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Background: Data on the burden of dyslipidaemia among people with HIV undergoing antiretroviral therapy (ART) in sub-Saharan Africa are limited and little is known about the factors contributing for poor lipid profiles. The aim of this study was to determine the prevalence of dyslipidaemia and factors associated with lipid levels among HIV-infected patients receiving first-line combination ART in North Shewa, Ethiopia., Methods: A cross-sectional study was conducted between April and December 2018 among 392 HIV-infected adults receiving first-line ART for at least six months at the ART clinic of Mehal Meda Hospital in North Shewa, Ethiopia. Blood samples were collected for determination of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and CD4 counts. Logistic regression analysis was used to determine factors associated with lipid abnormalities., Results: The prevalence of dyslipidaemia was 59.9% (95% CI 55.0-64.7%). High TC, high TG, low HDL-c, and high LDL-c were obtained in 47.3%, 30.9%, 19.4% and 29.6%, respectively. Fifty-four participants (13.8%) had high ratio of TC/HDL-c (TC/HDL-c ratio ≥ 5). Older age was independently associated with high TC (AOR = 2.51, 95% CI 1.64-3.84), high TG (AOR = 2.95, 95% CI 1.85-4.71), low HDL-c (AOR = 2.02, 95% CI 1.17-3.50), and high LDL-c (AOR = 3.37, 95% CI 2.08-5.47). Living in an urban area (AOR = 2.61, 95% CI 1.16-6.14) and smoking (AOR = 3.61, 95% CI 1.06-12.34) were associated with low HDL-c. Participants with longer duration of ART use were more likely to have high TG (AOR = 1.86, 95% CI: 1.13-3.07), low HDL-c (AOR = 3.47, 95% CI: 1.75-6.80), and high LDL-c (AOR = 2.20, 95% CI 1.30-3.71). High BMI was independently associated with higher TC (AOR = 2.43, 95% CI 1.19-4.97), high TG (AOR = 4.17, 95% CI 2.01-8.67) and high LDL-c (AOR = 6.53, 95% CI 3.05-13.98)., Conclusions: We found a high prevalence of dyslipidaemia among HIV-infected patients receiving first-line ART in North Shewa, Ethiopia. There is a need for monitoring of blood lipid levels in patients with HIV on long term first-line ART with a special attention to be focused on older age, urban residents, longer duration of ART use, high BMI and smokers., Competing Interests: The authors have declared that no competing interests exist.

Kidney transplant recipients (KTRs) abnormally replicate the Epstein Barr Virus (EBV). To better understand how long-term immunosuppression impacts the immune control of this EBV re-emergence, we systematically compared 10 clinically stable KTRs to 30 healthy controls (HCs). The EBV-specific T cell responses were determined in both groups by multiparameter flow cytometry with intra cellular cytokine staining (KTRs n = 10; HCs n = 15) and ELISpot-IFNγ assays (KTRs n = 7; HCs n = 7). The T/B/NK cell counts (KTRs n = 10; HCs n = 30) and the NK/T cell differentiation and activation phenotypes (KTRs n = 10; HCs n = 15/30) were also measured. We show that in KTRs, the Th1 effector CD4+ T cell responses against latent EBV proteins are weak (2/7 responders). Conversely, the frequencies total EBV-specific CD8+T cells are conserved in KTRs (n = 10) and span a wider range of EBNA-3A peptides (5/7responders) than in HCs (5/7responders). Those modifications of the EBV-specific T cell response were associated with a profound CD4+ T cell lymphopenia in KTRs compared to HCs, involving the naïve CD4+ T cell subset, and a persistent activation of highly-differentiated senescent CD8+ T cells. The proportion of total NK / CD8+ T cells expressing PD-1 was also increased in KTRs. Noteworthy, PD-1 expression on CD8+ T cells normalized with time after transplantation. In conclusion, we show modifications of the EBV-specific cellular immunity in long term transplant recipients. This may be the result of both persistent EBV antigenic stimulation and profound immunosuppression induced by anti-rejection treatments. These findings provide new insights into the immunopathology of EBV infection after renal transplantation., Competing Interests: The authors disclose no conflicts of interest except Martin Larsen who is the proprietary owner of Funky Cells software. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors have declared that no competing interests exist.

Background: HIV-positive patients are facing age-and disease-related comorbidities. Since gender differences in viro-immunological, clinical and therapeutic features have been described, aim of this analysis was to explore such differences in elderly HIV-positive females compared to males coming from the same cohort., Design: Cross-sectional study., Setting: Ten Infectious Diseases Center participating to a new multicenter Italian geriatric Cohort aiming at describing health transition over time in HIV-positive individuals., Participants: HIV-positive patients aged ≥65 years old., Measurements: We recorded clinical, viro-immunological and therapeutical data., Results: We included 210 women (17%) out of 1237 patients. Compared to males, elderly females were less likely to present a HIV-RNA <50 copies/mL (74.3% vs. 81.8%, OR 0.64, 95%CI 0.44-0.93); they showed higher CD4+/CD8+ ratio (p = 0.016). Combined antiretroviral therapy (cART) strategies were similar between genders (p>0.05), although women were less likely to be treated with protease Inhibitors (PIs) (p = 0.05); specifically, in triple-drug regimens females received less PIs (28% vs 38% p = 0.022) and more integrase inhibitors (30% vs. 20% p = 0.012). Bone disease was more common in females (p<0.001) while males presented more frequently cardiovascular disease (CVD) (p<0.001). In females with bone disease, PIs and boosted regimens (38% vs. 53.7% p = 0.026 and 30.4 vs 44.0% p = 0.048 respectively) were prescribed less frequently. Polypharmacy was common and similar in both genders (20% vs. 22.8%, p = >0.05). A higher use of lipid-lowering drugs (20.5% vs. 14.8%, p = 0.04) was observed in females and yet they were less likely to receive anti-thrombotic agents (18.6% vs. 26.3%, p = 0.019) even when CVD was recorded (57.1% vs. 83.1%, p = 0.018). In multivariate analysis, we found that female gender was independently associated with a higher CD4+/CD8+ ratio but not with virological suppression., Conclusions: Elderly HIV-positive women display a worse virologic response despite a better immune reconstitution compared to males. The burden of comorbidities as well as the medications received (including cART) may slightly differ according to gender. Our data suggest that more efforts and focused interventions are needed in this population., Competing Interests: EF has received travel grants, consultancy fees and speaker’s honoraria from Gilead Science, ViiV Healthcare, Janssen-Cilag and Merck Sharp & Dohme. GVDS has received travel grants and speaker’s honoraria from Gilead Science, ViiV Healthcare, Janssen-Cilag and Merck Sharp & Dohme. SN received travel grants, consultancy fees and speaker’s honoraria from Gilead Science, ViiV Healthcare, Janssen-Cilag and Merck Sharp & Dohme. AC received travel grants, consultancy fees and speaker’s honoraria from Gilead Science, ViiV Healthcare, Janssen-Cilag and Merck Sharp & Dohme. The other Authors have no conflicts to disclose. This work has been possible thanks to the unconditional grant by Viiv Healthcare Italy within the “Ageing and Frailty Working Group”. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Residual HIV-1 replication among individuals under antiretroviral therapy (ART) relates to HIV micro-inflammation., Objectives: To determine the levels of residual HIV replication markers among distinct subgroups of antiretroviral-treated individuals., Methods: One hundred sixteen patients were distributed into 5 treatment groups: first-line suppressive ART with a non-nucleoside analog reverse-transcriptase inhibitor (NNRTI) (n = 26), first-line suppressive ART with boosted protease inhibitors (PI-r) (n = 25), salvage therapy using PI-r (n = 27), salvage therapy with PI-r and raltegravir (n = 22) and virologic failure (n = 16). Episomal and total DNA quantitation was evaluated. ELISA was used for HIV antibody and LPS quantitation., Results: Episomal DNA was positive in 26% to 38% of individuals under suppressive ART, and it was higher among individuals experiencing ART virologic failure (p = 0.04). The HIV proviral load was higher among patients with detectable episomal DNA (p = 0.01). Individuals receiving initial PI-r treatment presented lower HIV antibody (p = 0.027) and LPS (p = 0.029) levels than individuals receiving NNRTI. There was a negative correlation between episomal DNA quantitation and the duration of suppressive ART (p = 0.04), CD4+ T-cell count (p = 0.08), and CD8+ T-cell count (p = 0.07)., Conclusions: Residual HIV replication has been inferred among individuals under suppressive ART according to episomal DNA detection. Residual replication may decrease with longer periods of suppressive ART and higher levels of CD4+ and CD8+ T cells. The relationship between episomal DNA and total DNA suggests there is a replenishment of the proviral reservoir with impacts on HIV persistence. Lower antibody and LPS levels among patients with initial PI-r ART suggest these regimens may more effectively suppress HIV and have a higher capacity to decrease the HIV antigenic component., Competing Interests: The authors have declared that no competing interests exist.

