HIV-1 Resistance Dynamics in Patients With Virologic Failure to Dolutegravir Maintenance Monotherapy.

Background: A high genetic barrier to resistance to the integrase strand transfer inhibitor (INSTI) dolutegravir has been reported in vitro and in vivo. We describe the dynamics of INSTI resistance-associated mutations (INSTI-RAMs) and mutations in the 3'-polypurine tract (3'-PPT) in relation to virologic failure (VF) observed in the randomized Dolutegravir as Maintenance Monotherapy for HIV-1 study (DOMONO, NCT02401828). Methods: From 10 patients with VF, plasma samples were collected before the start of cART and during VF, and were used to generate Sanger sequences of integrase, the 5' terminal bases of the 3' long terminal repeat (LTR), and the 3'-PPT. Results: Median human immunodeficiency virus RNA load at VF was 3490 copies/mL (interquartile range 1440-4990 copies/mL). INSTI-RAMs (S230R, R263K, N155H, and E92Q+N155H) were detected in 4 patients, no INSTI-RAMs were detected in 4 patients, and sequencing of the integrase gene was unsuccessful in 2 patients. The time to VF ranged from 4 weeks to 72 weeks. In 1 patient, mutations developed in the highly conserved 3'-PPT. No changes in the terminal bases of the 3'-LTR were observed. Conclusions: The genetic barrier to resistance is too low to justify dolutegravir maintenance monotherapy because single INSTI-RAMs are sufficient to cause VF. The large variation in time to VF suggests that stochastic reactivation of a preexisting provirus containing a single INSTI-RAM is the mechanism for failure. Changes in the 3'-PPT point to a new dolutegravir resistance mechanism in vivo. Clinical Trials Registration: NCT02401828.

The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors.

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

Specific quantification of inducible HIV-1 reservoir by RT-LAMP.

BACKGROUND: Strategies toward HIV-1 cure aim to clear, inactivate, reduce, or immunologically control the virus from a pool of latently infected cells such that combination antiretroviral therapy (cART) can be safely interrupted. In order to assess the impact of any putative curative interventions on the size and inducibility of the latent HIV-1 reservoir, robust and scalable assays are needed to precisely quantify the frequency of infected cells containing inducible HIV-1. METHODS: We developed Specific Quantification of Inducible HIV-1 by RT-LAMP (SQuHIVLa), leveraging the high sensitivity and specificity of RT-LAMP, performed in a single reaction, to detect and quantify cells expressing tat/rev HIV-1 multiply spliced RNA (msRNA) upon activation. The LAMP primer/probe used in SQuHIVLa was designed to exclusively detect HIV-1 tat/rev msRNA and adapted for different HIV-1 subtypes. RESULTS: Using SQuHIVLa, we successfully quantify the inducible viral reservoir in CD4+ T cells from people living with HIV-1 subtypes B and C on cART. The assay demonstrates high sensitivity, specificity, and reproducibility. CONCLUSIONS: SQuHIVLa offers a high throughput, scalable, and specific HIV-1 reservoir quantification tool that is amenable to resource-limited settings. This assay poses remarkable potential in facilitating the evaluation of potential interventional strategies toward achieving HIV-1 cure.

HIV infection remains challenging because the virus hides in certain cells, making it invisible to the immune system. This hidden virus forms what is called a latent HIV reservoir. If someone with HIV stops their antiviral therapy, the virus quickly re-emerges. Because of this, researchers are exploring various strategies to eliminate this reservoir and cure HIV. To evaluate these strategies, we need a method to measure the reservoir's size before and after trials. Our study introduces SQuHIVLa, a highly sensitive and specific method for quantifying the latent reservoir. SQuHIVLa could become a vital tool for monitoring HIV patients and assessing treatment effectiveness, bringing us closer to finding a cure.

Autologous dendritic cell vaccination against HIV-1 induces changes in natural killer cell phenotype and functionality.

Although natural killer (NK) cells have been studied in connection with dendritic cell (DC)-based vaccination in the field of cancer immunology, their role has barely been addressed in the context of therapeutic vaccination against HIV-1. In this study, we evaluated whether a therapeutic DC-based vaccine consisting of monocyte-derived DCs electroporated with Tat, Rev and Nef encoding mRNA affects NK cell frequency, phenotype and functionality in HIV-1-infected individuals. Although the frequency of total NK cells did not change, we observed a significant increase in cytotoxic NK cells following immunisation. In addition, significant changes in the NK cell phenotype associated with migration and exhaustion were observed together with increased NK cell-mediated killing and (poly)functionality. Our results show that DC-based vaccination has profound effects on NK cells, which highlights the importance of evaluating NK cells in future clinical trials looking at DC-based immunotherapy in the context of HIV-1 infection.

