Clinical trials aimed at HIV cure or remission: new pathways and lessons learned.

INTRODUCTION: The main barrier to finding a cure against HIV is the latent HIV reservoir, which persists in people living with HIV (PLWH) despite antiretroviral treatment (ART). Here, we discuss recent findings from interventional studies using mono- and combination therapies aimed at enhancing immune-mediated killing of the virus with or without activating HIV from latency. AREAS COVERED: We discuss latency reversal agents (LRAs), broadly neutralizing antibodies, immunomodulatory therapies, and studies aimed at inducing apoptosis. EXPERT OPINION: The landscape of clinical trials for HIV cure and remission has evolved considerably over the past 10 years. Several novel interventions such as immune checkpoint inhibitors, therapeutic vaccines, and broadly neutralizing antibodies have been tested either alone or in combination with LRAs but studies have so far not shown a meaningful impact on the frequency of latently infected cells. Immunomodulatory therapies could work differently in the setting of antigen expression, that is, during active viremia, and timing of interventions could therefore, be key to future therapeutic success. Lessons learned from clinical trials aimed at HIV cure indicate that while we are still far from reaching a complete eradication cure of HIV, clinical interventions capable of inducing enhanced control of HIV replication in the absence of ART might be a more feasible goal.

Clinical Interventions in HIV Cure Research.

Research over the past decade has resulted in a much-improved understanding of how and where HIV persists in patients on otherwise suppressive antiretroviral therapy (ART). It has become clear that the establishment of a latent infection in long-lived cells is the key barrier to curing HIV or allowing for sustained ART-free remission. Informed by in vitro and ex vivo studies, several therapeutic approaches aimed at depleting the pool of latently infected cells have been tested in small-scale experimental clinical trials including studies of ART intensification, genome editing, ART during acute/early infection and latency reversal. Many studies have focused on the use of latency-reversing agents (LRAs) to induce immune- or virus-mediated elimination of virus-producing cells. These trials have been instrumental in establishing safety and have shown that it is possible to impact the state HIV latency in patients on suppressive ART. However, administration of LRAs alone has thus far not demonstrated an effect on the frequency of latently infected cells or the time to virus rebound during analytical interruption of ART. More recently, there has been an enhanced focus on immune-based therapies in the onwards search for an HIV cure including therapeutic vaccines, toll-like receptor agonists, broadly neutralising antibodies, immune checkpoint inhibitors, interferon-α and interleukin therapy. In ongoing studies immunotherapy interventions are also tested in combination with latency reversal. In this chapter, the overall results of these clinical interventions ultimately aimed at a cure for HIV are presented and discussed.

Treatment of HIV-Infected Individuals with the Histone Deacetylase Inhibitor Panobinostat Results in Increased Numbers of Regulatory T Cells and Limits Ex Vivo Lipopolysaccharide-Induced Inflammatory Responses.

Histone deacetylase inhibitors (HDACi) modulate the transcriptional activity of all cells, including innate and adaptive immune cells. Therefore, we aimed to evaluate immunological effects of treatment with the HDACi panobinostat in HIV-infected patients during a clinical phase IIa latency reversal trial. Using flow cytometry, we investigated changes in T cell activation (CD69, CD38, HLA-DR) and the expression of CD39 and CTLA4 on regulatory T cells (Tregs). Whole-blood stimulations were performed and cytokine responses measured using Luminex. Gene expression in purified peripheral blood mononuclear cells (PBMCs) was evaluated using an Affymetrix HTA 2.0 gene chip. We found that proportions of CD4+ and CD8+ T cells expressing CD69 increased 24 h after initial panobinostat administration (P < 0.01), followed by an increase in the proportions of CD38+ HLA-DR+-coexpressing CD4+ T cells on day 4 (P = 0.02). Concurrently, proportions of Tregs increased by 40% (P = 0.003). Treg CTLA4 median fluorescent intensity (MFI) increased by 25% (P = 0.007), and CD39 MFI on CD39+ Treg increased by 12% (P = 0.02). Lipopolysaccharide (LPS)-induced inflammatory responses (interleukin-1ß [IL-1ß], IL-6, IL-12p40, and tumor necrosis factor alpha [TNF-α]) in whole blood were significantly downregulated 4 days after initial dosing. Lastly, panobinostat induced significant changes in the overall gene expression pattern (fold change, >1.5; false-discovery-rate [FDR]-corrected P, <0.05). Importantly, measures of immune function returned to baseline after panobinostat treatment and follow-up revealed no sustained effect on overall gene expression. IMPORTANCE The effect of treatment with histone deacetylase inhibitors on the immune system in HIV-infected individuals is not clear. Analysis of results from a clinical trial in which 15 HIV-infected individuals received 12 doses of panobinostat identified a significant impact on both T cell activation status and regulatory T cell suppressive marker expression and a reduced level of monocytic responsiveness to inflammatory stimuli. These changes were substantiated by global gene expression analysis. Collectively, the results suggest that panobinostat has multiple effects on innate and adaptive immune responses. Importantly, all the effects were transient, and further panobinostat treatment did not cause persistent long-term changes in gene expression patterns in HIV-infected individuals.