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA)., Competing Interests: Author PAG received funding from Debiopharm International. Authors NW and GV are employees of Debiopharm International. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Blockade of the programmed cell death protein/ligand 1 (PD-1/PD-L1) pathway with monoclonal antibodies (mAb) is now commonly used for cancer immunotherapy and has therapeutic potential in chronic viral infections including HIV-1. PD-1/PD-L1 blockade could augment HIV-1-specific immune responses and reverse HIV-1 latency, but the latter effect has not been clearly shown. We tested the ability of the human anti-PD-L1 mAb BMS-936559 and the human anti-PD-1 mAb nivolumab to increase HIV-1 virion production ex vivo from different peripheral blood mononuclear cell populations obtained from donors on suppressive antiretroviral therapy (ART). Fresh peripheral blood mononuclear cells (PBMC), CD8-depleted PBMC, total CD4+ T cells, and resting CD4+ T cells were purified from whole blood of HIV-1-infected donors and cultured in varying concentrations of BMS-936559 (20, 5, or 1.25μg/mL) or nivolumab (5 or 1.25μg/mL), with or without anti-CD3/CD28 stimulatory antibodies. Culture supernatants were assayed for virion HIV-1 RNA by qRT-PCR. Ex vivo exposure to BMS-936559 or nivolumab, with or without anti-CD3/CD28 stimulation, did not consistently increase HIV-1 virion production from blood mononuclear cell populations. Modest (2-fold) increases in virus production were observed in a subset of donors and in some cell types but were not reproducible in longitudinal samples. Cell surface expression of PD-1 and PD-L1 were not associated with changes in virus production. Ex vivo blockade of the PD-1 axis alone has limited effects on HIV-1 latency., Competing Interests: Research reported in this publication was supported by the University of Pittsburgh, Bristol-Myers Squibb Company, and the Howard Hughes Medical Institute. JWM is a consultant to Gilead Sciences and Merck, and has received grants from Bristol-Myers Squibb Company, Gilead Sciences and Janssen Pharmaceuticals, Inc. He owns stock options in Co-Crystal Pharma, Inc. JB. received support as a research fellow from the Howard Hughes Medical Institute. JC and EF report no competing interests. SC and SWM were employees of Bristol-Myers Squibb (BMS) Company at the time the work was performed. They are no longer affiliated with BMS. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: CD4 T-cell counts are still widely used to assess treatment eligibility and follow-up of HIV-infected patients. The World Health Organization (WHO) prequalification of in vitro diagnostics requested a manufacturer independent laboratory evaluation of the analytical performance at the Institute of Tropical Medicine (ITM) Antwerp, Belgium, of the Muse Auto CD4/CD4% system (Millipore), a new small capillary-flow cytometer dedicated to count absolute CD4-T cells and percentages in venous blood samples from HIV-infected patients., Methods: Two hundred and fifty (250) patients were recruited from the HIV outpatient clinic at ITM. Accuracy and precision of CD4 T cell counting on fresh EDTA anticoagulated venous blood samples were assessed in the laboratory on a Muse Auto CD4/CD4% system. Extensive precision analyses were performed both on fresh blood and on normal and low stabilized whole blood controls. Accuracy ((bias) was assessed by comparing results from Muse CD4/CD4% to the reference (single-platform FACSCalibur). Clinical misclassification was measured at 500, 350, 200 and 100 cells/μL thresholds., Results: Intra-assay precision was < 5%, and inter-assay was < 9%. CD4 T cell counts measured on Muse Auto CD4/CD4% System and on the reference instrument resulted in regression slopes of 0.97 for absolute counts and 1.03 for CD4 T cell percentages and a correlation coefficient of 0.99 for both. The average absolute bias as compared to the reference was negligible (4 cells/μL or 0.5%). The absolute average bias on CD4 T cell percentages was < 1%. Clinical misclassification at different CD4 T cell thresholds was small resulting in sensitivities and specificities equal or >90% at all thresholds except at 100 cells/μL (sensitivity = 87%). All samples could be analyzed as there was no repetitive rejection errors recorded., Conclusions: The Muse Auto CD4/CD4% System performed very well on fresh venous blood samples and met all WHO acceptance criteria for analytical performance of CD4 technologies., Competing Interests: The authors have declared that no competing interests exist.

HIV-1 co-infection is a leading cause of susceptibility to tuberculosis (TB), with the risk of TB being increased at all stages of HIV-1 infection. Antiretroviral treatment (ART) is the most effective way to reduce the risk of TB in HIV-1 co-infected people. Studying protective, ART-induced, immune restoration in HIV-1 infected individuals sensitised by Mycobacterium tuberculosis (Mtb) can thus help identify mechanisms of protection against TB. In order to understand ART-mediated prevention of TB in HIV-1 infected adults, we investigated the expression of 30 genes in whole blood from HIV-1 infected patients during the first 6 months of ART-induced immune reconstitution. The 30 selected genes were previously described to be differentially expressed between sorted Mtb specific central and effector memory CD4 T cells. HIV-1 infected persons sensitised by Mtb were recruited in Khayelitsha, South Africa, when initiating ART. RNA was extracted from whole blood at initiation and 1, 3 and 6 months of ART. qRT-PCR was used to determine gene expression and three reference 'housekeeping' genes were used to calculate the fold change in the expression of each gene relative to day 0 of ART. Results were assessed longitudinally. We observed a decrease in the expression of a number of genes at 6 months of ART, reflecting a decrease in immune activation. However, following correction for multiple comparisons and increasing CD4 counts, only the decrease in CD27 gene expression remained statistically significant. While not statistically significant, a number of genes also showed increased expression at various timepoints, illustrating the broad regeneration of the T cell pool in HIV-1 infected adults on ART. Our findings generate hypotheses underlying ART- induced protective immune reconstitution and may pave the way for future studies to evaluate ART mediated prevention of TB in HIV-1 infected persons., Competing Interests: The authors have declared that no competing interests exist.

Although anti-retroviral therapies have greatly extended the lives of HIV infected individuals, current treatments are unable to completely eliminate virally infected cells. A number of latency reversing agents have been proposed for use in a "shock and kill" strategy to reactivate latent HIV, thus making it vulnerable to killing mechanisms. Procyanidin trimer C1 (PC1) is a flavonoid found in multiple plant sources including grape, apple, and cacao, which has antioxidant and anti-inflammatory properties. We determined that PC1 reactivates latent HIV in cell line and primary cell models of HIV, through activation of the MAPK pathway. Notably, PC1 reactivates latent HIV without increasing surface markers of T cell activation. Combining several therapeutics, which activate HIV transcription through different mechanisms, is the most efficient approach to clinically reactivate latent reservoirs. We utilized PC1 (MAPK agonist), kansui (PKC agonist), and JQ1 (BET bromodomain inhibitor) in a triple combination approach to reactivate latent HIV in cell line and primary cell models of HIV latency. When used in combination, low concentrations which fail to reactivate HIV as single treatments, are effective. Thus, several mechanisms, using distinct activation pathways, act together to reactivate latent HIV., Competing Interests: The authors have declared that no competing interests exist.

The role of IL-27 in antiviral immunity is still incompletely understood, especially in the context of chronic viruses that induce a unique environment in their infected host. Cytomegalovirus (CMV) establishes a persistent, tissue localized infection followed by lifelong latency. CMV infects the majority of people and although asymptomatic in healthy individuals, can cause serious disease or death in those with naïve or compromised immune systems. Therefore, there is an urgent need to develop a protective CMV vaccine for people at-risk and identifying key regulators of the protective immune response towards CMV will be crucial. Here we studied mouse CMV (MCMV) in IL-27 receptor deficient animals (Il27ra-/-) to assess the role of IL-27 in regulating CMV immunity. We found that IL-27 enhanced the number of antiviral CD4 T cells upon infection. However, in contrast to a well-established role for CD4 T cells in controlling persistent replication and a positive effect of IL-27 on their numbers, IL-27 promoted MCMV persistence in the salivary gland. This coincided with IL-27 mediated induction of IL-10 production in CD4 T cells. Moreover, IL-27 reduced expression of the transcription factor T-bet and restricted a cytotoxic phenotype in antiviral CD4 T cells. This is a highly intriguing result given the profound cytotoxic phenotype of CMV-specific CD4 T cells seen in humans and we established that dendritic cell derived IL-27 was responsible for this effect. Together, these data show that IL-27 regulates the number and effector functions of MCMV-specific CD4 T cells and could be targeted to enhance control of persistent/latent infection., Competing Interests: The authors have declared that no competing interests exist.

Most HIV-infected patients who initiate combination antiretroviral therapy experience a viral load decline in several phases. These phases are characterized by different rates of viral load decay that decrease when transitioning from one phase to the next. There is no consensus as to the origin of these phases. One hypothesis put forward is that short- and long-lived infected cells are responsible for the first and second phases of decay, respectively. However, significant differences in drug concentrations are observed in monocytes from various tissues, suggesting the first two phases of decay in viral loads could instead be attributed to short-lived cells being differently exposed to drugs. Compared to a well-exposed compartment, new cell infection can be expected in a compartment with limited drug exposure, thus leading to a slower viral load decay with potential virologic failure and drug resistance. In the current study, the latter hypothesis was investigated using a model of viral kinetics. Empirical datasets were involved in model elaboration and parameter estimation. In particular, susceptibility assay data was used for an in vitro to in vivo extrapolation based on the expected drug concentrations inside physiological compartments. Results from numerical experiments of the short-term evolution of viral loads can reproduce the first two phases of viral decay when allowing new short-lived cell infections in an unidentified drug-limited compartment. Model long-term predictions are however less consistent with clinical observations. For the hypothesis to hold, efavirenz, tenofovir and emtricitabine drug exposure in the drug-limited compartment would have to be very low compared to exposure in peripheral blood. This would lead to significant long-term viral growth and the frequent development of resistant strains, a prediction not supported by clinical observations. This suggests that the existence of a drug-limited anatomical compartment is unlikely, by itself, to explain the second phase of viral load decay., Competing Interests: The authors confirm that there are no competing interests. The NSERC Industrial Research Chairs are funded jointly by NSERC (Natural Science and Engineering Research Council of Canada) and eligible partners Novartis, Pfizer and Inventiv Health. Partners are required by the government agency to comply with the rules of the agency. These rules imply that they have not interfered with the full and objective presentation, peer review, editorial decision-making, or publication of research articles.

