Maria J. Buzon

HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects

Highly active antiretroviral therapy (HAART) results in potent and durable suppression of HIV-1 viremia. However, HIV-1 replication resumes if therapy is interrupted. Although it is generally believed that active replication has been halted in individuals on HAART, immune activation and inflammation continue at abnormal levels, suggesting continued, low-level viral replication. To assess whether active replication might be driving immune activation in HAART, we examined the impact of treatment intensification with the integrase inhibitor raltegravir on viral complementary DNA and immune activation parameters. In the presence of raltegravir, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNAs. Raltegravir intensification of a three-drug suppressive HAART regimen resulted in a specific and transient increase in episomal DNAs in a large percentage of HAART-suppressed subjects. Furthermore, in subjects with these episomal DNAs, immune activation was higher at baseline and was subsequently normalized after raltegravir intensification. These results suggest that, despite suppressive HAART, active replication persists in some infected individuals and drives immune activation. The ability of raltegravir intensification to perturb the reservoir that supports active replication has implications for therapeutic strategies aimed at achieving viral eradication.

Panobinostat, a histone deacetylase inhibitor, for latent- virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial

BACKGROUND Activating the expression of latent virus is an approach that might form part of an HIV cure. We assessed the ability of the histone deacetylase inhibitor panobinostat to disrupt HIV-1 latency and the safety of this strategy. METHODS In this phase 1/2 clinical trial, we included aviraemic adults with HIV treated at Aarhus University Hospital, Denmark. Participants received oral panobinostat (20 mg) three times per week every other week for 8 weeks while maintaining combination antiretroviral therapy. The primary outcome was change from baseline of cell-associated unspliced HIV RNA. Secondary endpoints were safety, plasma HIV RNA, total and integrated HIV DNA, infectious units per million CD4 T cells, and time to viral rebound during an optional analytical treatment interruption of antiretroviral therapy. This trial is registered with ClinicalTrial.gov, number NCT01680094. FINDINGS We enrolled 15 patients. The level of cell-associated unspliced HIV RNA increased significantly at all timepoints when patients were taking panobinostat (p < 0·0001). The median maximum increase in cell-associated unspliced HIV RNA during panobinostat treatment was 3·5-fold (range 2·1-14·4). Panobinostat induced plasma viraemia with an odds ratio of 10·5 (95% CI 2·2-50·3; p = 0·0002) compared with baseline. We recorded a transient decrease in total HIV DNA, but no cohort-wide reduction in total HIV DNA, integrated HIV DNA, or infectious units per million. Nine patients participated in the analytical treatment interruption, median time to viral rebound was 17 days (range 14-56). Panobinostat was well tolerated. 45 adverse events were reported, but only 16 (all grade 1) were presumed related to panobinostat. INTERPRETATION Panobinostat effectively disrupts HIV latency in vivo and is a promising candidate for future combination clinical trials aimed at HIV eradication. However, panobinostat did not reduce the number of latently infected cells and this approach may need to be combined with others to significantly affect the latent HIV reservoir. FUNDING The Danish Council for Strategic Research and Aarhus University.

HIV-1 persistence in CD4 + T cells with stem cell-like properties

Cellular HIV-1 reservoirs that persist despite antiretroviral treatment are incompletely defined. We show that during suppressive antiretroviral therapy, CD4+ T memory stem cells (TSCM cells) harbor high per-cell levels of HIV-1 DNA and make increasing contributions to the total viral CD4+ T cell reservoir over time. Moreover, we conducted phylogenetic studies that suggested long-term persistence of viral quasispecies in CD4+ TSCM cells. Thus, HIV-1 may exploit the stem cell characteristics of cellular immune memory to promote long-term viral persistence.

Long-Term Antiretroviral Treatment Initiated at Primary HIV-1 Infection Affects the Size, Composition, and Decay Kinetics of the Reservoir of HIV-1-Infected CD4 T Cells

ABSTRACT Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

Increased coronary atherosclerosis and immune activation in HIV-1 elite controllers.

HIV-1 elite controllers spontaneously maintain suppressed levels of viremia, but exhibit significant immune activation. We investigated coronary atherosclerosis by coronary computed tomography angiography (CTA) in elite controllers, nonelite controller, chronically HIV-1 infected, antiretroviral therapy (ART)-treated patients with undetectable viral load (‘chronic HIV’), and HIV-negative controls. Prevalence of atherosclerosis (78 vs. 42%, P < 0.05) and markers of immune activation were increased in elite controllers compared with HIV-negative controls. sCD163, a monocyte activation marker, was increased in elite controllers compared with chronic HIV-1 (P < 0.05) and compared with HIV-negative controls (P < 0.05). These data suggest a significant degree of coronary atherosclerosis and monocyte activation among elite controllers.

Treatment intensification with raltegravir in subjects with sustained HIV-1 viraemia suppression: a randomized 48-week study.

BACKGROUND Residual viraemia is a major obstacle to HIV-1 eradication in subjects receiving HAART. The intensification with raltegravir could impact latent reservoirs and might lead to a reduction of plasma HIV-1 viraemia (viral load [VL]), complementary DNA intermediates and immune activation. METHODS This was a prospective, open-label, randomized study comprising 69 individuals on suppressive HAART randomly assigned 2:1 to add raltegravir during 48 weeks. RESULTS Total and integrated HIV-1 DNA, and ultrasensitive VL remained stable despite intensification. There was a significant increase in episomal HIV DNA at weeks 2-4 in the raltegravir group returning to baseline levels at week 48. Median CD4(+) T-cell counts increased 124 and 80 cells/µl in the intensified and control groups after 48 weeks (P=0.005 and P=0.027, respectively), without significant differences between groups. No major changes were observed in activation of CD4(+) T-cells. Conversely, raltegravir intensification significantly reduced activation of CD8(+) T-cells at week 48 (HLA-DR(+)CD38(+), P=0.005), especially in the memory compartment (CD38(+) of CD8(+)CD45RO(+), P<0.0001). Linear mix models also depicted a larger decrease in CD8(+) T-cell activation in the intensification group (P=0.036 and P=0.010, respectively). Raltegravir intensification was not associated to any particular adverse event. CONCLUSIONS Intensification of HAART with raltegravir during 48 weeks was safe and associated with a significant decrease in CD8(+) T-cell activation, and a transient increase of episomal HIV-1 DNA. However, raltegravir did not significantly contribute to changes in CD4(+) T-cell counts, ultrasensitive VL, and total and integrated HIV-1 DNA. These findings suggest that raltegravir impacts residual HIV-1 replication and support new strategies to impair HIV-1 persistence. ClinicalTrials.gov identifier: NCT00554398.

Infrequent Recovery of HIV from but Robust Exogenous Infection of Activated CD4+ T Cells in HIV Elite Controllers

BACKGROUND. Human immunodeficiency virus (HIV) elite controllers are able to control infection with HIV-1 spontaneously to undetectable levels in the absence of antiretroviral therapy, but the mechanisms leading to this phenotype are poorly understood. Although low frequencies of HIV-infected peripheral CD4(+) T cells have been reported in this group, it remains unclear to what extent these are due to viral attenuation, active immune containment, or intracellular host factors that restrict virus replication. METHODS. We assessed proviral DNA levels, autologous viral growth from and infectability of in vitro activated, CD8(+) T cell-depleted CD4(+) T cells from HIV elite controllers (mean viral load, <50 copies/mL), viremic controllers (mean viral load, <2000 copies/mL), chronic progressors, and individuals receiving highly active antiretroviral therapy. RESULTS. Although we successfully detected autologous virus production in ex vivo activated CD4(+) T cells from all chronic progressors and from most of the viremic controllers, we were able to measure robust autologous viral replication in only 2 of 14 elite controllers subjected to the same protocol. In vitro activated autologous CD4(+) T cells from elite controllers, however, supported infection with both X4 and R5 tropic HIV strains at comparable levels to those in CD4(+) T cells from HIV-uninfected subjects. Proviral DNA levels were the lowest in elite controllers, suggesting that extremely low frequencies of infected cells contribute to difficulty in isolation of virus. CONCLUSIONS. These data indicate that elite control is not due to inability of activated CD4(+) T cells to support HIV infection, but the relative contributions of host and viral factors that account for maintenance of low-level infection remain to be determined.

Transcriptional Profiling of CD4 T Cells Identifies Distinct Subgroups of HIV-1 Elite Controllers

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) elite controllers maintain undetectable levels of viral replication in the absence of antiretroviral therapy (ART), but their underlying immunological and virological characteristics may vary. Here, we used a whole-genome transcriptional profiling approach to characterize gene expression signatures of CD4 T cells from an unselected cohort of elite controllers. The transcriptional profiles for the majority of elite controllers were similar to those of ART-treated patients but different from those of HIV-1-negative persons. Yet, a smaller proportion of elite controllers showed an alternative gene expression pattern that was indistinguishable from that of HIV-1-negative persons but different from that of highly active antiretroviral therapy (HAART)-treated individuals. Elite controllers with the latter gene expression signature had significantly higher CD4 T cell counts and lower levels of HIV-1-specific CD8+ T cell responses but did not significantly differ from other elite controllers in terms of HLA class I alleles, HIV-1 viral loads determined by ultrasensitive single-copy PCR assays, or chemokine receptor polymorphisms. Thus, these data identify a specific subgroup of elite controllers whose immunological and gene expression characteristics approximate those of HIV-1-negative persons.

Reactivation of latent HIV-1 in central memory CD4 + T cells through TLR-1/2 stimulation

BackgroundToll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4+ T cells. Memory CD4+ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4+ T cells has not been studied.ResultsWe evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4+ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation.ConclusionsOur findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs.

Deep Molecular Characterization of HIV-1 Dynamics under Suppressive HAART

In order to design strategies for eradication of HIV-1 from infected individuals, detailed insight into the HIV-1 reservoirs that persist in patients on suppressive antiretroviral therapy (ART) is required. In this regard, most studies have focused on integrated (proviral) HIV-1 DNA forms in cells circulating in blood. However, the majority of proviral DNA is replication-defective and archival, and as such, has limited ability to reveal the dynamics of the viral population that persists in patients on suppressive ART. In contrast, extrachromosomal (episomal) viral DNA is labile and as a consequence is a better surrogate for recent infection events and is able to inform on the extent to which residual replication contributes to viral reservoir maintenance. To gain insight into the diversity and compartmentalization of HIV-1 under suppressive ART, we extensively analyzed longitudinal peripheral blood mononuclear cells (PBMC) samples by deep sequencing of episomal and integrated HIV-1 DNA from patients undergoing raltegravir intensification. Reverse-transcriptase genes selectively amplified from episomal and proviral HIV-1 DNA were analyzed by deep sequencing 0, 2, 4, 12, 24 and 48 weeks after raltegravir intensification. We used maximum likelihood phylogenies and statistical tests (AMOVA and Slatkin-Maddison (SM)) in order to determine molecular compartmentalization. We observed low molecular variance (mean variability ≤0.042). Although phylogenies showed that both DNA forms were intermingled within the phylogenetic tree, we found a statistically significant compartmentalization between episomal and proviral DNA samples (P<10−6 AMOVA test; P = 0.001 SM test), suggesting that they belong to different viral populations. In addition, longitudinal analysis of episomal and proviral DNA by phylogeny and AMOVA showed signs of non-chronological temporal compartmentalization (all comparisons P<10−6) suggesting that episomal and proviral DNA forms originated from different anatomical compartments. Collectively, this suggests the presence of a chronic viral reservoir in which there is stochastic release of infectious virus and in which there are limited rounds of de novo infection. This could be explained by the existence of different reservoirs with unique pharmacological accessibility properties, which will require strategies that improve drug penetration/retention within these reservoirs in order to minimise maintenance of the viral reservoir by de novo infection.