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Background: Optimization of combination antiretroviral therapy (cART) can impact the human immunodeficiency virus (HIV) reservoir. We evaluated the effect on the HIV reservoir in peripheral blood and ileum biopsies in patients switching from boosted protease inhibitor (PI/r)-based therapy to dolutegravir (DTG)-based therapy., Methods: Impact of Integrase-inhibitor DOlutegravir On the viral Reservoir (INDOOR) is a phase 4 open-label clinical trial that randomly included 42 HIV type 1-infected individuals on effective cART: 20 who switched from PI/r-based to DTG-based cART (switch group), and 22 who remained in PI/r-based regimens (control group). We analyzed blood and ileum biopsies to quantify episomal, total, and integrated HIV DNA, cell-associated HIV RNA, residual plasma viremia, T-cell subsets, cell activation, and inflammation markers., Results: There were no related adverse events or treatment discontinuations due to drug intolerance. The HIV reservoir was consistently larger in ileal than in peripheral CD4+ T cells in both groups (P < .01). Residual viremia in plasma decreased in the switch group (P = .03). However, we did not observe significant longitudinal changes in low-level viral replication, total and integrated HIV reservoir, HIV transcription, T-cell maturation subsets, immunoactivation markers, inflammatory soluble proteins, or cellular markers of latently infected cells., Conclusions: The INDOOR study is the first evaluation of changes in HIV reservoir size in ileum biopsies and in peripheral blood in individuals switched from PI/r- to DTG-based cART. Although this switch was safe and well tolerated, it had no impact on a large array of immunological and inflammatory markers or on HIV reservoir markers in peripheral or in ileal CD4+ T cells., Clinical Trials Registration: EudraCT 2014-004331-39., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.)

The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles. Reactivating latent HIV reservoirs could be beneficial towards a functional cure. Here, the authors show that Tat-LNP effectively reactivates HIV while preserving the cell transcriptome. Upon reactivation, p24+ cells exhibit distinct genes and pathways potentially contributing to their persistence. [ABSTRACT FROM AUTHOR]

Long-term changes in the immune system of successfully treated people living with HIV (PLHIV) remain incompletely understood. In this study, we assessed 108 white blood cell (WBC) populations in a cohort of 211 PLHIV on stable antiretroviral therapy and in 56 HIV-uninfected controls using flow cytometry. We show that marked differences exist in T cell maturation and differentiation between PLHIV and HIV-uninfected controls: PLHIV had reduced percentages of CD4+ T cells and naïve T cells and increased percentages of CD8+ T cells, effector T cells, and T helper 17 (Th17) cells, together with increased Th17/regulatory T cell (Treg) ratios. PLHIV also exhibited altered B cell maturation with reduced percentages of memory B cells and increased numbers of plasmablasts. Determinants of the T and B cell composition in PLHIV included host factors (age, sex, and smoking), markers of the HIV reservoir, and CMV serostatus. Moreover, higher circulating Th17 percentages were associated with higher plasma concentrations of interleukin (IL) 6, soluble CD14, the gut homing chemokine CCL20, and intestinal fatty acid binding protein (IFABP). The changes in circulating lymphocytes translated into functional changes with reduced interferon (IFN)- γ responses of peripheral blood mononuclear cells to stimulation with Candida albicans and Mycobacterium tuberculosis. In conclusion, this comprehensive analysis confirms the importance of persistent abnormalities in the number and function of circulating immune cells in PLHIV on stable treatment. [ABSTRACT FROM AUTHOR]

Background: Validated biomarkers to evaluate HIV-1 cure strategies are currently lacking, therefore requiring analytical treatment interruption (ATI) in study participants. Little is known about the safety of ATI and its long-term impact on patient health.Objectives: ATI safety was assessed and potential biomarkers predicting viral rebound were evaluated.Methods: PBMCs, plasma and CSF were collected from 11 HIV-1-positive individuals at four different timepoints during ATI (NCT02641756). Total and integrated HIV-1 DNA, cell-associated (CA) HIV-1 RNA transcripts and restriction factor (RF) expression were measured by PCR-based assays. Markers of neuroinflammation and neuronal injury [neurofilament light chain (NFL) and YKL-40 protein] were measured in CSF. Additionally, neopterin, tryptophan and kynurenine were measured, both in plasma and CSF, as markers of immune activation.Results: Total HIV-1 DNA, integrated HIV-1 DNA and CA viral RNA transcripts did not differ pre- and post-ATI. Similarly, no significant NFL or YKL-40 increases in CSF were observed between baseline and viral rebound. Furthermore, markers of immune activation did not increase during ATI. Interestingly, the RFs SLFN11 and APOBEC3G increased after ATI before viral rebound. Similarly, Tat-Rev transcripts were increased preceding viral rebound after interruption.Conclusions: ATI did not increase viral reservoir size and it did not reveal signs of increased neuronal injury or inflammation, suggesting that these well-monitored ATIs are safe. Elevation of Tat-Rev transcription and induced expression of the RFs SLFN11 and APOBEC3G after ATI, prior to viral rebound, indicates that these factors could be used as potential biomarkers predicting viral rebound. [ABSTRACT FROM AUTHOR]

