Specific plasma microRNAs are associated with CD4 + T-cell recovery during suppressive antiretroviral therapy for HIV-1.

OBJECTIVE: This study investigated the association of plasma microRNAs before and during antiretroviral therapy (ART) with poor CD4 + T-cell recovery during the first year of ART. DESIGN: MicroRNAs were retrospectively measured in stored plasma samples from people with HIV (PWH) in sub-Saharan Africa who were enrolled in a longitudinal multicountry cohort and who had plasma viral-load less than 50 copies/ml after 12 months of ART. METHODS: First, the levels of 179 microRNAs were screened in a subset of participants from the lowest and highest tertiles of CD4 + T-cell recovery (ΔCD4) ( N  = 12 each). Next, 11 discordant microRNAs, were validated in 113 participants (lowest tertile ΔCD4: n  = 61, highest tertile ΔCD4: n  = 52). For discordant microRNAs in the validation, a pathway analysis was conducted. Lastly, we compared microRNA levels of PWH to HIV-negative controls. RESULTS: Poor CD4 + T-cell recovery was associated with higher levels of hsa-miR-199a-3p and hsa-miR-200c-3p before ART, and of hsa-miR-17-5p and hsa-miR-501-3p during ART. Signaling by VEGF and MET, and RNA polymerase II transcription pathways were identified as possible targets of hsa-miR-199a-3p, hsa-200c-3p, and hsa-miR-17-5p. Compared with HIV-negative controls, we observed lower hsa-miR-326, hsa-miR-497-5p, and hsa-miR-501-3p levels before and during ART in all PWH, and higher hsa-miR-199a-3p and hsa-miR-200c-3p levels before ART in all PWH, and during ART in PWH with poor CD4 + T-cell recovery only. CONCLUSION: These findings add to the understanding of pathways involved in persistent HIV-induced immune dysregulation during suppressive ART.

Noncanonical-NF-κB activation and DDX3 inhibition reduces the HIV-1 reservoir by elimination of latently infected cells ex-vivo.

IMPORTANCE: HIV-1 continues to be a major global health challenge. Current HIV-1 treatments are effective but need lifelong adherence. An HIV-1 cure should eliminate the latent viral reservoir that persists in people living with HIV-1. Different methods have been investigated that focus on reactivation and subsequent elimination of the HIV-1 reservoir, and it is becoming clear that a combination of compounds with different mechanisms of actions might be more effective. Here, we target two host factors, inhibitor of apoptosis proteins that control apoptosis and the DEAD-box helicase DDX3, facilitating HIV mRNA transport/translation. We show that targeting of these host factors with SMAC mimetics and DDX3 inhibitors induce reversal of viral latency and eliminate HIV-1-infected cells in vitro and ex vivo.

Long-term HIV treatment outcomes and associated factors in sub-Saharan Africa: multicountry longitudinal cohort analysis.

OBJECTIVE: In a multicountry prospective cohort of persons with HIV from six countries between 2007 and 2015, we evaluated long-term outcomes of first-line non-nucleoside reverse-transcriptase inhibitor-based antiretroviral therapy (ART), and risk factors for loss-to-follow-up, mortality, virological failure, and incomplete CD4 + T-cell recovery. METHODS: We calculated cumulative incidence of lost-to-follow-up, death, virological failure (VL ≥ 1000 cps/ml) and incomplete CD4 + T-cell recovery (<500 cells/µl) at successive years, using Kaplan-Meier and Cox regression. RESULTS: Of 2735 participants, 58.0% were female, median age was 37 (interquartile range [IQR] 32-43) years, and median pre-ART CD4 + T-cell count was 135 (IQR 63-205)/µl. Total follow-up time was 7208 person-years (median 24.3 months, IQR 18.7-58.3). Deaths by any cause and loss to follow-up occurred mostly during the first year of ART (84%, 201/240 and 56%, 199/353, respectively). During their first 6 years of ART, 71% (95% confidence interval [CI] 69.0-73.7) were retained on first-line, and among those 90-93% sustained viral suppression (<1000 cps/ml); CD4 + T-cell recovery was incomplete in 60% (220/363) of participants. The risk factors associated with poor outcomes during long-term ART were: for loss-to-follow-up, recent VL ≥1000 cps/ml, recent CD4 + T-cell count ≤50 cells/µl, age <30 years, being underweight; for mortality, recent CD4 + T-cell count ≤50 cells/µl; and, for virological failure, age <40 years, recent CD4 + T-cell count ≤200 cells/µl, poor adherence, male sex, and low-level viremia. CONCLUSION: To achieve long-term ART success towards the UNAIDS targets, early ART initiation is crucial, coupled with careful monitoring and retention support, particularly in the first year of ART. Male and youth-centred care delivery models are needed to improve outcomes for those vulnerable groups.

