Despite antiretroviral therapy, proviral latency of human immunodeficiency virus type 1 (HIV-1) remains a principal obstacle to curing the infection. Inducing the expression of latent genomes within ...resting CD4(+) T cells is the primary strategy to clear this reservoir. Although histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (also known as vorinostat, VOR) can disrupt HIV-1 latency in vitro, the utility of this approach has never been directly proven in a translational clinical study of HIV-infected patients. Here we isolated the circulating resting CD4(+) T cells of patients in whom viraemia was fully suppressed by antiretroviral therapy, and directly studied the effect of VOR on this latent reservoir. In each of eight patients, a single dose of VOR increased both biomarkers of cellular acetylation, and simultaneously induced an increase in HIV RNA expression in resting CD4(+) cells (mean increase, 4.8-fold). This demonstrates that a molecular mechanism known to enforce HIV latency can be therapeutically targeted in humans, provides proof-of-concept for histone deacetylase inhibitors as a therapeutic class, and defines a precise approach to test novel strategies to attack and eradicate latent HIV infection directly.
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.
Despite the extraordinary success of HIV-1 antiretroviral therapy in prolonging life, infected individuals face lifelong therapy because of a reservoir of latently-infected cells that harbor ...replication competent virus. Recently, compounds have been identified that can reverse HIV-1 latency in vivo. These latency- reversing agents (LRAs) could make latently-infected cells vulnerable to clearance by immune cells, including cytolytic CD8+ T cells. We investigated the effects of two leading LRA classes on CD8+ T cell phenotype and function: the histone deacetylase inhibitors (HDACis) and protein kinase C modulators (PKCms). We observed that relative to HDACis, the PKCms induced much stronger T cell activation coupled with non-specific cytokine production and T cell proliferation. When examining antigen-specific CD8+ T cell function, all the LRAs except the HDACi Vorinostat reduced, but did not abolish, one or more measurements of CD8+ T cell function. Importantly, the extent and timing of these effects differed between LRAs. Panobinostat had detrimental effects within 10 hours of drug treatment, whereas the effects of the other LRAs were observed between 48 hours and 5 days. These observations suggest that scheduling of LRA and CD8+ T cell immunotherapy regimens may be critical for optimal clearance of the HIV-1 reservoir.
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.
In 1998 a collaboration between Duke University and the University of North Carolina, Chapel Hill (UNC) was founded to enhance identification of persons with acute HIV-1 infection (AHI). The Duke-UNC ...AHI Research Consortium Cohort consists of patients ≥18 years old with a positive nucleic acid amplification test (NAAT) and either a negative enzyme immunoassay (EIA) test or a positive EIA with a negative/indeterminate Western blot. Patients were referred to the cohort from acute care settings and state-funded HIV testing sites that use NAAT testing on pooled HIV-1 antibody-negative samples. Between 1998 and 2010, 155 patients with AHI were enrolled: 81 (52%) African-Americans, 63 (41%) white, non-Hispanics, 137 (88%) males, 108 (70%) men who have sex with men (MSM), and 18 (12%) females. The median age was 27 years (IQR 22-38). Most (n=138/155) reported symptoms with a median duration of 17.5 days. The median nadir CD4 count was 408 cells/mm(3) (IQR 289-563); the median observed peak HIV-1 level was 726,859 copies/ml (IQR 167,585-3,565,728). The emergency department was the most frequent site of initial presentation (n=55/152; 3 missing data). AHI diagnosis was made at time of first contact in 62/137 (45%; 18 missing data) patients. This prospectively enrolled cohort is the largest group of patients with AHI reported from the Southeastern United States. The demographics reflect the epidemic of this geographic area with a high proportion of African-Americans, including young black MSM. Highlighting the challenges of diagnosing AHI, less than half of the patients were diagnosed at the first healthcare visit. Women made up a small proportion despite increasing numbers in our clinics.
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.
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.
Acute 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.
Cross-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.
Thirty-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.
The 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.
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.
PDF
