HIV Replication Increases the Mitochondrial DNA Content of Plasma Extracellular Vesicles.

Extracellular vesicles (EVs) and their cargo have been studied intensively as potential sources of biomarkers in HIV infection; however, their DNA content, particularly the mitochondrial portion (mtDNA), remains largely unexplored. It is well known that human immunodeficiency virus (HIV) infection and prolonged antiretroviral therapy (ART) lead to mitochondrial dysfunction and reduced mtDNA copy in cells and tissues. Moreover, mtDNA is a well-known damage-associated molecular pattern molecule that could potentially contribute to increased immune activation, oxidative stress, and inflammatory response. We investigated the mtDNA content of large and small plasma EVs in persons living with HIV (PLWH) and its implications for viral replication, ART use, and immune status. Venous blood was collected from 196 PLWH, ART-treated or ART-naïve (66 with ongoing viral replication, ≥20 copies/mL), and from 53 HIV-negative persons, all recruited at five HIV testing or treatment centers in Burkina Faso. Large and small plasma EVs were purified and counted, and mtDNA level was measured by RT-qPCR. Regardless of HIV status, mtDNA was more abundant in large than small EVs. It was more abundant in EVs of viremic than aviremic and control participants and tended to be more abundant in participants treated with Tenofovir compared with Zidovudine. When ART treatment was longer than six months and viremia was undetectable, no variation in EV mtDNA content versus CD4 and CD8 count or CD4/CD8 ratio was observed. However, mtDNA in large and small EVs decreased with years of HIV infection and ART. Our results highlight the impact of viral replication and ART on large and small EVs' mtDNA content. The mechanisms underlying the differential incorporation of mtDNA into EVs and their effects on the surrounding cells warrant further investigation.

Immunovirological discordance among female sex workers who start antiretroviral therapy in Burkina Faso.

BACKGROUND: In people living with HIV/AIDS (PLWHA), initiation of antiretroviral therapy (ART) leads to sustained effective suppression of viral replication and increasing CD4 + T cell count. However, a fraction of ART-treated patients still fail to reach adequate CD4 + T cell number despite a suppressed viral load (VL), and this phenomenon is defined as immunovirological discordance (IVD). In Africa, several studies have reported immunovirological outcomes of antiretroviral therapy, but little is known about IVD occurrence in Female sex workers (FSW). This study aimed to assess the prevalence of IVD and associated factors among a cohort of HIV infected FSW in Burkina Faso. METHODS: We conducted a cohort study from December 2003 to October 2016. Immunovirological discordance was defined as CD4 + T cell gain < 100 cells/µL despite a suppressed VL (VL < 1000 copies/mL) 12 months after ART initiation. The CD4 + T cells were counted using BD FACSCount™ System and point of care Pima™ CD4 + Analyzer. HIV-1 RNA was quantified by real-time polymerase-chain-reaction assay with the use of the ABI 7000 system. We conducted a logistic regression to identify factors associated with discordant responses. RESULTS: Among the 123 HIV-1 infected FSW having at least 12 months follow-up on ART, 105 (85.4%) achieved HIV-1 RNA suppression. Among the latter 25 gained less than 100 CD4 + T cells within 12 months follow-up. The IVD rate was 23.8% (95%CI 16.04%-33.11%). After adjustment for age, WHO clinical stage and ART regimen including nucleoside/nucleotide reverse transcriptase inhibitors, only baseline CD4 + T cell count between 200 to 350 cells/µL (adjusted OR: 4.15; 95%CI 1.13-15.22) and 350 to 500 cells/µL (adjusted OR: 17.50; 95%CI 2.68-114.31) remain significantly associated with IVD occurrence. CONCLUSIONS: Immunovirological discordance response was common in FSW with proportions close to those observed in the general population. A diagnosis and personalized follow-up of patients who do not achieve full immune reconstitution would make it possible to avoid complications in terms of morbidity and mortality.

Vesicular MicroRNA as Potential Biomarkers of Viral Rebound.