Patient-reported outcomes in daily clinical practise in HIV outpatient care.

INTRODUCTION: The use of patient-reported outcomes (PROs) in outpatient care holds promise as a tool to enhance the quality of care. The management of chronic HIV infection is multidimensional, and clinical assessment includes broad screening to identify complications. With growing constraints on time and resources, the use of PROs may provide a much-needed tool to ensure optimal HIV care. The aim of this study was to evaluate the clinical implementation and use of a Web-based tool to collect PROs in a cohort of HIV-infected individuals. METHODS: In December 2015, the PRO system AmbuFlex, a Web-based tool for self-reporting of clinical symptoms, was implemented in HIV outpatient care at Aarhus University Hospital. The HIV-specific questionnaire was designed to cover items in the European AIDS Clinical Society guidelines. Patients responded through a Web-based system from home. Based on an HIV-specific algorithm, responses were automatically assigned a green, yellow, or red colour code reflecting the severity of the symptom. HIV-related data from the electronic hospital management system were used to compare respondents and non-respondents. For cognitive and red symptoms, patient records were accessed to address whether PRO provided new information. Furthermore, it was sought to determine whether implementing PROs in clinical care can help focus the consultation on current needs. This was done by checking if a flagged symptom was assessed clinically at the following consultation. RESULTS: Five hundred and five HIV patients were invited to participate and 277 (55%) accepted the invitation. Compared to respondents, non-respondents were significantly younger and more often female, born outside Denmark, newly diagnosed, and with a plasma viral load >50 copies/ml. Among the 262 correctly received PRO questionnaires, 104 (39%) had solely green colour-coded responses, whereas 59 (23%) had one or more red colour-coded responses. Of 69 red symptoms, 28 (41%) led to a specific clinical assessment. In many cases, PROs appeared to provide new information on cognitive (76%) and red-coded symptoms (42%). CONCLUSIONS: The use of PROs identified several cases where physical or cognitive symptoms appeared to have been unnoticed. A substantial proportion of patients reported no symptoms requiring medical attention, suggesting a potential to individualize outpatient care and redistribute resource utilization.

Broad activation of latent HIV-1 in vivo.

The 'shock and kill' approach to cure human immunodeficiency virus (HIV) includes transcriptional induction of latent HIV-1 proviruses using latency-reversing agents (LRAs) with targeted immunotherapy to purge infected cells. The administration of LRAs (panobinostat or vorinostat) to HIV-1-infected individuals on antiretroviral therapy induces a significant increase in cell-associated unspliced (CA-US) HIV-1 RNA from CD4(+) T cells. However, it is important to discern whether the increases in CA-US HIV-1 RNA are due to limited or broad activation of HIV-1 proviruses. Here we use single-genome sequencing to find that the RNA transcripts observed following LRA administration are genetically diverse, indicating activation of transcription from an extensive range of proviruses. Defective sequences are more frequently found in CA HIV-1 RNA than in HIV-1 DNA, which has implications for developing an accurate measure of HIV-1 reservoir size. Our findings provide insights into the effects of panobinostat and vorinostat as LRAs for latent HIV-1.

HIV-1 transcriptional activity during frequent longitudinal sampling in aviremic patients on antiretroviral therapy.

BACKGROUND: HIV-1 transcription during suppressive antiretroviral therapy (ART) is not well understood. This is problematic as latency-reactivating agent-based HIV-1 eradication trials utilize changes in viral transcription as an efficacy biomarker. METHODS: We conducted an observational cohort study enrolling aviremic, HIV-1-infected adults on long-term ART. Cell-associated unspliced (CA-US) HIV-1 RNA and total HIV-1 DNA were quantified in unfractionated CD4 T cells monthly for a total of six consecutive visits. Random-effects models were used to determine the following: (i) proportion of variation attributable to intra-individual versus inter-individual changes; (ii) range estimate for random samples from any participant or cohort-matched individual (95% prediction interval); and (iii) range estimate for random samples from the same person (95% variation intervals expressed as fold change). RESULTS: Among our cohort of 26 HIV-1 patients, 10.4% of variation in CA-US HIV-1 RNA was attributable to intra-individual fluctuations. Similarly, intra-individual changes also accounted for minor proportions of the variation in total HIV-1 DNA (5.1%) and RNA/DNA (28.3%). The 95% prediction interval (per 10 CD4 T cells) for CA-US HIV-1 RNA and HIV-1 DNA were each approximately 2 log10. Finally, model-derived 95% variation intervals indicate that spontaneous changes above 2.11-fold in CA-US HIV-1 RNA would occur in less than 5% of repeated measurements in an individual on long-term ART. CONCLUSION: The individual CA-US HIV-1 RNA levels are remarkably stable during ART. Importantly, the observed variations were less than the reported changes for latency-reactivating agent trials. These data will serve as a foundation for planning and interpreting future eradication trials.

Reversal of Latency as Part of a Cure for HIV-1.

Here, the use of pharmacological agents to reverse HIV-1 latency will be explored as a therapeutic strategy towards a cure. However, while clinical trials of latency-reversing agents LRAs) have demonstrated their ability to increase production of latent HIV-1, such interventions have not had an effect on the size of the latent HIV-1 reservoir. Plausible explanations for this include insufficient host immune responses against virus-expressing cells, the presence of escape mutations in archived virus, or an insufficient scale of latency reversal. Importantly, these early studies of LRAs were primarily designed to investigate their ability to perturb the state of HIV-1 latency; using the absence of an impact on the size of the HIV-1 reservoir to discard their potential inclusion in curative strategies would be erroneous and premature.

The histone deacetylase inhibitor panobinostat lowers biomarkers of cardiovascular risk and inflammation in HIV patients.

OBJECTIVE: To investigate the effect of the histone deacetylase inhibitor panobinostat on HIV-associated inflammation. DESIGN: Sub-study of a single-arm, phase I/II clinical trial. METHODS: HIV-infected adults on suppressive antiretroviral therapy received oral panobinostat 20 mg three times per week, every other week, for 8 weeks, that is, four cycles of treatment. Plasma levels of high-sensitivity C-reactive protein, matrix metalloproteinase 9, soluble CD40 ligand and interleukin-6 were determined using human ELISA kits. Soluble endothelia selectin (E-selectin) was measured by a multiplex immunoassay. Total monocyte count, phenotype changes on monocytes and monocyte histone acetylation were analyzed using flow cytometry. Whole-genome expression in peripheral blood mononuclear cells was analyzed at baseline and on-panobinostat employing the Affymetrix Human Transcriptome Array 2.0 microarray assay. Changes from baseline were analyzed using Wilcoxon signed-rank test. For the gene-expression analyses, fold-changes, P values and false detection rate were computed using TAC software. RESULTS: Panobinostat treatment led to significant reductions in multiple established plasma markers of inflammation. Notably, high-sensitivity C-reactive protein decreased by a median of 58% during treatment and this change persisted for 4 weeks after treatment. Plasma levels of interleukin-6, matrix metalloproteinase 9, E-selectin and soluble CD40 ligand also significantly decreased on and/or postpanobinostat. Additionally, we observed a significant reduction in the proportions of intermediate monocytes and tissue factor-positive monocytes. This suppression of cardiovascular risk biomarkers was associated with a prominent reduction in the expression of genes related to inflammation and atherosclerosis. CONCLUSION: Collectively, these data indicate that panobinostat may have therapeutic potential to target excess inflammation in HIV patients with high cardiovascular risk.

Comparison of HDAC inhibitors in clinical development: effect on HIV production in latently infected cells and T-cell activation.

OBJECTIVE: We aimed to compare the potential for inducing HIV production and the effect on T-cell activation of potent HDAC inhibitors undergoing clinical investigation. DESIGN: In vitro study RESULTS: The various HDAC inhibitors displayed significant potency differences in stimulating HIV-1 expression from the latently infected cell lines with panobinostat>givinostat ≈belinostat>vorinostat>valproic acid. Panobinostat was significantly more potent than all other HDAC inhibitors and induced virus production even in the very low concentration range 8-31 nM. The proportion of primary T-cells expressing the early activation marker CD69 increased moderately in all HDAC inhibitor-treated cells compared with untreated cells. Finally, proof was obtained that panobinostat, givinostat and belinostat induce virus production in latently infected primary cells at therapeutic concentrations with panobinostat being the most potent stimulator. METHODS: The latently infected cell lines ACH2 and U1 were treated with the HDAC inhibitors panobinostat, givinostat, belinostat, vorinostat and valproic acid. Viral induction was estimated by p24 production. Peripheral blood mononuclear cells from uninfected donors were treated with the HDAC inhibitors and the expression of activation markers on T-cell phenotypes was measured using flow cytometry. Finally, the ability of givinostat, belinostat and panobinostat to reactivate latent HIV-1 expression in primary T-cells was investigated employing a CCL19-induced latent primary CD4+ T cell infection model. CONCLUSION: At therapeutic concentrations panobinostat stimulate HIV-1 expression in latently infected cells with greater potency than other HDAC inhibitors undergoing clinical investigation. These findings warrant further investigation and panobinostat is now being advanced into clinical testing against latent HIV infection.