Background: A blood-based assay that could quantify HIV susceptibility would be very valuable for HIV prevention research. Previously, we developed and validated an ex vivo, flow-based, HIV entry assay to assess genital HIV susceptibility in endocervical CD4+ T cells., Methods: Here we assessed whether this tool could be used to predict HIV risk using blood-derived CD4+ T cells in a rigorously-blinded, nested case-control study using blood samples collected from high-risk, HIV-uninfected South African women enrolled in the CAPRISA 004 clinical trial. Cases, subsequently acquiring HIV were sampled prior to HIV infection and compared with controls, who remained HIV-uninfected. The primary endpoint was ex vivo entry of a CCR5-tropic HIV founder virus into blood CD4+ T cells. Secondary endpoints included HIV entry into CD4+ central (TCM) and effector (TEM) memory T cells, and into CD4+ T cell subsets expressing CCR5, CD69, CCR6, α4β1 or α4β7., Results: Compared to bulk CD4+ T cells (4.9% virus entry), CD4+ T cells expressing CCR5, CCR6 or α4β1 and TEM were highly susceptible (15.5%, 8.8%, 8.2% and 10.8% entry, respectively, all p<0.0001), while TCM, CD69+ or α4β7+ CD4+ cells were moderately susceptible (6.4%, 6.0% and 5.8% respectively, p ≤ 0.003). While the proportion of the aforementioned highly susceptible cells correlated with overall virus entry into CD4+ T cells within an individual (r = 0.68, 0.47, 0.67, and 0.60 respectively, p<0.0001), blood virus entry did not predict subsequent mucosal HIV acquisition after controlling for sexual behaviour and condom use (OR 0.92, 95% CI 0.77-1.11, p = 0.40)., Conclusions: Although virus entry identified several previously known highly susceptible cellular HIV targets, blood HIV entry did not predict subsequent heterosexual HIV acquisition. Assessment of mucosal HIV susceptibility may require sampling at the site of HIV exposure., Competing Interests: The authors have declared that no competing interests exist.

Background: Human immunodeficiency virus (HIV) promotes an inflammatory process, leading to the progressive loss of the functional capacity of the immune system. The HIV infection induces alterations in several tissues, but mainly in the gut-associated lymphoid tissue (GALT). However, the degree of GALT deterioration varies among infected individuals. In fact, it has been shown that HIV-controllers, who spontaneously control viral replication, exhibit a lower inflammatory response, and a relative normal frequency and function of most of the immune cells. Inflammasomes are molecular complexes involved in the inflammatory response, being NLRP1, NLRP3, NLRC4, AIM2 and Pyrin inflammasomes, the best characterized so far. These complexes regulate the maturation of cytokines of the IL-1 family, including IL-1β and IL-18. These cytokines have been associated with immune activation and expansion of HIV target cells, promoting viral replication. Interesting, some reports indicate that HIV induces the activation of the NLRP3 inflammasome, but the role of this, and other inflammasomes during HIV infection, especially in GALT, remains unclear., Objective: To compare the relative expression of inflammasome components and the proinflammatory response related to their activity, between HIV-progressors and HIV-controllers., Methods: GALT biopsies and peripheral blood mononuclear cells (PBMCs) from 15 HIV-controllers and 15 HIV-progressors were obtained. The relative expression of the following inflammasome components were evaluated by RT-PCR: NLRP3, NLRC4, NLRP1, AIM2, ASC, Caspase-1, IL-1β and IL-18. In addition, plasma concentration of IL-18 was evaluated as an indicator of baseline proinflammatory status. Finally, in supernatants of PBMCs in vitro stimulated with inflammasome agonists, the concentrations of IL-1β and IL-18 were quantified by ELISA., Results: HIV-progressors exhibited higher expression of IL-1β, IL-18 and caspase-1 genes in GALT and PBMCs compared with HIV-controllers. In addition, HIV-progressors had also increased expression of ASC in PBMCs. When plasma levels were evaluated, IL-18 was increased in HIV-progressors. Interesting, these patients also showed an increased production of IL-1β in supernatants of PBMCs stimulated in vitro with the agonists of AIM2, NLRP1 and NLRC4 inflammasomes. Finally, the expression of caspase-1, NLRP1, IL-1β and IL-18 in GALT or peripheral blood was correlated with CD4+ T-cell count and viral load., Conclusion: Our results suggest that during HIV-infection, the required signals to induce the expression of different components of the inflammasomes are produced, both in GALT and in periphery. The activation of these molecular complexes could increase the number of target cells, favoring HIV replication and cell death, promoting the disease progression.

Objectives: Despite successful virological suppression, HIV transcription frequently persists intracellularly. In this study, we hypothesize that HIV persistent transcription(HIVpt) may affect to a different extent patients on stable efavirenz(EFV) versus atazanavir(ATV)-based regimens. The role of the expression of drug efflux transporters in HIVpt was also investigated., Methods: We prospectively enrolled 51 virologically suppressed patients on first-line treatment for one year with EFV or ATV combined with emtricitabine and tenofovir and followed them up for one year. Simultaneous ultrasensitive subpopulation staining/hybridization in situ(SUSHI) was performed to identify HIVpt in CD4+ T-cells and in the CD4+CD45RO+ T-cell subpopulation. The differential mRNA expression of P-glycoprotein(P-gp/ABCB1) and multidrug resistance-associated protein-1(MRP1/ABCC1) was also evaluated. Univariate logistic regression models were used to evaluate predictors of HIVpt., Results: In the CD4+ T-cell population, HIVpt affected 13/30 of patients on EFV versus 10/21 on ATV. In the CD4+CD45RO+ T-cell population, HIVpt was present in 14/30 of patients on EFV versus 15/21 on ATV. A trend for association was observed between the risk of HIVpt and ATV treatment in the CD4+CD45RO+ T-cell population (OR 2.86, 95% CI 0.87-9.37, p = 0.083). HIVpt status was not associated with loss of virological suppression or CD4 evolution. We found no evidence of differential expression of the drug efflux transporters P-gp and MRP1., Conclusions: Further study is required to evaluate whether the HIVpt profile in specific cell populations may differ across different antiretroviral regimens and to elucidate the potential clinical impact.

The dynamics of latent HIV is linked to infection and clearance of resting memory CD4+ T cells. Infection also resides within activated, non-dividing memory cells and can be impacted by antigen-driven and homeostatic proliferation despite suppressive antiretroviral therapy (ART). We investigated whether plasma viral level (pVL) and HIV DNA dynamics could be explained by HIV's impact on memory CD4+ T cell homeostasis. Median total, 2-LTR and integrated HIV DNA levels per μL of peripheral blood, for 8 primary (PHI) and 8 chronic HIV infected (CHI) individuals enrolled on a raltegravir (RAL) based regimen, exhibited greatest changes over the 1st year of ART. Dynamics slowed over the following 2 years so that total HIV DNA levels were equivalent to reported values for individuals after 10 years of ART. The mathematical model reproduced the multiphasic dynamics of pVL, and levels of total, 2-LTR and integrated HIV DNA in both PHI and CHI over 3 years of ART. Under these simulations, residual viremia originated from reactivated latently infected cells where most of these cells arose from clonal expansion within the resting phenotype. Since virion production from clonally expanded cells will not be affected by antiretroviral drugs, simulations of ART intensification had little impact on pVL. HIV DNA decay over the first year of ART followed the loss of activated memory cells (120 day half-life) while the 5.9 year half-life of total HIV DNA after this point mirrored the slower decay of resting memory cells. Simulations had difficulty reproducing the fast early HIV DNA dynamics, including 2-LTR levels peaking at week 12, and the later slow loss of total and 2-LTR HIV DNA, suggesting some ongoing infection. In summary, our modelling indicates that much of the dynamical behavior of HIV can be explained by its impact on memory CD4+ T cell homeostasis.

HIV-1 induces changes in the miRNA expression profile of infected CD4+ T cells that could improve viral replication. HIV-1 regulator Tat modifies the cellular gene expression and has been appointed as an RNA silencing suppressor. Tat is a 101-residue protein codified by two exons that regulates the elongation of viral transcripts. The first exon of Tat (amino acids 1-72) forms the transcriptionally active protein Tat72, but the presence of the second exon (amino acids 73-101) results in a more competent regulatory protein (Tat101) with additional functions. Intracellular, full-length Tat101 induces functional and morphological changes in CD4+ T cells that contribute to HIV-1 pathogenesis such as delay in T-cell proliferation and protection against FasL-mediated apoptosis. But the precise mechanism by which Tat produces these changes remains unknown. We analyzed how the stable expression of intracellular Tat101 and Tat72 modified the miRNA expression profile in Jurkat cells and if this correlated with changes in apoptotic pathways and cell cycle observed in Tat-expressing cells. Specifically, the enhanced expression of hsa-miR-21 and hsa-miR-222 in Jurkat-Tat101 cells was associated with the reduced expression of target mRNAs encoding proteins related to apoptosis and cell cycle such as PTEN, PDCD4 and CDKN1B. We developed Jurkat cells with stable expression of hsa-miR-21 or hsa-miR-222 and observed a similar pattern to Jurkat-Tat101 in resistance to FasL-mediated apoptosis, cell cycle arrest in G2/M and altered cell morphology. Consequently, upregulation of hsa-miR-21 and hsa-miR-222 by Tat may contribute to protect against apoptosis and to anergy observed in HIV-infected CD4+ T cells.

Diarrheal diseases (DD) have distinct etiological profiles in immune-deficient and immune-competent patients. This study compares detection rates, genotype distribution and viral loads of different enteric viral agents in HIV-1 seropositive (n = 200) and HIV-1 seronegative (n = 125) children hospitalized with DD in Rio de Janeiro, Brazil. Except for group A rotavirus (RVA), which were detected through enzyme immunoassay, the other enteric viruses (norovirus [NoV], astrovirus [HAstV], adenovirus [HAdV] and bocavirus [HBoV]) were detected through PCR or RT-PCR. A quantitative PCR was performed for RVA, NoV, HAstV, HAdV and HBoV. Infections with NoV (19% vs. 9.6%; p<0.001), HBoV (14% vs. 7.2%; p = 0.042) and HAdV (30.5% vs. 14.4%; p<0.001) were significantly more frequent among HIV-1 seropositive children. RVA was significantly less frequent among HIV-1 seropositive patients (6.5% vs. 20%; p<0.001). Similarly, frequency of infection with HAstV was lower among HIV-1 seropositive children (5.5% vs. 12.8%; p = 0.018). Among HIV-1 seropositive children 33 (16.5%) had co-infections, including three enteric viruses, such as NoV, HBoV and HAdV (n = 2) and NoV, HAstV and HAdV (n = 2). The frequency of infection with more than one virus was 17 (13.6%) in the HIV-1 negative group, triple infection (NoV + HAstV + HBoV) being observed in only one patient. The median viral load of HAstV in feces was significantly higher among HIV-1 positive children compared to HIV-1 negative children. Concerning children infected with RVA, NoV, HBoV and HAdV, no statistically significant differences were observed in the medians of viral loads in feces, comparing HIV-1 seropositive and HIV-1 seronegative children. Similar detection rates were observed for RVA, HAstV and HAdV, whilst NoV and HBoV were significantly more prevalent among children with CD4+ T lymphocyte count below 200 cells/mm3. Enteric viruses should be considered an important cause of DD in HIV-1 seropositive children, along with pathogens more classically associated with intestinal infections in immunocompromised hosts.

Objective: To estimate patients enrolling on antiretroviral therapy (ART) over time; describe trends in baseline characteristics; and compare immunological response, loss to follow-up (LTFU), and mortality by three age groups (25-39, 40-49 and ≥50 years)., Design: A retrospective observation cohort study., Methods: This study used routine ART data from two public clinics in Lilongwe, Malawi. All HIV-infected individuals, except pregnant or breastfeeding women, aged ≥ 25 years at ART initiation between 2006 and 2015 were included. Poisson regression models estimated risk of mortality, stratified by age groups., Results: Of 37,378 ART patients, 3,406 were ≥ 50 years old. Patients aged ≥ 50 years initiated ART with more advanced WHO clinical stage and lower CD4 cell count than their younger counterparts. Older patients had a significantly slower immunological response to ART in the first 18 months on ART compared to patients aged 25-39 years (p = 0.04). Overall mortality rates were 2.3 (95% confidence Interval (CI) 2.2-2.4), 2.9 (95% CI 2.7-3.2) and 4.6 (95% CI 4.2-5.1) per 100 person-years in patients aged 25-39 years, 40-49 years and 50 years and older, respectively. Overall LTFU rates were 6.3 (95% CI 6.1-6.5), 4.5 (95% CI 4.2-4.7), and 5.6 (95% CI 5.1-6.1) per 100 person years among increasing age cohorts. The proportion of patients aged ≥ 50 years and newly enrolling into ART care remained stable at 9% while the proportion of active ART patients aged ≥50 years increased from 10% in 2006 to 15% in 2015., Conclusion: Older people had slower immunological response and higher mortality. Malawi appears to be undergoing a demographic shift in people living with HIV. Increased consideration of long-term ART-related problems, drug-drug interactions and age-related non-communicable diseases is warranted.

HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018) without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches.

Objective: HIV controllers (HICs) spontaneously maintain HIV viral replication at low level without antiretroviral therapy (ART), a small number of whom will eventually lose this ability to control HIV viremia. The objective was to identify factors associated with loss of virological control., Methods: HICs were identified in COHERE on the basis of ≥5 consecutive viral loads (VL) ≤500 copies/mL over ≥1 year whilst ART-naive, with the last VL ≤500 copies/mL measured ≥5 years after HIV diagnosis. Loss of virological control was defined as 2 consecutive VL >2000 copies/mL. Duration of HIV control was described using cumulative incidence method, considering loss of virological control, ART initiation and death during virological control as competing outcomes. Factors associated with loss of virological control were identified using Cox models. CD4 and CD8 dynamics were described using mixed-effect linear models., Results: We identified 1067 HICs; 86 lost virological control, 293 initiated ART, and 13 died during virological control. Six years after confirmation of HIC status, the probability of losing virological control, initiating ART and dying were 13%, 37%, and 2%. Current lower CD4/CD8 ratio and a history of transient viral rebounds were associated with an increased risk of losing virological control. CD4 declined and CD8 increased before loss of virological control, and before viral rebounds., Discussion: Expansion of CD8 and decline of CD4 during HIV control may result from repeated low-level viremia. Our findings suggest that in addition to superinfection, other mechanisms, such as low grade viral replication, can lead to loss of virological control in HICs.

The latent reservoir is a major barrier to HIV eradication. Reservoir size is emerging as an important biomarker to assess the likelihood of HIV remission in the absence of antiretroviral therapy (ART) and may be reduced by earlier initiation of ART that restricts HIV spread into CD4+ T cells. Reservoir size is traditionally measured with a quantitative viral outgrowth assay (QVOA) that induces replication-competent HIV production through in vitro stimulation of resting CD4+ T cells. However, the recent identification of replication-intact, non-induced proviral genomes (NIPG) suggests the QVOA significantly underestimates (by 62-fold) latent reservoir size in chronically-infected adults. Whether formation and persistence of Intact, NIPG is thwarted by early ART initiation and long-term virologic suppression in perinatal infection is unclear. Here, we show that the latent reservoir in 11 early treated, long-term suppressed perinatally infected children and adolescents was not inducible by QVOA and dominated by defective, NIPG. Single genome analysis of 164 NIPG from 232 million cultured resting CD4+ T cells revealed no replication-intact, near-full length sequences. Forty-three (26%) NIPG contained APOBEC3G-mediated hypermutation, 115 (70%) NIPG contained large internal deletions, one NIPG contained nonsense mutations and indels, and 5 (3%) NIPG were assigned as "Not Evaluable" due to multiple failed sequencing attempts that precluded further classification. The lack of replication competent inducible provirus and intact NIPG in this cohort indicate early, long-term ART of perinatal infection leads to marked diminution of replication-competent HIV-1 reservoirs, creating a favorable state towards interventions aimed at virologic remission., Competing Interests: SL is employed by a commercial company: Bioinfoexperts LLC. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: To investigate if plasma HIV-1 tropism testing could identify subjects at higher risk for clinical progression and death in routine clinical management., Design: Nested case-control study within the EuroSIDA cohort., Methods: Cases were subjects with AIDS or who died from any cause, with a plasma sample with HIV-1 RNA >1000 copies/mL available for tropism testing 3 to 12 months prior to the event. At least 1 control matched for age, HIV-1 RNA and HCV status at the time of sampling were selected per each case. Conditional logistic regression was used to investigate exposures associated with clinical progression to AIDS or death. A linear mixed model with random intercept was used to compare CD4+T-cell slopes by HIV tropism over the 12 months following the date of sampling., Results: The study included 266 subjects, 100 cases and 166 controls; one quarter had X4 HIV; 26% were ART-naïve. Baseline factors independently associated with clinical progression or death were female gender (OR = 2.13 vs. male, 95CI = 1.04, 4.36), p = 0.038), CD4+T-cell count (OR = 0.90 (95CI = 0.80, 1.00) per 100 cells/mm3 higher, p = 0.058), being on ART (OR = 2.72 vs. being off-ART (95CI = 1.15, 6.41), p = 0.022) and calendar year of sample [OR = 0.84 (95CI = 0.77, 0.91) per more recent year, p<0.001). Baseline tropism was not associated with the risk of clinical progression or death. CD4+T-cell slopes did not differ within or between tropism groups., Conclusions: The predictive role of plasma tropism determined using 454 sequencing in the context of people receiving cART with detectable VL is not helpful to identify subjects at higher risk for clinical progression to AIDS or death., Competing Interests: The authors have declared that no competing interests exist.

Background: CD4 T-cell counts are widely used to assess treatment eligibility and to follow-up HIV-infected patients. The World Health Organization prequalification of in vitro diagnostics program conducted a performance evaluation of the FACSPresto (BD Biosciences), a new point-of-care instrument to measure absolute CD4-T cell (CD4) counts and percentages in venous and capillary blood samples from HIV-infected patients., Methods: Patients were recruited in Belgium (200 patients) and in Tanzania (247 patients). Venous blood samples were analyzed in two nearby reference laboratories. In addition, nurses/technicians collected a capillary blood sample by finger prick directly into a FACSPresto CD4 cartridge. Assay precision was assessed on fresh blood and on external quality control samples. Trueness (bias) was assessed by comparing results from FACSPresto with the reference (single-platform FACSCalibur). Clinical misclassification was measured at 200, 350 and 500 cells/μL thresholds., Results: Intra-assay precision was < 6%, and inter-assay < 8%. CD4 results from FACSPresto and reference method resulted in regression slopes of 0.99-1.11 using either venous or capillary blood. Correlation was better for venous than for capillary blood (minimum 0.97 vs 0.93 respectively). Capillary blood resulted in a larger bias than venous blood, with 24 and 83 cells/μL for absolute CD4 counts on capillary blood in Antwerp and Dar es Salaam respectively, vs 12 and 41 cells/μL on venous blood. Bias on CD4% was < 1% on both venous and capillary blood, and was proportionally better than for absolute CD4 counts. Clinical misclassification was in line with the average overestimation, showing a very good specificity, but sensitivity around 70-90%. The rejection rate was 11% on first reading, leading to 6% of all samples without final result after a second reading., Conclusions: The FACSPresto performed very well on venous blood samples, and well on capillary blood samples., Competing Interests: The authors have declared that no competing interests exist.

Humanized pigs have been developed to reduce the incidence of immune rejection in xenotransplantation, but significant concerns remain, such as transmission of viral zoonosis. Porcine endogenous retroviruses (PERV), which exist in the genome of pigs, are produced as infectious virions from all porcine cells and cause zoonosis. Here, we examined the possibility of zoonosis of hosts under conditions of immune suppression or xenotransplantation of cells producing host-adapted viruses. Upon transplantation of PERV-producing porcine cells into mice, no transmission of PERV was detected, whereas, transmission of PERV from mice transplanted with mouse-adapted PERV-producing cells was detected. In addition, the frequency of PERV transmission was increased in CsA treated mice transplanted with PERV-producing murine cells, compared with PERV-producing porcine cells. Transmission of PERV to host animals did not affect weight but immune responses, in particular, the number of T cells from PERV-transmitted mice, were notably reduced. The observed risk of PERV zoonosis highlights the requirement for thorough evaluation of viral zoonosis under particular host conditions, such as immunosuppressive treatment and transplantation with host-adapted virus-producing cells., Competing Interests: The authors have declared that no competing interests exist.

Background: The Nigerian Antiretroviral therapy (ART) program started in 2004 and now ranks among the largest in Africa. However, nationally representative data on outcomes have not been reported., Methods: We evaluated retrospective cohort data from a nationally representative sample of adults aged ≥15 years who initiated ART during 2004 to 2012. Data were abstracted from 3,496 patient records at 35 sites selected using probability-proportional-to-size (PPS) sampling. Analyses were weighted and controlled for the complex survey design. The main outcome measures were mortality, loss to follow-up (LTFU), and retention (the proportion alive and on ART). Potential predictors of attrition were assessed using competing risk regression models., Results: At ART initiation, 66.4 percent (%) were females, median age was 33 years, median weight 56 kg, median CD4 count 161 cells/mm3, and 47.1% had stage III/IV disease. The percentage of patients retained at 12, 24, 36 and 48 months was 81.2%, 74.4%, 67.2%, and 61.7%, respectively. Over 10,088 person-years of ART, mortality, LTFU, and overall attrition (mortality, LTFU, and treatment stop) rates were 1.1 (95% confidence interval (CI): 0.7-1.8), 12.3 (95%CI: 8.9-17.0), and 13.9 (95% CI: 10.4-18.5) per 100 person-years (py) respectively. Highest attrition rates of 55.4/100py were witnessed in the first 3 months on ART. Predictors of LTFU included: lower-than-secondary level education (reference: Tertiary), care in North-East and South-South regions (reference: North-Central), presence of moderate/severe anemia, symptomatic functional status, and baseline weight <45kg. Predictor of mortality was WHO stage higher than stage I. Male sex, severe anemia, and care in a small clinic were associated with both mortality and LTFU., Conclusion: Moderate/Advanced HIV disease was predictive of attrition; earlier ART initiation could improve program outcomes. Retention interventions targeting men and those with lower levels of education are needed. Further research to understand geographic and clinic size variations with outcome is warranted., Competing Interests: The authors have declared that no competing interests exist.

The cytoplasmic tail (gp41CT) of the HIV-1 envelope (Env) mediates Env incorporation into virions and regulates Env intracellular trafficking. Little is known about the functional impact of variability in this domain. To address this issue, we compared the replication of recombinant virus pairs carrying the full Env (Env viruses) or the Env ectodomain fused to the gp41CT of NL4.3 (EnvEC viruses) (12 subtype C and 10 subtype B pairs) in primary CD4+ T-cells and monocyte-derived-macrophages (MDMs). In CD4+ T-cells, replication was as follows: B-EnvEC = B-Env>C-EnvEC>C-Env, indicating that the gp41CT of subtype C contributes to the low replicative capacity of this subtype. In MDMs, in contrast, replication capacity was comparable for all viruses regardless of subtype and of gp41CT. In CD4+ T-cells, viral entry, viral release and viral gene expression were similar. However, infectivity of free virions and cell-to-cell transmission of C-Env viruses released by CD4+ T-cells was lower, suggestive of lower Env incorporation into virions. Subtype C matrix only minimally rescued viral replication and failed to restore infectivity of free viruses and cell-to-cell transmission. Taken together, these results show that polymorphisms in the gp41CT contribute to viral replication capacity and suggest that the number of Env spikes per virion may vary across subtypes. These findings should be taken into consideration in the design of vaccines., Competing Interests: The authors have declared that no competing interests exist.

Background: South African HIV treatment guidelines call for patients who fail first-line antiretroviral therapy (ART) to be switched to second-line ART, yet logistical issues, clinician decisions and patient preferences make delay in switching to second-line likely. We explore the impact of delaying second-line ART after first-line treatment failure on rates of death and virologic failure., Methods: We include patients with documented virologic failure on first-line ART from an observational cohort of 9 South African clinics. We explored predictors of delayed second-line switch and used marginal structural models to analyze rates of death following first-line failure by categorical time to switch to second-line. Cox proportional hazards models were used to examine virologic failure on second-line ART among patients who switched to second-line., Results: 5895 patients failed first-line ART, and 63% switched to second-line. Among patients who switched, median time to switch was 3.4 months (IQR: 1.1-8.7 months). Longer time to switch was associated with higher CD4 counts, lower viral loads and more missed visits prior to first-line failure. Worse outcomes were associated with delay in second-line switch among patients with a peak CD4 count on first-line treatment ≤100 cells/mm3. Among these patients, marginal structural models showed increased risk of death (adjusted HR for switch in 6-12 months vs. 0-1.5 months = 1.47 (95% CI: 0.94-2.29), and Cox models showed increased rates of second-line virologic failure despite the presence of survivor bias (adjusted HR for switch in 3-6 months vs. 0-1.5 months = 2.13 (95% CI: 1.01-4.47))., Conclusions: Even small delays in switch to second-line ART were associated with increased death and second-line failure among patients with low CD4 counts on first-line. There is opportunity for healthcare providers to switch patients to second-line more quickly., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To study the association between CD4/CD8 ratio and morbidity in HIV-infected patients on antiretroviral therapy (ART)., Methods: The APROCO/COPILOTE cohort enrolled patients initiating a protease inhibitor-containing ART in 1997-1999. The association between occurrence of first non AIDS-defining severe events (NADE) and time-dependent measures of immune restoration was assessed by 4 Cox models with different definitions of restoration, CD4+ cell counts (CD4), CD4/CD8 ratio, both CD4 and CD4/CD8 ratio, or a composite variable (CD4< 500/mm3, CD4 > 500/mm3 and CD4/CD8 ratio < 1, CD4 > 500/mm3 and CD4/CD8 ratio > 1). Models adjusted on baseline characteristics and time-dependent viral load were compared using Akaike Information Criterion., Results: We included 1227 patients. Median duration of follow-up was 9.2 years (IQR: 4.2-11.4). Median CD4 was 530/mm3 at 9 years. Median CD4/CD8 ratio was 0.3 (IQR: 0.2-0.5) at baseline and 0.6 (IQR: 0.4-0.9) after 9 years. Incidence of first NADE was 7.4/100 person-years, the most common being bacterial infections (21%), cardiovascular events (14%) and cancers (10%). For both bacterial infections and cardiovascular events, the CD4/CD8 ratio did not add predictive information to the CD4 cell count. However, low CD4/CD8 ratio was the best predictor of non-AIDS cancers (adjusted HR = 2.13 for CD4/CD8 < 0.5; 95% CI = 1.32-3.44)., Conclusions: CD4/CD8 ratio remains < 1 in most HIV-infected patients despite long-term CD4+ cell counts restoration on ART. A CD4/CD8 ratio < 0.5 could identify patients who require a more intensive strategy of cancer prevention or screening., Competing Interests: The authors have declared that no competing interests exist.

Background: A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir"). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART)., Methods: We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (Alu PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables., Results: Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results., Conclusions: Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.

Introduction: Enumeration of CD4+ T lymphocytes is important for pre-ART disease staging and screening for opportunistic infections, however access to CD4 testing in resource limited settings is poor. Point of care (POC) technologies can facilitate improved access to CD4 testing. We evaluated the analytical performance of a novel POC device the FACSPresto compared to the FACSCalibur as a reference standard and to the PIMA, a POC device in widespread use in sub-Saharan Africa., Method: Specimens were obtained from 253 HIV infected adults. Venous blood samples were analyzed on the FACSPresto and the FACSCalibur, in a subset of 41 samples additional analysis was done on the PIMA., Results: The absolute CD4 count results obtained on the FACSPresto were comparable to those on the FACSCalibur with low absolute (9.5cells/μl) and relative bias (3.2%). Bias in CD4% values was also low (1.06%) with a relative bias of 4.9%. The sensitivity was lower at a CD4 count threshold of ≤350cells/μl compared with ≤500cells/μl (84.9% vs. 92.8%) resulting in a high upward misclassification rate at low CD4 counts. Specificity at thresholds of ≤350cells/μl and ≤500cells/μl were 96.6% and 96.8% respectively. The PIMA had a high absolute (-68.6cells/μl) and relative bias (-10.5%) when compared with the FACSCalibur. At thresholds of ≤350cells/μl and ≤500cells/μl the sensitivity was 100% and 95.5% respectively; specificity was 85.7% and 84.2% respectively. The coefficients of repeatability were 4.13%, 5.29% and 9.8% respectively., Discussion: The analytic performance of the FACSPresto against the reference standard was very good with better agreement and precision than the PIMA. The FACSPresto had comparable sensitivity at a threshold of 500 cells/μl and better specificity than the PIMA. However the FACSPresto showed reduced sensitivity at low CD4 count thresholds., Conclusion: The FACSPresto can be reliably used as a POC device for enumerating absolute CD4 count and CD4% values.

Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability.

To explore the differences between the extreme SIV infection phenotypes, nonprogression (BEN: benign) to AIDS in sooty mangabeys (SMs) and progression to AIDS (MAL: malignant) in rhesus macaques (RMs), we performed an integrated dual positive-negative connectivity (DPNC) analysis of gene coexpression networks (GCN) based on publicly available big data sets in the GEO database of NCBI. The microarray-based gene expression data sets were generated, respectively, from the peripheral blood of SMs and RMs at several time points of SIV infection. Significant differences of GCN changes in DPNC values were observed in SIV-infected SMs and RMs. There are three groups of enriched genes or pathways (EGPs) that are associated with three SIV infection phenotypes (BEN+, MAL+ and mixed BEN+/MAL+). The MAL+ phenotype in SIV-infected RMs is specifically associated with eight EGPs, including the protein ubiquitin proteasome system, p53, granzyme A, gramzyme B, polo-like kinase, Glucocorticoid receptor, oxidative phosyphorylation and mitochondrial signaling. Mitochondrial (endosymbiotic) dysfunction is solely present in RMs. Specific BEN+ pattern changes in four EGPs are identified in SIV-infected SMs, including the pathways contributing to interferon signaling, BRCA1/DNA damage response, PKR/INF induction and LGALS8. There are three enriched pathways (PRR-activated IRF signaling, RIG1-like receptor and PRR pathway) contributing to the mixed (BEN+/MAL+) phenotypes of SIV infections in RMs and SMs, suggesting that these pathways play a dual role in the host defense against viral infections. Further analysis of Hub genes in these GCNs revealed that the genes LGALS8 and IL-17RA, which positively regulate the barrier function of the gut mucosa and the immune homeostasis with the gut microbiota (exosymbiosis), were significantly differentially expressed in RMs and SMs. Our data suggest that there exists an exo- (dysbiosis of the gut microbiota) and endo- (mitochondrial dysfunction) symbiotic imbalance (EESI) in HIV/SIV infections. Dissecting the mechanisms of the exo-endo symbiotic balance (EESB) that maintains immune homeostasis and the EESI problems in HIV/SIV infections may lead to a better understanding of the pathogenesis of AIDS and the development of novel interventions for the rational control of this disease.

Aberrant turnover of memory CD4+ T-cells is central to Acquired Immunodeficiency Syndrome (AIDS) progression. Understanding the relationship between the turnover of CD4+ subsets and immunological homeostasis during simian immunodeficiency virus (SIV) infection in natural hosts may provide insight into mechanisms of immune regulation that may serve as models for therapeutic intervention in Human Immunodeficiency Virus (HIV)-infected persons. Sooty mangabeys (SMs) have naturally evolved with SIV to avoid AIDS progression while maintaining healthy peripheral CD4+ T-cell counts and thus represent a model by which therapeutic interventions for AIDS progression might be elucidated. To assess the relationship between the turnover of CD4+ subsets and immunological homeostasis during SIV infection in non-progressive hosts, we treated 6 SIV-uninfected and 9 SIV-infected SMs with 2'-bromo-5'-deoxyuridine (BrdU) for 14 days and longitudinally assessed CD4+ T-cell subset turnover by polychromatic flow cytometry. We observed that, in SIV-infected SMs, turnover of CD4+ T-cell naïve and central, transitional, and effector memory subsets is comparable to that in uninfected animals. Comparable turnover of CD4+ T-cell subsets irrespective of SIV-infection status likely contributes to the lack of aberrant immune activation and disease progression observed after infection in non-progressive hosts.

Background: HIV infection is a concern in the army troupes because of the risk behaviour of the military population. In order to allow regular access to CD4+ T cell enumeration of military personnel as well as their dependents and civilians living with HIV, the Senegalese Army AIDS program is implementing PIMATM Alere technology in urban and semi-urban military medical centres. Validation such device is therefore required prior their wide implementation. The purpose of this study was to compare CD4+ T cell count measurements between the PIMATM Alere to the BD FACSCountTM., Methodology: We selected a total of 200 subjects including 50 patients with CD4+ T-cells below 200/mm3, 50 between 200 and 350/mm3, 50 between 351 and 500/mm3, and 50 above 500/mm3. CD4+ T-cell count was performed on venous blood using the BD FASCountTM as reference method and the PIMATM Point of Care technology. The mean biases and limits of agreement between the PIMATM Alere and BD FACSCountTM were assessed with the Bland-Altman analysis, the linear regression performed using the Passing-Bablok regression analysis, and the percent similarity calculated using the Scott method., Results: Our data have shown a mean difference of 22.3 cells/mm3 [95%CI:9.1-35.5] between the BD FACSCountTM and PIMATM Alere CD4 measurements. However, the mean differences of the two methods was not significantly different to zero when CD4+ T-cell count was below 350/mm3 (P = 0.76). The Passing-Bablok regression in categorized CD4 counts has also showed concordance correlation coefficient of 0.89 for CD4+ T cell counts below 350/mm3 whilst it was 0.5 when CD4 was above 350/mm3., Conclusion: Overall, our data have shown that for low CD4 counts, the results from the PIMATM Alere provided accurate CD4+ T cell counts with a good agreement compared to the FACSCountTM.

Background: Decreased hepatitis C virus (HCV) clearance, faster cirrhosis progression and higher HCV RNA levels are associated with Human Immunodeficiency virus (HIV) coinfection. The CD4+ T helper cytokines interleukin (IL)-21 and IL-17A are associated with virus control and inflammation, respectively, both important in HCV and HIV disease progression. Here, we examined how antigen-specific production of these cytokines during HCV mono and HIV/HCV coinfection was associated with HCV virus control., Methods: We measured HCV-specific IL-21 and IL-17A production by transwell cytokine secretion assay in PBMCs from monoinfected and coinfected individuals. Viral control was determined by plasma HCV RNA levels., Results: In acutely infected individuals, those able to establish transient/complete HCV viral control tended to have stronger HCV-specific IL-21-production than non-controllers. HCV-specific IL-21 production also correlated with HCV viral decline in acute infection. Significantly stronger HCV-specific IL-21 production was detected in HAART-treated coinfected individuals. HCV-specific IL-17A production was not associated with lower plasma HCV RNA levels in acute or chronic HCV infection and responses were stronger in HIV coinfection. HCV-specific IL-21/ IL-17A responses did not correlate with microbial translocation or fibrosis. Exogenous IL-21 treatment of HCV-specific CD8+ T cells from monoinfected individuals enhanced their function although CD8+ T cells from coinfected individuals were somewhat refractory to the effects of IL-21., Conclusions: These data show that HCV-specific IL-21 and IL-17A-producing T cells are induced in HIV/HCV coinfection. In early HIV/HCV coinfection, IL-21 may contribute to viral control, and may represent a novel tool to enhance acute HCV clearance in HIV/HCV coinfected individuals.

The large majority of human immunodeficiency virus type 1 (HIV-1) markers of disease progression/severity previously identified have been associated with alterations in host genetic and immune responses, with few studies focused on viral genetic markers correlate with changes in disease severity. This study presents a cross-sectional/longitudinal study of HIV-1 single nucleotide polymorphisms (SNPs) contained within the viral promoter or long terminal repeat (LTR) in patients within the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. HIV-1 LTR SNPs were found to associate with the classical clinical disease parameters CD4+ T-cell count and log viral load. They were found in both defined and undefined transcription factor binding sites of the LTR. A novel SNP identified at position 108 in a known COUP (chicken ovalbumin upstream promoter)/AP1 transcription factor binding site was significantly correlated with binding phenotypes that are potentially the underlying cause of the associated clinical outcome (increase in viral load and decrease in CD4+ T-cell count).

Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses.