Changes in the cellular microRNA (miRNA) expression profile in response to HIV infection, replication or latency have been reported. Nevertheless, little is known concerning the abundance of miRNA in extracellular vesicles (EVs). In the search for a reliable predictor of viral rebound, we quantified the amount of miR-29a, miR-146a, and miR-155 in two types of plasma extracellular vesicles. Venous blood was collected from 235 ART-treated and ART-naive persons living with HIV (85 with ongoing viral replication, ≥20 copies/mL) and 60 HIV-negative participants at five HIV testing or treatment centers in Burkina Faso. Large and small plasma EVs were purified and counted, and mature miRNA miR-29a, miR-146a, and miR-155 were measured by RT-qPCR. Diagnostic performance of miRNA levels in large and small EVs was evaluated by a receiver operating characteristic curve analysis. The median duration of HIV infection was 36 months (IQR 14-117). The median duration of ART was 34 months (IQR 13-85). The virus was undetectable in 63.8% of these persons. In the others, viral load ranged from 108 to 33,978 copies/mL (median = 30,032). Large EVs were more abundant in viremic participants than aviremic. All three miRNAs were significantly more abundant in small EVs in persons with detectable HIV RNA, and their expression levels in copies per vesicle were a more reliable indicator of viral replication in ART-treated patients with low viremia (20-1000 copies/mL). HIV replication increased the production of large EVs more than small EVs. Combined with viral load measurement, quantifying EV-associated miRNA abundance relative to the number of vesicles provides a more reliable marker of the viral status. The expression level as copies per small vesicle could predict the viral rebound in ART-treated patients with undetectable viral loads.

Plasma vesicular miR-155 as a biomarker of immune activation in antiretroviral treated people living with HIV.

People living with HIV (PLWH), despite suppression of viral replication with antiretroviral therapy (ART), have high morbidity and mortality due to immune activation and chronic inflammation. Discovering new biomarkers of immune activation status under ART will be pertinent to improve PLWH quality of life when the majority will be treated. We stipulate that plasma large and small extracellular vesicle (EVs) and their microRNA content could be easily measured biomarkers to monitor immune activation in PLWH. Venous blood samples from n = 128 ART-treated PLWH with suppressed viral load (≤ 20 copies/mL) and n = 60 HIV-uninfected participants were collected at five testing or treatment centers of PLWH in Burkina Faso. Large and small plasma EVs were purified, counted, and the mature miRNAs miR-29a, miR-146a, and miR-155 were quantified by RT-qPCR. Diagnostic performances of large and small EVs miRNAs level were evaluated by receiver operating characteristic (ROC) curve analysis and principal component analysis (PCA). Among the EVs microRNA measured, only large EVs miR-155 copies distinguished PLWH with immune activation, with AUC of 0.75 for CD4/CD8 < 1 (95% CI: 0.58-0.91, P = 0.0212), and 0.77 for CD8 T cells ≥ 500/µL (95% CI: 0.63-0.92, P = 0.0096). In addition, PCA results suggest that large EVs miR-155 copies may be a biomarker of immune activation. Since miR-155 may influence immune cell function, its enrichment in large EV subpopulations could be a functional biomarker of immune activation in PLWH on ART. This measure could help to monitor and diagnose the immune activation with more accuracy.

Prevalence and factors associated of Trichomonas vaginalis infection among pregnant women in Bobo-Dioulasso, Burkina Faso.

Epidemiological studies of vaginalis trichomonosis, especially in pregnant women are rare in Africa due to the lack of screening programs. The present study aimed to assess the prevalence of T. vaginalis infection and its associated factors in pregnant women who attended the antenatal care clinics in three primary health centers of Bobo-Dioulasso. We carried out a cross-sectional study for descriptive and analytical purposes from February to April 2015 in pregnant women seen in prenatal consultations. The study took place in 3 primary public health centers: Guimbi (Central Urban), Bolomakoté (Peri-urban) and Yéguérésso (rural). The trophozoites of Trichomonas vaginalis was carried out by microscopy on vaginal swabs and urine samples. Sociodemographic, obstetric and biological variables were also collected. A total of 315 pregnant women were included in the study. The overall prevalence of urogenital trichomonosis was 3.2%. It was 1.9% in Guimbi, 2.9% in Bolomakoté, and 4.7% in Yéguérésso. The prevalence of HIV infection was 2.2%. Married women were less exposed to T. vaginalis infection than single women (p=0.03). The prevalence of urogenital trichomonosis obtained was considered lower compared to the previously reported from Burkina Faso. Thus, it is essential to extend this study to the whole country periodically by integrating other STIs not subject to a surveillance system and by integrating molecular epidemiology tools.

Diurnal Variation of Plasma Extracellular Vesicle Is Disrupted in People Living with HIV.

BACKGROUND: Several types of extracellular vesicles (EVs) secreted by various immune and non-immune cells are present in the human plasma. We previously demonstrated that EV abundance and microRNA content change in pathological conditions, such as HIV infection. Here, we investigated daily variations of large and small EVs, in terms of abundance and microRNA contents in people living with HIV (PLWH) receiving antiretroviral therapy (HIV+ART) and uninfected controls (HIV-). METHODS: Venous blood samples from n = 10 HIV+ART and n = 10 HIV- participants were collected at 10:00 and 22:00 the same day. Large and small plasma EVs were purified, counted, and the mature miRNAs miR-29a, miR-29b, miR-92, miR-155, and miR-223 copies were measured by RT-PCR. RESULTS: Large EVs were significantly bigger in the plasma collected at 10:00 versus 22:00 in both groups. There was a significant day-night increase in the quantity of 5 miRNAs in HIV- large EVs. In HIV+ART, only miR-155 daily variation has been observed in large EVs. Finally, EV-miRNA content permits to distinguish HIV- to HIV+ART in multivariate analysis. CONCLUSION: These results point that plasma EV amount and microRNA contents are under daily variation in HIV- people. This new dynamic measure is disrupted in PLWH despite viral-suppressive ART. This study highlights a significant difference concerning EV abundance and their content measured at 22:00 between both groups. Therefore, the time of blood collection must be considered in the future for the EV as biomarkers.

Velocity Gradient Separation Reveals a New Extracellular Vesicle Population Enriched in miR-155 and Mitochondrial DNA.

Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.

Plasma Extracellular Vesicle Subtypes May be Useful as Potential Biomarkers of Immune Activation in People With HIV.

BACKGROUND: Extracellular vesicles (EVs) are intercellular messengers with epigenetic potential since they can shuttle microRNA (miRNA). EVs and miRNA play a role in human immunodeficiency virus (HIV) infection immunopathogenesis. Chronic immune activation and systemic inflammation during HIV infection despite effective antiretroviral therapy (ART) are associated with non-acquired immunodeficiency syndrome (AIDS) comorbidities in people living with HIV (PLWH). Analysis of plasma EVs and their miRNA content may be useful as immune activation or inflammatory biomarkers in PLWH receiving ART. In this study, we hypothesized that the number, size, and miRNA of large and small EVs could reflect immune activation associated with an elevated CD8 T-cell count or a low CD4/CD8 ratio in PLWH. METHODS: Plasma EVs subtype purified from PLWH and uninfected controls were sized using dynamic light scattering and quantified using flow cytometry and acetylcholine esterase (AChE) activity. Expression of mature miRNAs miR-92, miR-155, miR-223 was measured by quantitative reverse-transcriptase polymerase chain reaction in EVs and leucocytes. RESULTS: HIV infection induces increased production of small EVs in plasma. EV subtypes were differentially enriched in miR-92, miR-155, and miR-223. Positive correlations between CD8 T-cell count and large EVs abundance and small EVs AChE activity were observed. CD4/CD8 ratio was negatively correlated with small EV AChE activity, and miRNA-155 level per small EV was negatively correlated with CD8 T-cell count. CONCLUSIONS: These findings suggest that quantifying large or small EVs and profiling miRNA content per EV might provide new functional biomarkers of immune activation and inflammation.