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.
Abstract
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.
Decay of the HIV reservoir in resting CD4 T cells measured using the intact proviral DNA assay closely resembles that measured using the quantitative viral outgrowth assay.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The histone deacetylase (HDAC) inhibitor vorinostat (VOR) can increase HIV RNA expression in vivo within resting CD4+ T cells of aviremic HIV+ individuals. However, while studies of VOR or other HDAC ...inhibitors have reported reversal of latency, none has demonstrated clearance of latent infection. We sought to identify the optimal dosing of VOR for effective serial reversal of HIV latency.
In a study of 16 HIV-infected, aviremic individuals, we measured resting CD4+ T cell-associated HIV RNA ex vivo and in vivo following a single exposure to VOR, and then in vivo after a pair of doses separated by 48 or 72 hours, and finally following a series of 10 doses given at 72-hour intervals.
Serial VOR exposures separated by 72 hours most often resulted in an increase in cell-associated HIV RNA within circulating resting CD4+ T cells. VOR was well tolerated by all participants. However, despite serial reversal of latency over 1 month of VOR dosing, we did not observe a measurable decrease (>0.3 log10) in the frequency of latent infection within resting CD4+ T cells.
These findings outline parameters for the experimental use of VOR to clear latent infection. Latency reversal can be achieved by VOR safely and repeatedly, but effective depletion of persistent HIV infection will require additional advances. In addition to improvements in latency reversal, these advances may include the sustained induction of potent antiviral immune responses capable of recognizing and clearing the rare cells in which HIV latency has been reversed.
Clinicaltrials.gov NCT01319383.
NIH grants U01 AI095052, AI50410, and P30 CA016086 and National Center for Advancing Translational Sciences grant KL2 TR001109.
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.
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.
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 mononudear 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.
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.
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.
Abstract
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.
The HIV latency reversal agent vorinostat was well tolerated when given with expanded, autologous, HIV-specific cytotoxic T cells, but only modest effects were seen on the pool of latently infected cells that persist in people with HIV on therapy.
HIV transmission is influenced by status awareness and receipt of care and treatment. We analyzed these attributes of named partners of persons with acute HIV infection (index AHI cases) to ...characterize the transmission landscape in North Carolina (NC).
Secondary analysis of programmatic data.
We used data from the NC Screening and Tracing of Active Transmission Program (2002-2013) to determine HIV status (uninfected, AHI, or chronic HIV infection CHI), diagnosis status (new or previously-diagnosed), and care and treatment status (not in care, in care and not on treatment, in care and on treatment) of index AHI cases' named partners. We developed an algorithm identifying the most likely transmission source among known HIV-infected partners to estimate the proportion of transmissions arising from contact with persons at different HIV continuum stages. We conducted a complementary analysis among a subset of index AHI cases and partners with phylogenetically-linked viruses.
Overall, 358 index AHI cases named 932 partners, of which 218 were found to be HIV-infected (162 (74.3%) previously-diagnosed, 11 (5.0%) new AHI, 45 (20.6%) new CHI). Most transmission events appeared attributable to previously-diagnosed partners (77.4%, 95% confidence interval 69.4-85.3%). Among these previously-diagnosed partners, 23.2% (14.0-32.3%) were reported as in care and on treatment near the index AHI case diagnosis date. In the subset study of 33 phylogenetically-linked cases and partners, 60.6% of partners were previously diagnosed (43.9-77.3%).
A substantial proportion of HIV transmission in this setting appears attributable to contact with previously-diagnosed partners, reinforcing the need for improved engagement in care after diagnosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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