The presence of replication-competent HIV-1 -which resides mainly in resting CD4+ T cells--is a major hurdle to its eradication. While pharmacological approaches have been useful for inducing the expression of this latent population of virus, they have been unable to purge HIV-1 from all its reservoirs. Additionally, many of these strategies have been associated with adverse effects, underscoring the need for alternative approaches capable of reactivating viral expression. Here we show that engineered transcriptional modulators based on customizable transcription activator-like effector (TALE) proteins can induce gene expression from the HIV-1 long terminal repeat promoter, and that combinations of TALE transcription factors can synergistically reactivate latent viral expression in cell line models of HIV-1 latency. We further show that complementing TALE transcription factors with Vorinostat, a histone deacetylase inhibitor, enhances HIV-1 expression in latency models. Collectively, these findings demonstrate that TALE transcription factors are a potentially effective alternative to current pharmacological routes for reactivating latent virus and that combining synthetic transcriptional activators with histone deacetylase inhibitors could lead to the development of improved therapies for latent HIV-1 infection.

The goal of this research was to establish a new and innovative framework for cost-effectiveness modeling of HIV-1 treatment, simultaneously considering both clinical and epidemiological outcomes. EPICE-HIV is a multi-paradigm model based on a within-host micro-simulation model for the disease progression of HIV-1 infected individuals and an agent-based sexual contact network (SCN) model for the transmission of HIV-1 infection. It includes HIV-1 viral dynamics, CD4+ T cell infection rates, and pharmacokinetics/pharmacodynamics modeling. Disease progression of HIV-1 infected individuals is driven by the interdependent changes in CD4+ T cell count, changes in plasma HIV-1 RNA, accumulation of resistance mutations and adherence to treatment. The two parts of the model are joined through a per-sexual-act and viral load dependent probability of disease transmission in HIV-discordant couples. Internal validity of the disease progression part of the model is assessed and external validity is demonstrated in comparison to the outcomes observed in the STaR randomized controlled clinical trial. We found that overall adherence to treatment and the resulting pattern of treatment interruptions are key drivers of HIV-1 treatment outcomes. Our model, though largely independent of efficacy data from RCT, was accurate in producing 96-week outcomes, qualitatively and quantitatively comparable to the ones observed in the STaR trial. We demonstrate that multi-paradigm micro-simulation modeling is a promising tool to generate evidence about optimal policy strategies in HIV-1 treatment, including treatment efficacy, HIV-1 transmission, and cost-effectiveness analysis.

Co-infection with Mycobacterium tuberculosis accelerates progression from HIV to AIDS. Our previous studies showed that M. tuberculosis complex, unlike M. smegmatis, enhances TLR2-dependent susceptibility of CD4+ T cells to HIV. The M. tuberculosis complex produces multiple TLR2-stimulating lipoproteins, which are absent in M. smegmatis. M. tuberculosis production of mature lipoproteins and TLR2 stimulation is dependent on cleavage by lipoprotein signal peptidase A (LspA). In order to determine the role of potential TLR2-stimulating lipoproteins on mycobacterial-mediated HIV infectivity of CD4+ T cells, we generated M. smegmatis recombinant strains overexpressing genes encoding various M. bovis BCG lipoproteins, as well as a Mycobacterium bovis BCG strain deficient in LspA (ΔlspA). Exposure of human peripheral blood mononuclear cells (PBMC) to M. smegmatis strains overexpressing the BCG lipoproteins, LprF (p<0.01), LprH (p<0.05), LprI (p<0.05), LprP (p<0.001), LprQ (p<0.005), MPT83 (p<0.005), or PhoS1 (p<0.05), resulted in increased HIV infectivity of CD4+ T cells isolated from these PBMC. Conversely, infection of PBMC with ΔlspA reduced HIV infectivity of CD4+ T cells by 40% relative to BCG-infected cells (p<0.05). These results may have important implications for TB vaccination programs in areas with high mother-to-child HIV transmission.

Background: Examination of a cohort of cats experimentally infected with feline immunodeficiency virus (FIV) for 5.75 years revealed detectable proviral DNA in peripheral blood mononuclear cells (PBMCs) harvested during the asymptomatic phase, undetectable plasma viral RNA (FIV gag), and rarely detectable cell-associated viral RNA. Despite apparent viral latency in peripheral CD4+ T cells, circulating CD4+ T cell numbers progressively declined in progressor animals. The aim of this study was to explore this dichotomy of peripheral blood viral latency in the face of progressive immunopathology. The viral replication status, cellular immunophenotypes, and histopathologic features were compared between popliteal lymph nodes (PLNs) and peripheral blood. Also, we identified and further characterized one of the FIV-infected cats identified as a long-term non-progressor (LTNP)., Results: PLN-derived leukocytes from FIV-infected cats during the chronic asymptomatic phase demonstrated active viral gag transcription and FIV protein translation as determined by real-time RT-PCR, Western blot and in situ immunohistochemistry, whereas viral RNA in blood leukocytes was either undetectable or intermittently detectable and viral protein was not detected. Active transcription of viral RNA was detectable in PLN-derived CD4+ and CD21+ leukocytes. Replication competent provirus was reactivated ex vivo from PLN-derived leukocytes from three of four FIV-infected cats. Progressor cats showed a persistent and dramatically decreased proportion and absolute count of CD4+ T cells in blood, and a decreased proportion of CD4+ T cells in PLNs. A single long-term non-progressor (LTNP) cat persistently demonstrated an absolute peripheral blood CD4+ T cell count indistinguishable from uninfected animals, a lower proviral load in unfractionated blood and PLN leukocytes, and very low amounts of viral RNA in the PLN., Conclusion: Collectively our data indicates that PLNs harbor important reservoirs of ongoing viral replication during the asymptomatic phase of infection, in spite of undetectable viral activity in peripheral blood. A thorough understanding of tissue-based lentiviral reservoirs is fundamental to medical interventions to eliminate virus or prolong the asymptomatic phase of FIV infection.

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIVBaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.

Objective: To determine immunologic, virologic outcomes and drug resistance among children and adolescents receiving care during routine programmatic implementation in a low-income country., Methods: A cross-sectional evaluation with collection of clinical and laboratory data for children (0-<10 years) and adolescents (10-19 years) attending a public ART program in Harare providing care for pediatric patients since 2004, was conducted. Longitudinal data for each participant was obtained from the clinic based medical record., Results: Data from 599 children and adolescents was evaluated. The participants presented to care with low CD4 cell count and CD4%, median baseline CD4% was lower in adolescents compared with children (11.0% vs. 15.0%, p<0.0001). The median age at ART initiation was 8.0 years (IQR 3.0, 12.0); median time on ART was 2.9 years (IQR 1.7, 4.5). On ART, median CD4% improved for all age groups but remained below 25%. Older age (≥ 5 years) at ART initiation was associated with severe stunting (HAZ <-2: 53.3% vs. 28.4%, p<0.0001). Virologic failure rate was 30.6% and associated with age at ART initiation. In children, nevirapine based ART regimen was associated with a 3-fold increased risk of failure (AOR: 3.5; 95% CI: 1.3, 9.1, p = 0.0180). Children (<10 y) on ART for ≥4 years had higher failure rates than those on ART for <4 years (39.6% vs. 23.9%, p = 0.0239). In those initiating ART as adolescents, each additional year in age above 10 years at the time of ART initiation (AOR 0.4 95%CI: 0.1, 0.9, p = 0.0324), and each additional year on ART (AOR 0.4, 95%CI 0.2, 0.9, p = 0.0379) were associated with decreased risk of virologic failure. Drug resistance was evident in 67.6% of sequenced virus isolates., Conclusions: During routine programmatic implementation of HIV care for children and adolescents, delayed age at ART initiation has long-term implications on immunologic recovery, growth and virologic outcomes.

Objective: To evaluate the Fibrosis (FIB)-4 index as a predictor of major liver-related events (LRE) and liver-related death (LRD) in human immunodeficiency virus (HIV) type-1 patients initiating combination antiretroviral therapy (cART)., Design: Retrospective analysis of a prospective cohort study., Setting: Italian HIV care centers participating to the ICONA Foundation cohort., Participants: Treatment-naive patients enrolled in ICONA were selected who: initiated cART, had hepatitis C virus (HCV) serology results, were HBsAg negative, had an available FIB-4 index at cART start and during follow up., Methods: Cox regression models were used to determine the association of FIB4 with the risk of major LRE (gastrointestinal bleeding, ascites, hepatic encephalopathy, hepato-renal syndrome or hepatocellular carcinoma) or LRD., Results: Three-thousand four-hundred seventy-five patients were enrolled: 73.3% were males, 27.2% HCV seropositive. At baseline (time of cART initiation) their median age was 39 years, had a median CD4+ T cell count of 260 cells/uL, and median HIV RNA 4.9 log copies/mL, 65.9% had a FIB-4 <1.45, 26.4% 1.45-3.25 and 7.7% >3.25. Over a follow up of 18,662 person-years, 41 events were observed: 25 major LRE and 16 LRD (incidence rate, IR, 2.2 per 1,000 PYFU [95% confidence interval, CI 1.6-3.0]). IR was higher in HCV seropositives as compared to negatives (5.9 vs 0.5 per 1,000 PYFU). Higher baseline FIB-4 category as compared to <1.45 (FIB-4 1.45-3.25: HR 3.55, 95% CI 1.09-11.58; FIB-4>3.25: HR 4.25, 1.21-14.92) and time-updated FIB-4 (FIB-4 1.45-3.25: HR 3.40, 1.02-11.40; FIB-4>3.25: HR 21.24, 6.75-66.84) were independently predictive of major LRE/LRD, after adjusting for HIV- and HCV-related variables, alcohol consumption and type of cART., Conclusions: The FIB-4 index at cART initiation, and its modification over time are risk factors for major LRE or LRD, independently of infection with HCV and could be used to monitor patients on cART.