Introduction: Viral remission after analytical treatment interruption (ATI), termed post‐treatment control, has been described in a small proportion of HIV‐positive patients. This phenomenon has been separately associated to both low levels of HIV‐1 proviral DNA as well as cell‐associated RNA. We investigated whether the combination of both parameters could help predict delayed viral rebound after treatment interruption (TI). Methods: We conducted an open single‐arm ATI study in four Belgian HIV reference centres from January 2016 to July 2018. Eligible participants were adults who had fewer than 50 HIV‐1 RNA copies/mL for more than two years, more than 500 CD4 cells/µL for more than three months, and were in general good health. Consenting participants who had fewer than 66 copies total HIV‐1 DNA (t‐DNA) and fewer than 10 copies cell‐associated HIV‐1 unspliced RNA (US‐RNA) per million peripheral blood mononuclear cells (PBMCs), interrupted therapy and were monitored closely. Antiretroviral therapy (ART) was resumed after two consecutive viral loads exceeding 1000 copies or one exceeding 10,000 copies/mL. The primary outcome was the proportion of participants with fewer than 50 HIV‐1 RNA copies/mL 48 weeks after TI. Secondary outcomes were time to viral rebound, the frequency of serious adverse events (AEs) and evolution of t‐DNA and US‐RNA after TI. Results: All 16 consenting participants who interrupted therapy experienced rapid viral rebound two to eight weeks after TI. No serious AEs were observed. Levels of t‐DNA and US‐RNA increased after TI but returned to pre‐ATI levels after treatment restart. None of the studied demographic, clinical and biological parameters were predictive of time of viral rebound. Conclusions: The combination of low levels of t‐DNA and US‐RNA in PBMCs, corresponding respectively to a small and transcriptionally silent viral reservoir, is not predictive of viral remission after TI in patients on ART. [ABSTRACT FROM AUTHOR]

Background: The gold standard for HIV-1 treatment is to administer triple antiretroviral therapy, but a shift to simplified regimens is being explored. Boosted darunavir monotherapy can be considered for patients who are for specific reasons not good candidates for dual or triple therapy. Still, a number of patients fail virologically or need to switch treatment.Objectives: To identify predictive markers for those patients that are more likely to sustain virological control under monotherapy, virological and immunological markers were explored in HIV-1-positive patients that experienced virological failure on ritonavir-boosted darunavir monotherapy in the PROTEA trial.Methods: As a retrospective nested study of the PROTEA study (NCT01448707), we analysed 77 HIV-1-infected patients who were on darunavir/ritonavir 800/100 mg monotherapy up to 96 weeks. Patients were appointed to three distinct cohorts based on viral loads (VLs): (i) undetectable VL after 96 weeks; (ii) very-low-level viraemia (5-39 copies/mL); and (iii) failing treatment. Total HIV-1 DNA, integrated HIV-1 DNA and 2-long terminal repeat circular HIV-1 DNA (2LTR circles) were measured in PBMCs at baseline, week 48 and week 96.Results: Total HIV-1 DNA and integrated HIV-1 DNA at baseline differed significantly between patients who experienced virological failure on monotherapy (P < 0.01 and P < 0.001). Although a higher level of HIV-1 DNA was measured in failures, this marker by itself does not provide enough predictive value to prospectively predict virological failure in patients on monotherapy.Conclusions: HIV-1 reservoir markers correlate with therapy failure in ritonavir-boosted darunavir monotherapy. However, their role as a predictive marker combined with other markers in a routine clinical setting should be further explored. [ABSTRACT FROM AUTHOR]

Although antiretroviral therapy is able to suppress HIV replication in infected patients, the virus persists and rebounds when treatment is stopped. In order to find a cure that can eradicate the latent reservoir, one must be able to quantify the persisting virus. Traditionally, HIV persistence studies have used real-time PCR (qPCR) to measure the viral reservoir represented by HIV DNA and RNA. Most recently, digital PCR is gaining popularity as a novel approach to nucleic acid quantification as it allows for absolute target quantification. Various commercial digital PCR platforms are nowadays available that implement the principle of digital PCR, of which Bio-Rad's QX200 ddPCR is currently the most used platform in HIV research. Quantification of HIV by digital PCR is proving to be a valuable improvement over qPCR as it is argued to have a higher robustness to mismatches between the primers-probe set and heterogeneous HIV, and forfeits the need for a standard curve, both of which are known to complicate reliable quantification. However, currently available digital PCR platforms occasionally struggle with unexplained false-positive partitions, and reliable segregation between positive and negative droplets remains disputed. Future developments and advancements of the digital PCR technology are promising to aid in the accurate quantification and characterization of the persistent HIV reservoir. [ABSTRACT FROM AUTHOR]

Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1–12) and 14 (5–26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2–5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an "integrated standard"; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies. [ABSTRACT FROM AUTHOR]

• We present a detailed protocol for a novel triplex dPCR assay for the quantification of the intact HIV-1 DNA reservoir. • We assessed and evaluated the performance of these assays on HIV-1 control cell line material and HIV-1 patient samples. • The triplex assays were implemented on the Naica dPCR platform (Stilla). The development of an HIV-1 cure is hampered by the existence of a persistent (latent) reservoir that contains a small proportion of replication-competent intact proviruses which refuels viral replication upon treatment discontinuation. Therefore, an accurate evaluation and quantification of these (intact) proviruses is essential to determine the efficacy of HIV-1 cure strategies which aim to eliminate this reservoir. Here, we present two triplex digital PCR assays which resulted from a combination of two existing methods, the IPDA (a 2-colour digital PCR based method) and Q4PCR assays (4 colour qPCR method), and tested the functionality on a three-colour digital PCR platform. In the present paper, we provide a step-by-step experimental protocol for these triplex digital PCR assays and validate their performance on a latently infected Jurkat cell-line model and HIV-1 patient samples. Our data demonstrates the potential and flexibility of increasing the number of subgenomic regions of HIV-1 within the IPDA to acquire sensitive detection of the HIV-1 reservoir while benefitting from the advantages of a dPCR setup. [ABSTRACT FROM AUTHOR]