Genotypic and Phenotypic Characterization of Replication-Competent HIV-2 Isolated from Controllers and Progressors.

Although some individuals with HIV-2 develop severe immunodeficiency and AIDS-related complications, most may never progress to AIDS. Replication-competent HIV-2 isolated from asymptomatic long-term non-progressors (controllers) have lower replication rates than viruses from individuals who progress to AIDS (progressors). To investigate potential retroviral factors that correlate with disease progression in HIV-2, we sequenced the near full-length genomes of replication-competent viruses previously outgrown from controllers and progressors and used phylogeny to seek genotypic correlates of disease progression. We validated the integrity of all open reading frames and used cell-based assays to study the retroviral transcriptional activity of the long terminal repeats (LTRs) and Tat proteins of HIV-2 from controllers and progressors. Overall, we did not identify genotypic defects that may contribute to HIV-2 non-progression. Tat-induced, LTR-mediated transcription was comparable between viruses from controllers and progressors. Our results were obtained from a small number of participants and should be interpreted accordingly. Overall, they suggest that progression may be determined before or during integration of HIV-2.

The BAF complex inhibitor pyrimethamine reverses HIV-1 latency in people with HIV-1 on antiretroviral therapy.

Reactivation of the latent HIV-1 reservoir is a first step toward triggering reservoir decay. Here, we investigated the impact of the BAF complex inhibitor pyrimethamine on the reservoir of people living with HIV-1 (PLWH). Twenty-eight PLWH on suppressive antiretroviral therapy were randomized (1:1:1:1 ratio) to receive pyrimethamine, valproic acid, both, or no intervention for 14 days. The primary end point was change in cell-associated unspliced (CA US) HIV-1 RNA at days 0 and 14. We observed a rapid, modest, and significant increase in (CA US) HIV-1 RNA in response to pyrimethamine exposure, which persisted throughout treatment and follow-up. Valproic acid treatment alone did not increase (CA US) HIV-1 RNA or augment the effect of pyrimethamine. Pyrimethamine treatment did not result in a reduction in the size of the inducible reservoir. These data demonstrate that the licensed drug pyrimethamine can be repurposed as a BAF complex inhibitor to reverse HIV-1 latency in vivo in PLWH, substantiating its potential advancement in clinical studies.

TILDA: Tat/Rev Induced Limiting Dilution Assay.

Recently the Tat/rev Induced Limiting Dilution Assay, or TILDA, has been proposed as a possible alternative method to quantify the HIV-1 reservoir. TILDA estimates the frequency of latently infected cells by probing, in a limiting dilution format, the presence or inducibility of tat and rev multiply spliced HIV-1 RNA. In doing so, TILDA reduces overestimation of reservoir size compared to HIV-1 DNA measurements because multiply spliced HIV-1 RNA is less likely to be transcribed from dysfunctional genomes with replication defects. TILDA is easy to perform, requires a very low input number of cells and has a fast turnaround time, making it ideal for use in clinical settings. Here we describe the execution of TILDA with particular emphasis on cell preparation and the limiting dilution scheme.

HIV-1 resistance against dolutegravir fluctuates rapidly alongside erratic treatment adherence: a case report.

OBJECTIVES: We report a case of incomplete HIV-1 suppression on a dolutegravir, lamivudine, and abacavir single-tablet regimen with the emergence of the H51Y and G118R integrase resistance mutations. METHODS: Integrase sequencing was performed retrospectively by Sanger and next-generation sequencing. Rates of emergence and decline of resistance mutations were calculated using next-generation sequencing data. Dolutegravir plasma concentrations were measured by ultra-performance liquid chromatography-tandem mass spectrometry. The effects of H51Y and G118R on infectivity, fitness, and susceptibility to dolutegravir were quantified using cell-based assays. RESULTS: During periods of non-adherence to treatment, mutations were retrospectively documented only by next-generation sequencing. Misdiagnosis by Sanger sequencing was caused by the rapid decline of mutant strains within the retroviral population. This observation was also true for a M184V lamivudine-resistant reverse transcriptase mutation found in association with integrase mutations on single HIV genomes. Resistance rebound upon treatment re-initiation was swift (>8000 copies per day). Next-generation sequencing indicated cumulative adherence to treatment. Compared to WT HIV-1, relative infectivity was 73%, 38%, and 43%; relative fitness was 100%, 35%, and 10% for H51Y, G118R, and H51Y+G118R viruses, respectively. H51Y did not change the susceptibility to dolutegravir, but G188R and H51Y+G118R conferred 7- and 28-fold resistance, respectively. CONCLUSION: This case illustrates how poorly-fit drug-resistant viruses wax and wane alongside erratic treatment adherence and are easily misdiagnosed by Sanger sequencing. We recommend next-generation sequencing to improve the clinical management of incomplete virological suppression with dolutegravir.