Plasma Inflammatory Biomarkers Predict CD4+ T-cell Recovery and Viral Rebound in HIV-1 Infected Africans on Suppressive Antiretroviral Therapy.

This multicountry prospective study investigated whether persistent systemic inflammation, measured by 8 plasma biomarkers, in HIV-1-infected Africans during suppressive antiretroviral therapy (ART) (viral load <50 copies/mL), was associated with CD4+ T-cell recovery and viral rebound (>1000 copies/mL) during long-term treatment. On-ART sCD14 and C-reactive protein concentrations were inversely associated with subsequent CD4+ T-cell counts. Risk of viral rebound was increased for participants with higher on-ART CXCL10 concentrations and reduced for those with a greater sCD163 decline during the first year of ART. Persistent systemic inflammation predicted CD4+ T-cell recovery and viral rebound, warranting further mechanistic research in relation to clinical outcomes.

Access to HIV Viral Load Testing and Antiretroviral Therapy Switch Practices: A Multicountry Prospective Cohort Study in Sub-Saharan Africa.

Poor access to HIV viral load (VL) testing prevents the timely monitoring of HIV treatment adherence and efficacy. Factors enabling clinical benefits of VL testing when added to local standards of care, can inform the development of more cost-effective routine VL scale-up plans. We compared antiretroviral therapy (ART) switch practices in 13 clinics across 6 countries, with full (N = 8), phasing-in (N = 3) or no onsite access (N = 2) to VL. The analysis used data from the Pan-African Studies to Evaluate Resistance (PASER), observing virological and drug resistance outcomes among adults receiving first- or second-line ART between 2008 and 2015. Study plasma viral load (sVL) determined at baseline, every 12 months thereafter and at the time of switch served for retrospectively validating switch decisions, categorized into "necessary," "unnecessary," and "missed." Virological failure was defined as two consecutive sVL ≥1,000 HIV-RNA copies/mL. One thousand nine hundred ninety-five of the 2,420 (82.4%) study participants had continuous virological suppression during the median 30 months of follow-up. Among the 266 virological failures (11.0%), the proportion of necessary switches were similar in clinics with full (37%), phasing-in (25%), or no access (39%) to local VL testing. Documented utilization of local VL results for the switch decision was associated with higher percentage of necessary switch (87.6% vs. 67.9%). Shorter time to necessary switch was associated with higher rates of long-term virological suppression, regardless of access to local viral load. Availability of HIV VL testing capacity does not systematically result in adequate switch practices or better virological outcomes. Systems supporting sufficient test demand execution, and actual utilization of results for patient management need strengthening.

Plasma Biomarkers of Human Immunodeficiency Virus-Related Systemic Inflammation and Immune Activation in Sub-Saharan Africa Before and During Suppressive Antiretroviral Therapy.

We evaluated immune biomarker profiles in human immunodeficiency virus (HIV)-infected adults (n = 398) from 5 African countries. Although all biomarkers decreased after antiretroviral therapy (ART) initiation, levels of C-X-C chemokine ligand 10 (CXCL10), lipopolysaccharide-binding protein, C-reactive protein, soluble CD163, and soluble scavenger receptor CD14 were significantly higher during ART than in an HIV-uninfected reference group (n = 90), indicating persistent monocyte/macrophage activation, inflammation, and microbial translocation. Before ART initiation, high HIV viral load was associated with elevated CXCL10 and tuberculosis coinfection was associated with elevated soluble CD14. High pre-ART levels of each biomarker strongly predicted residual immune activation during ART. Chemokine (C-C motif) ligand 2, lipopolysaccharide-binding protein, C-reactive protein, and interleukin 6 were differentially expressed between countries. Further research is needed on the clinical implications of residual immune dysregulation.

