Gilles Darcis

An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression.

The persistence of latently infected cells in patients under combinatory antiretroviral therapy (cART) is a major hurdle to HIV-1 eradication. Strategies to purge these reservoirs are needed and activation of viral gene expression in latently infected cells is one promising strategy. Bromodomain and Extraterminal (BET) bromodomain inhibitors (BETi) are compounds able to reactivate latent proviruses in a positive transcription elongation factor b (P-TEFb)-dependent manner. In this study, we tested the reactivation potential of protein kinase C (PKC) agonists (prostratin, bryostatin-1 and ingenol-B), which are known to activate NF-κB signaling pathway as well as P-TEFb, used alone or in combination with P-TEFb-releasing agents (HMBA and BETi (JQ1, I-BET, I-BET151)). Using in vitro HIV-1 post-integration latency model cell lines of T-lymphoid and myeloid lineages, we demonstrated that PKC agonists and P-TEFb-releasing agents alone acted as potent latency-reversing agents (LRAs) and that their combinations led to synergistic activation of HIV-1 expression at the viral mRNA and protein levels. Mechanistically, combined treatments led to higher activations of P-TEFb and NF-κB than the corresponding individual drug treatments. Importantly, we observed in ex vivo cultures of CD8+-depleted PBMCs from 35 cART-treated HIV-1+ aviremic patients that the percentage of reactivated cultures following combinatory bryostatin-1+JQ1 treatment was identical to the percentage observed with anti-CD3+anti-CD28 antibodies positive control stimulation. Remarkably, in ex vivo cultures of resting CD4+ T cells isolated from 15 HIV-1+ cART-treated aviremic patients, the combinations bryostatin-1+JQ1 and ingenol-B+JQ1 released infectious viruses to levels similar to that obtained with the positive control stimulation. The potent effects of these two combination treatments were already detected 24 hours post-stimulation. These results constitute the first demonstration of LRA combinations exhibiting such a potent effect and represent a proof-of-concept for the co-administration of two different types of LRAs as a potential strategy to reduce the size of the latent HIV-1 reservoirs.

Sequential treatment with 5-aza-2′-deoxycytidine and deacetylase inhibitors reactivates HIV-1

Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency‐reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5‐AzadC) in combination with clinically tolerable HDACIs in reactivating HIV‐1 from latency first in vitro and next ex vivo. We showed that a sequential treatment with 5‐AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo. Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV‐1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5‐AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV.

Preclinical shock strategies to reactivate latent HIV-1: an update.

Purpose of reviewThe ‘shock and kill’ strategy consists of activating HIV-1 expression to allow latently infected cells to die from viral cytopathic effects or host cytolytic immune effectors. This strategy relies on small molecules, called latency reversing agents, which activate HIV transcription. Recent findingsSeveral mechanisms operating at the transcriptional level are involved in the establishment and maintenance of HIV-1 latency, including the absence of crucial inducible host transcription factors, epigenetic silencing, and the sequestration of the positive transcription elongation factor B. Progresses made toward the understanding of the molecular mechanisms of HIV-1 transcriptional repression have led to the identification of latency reversing agents that activate HIV transcription, such as histone deacetylase inhibitors or protein kinase C agonists. Multiple studies have recently pointed interesting ways to optimize the shock strategy by using combinations of latency reversing agents with an appropriate time schedule. SummaryCombining latency reversing agents appears as one potential strategy for therapy against HIV-1 latency.

Reactivation capacity by latency-reversing agents ex vivo correlates with the size of the HIV-1 reservoir.

Objective: HIV-1 reservoirs are the major hurdle to virus clearance in combination antiretroviral therapy (cART)-treated patients. An approach to eradicating HIV-1 involves reversing latency in cART-treated patients to make latent cells visible to the host immune system. Stimulation of patient cell cultures with latency-reversing agents (LRAs) ex vivo results in heterogeneous responses among HIV-infected patients. Determinants of this heterogeneity are unknown and consequently important to determine. Design and methods: Here, we grouped and retrospectively analyzed the data from our two recent HIV-1 reactivation studies to investigate the role of the HIV-1 reservoir size in the reactivation capacity by LRAs in ex vivo cultures of CD8+-depleted peripheral blood mononuclear cells (PBMCs) isolated from 54 cART-treated patients and of resting CD4+ T cells isolated from 30 cART-treated patients. Results: Our results established a statistically relevant positive correlation between the HIV-1 reservoir size measured by total cell-associated HIV-1 DNA and the frequency of positive HIV-1 recovery measurements in response to various LRAs in ex vivo cultures of cells isolated from cART-treated HIV+ aviremic patients. HIV-1 reservoir size also correlated with the extracellular HIV-1 RNA median level measured in supernatants of cell cultures following LRA treatments. However, we identified HIV+ patients whose positive measurements frequency and median level of extracellular HIV-1 RNA deviated from linearity relative to their corresponding HIV reservoir size. Conclusion: We demonstrated that the reservoir size is one predictive marker of LRA effectiveness but this parameter alone is not sufficient. The identification of other predictive markers is necessary to predict the success of HIV anti-latency approaches.

Louse-borne relapsing fever in a refugee from Somalia arriving in Belgium.

We report a case of louse-borne relapsing fever (LBRF) in a refugee from Somalia who had arrived in Belgium a few days earlier. He complained of myalgia and secondarily presented fever. Blood smears revealed spirochetes later identified as Borrelia recurrentis. LBRF should be considered in countries hosting refugees, particularly those who transit through endemic regions.

Molecular Control of HIV and SIV Latency.

The HIV latent reservoirs are considered as the main hurdle to viral eradication. Numerous mechanisms lead to the establishment of HIV latency and act at the transcriptional and post-transcriptional levels. A better understanding of latency is needed in order to ultimately achieve a cure for HIV. The mechanisms underlying latency vary between patients, tissues, anatomical compartments, and cell types. From this point of view, simian immunodeficiency virus (SIV) infection and the use of nonhuman primate (NHP) models that recapitulate many aspects of HIV-associated latency establishment and disease progression are essential tools since they allow extensive tissue sampling as well as a control of infection parameters (virus type, dose, route, and time).

Exploring the anatomical HIV reservoirs: Role of the testicular tissue

Interruption of combination antiretroviral therapy (cART) almost invariably leads to the re-emergence of detectable HIV replication, typically in about 2 weeks [1]. The source of persistent viremia or viral rebound following cART interruption is likely multifactorial. It could arise from residual ongoing viral replication during cART and/or from reactivation of HIV expression from viral compartments and reservoirs.

Tackling HIV Persistence: Pharmacological versus CRISPR-Based Shock Strategies

Jan Svoboda studied aspects of viral latency, in particular with respect to disease induction by avian RNA tumor viruses, which were later renamed as part of the extended retrovirus family. The course of retroviral pathogenesis is intrinsically linked to their unique property of integrating the DNA copy of the retroviral genome into that of the host cell, thus forming the provirus. Retroviral latency has recently become of major clinical interest to allow a better understanding of why we can effectively block the human immunodeficiency virus type 1 (HIV-1) in infected individuals with antiviral drugs, yet never reach a cure. We will discuss HIV-1 latency and its direct consequence—the formation of long-lasting HIV-1 reservoirs. We next focus on one of the most explored strategies in tackling HIV-1 reservoirs—the “shock and kill” strategy—which describes the broadly explored pharmacological way of kicking the latent provirus, with subsequent killing of the virus-producing cell by the immune system. We furthermore present how the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system can be harnessed to reach the same objective by reactivating HIV-1 gene expression from latency. We will review the benefits and drawbacks of these different cure strategies.

Wild-type HIV infection despite PrEP: a lot to learn from a case report

We read with interest the case report by Hoornenborg and colleagues about the acquisition of wild-type HIV-1 infection in a patient on pre-exposure prophylaxis (PrEP) despite high intracellular concentrations of tenofovir diphosphate. This case is all the more important given that PrEP roll-out is a work in progress, with regulatory approvals for its use for HIV prevention completed in 15 countries and dossiers under review in many others. Despite its well demonstrated efficacy for the prevention of HIV acquisition, PrEP’s widespred uptake means that this particular and unprecedented event might happen again. After seroconversion in the reported case, health-care workers decided to interrupt PrEP but not to start combination antiretroviral therapy (ART) because of the un certainty on an actual infection. The atypical seroconversion pattern, with initially only gp160 detected in the western blot, contributed to questioning of the diagnosis of acute HIV infection. This strategy has the indisputable advantage of confirming what was strongly suspected. However, as suggested by the impossibility to detect any HIV proviruses in peripheral blood mononuclear cells, the HIV infection was probably mucosally contained. The HIV infection was thus characterised by an extremely low and localised viral reservoir and most likely a very limited viral diversity. This unique profile is associated with favourable outcomes such as a low or even absent immune activation. Limiting the viral reservoir size and diversity also could impede acquisition of mutations to evade cytotoxic T cells thereby conserving a better chance of response to potential cure strategies. Retrospectively, not starting ART at seroconversion was therefore a missed opportunity. We learned a lot from the case reported by Hoornenborg and colleagues and can use this knowledge to guide our therapeutic strategy should such cases arise again. Starting ART at seroconversion seems the best option considering all potential favourable outcomes. Concern about toxicity is a weak argument in the era of modern ART and for a patient already exposed to PrEP. If desired by the patient and considering the pros and cons, a structured treatment interruption might still be considered subsequently.

Factors associated with late presentation for HIV care in a single Belgian reference center: 2006–2017

Late presentation for HIV care is a major issue and the cause of higher morbidity, mortality and transmission. In this regard, we analyzed the characteristics of patients presenting for care at our center from January 2006 to July 2017 (n = 687). The majority of the studied population was of African origin (54.3%) with heterosexual women representing the main group (n = 292; 42.5%). 44% of the patients were late presenters (LP) (presenting for care with CD4 T cells <350/mm3 or an AIDS defining event) and 24% were late presenters with advanced disease (LP-AD) (presenting for care with CD4 T cells <200/mm3 or an AIDS defining event). A very high risk of being LP and LP-AD was associated with Sub-Saharan origin (OR 3.4 and 2.6 respectively). Other factors independently associated with LP or LP-AD were age (OR 1.3), male gender (OR 2.0 and 1.5 respectively) and heterosexual route of transmission (OR 2.4 and 2.3 respectively). A significant increase in HIV screening without forgetting those groups would contribute to earlier HIV diagnosis, a key element to end the HIV epidemic. To achieve this goal, addressing the specific hurdles to HIV testing in the migrant population is critical.