Unlike resting CD4+ T cells, activated CD4+T cells are highly susceptible to infection of human immunodeficiency virus 1 (HIV-1). HIV-1 infects T cells and macrophages without activating the nucleic acid sensors and the anti-viral type I interferon response. Adenosine deaminase acting on RNA 1 (ADAR1) is an RNA editing enzyme that displays antiviral activity against several RNA viruses. Mutations in ADAR1 cause the autoimmune disorder Aicardi-Goutieères syndrome (AGS). This disease is characterized by an inappropriate activation of the interferon-stimulated gene response. Here we show that HIV-1 replication, in ADAR1-deficient CD4+T lymphocytes from AGS patients, is blocked at the level of protein translation. Furthermore, viral protein synthesis block is accompanied by an activation of interferon-stimulated genes. RNA silencing of ADAR1 in Jurkat cells also inhibited HIV-1 protein synthesis. Our data support that HIV-1 requires ADAR1 for efficient replication in human CD4+T cells.

Background: Measurement of HIV DNA-bearing cells in cerebrospinal fluid (CSF) is challenging because few cells are present. We present a novel application of the sensitive droplet digital (dd)PCR in this context., Methods: We analyzed CSF cell pellets and paired peripheral blood mononuclear cells (PBMC) from 28 subjects, 19 of whom had undetectable HIV RNA (<48 copies/mL) in both compartments. We extracted DNA from PBMC using silica-based columns and used direct lysis on CSF cells. HIV DNA and the host housekeeping gene (RPP30) were measured in CSF and PBMC by (dd)PCR. We compared HIV DNA levels in virally-suppressed and-unsuppressed subgroups and calculated correlations between HIV DNA and RNA levels in both compartments using non-parametric tests., Results: HIV DNA was detected in 18/28 (64%) CSF cell pellets, including 10/19 (53%) samples with undetectable HIV RNA. HIV DNA levels in CSF cell pellets were not correlated with RPP30 (p = 0.3), but correlated positively with HIV RNA in CSF (p = 0.04) and HIV DNA in PBMC (p = 0.03). Cellular HIV DNA in CSF was detected in comparable levels in HIV RNA-suppressed and unsuppressed subjects (p = 0.14). In contrast, HIV DNA levels in PBMC were significantly lower in HIV RNA-suppressed than in unsuppressed subjects (p = 0.014). Among subjects with detectable HIV DNA in both compartments, HIV DNA levels in CSF were significantly higher than in PBMC (p<0.001)., Conclusions: Despite low mononuclear cell numbers in CSF, HIV DNA was detected in most virally suppressed individuals. In contrast to PBMC, suppressive ART was not associated with lower HIV DNA levels in CSF cells, compared to no ART, perhaps due to poorer ART penetration, slower decay of HIV DNA, or enrichment of HIV DNA-bearing mononuclear cells into the CSF, compared to blood. Future studies should determine what fraction of HIV DNA is replication-competent in CSF leukocytes, compared to PBMC.

Unlabelled: The hepatitis B virus (HBV) and the human immunodeficiency virus type 1 (HIV-1) can infect cells of the lymphatic system. It is unknown whether HIV-1 co-infection impacts infection of peripheral blood mononuclear cell (PBMC) subsets by the HBV., Aims: To compare the detection of HBV genomes and HBV sequences in unsorted PBMCs and subsets (i.e., CD4+ T, CD8+ T, CD14+ monocytes, CD19+ B, CD56+ NK cells) in HBV mono-infected vs. HBV/HIV-1 co-infected individuals., Methods: Total PBMC and subsets isolated from 14 HBV mono-infected (4/14 before and after anti-HBV therapy) and 6 HBV/HIV-1 co-infected individuals (5/6 consistently on dual active anti-HBV/HIV therapy) were tested for HBV genomes, including replication indicative HBV covalently closed circular (ccc)-DNA, by nested PCR/nucleic hybridization and/or quantitative PCR. In CD4+, and/or CD56+ subsets from two HBV monoinfected cases, the HBV polymerase/overlapping surface region was analyzed by next generation sequencing., Results: All analyzed whole PBMC from HBV monoinfected and HBV/HIV coinfected individuals were HBV genome positive. Similarly, HBV DNA was detected in all target PBMC subsets regardless of antiviral therapy, but was absent from the CD4+ T cell subset from all HBV/HIV-1 positive cases (P<0.04). In the CD4+ and CD56+ subset of 2 HBV monoinfected cases on tenofovir therapy, mutations at residues associated with drug resistance and/or immune escape (i.e., G145R) were detected in a minor percentage of the population., Summary: HBV genomes and drug resistant variants were detectable in PBMC subsets from HBV mono-infected individuals. The HBV replicates in PBMC subsets of HBV/HIV-1 patients except the CD4+ T cell subpopulation.

Background: Blood collected in conventional EDTA tubes requires laboratory analysis within 48 hours to provide valid CD4 cell count results. This restricts access to HIV care for patients from rural areas in resource-constraint settings due to sample transportation problems. Stabilization Tubes with extended storage duration have been developed but not yet evaluated comprehensively., Objective: To investigate stability of absolute CD4 cell count measurement of samples in BD Vacutainer CD4 Stabilization Tubes over the course of 30 days., Methods: This was a laboratory-based method comparison study conducted at a rural district hospital in Beitbridge, Zimbabwe. Whole peripheral blood from 88 HIV positive adults was drawn into BD Vacutainer CD4 Stabilization Tubes and re-tested 1, 2, 3, 5, 7, 14 and 30 days after collection on BD FacsCount and Partec Cyflow cytometers in parallel. Absolute CD4 cell levels were compared to results from paired samples in EDTA tubes analysed on BD FacsCount at the day of sample collection (references methodology). Bland-Altman analysis based on ratios of the median CD4 counts was used, with acceptable variation ranges for Limits of Agreements of +/-20%., Results: Differences in ratios of the medians remained below 10% until day 21 on BD FacsCount and until day 5 on Partec Cyflow. Variations of Limits of Agreement were beyond 20% after day 1 on both cytometers. Specimen quality decreased steadily after day 5, with only 68% and 40% of samples yielding results on BD FacsCount and Partec Cyflow at day 21, respectively., Conclusions: We do not recommend the use of BD Vacutainer CD4 Stabilization Tubes for absolute CD4 cell count measurement on BD FacsCount or Partec Cyflow due to large variation of results and decay of specimen quality. Alternative technologies for enhanced CD4 testing in settings with limited laboratory and sample transportation capacity still need to be developed.

Background: 2013 WHO guidelines recommend starting ART at CD4+ T-cell counts ≤500 cells/μL. We present the T-cell counts from adult Africans with HIV shortly following transmission to their sexual partners., Methods: HIV-discordant couples in Zambia, Uganda and Rwanda were followed prospectively and received couples counseling and condoms. HIV uninfected partners were tested for HIV at least quarterly and HIV-infected partners received HIV care and referral for ART per national guidelines. Upon diagnosis of incident HIV infection in the previously HIV-uninfected partner, a blood sample was collected from both partners to measure CD4+ T-cells and perform viral linkage. The estimated date of infection (EDI) of the incident case was calculated based on testing history. EDI was unknown for suspected transmitting partners., Results: From 2006-2011, 4,705 HIV-discordant couples were enrolled in this cohort, and 443 cases of incident HIV infection were documented. Virus linkage analysis was performed in 374 transmission pairs, and 273 (73%) transmissions were linked genetically. CD4 counts in the transmitting partner were measured a median of 56 days after EDI (mean:90.5, min:10, max:396). The median CD4 count was 339 cells/μl (mean:386.4, min:15, max:1,434), and the proportion of partners with a CD4+ T-cell count above 500/μl was 25% (95% CI:21, 31)., Conclusions: In our cohort of discordant couples, 73% of HIV transmissions occurred within the relationship, and the transmitter CD4+ T cell count shortly after the transmission event was frequently higher than the WHO 2013 ART-initiation guidelines.

Studies have shown that the transmission of HIV is most likely to occur via rectal or vaginal routes, and rarely through oral exposure. However, the mechanisms of virus entry at mucosal surfaces remain incompletely understood. Prophylactic strategies against HIV infection may be attainable once gaps in current knowledge are filled. To address these gaps, we evaluated essentially normal epithelial surfaces and mapped the periluminal distribution of CD4+ HIV target cells, including T cells and antigen-presenting cells, and an HIV-binding molecule gp340 that can be expressed by epithelial cells in secreted and cell-associated forms. Immunohistochemistry for CD4, CD16, CD3, CD1a and gp340 in human oral, rectal/sigmoid and cervical mucosal samples from HIV-negative subjects demonstrated that periluminal HIV target cells were more prevalent at rectal/sigmoid and endocervical surfaces lined by simple columnar epithelium, than at oral and ectocervical surfaces covered by multilayered stratified squamous epithelium (p<0.001). gp340 expression patterns at these sites were also distinct and strong in oral minor salivary gland acini and ducts, including ductal saliva, in individual rectum/sigmoid and endocervix periluminar columnar cells, and in ectocervix squamous cells. Only weak expression was noted in the oral non-ductal squamous epithelium. We conclude that periluminal HIV target cells, together with periluminal epithelial cell-associated gp340 appear to be most accessible for HIV transmission at rectal/sigmoid and endocervical surfaces. Our data help define vulnerable structural features of mucosal sites exposed to HIV.