Suboptimal immune recovery during antiretroviral therapy with sustained HIV suppression in sub-Saharan Africa.

OBJECTIVE: To assess incidence, determinants and clinical consequences of suboptimal immune recovery in HIV-1 infected adults in sub-Saharan Africa with sustained viral suppression on antiretroviral therapy (ART). DESIGN: Multicountry prospective cohort. METHODS: Suboptimal immune recovery was defined as proportions of participants who failed to attain clinically relevant CD4+ cell count thresholds (>200, >350 and >500 cells/µl) despite sustained viral suppression on continuous first-line ART. Participants were censored at the earliest of death, loss to follow-up, last viral load less than 50 copies/ml, or database closure. Determinants of immune recovery were assessed using multivariable Cox regression. We estimated incidence rates of AIDS, pulmonary tuberculosis and all-cause mortality for CD4+ strata. RESULTS: One thousand, five hundred and ninety-two participants were included; 60% were women, median age was 37 years (IQR 31-43) and median pre-ART CD4+ cell count was 147 cells/µl (IQR 76-215). After 6 years of ART, suboptimal immune recovery at CD4+ cell counts less than 200 cells/µl, less than 350  cells/µl, and less than 500 cells/µl occurred in 7, 27, and 57% of participants, respectively. Compared with participants with CD4+ cell count greater than 500 cells/µl, on-ART incidence rates were 12.5, 4.1, 0.9 times higher for AIDS and 16.9, 3.5, and 2.3 times higher for pulmonary tuberculosis in participants with CD4+ cell count less than 200, 200-349, and 350-499 cells/µl, respectively. All-cause mortality was highest in participants with CD4+ cell count less than 200 cells/µl, and comparable across the higher CD4+ strata. Older age, male sex, and lower pre-ART CD4+ cell count were strongly associated with suboptimal immune recovery. CONCLUSION: These findings warrant close clinical and laboratory monitoring until adequate immune reconstitution is achieved and support early ART initiation before decline of CD4+ cell count.

Exosomal sorting of the viral oncoprotein LMP1 is restrained by TRAF2 association at signalling endosomes.

The Epstein-Barr virus (EBV)-encoded oncoprotein latent membrane protein 1 (LMP1) constitutively activates nuclear factor κB (NFκB) from intracellular membranes to promote cell growth and survival. LMP1 associates with CD63 in intracellular membranes and is released via exosomes. Whether tumour necrosis factor (TNF) receptor-associated factors (TRAFs) mediate LMP1 NFκB signalling from endosomes and modulate exosomal sorting is unknown. In this article, we show that LMP1-TRAF2 signalling complexes accumulate at endosomes in a palmitoylation-dependent manner, thereby driving LMP1-dependent oncogenicity. Palmitoylation is a reversible post-translational modification and is considered to function as a membrane anchor for proteins. Mutagenesis studies showed that LMP1-TRAF2 trafficking to endosomes is dependent on one single cysteine residue (C78), a known palmitoylation site of LMP1. Notably, growth assays in soft agar revealed that oncogenic properties of the palmitoylation-deficient LMP1 mutant C78A were diminished compared to wild-type LMP1. Since LMP1 recruitment of TRAF2 and downstream NFκB signalling were not affected by a disturbance in palmitoylation, the specific localization of LMP1 at endosomal membranes appears crucial for its transforming potential. The importance of palmitoylation for trafficking to and signalling from endosomal membranes was not restricted to LMP1, as similar observations were made for the cellular oncoproteins Src and Fyn. Despite abundant LMP1-TRAF2 association at endosomal membranes TRAF2 could not be detected in exosomes by Western blotting or proteomics. Interestingly, point mutations that prevented TRAF binding strongly promoted the sorting and release of LMP1 via exosomes. These observations reveal that LMP1-TRAF2 complexes at endosomes support oncogenic NFκB activation and suggest that LMP1 dissociates from the activated signalling complexes upon sorting into intraluminal vesicles. We propose that "signalling endosomes" in EBV-infected tumour cells can fuse with the plasma membrane, explaining LMP1 release via exosomes.