Inducible HIV-1 Reservoir Quantification: Clinical Relevance, Applications and Advancements of TILDA.

The presence of a stable HIV-1 reservoir persisting over time despite effective antiretroviral suppression therapy precludes a cure for HIV-1. Characterizing and quantifying this residual reservoir is considered an essential prerequisite to develop and validate curative strategies. However, a sensitive, reproducible, cost-effective, and easily executable test is still needed. The quantitative viral outgrowth assay is considered the gold standard approach to quantify the reservoir in HIV-1-infected patients on suppressive ART, but it has several limitations. An alternative method to quantify the viral reservoir following the reactivation of latent HIV-1 provirus detects multiply-spliced tat/rev RNA (msRNA) molecules by real-time PCR [tat/rev induced limiting dilution assay (TILDA)]. This article provides a perspective overview of the clinical relevance, various applications, recent advancements of TILDA, and how the assay has contributed to our understanding of the HIV-1 reservoir.

Selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir.

An innovative approach to eliminate HIV-1-infected cells emerging out of latency, the major hurdle to HIV-1 cure, is to pharmacologically reactivate viral expression and concomitantly trigger intracellular pro-apoptotic pathways in order to selectively induce cell death (ICD) of infected cells, without reliance on the extracellular immune system. In this work, we demonstrate the effect of DDX3 inhibitors on selectively inducing cell death in latent HIV-1-infected cell lines, primary CD4+ T cells and in CD4+ T cells from cART-suppressed people living with HIV-1 (PLWHIV). We used single-cell FISH-Flow technology to characterise the contribution of viral RNA to inducing cell death. The pharmacological targeting of DDX3 induced HIV-1 RNA expression, resulting in phosphorylation of IRF3 and upregulation of IFNß. DDX3 inhibition also resulted in the downregulation of BIRC5, critical to cell survival during HIV-1 infection, and selectively induced apoptosis in viral RNA-expressing CD4+ T cells but not bystander cells. DDX3 inhibitor treatment of CD4+ T cells from PLWHIV resulted in an approximately 50% reduction of the inducible latent HIV-1 reservoir by quantitation of HIV-1 RNA, by FISH-Flow, RT-qPCR and TILDA. This study provides proof of concept for pharmacological reversal of latency coupled to induction of apoptosis towards the elimination of the inducible reservoir.

Inter-Laboratory Reproducibility of Inducible HIV-1 Reservoir Quantification by TILDA.

Substantial efforts to eliminate or reduce latent HIV-1 reservoirs are underway in clinical trials and have created a critical demand for sensitive, accurate, and reproducible tools to evaluate the efficacy of these strategies. Alternative reservoir quantification assays have been developed to circumvent limitations of the quantitative viral outgrowth assay. One such assay is tat/rev induced limiting dilution assay (TILDA), which measures the frequency of CD4+ T cells harboring inducible latent HIV-1 provirus. We modified pre-amplification reagents and conditions (TILDA v2.0) to improve assay execution and first internally validated assay performance using CD4+ T cells obtained from cART-suppressed HIV-1-infected individuals. Detection of tat/rev multiply spliced RNA was not altered by modifying pre-amplification conditions, confirming the robustness of the assay, and supporting the technique's amenability to limited modifications to ensure better implementation for routine use in clinical studies of latent HIV-1 reservoirs. Furthermore, we cross-validated results of TILDA v2.0 and the original assay performed in two separate laboratories using samples from 15 HIV-1-infected individuals. TILDA and TILDA v2.0 showed a strong correlation (Lin's Concordance Correlation Coefficient = 0.86). The low inter-laboratory variability between TILDAs performed at different institutes further supports use of TILDA for reservoir quantitation in multi-center interventional HIV-1 Cure trials.

Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal.

Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal.