediated antiviral activity following the two infusions in some, but not all, assays. As expected, however, in the absence of a latency reversing agent, no meaningful decline in the frequency of resting CD4 T cell infection was detected. HXTC therapy in ART-suppressed, HIV-infected individuals appears safe and well tolerated, without any clinical signs of immune activation, likely due to the low residual HIV antigen burden present during ART.

Integration of Contact Tracing and Phylogenetics in an Investigation of Acute HIV Infection.

BACKGROUND: The integration of traditional contact tracing with HIV sequence analyses offers opportunities to mitigate some of the barriers to effective network construction. We used combined analyses during an outbreak investigation of spatiotemporally clustered acute HIV infections to evaluate if the observed clustering was the product of a single outbreak. METHODS: We investigated acute and recent HIV index cases reported in North Carolina from 2013 to 2014 and their reported contacts. Contact tracing networks were constructed with surveillance data and compared with phylogenetic transmission clusters involving an index case using available HIV-1 pol sequences including 1672 references. Clusters were defined as clades of 2 or more sequences with a less than 1.5% genetic distance and a bootstrap of at least 98% on maximum-likelihood phylogenies. RESULTS: In total, 68 index cases and 210 contacts (71 HIV infected) were reported. The contact tracing network involved 58 components with low overall density (1.2% statewide); 33% of first-degree contacts could not be located. Among 38 (56%) of 68 index cases and 34 (48%) of 71 contacts with sequences, 13 phylogenetic clusters were identified (size 2-4 members). Four clusters connected network components that were not linked in contact tracing. The largest component (n = 28 cases) included 2 distinct phylogenetic clusters and spanned 2 regions. CONCLUSIONS: We identified the concurrent expansion of multiple small transmission clusters rather than a single outbreak in a largely disconnected contact tracing network. Integration of phylogenetic analyses provided timely information on transmission networks during the investigation. Our findings highlight the potential of combined methods to better identify high-risk networks for intervention.

Peripheral Vγ9Vδ2 T Cells Are a Novel Reservoir of Latent HIV Infection.

Eradication of HIV infection will require the identification of all cellular reservoirs that harbor latent infection. Despite low or lack of CD4 receptor expression on Vδ2 T cells, infection of these cells has previously been reported. We found that upregulation of the CD4 receptor may render primary Vδ2 cells target for HIV infection in vitro and we propose that HIV-induced immune activation may allow infection of γδ T cells in vivo. We assessed the presence of latent HIV infection by measurements of DNA and outgrowth assays within Vδ2 cells in 18 aviremic patients on long-standing antiretroviral therapy. In 14 patients we recovered latent but replication-competent HIV from highly purified Vδ2 cells demonstrating that peripheral Vδ2 T cells are a previously unrecognized reservoir in which latent HIV infection is unexpectedly frequent.

Broadly-specific cytotoxic T cells targeting multiple HIV antigens are expanded from HIV+ patients: implications for immunotherapy.

Antiretroviral therapy (ART) is unable to eradicate human immunodeficiency virus type 1 (HIV-1) infection. Therefore, there is an urgent need to develop novel therapies for this disease to augment anti-HIV immunity. T cell therapy is appealing in this regard as T cells have the ability to proliferate, migrate, and their antigen specificity reduces the possibility of off-target effects. However, past human studies in HIV-1 infection that administered T cells with limited specificity failed to provide ART-independent, long-term viral control. In this study, we sought to expand functional, broadly-specific cytotoxic T cells (HXTCs) from HIV-infected patients on suppressive ART as a first step toward developing cellular therapies for implementation in future HIV eradication protocols. Blood samples from seven HIV+ patients on suppressive ART were used to derive HXTCs. Multiantigen specificity was achieved by coculturing T cells with antigen-presenting cells pulsed with peptides representing Gag, Pol, and Nef. All but two lines were multispecific for all three antigens. HXTCs demonstrated efficacy as shown by release of proinflammatory cytokines, specific lysis of antigen-pulsed targets, and the ability to suppress HIV replication in vitro. In conclusion, we are able to generate broadly-specific cytotoxic T cell lines that simultaneously target multiple HIV antigens and show robust antiviral function.

Precise Quantitation of the Latent HIV-1 Reservoir: Implications for Eradication Strategies.

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.

Quantitation of replication-competent HIV-1 in populations of resting CD4+ T cells.

UNLABELLED: Central memory (TCM) 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 TCM 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.

Immediate antiviral therapy appears to restrict resting CD4+ cell HIV-1 infection without accelerating the decay of latent infection.

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-1 expression within resting CD4+ T cells after multiple doses of vorinostat.

BACKGROUND: A single dose of the histone deacetylase inhibitor vorinostat (VOR) up-regulates HIV RNA expression within resting CD4(+) T cells of treated, aviremic human immunodeficiency virus (HIV)-positive participants. The ability of multiple exposures to VOR to repeatedly disrupt latency has not been directly measured, to our knowledge. METHODS: Five participants in whom resting CD4(+) T-cell-associated HIV RNA (rc-RNA) increased after a single dose of VOR agreed to receive daily VOR Monday through Wednesday for 8 weekly cycles. VOR serum levels, peripheral blood mononuclear cell histone acetylation, plasma HIV RNA single-copy assays, rc-RNA, total cellular HIV DNA, and quantitative viral outgrowth assays from resting CD4(+) T cells were assayed. RESULTS: VOR was well tolerated, with exposures within expected parameters. However, rc-RNA measured after dose 11 (second dose of cycle 4) or dose 22 (second dose of cycle 8) increased significantly in only 3 of the 5 participants, and the magnitude of the rc-RNA increase was much reduced compared with that after a single dose. Changes in histone acetylation were blunted. Results of quantitative viral outgrowth and other assays were unchanged. CONCLUSIONS: Although HIV latency is disrupted by an initial VOR dose, the effect of subsequent doses in this protocol was much reduced. We hypothesize that the global effect of VOR results in a refractory period of ≥ 24 hours. The optimal schedule for VOR administration is still to be defined.

"No one's at home and they won't pick up the phone": using the Internet and text messaging to enhance partner services in North Carolina.

BACKGROUND: The Internet and mobile devices are increasingly used by men who have sex with men to find potential partners. Lack of partner information, besides e-mail addresses or user profiles, limits the ability to adequately perform partner notification by traditional means and test those at high risk. To streamline North Carolina Internet Partner Notification (IPN) services, University of North Carolina at Chapel Hill collaborated with the North Carolina Division of Public Health beginning in July 2011 to formalize state IPN and text messaging for partner notification (txtPN) policies and centralize notification practices by designating a single IPN/txtPN field coordinator within the University of North Carolina at Chapel Hill. METHODS: We compared the number of IPN and txtPN contacts initiated and their outcomes in July 1, 2011, to June 30, 2012, and compared with outcomes in January 1, 2010, to December 31, 2010, the year before the collaboration. RESULTS: Overall, 362 IPN contacts were initiated compared with 133 initiated in 2010. More than half (59.1%) were black; mean age was 28.8 years. Almost all were men who have sex with men (83.7%). Approximately two-thirds (n = 230; 63.5%) of contacts were successfully notified using centralized IPN. Seven new cases of HIV infection, 11 new cases of syphilis, and 19 known previous HIV-positive persons were identified. Text messaging for partner notification was used for 29 contacts who did not initially respond to traditional notification or IPN; 14 (48%) responded to txtPN in a median time of 57.5 minutes (interquartile range, 9-2708). CONCLUSIONS: Centralization of IPN services augmented partner detection of new HIV and syphilis diagnoses. Text messaging for partner notification represents a potentially effective method for augmenting traditional partner services. In addition, IPN and txtPN allow identification of HIV-infected persons in need of linkage to care.

Incident sexually transmitted infection as a biomarker for high-risk sexual behavior after diagnosis of acute HIV.

BACKGROUND: Sexually transmitted infection (STI) diagnosis after diagnosis of acute HIV infection (AHI) indicates ongoing high-risk sexual behavior and possible risk of HIV transmission. We assessed predictors of STI acquisition and the effect of time since care entry on STI incidence in patients with AHI in care and receiving consistent risk-reduction messaging. METHODS: Data on incident gonorrhea, chlamydia, trichomoniasis, primary/secondary syphilis, demographic, and clinical risk factors were abstracted from medical charts for patients diagnosed as having AHI and engaged in care. Poisson regression models using generalized estimating equations were fit to estimate incidence rates (IRs), IR ratios, and robust 95% confidence intervals. RESULTS: Among 185 patients with AHI, 26 (14%) were diagnosed as having at least 1 incident STI over 709.4 person-years; 46 STIs were diagnosed during follow-up (IR, 6.8/100 person-years). The median time from HIV care entry to first STI diagnosis was 609 days (range, 168-1681 days). Men who have sex with men (P = 0.03), a shorter time between presentation to medical care and AHI diagnosis (P = 0.06), and STI diagnosis before AHI diagnosis (P = 0.0003) were predictors of incident STI. Sexually transmitted infection IR greater than 1 year after entering care was double that of patients in care 1 year or less (IR ratio, 2.0; 95% confidence interval, 0.8-4.9). HIV viral load was above the limits of detection within 1 month of 11 STI diagnoses in 6 patients (23.1%) (median, 15,898 copies/mL; range, 244-152,000 copies/mL). CONCLUSIONS: Despite regular HIV care, STI incidence was high among this primarily young, men who have sex with men AHI cohort. Early antiretroviral initiation may decrease HIV transmission given ongoing risk behaviors despite risk-reduction messaging.

CD4+CD8+ T cells represent a significant portion of the anti-HIV T cell response to acute HIV infection.

Previous studies have revealed that HIV-infected individuals possess circulating CD4(+)CD8(+) double-positive (DP) T cells specific for HIV Ags. In the present study, we analyzed the proliferation and functional profile of circulating DP T cells from 30 acutely HIV-infected individuals and 10 chronically HIV-infected viral controllers. The acutely infected group had DP T cells that showed more proliferative capability and multifunctionality than did both their CD4(+) and CD8(+) T cells. DP T cells were found to exhibit greater proliferation and higher multifunctionality compared with CD4 T cells in the viral controller group. The DP T cell response represented 16% of the total anti-HIV proliferative response and >70% of the anti-HIV multifunctional response in the acutely infected subjects. Proliferating DP T cells of the acutely infected subjects responded to all HIV Ag pools with equal magnitude. Conversely, the multifunctional response was focused on the pool representing Nef, Rev, Tat, VPR, and VPU. Meanwhile, the controllers' DP T cells focused on Gag and the Nef, Rev, Tat, VPR, and VPU pool for both their proliferative and multifunctional responses. Finally, we show that the presence of proliferating DP T cells following all HIV Ag stimulations is well correlated with proliferating CD4 T cells whereas multifunctionality appears to be largely independent of multifunctionality in other T cell compartments. Therefore, DP T cells represent a highly reactive cell population during acute HIV infection, which responds independently from the traditional T cell compartments.

Sequence Analysis of Inducible, Replication-Competent Virus Reveals No Evidence of HIV-1 Evolution During Suppressive Antiviral Therapy, Indicating a Lack of Ongoing Viral Replication.

Background: Persistence of HIV-1 in reservoirs necessitates life-long antiretroviral therapy (ART). There are conflicting data using genetic analysis on whether persistence includes an actively replicating reservoir with strong evidence arguing against replication. Methods: We investigated the possibility of ongoing viral evolution during suppressive therapy by comparing near full-length viral genomic sequences using phylogenetic analysis of viral RNA in plasma before therapy initiation early after infection and from virus induced to grow from the latent reservoir after a period of suppressive ART. We also focused our analysis on evidence of selective pressure by drugs in the treatment regimen and at sites of selective pressure by the adaptive immune response. Results: Viral genomes induced to grow from the latent reservoir from 10 participants with up to 9 years on suppressive ART were highly similar to the nearly homogeneous sequences in plasma taken early after infection at ART initiation. This finding was consistent across the entire genome and when the analysis focused on sites targeted by the drug regimen and by host selective pressure of antibody and cytotoxic T cells. The lack of viral evolution away from pretherapy sequences in spite of demonstrated selective pressure is most consistent with a lack of viral replication during reservoir persistence. Conclusions: These results do not support ongoing viral replication as a mechanism of HIV-1 persistence during suppressive ART.

Dominant CD4+ T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV.

In people with HIV (PWH), the post-antiretroviral therapy (ART) window is critical for immune restoration and HIV reservoir stabilization. We employ deep immune profiling and T cell receptor (TCR) sequencing and examine proliferation to assess how ART impacts T cell homeostasis. In PWH on long-term ART, lymphocyte frequencies and phenotypes are mostly stable. By contrast, broad phenotypic changes in natural killer (NK) cells, γδ T cells, B cells, and CD4+ and CD8+ T cells are observed in the post-ART window. Whereas CD8+ T cells mostly restore, memory CD4+ T subsets and cytolytic NK cells show incomplete restoration 1.4 years post ART. Surprisingly, the hierarchies and frequencies of dominant CD4 TCR clonotypes (0.1%-11% of all CD4+ T cells) remain stable post ART, suggesting that clonal homeostasis can be independent of homeostatic processes regulating CD4+ T cell absolute number, phenotypes, and function. The slow restoration of host immunity post ART also has implications for the design of ART interruption studies.

Clonal sequences recovered from plasma from patients with residual HIV-1 viremia and on intensified antiretroviral therapy are identical to replicating viral RNAs recovered from circulating resting CD4+ T cells.

Despite successful antiretroviral therapy (ART), low-level viremia (LLV) may be intermittently detected in most HIV-infected patients. Longitudinal blood plasma and resting CD4(+) T cells were obtained from two patients on suppressive ART to investigate the source of LLV. Single-genome sequencing of HIV-1 env from LLV plasma was performed, and the sequences were compared to sequences recovered from limiting-dilution outgrowth assays of resting CD4(+) T cells. The circulating LLV virus clone was identical to virus recovered from outgrowth assays from pools of millions of resting CD4(+) T cells. Understanding the sources of LLV requires evaluation of all possible reservoirs of persistent HIV infection.