HIV-1 RNA monitoring with a dual-target diagnostic assay: A case report.

In a restricted subset of people living with HIV-1 (PLWH) on antiretroviral therapy (ART) with persistent suppressed viral load (i.e., pol-based HIV-RNA repeatedly undetected), a dual-target (pol and LTR) diagnostic assay for HIV-RNA monitoring can measure quantifiable levels of viral loads (VL) above 30 copies/mL exclusively through the amplification of the LTR region, while the pol target results undetected. We report a patient who shows high levels of HIV-RNA detected exclusively through amplification of the LTR region while undetected by the pol region, during a long monitoring period, from 2018 to date. In this follow-up, the ART was modified without reaching LTR-based undetected HIV-RNA values. Immunological and virological parameters remained optimal with a progressive and steady gain of the CD4/CD8 ratio. The clinical history of this patient, shows that LTR-based viremia above 50 copies/mL can be found occasionally or persistently in the plasma of PLWH under suppressive ART, even at high levels. Based on previous studies, VL detected and quantified exclusively through the amplification of the LTR region corresponds to partial or incomplete HIV-RNA transcripts, which cannot trigger new infections. Interestingly, changes in ART do not eliminate repeated findings of these unusual viral elements.

Evaluation of HIV-1 DNA levels among adolescents living with perinatally acquired HIV-1 in Yaounde, Cameroon: A contribution to paediatric HIV cure research in Sub-Saharan Africa.

Background: With the advent of antiretroviral therapy (ART), most children living with HIV in sub-Saharan Africa (SSA) are growing toward adolescence, with scarcity of evidence on the size of viral reservoirs to enhance paediatric cure research strategies. This study aims to compare HIV-1 proviral DNA levels according to virological response among adolescents living with perinatally acquired HIV-1 (ALPHIV) and identify associated-factors in the Cameroonian context. Methods: In this observational cohort study, HIV-1 RNA viremia and CD4+ T-cell count were assessed through RT-PCR and flow cytometry respectively at three time-points over 18 months of observation. At the third time-point, 80 randomly-selected participants were classified as with viremia (≥50 HIV-1 copies/mL; n = 40) or without viremia (<50 HIV-1 copies/mL; n = 40); immune-competent (≥500 CD4+ T cells/mm3) or immunocompromised (<500 CD4+ T cells/mm3). Among these participants, total HIV-1 DNA load was quantified through droplet digital PCR using Bio-Rad QX200. Results: Of the 80 randomly-selected adolescents, median [IQR] age was 15 (13-17) years, 56.2% were female, duration on ART was 9.3 [5.4-12.2] years. Among the 40 viremic ones (median viremia 7312 [283-71482]) HIV-1 copies/ml, 75.0% (30/40) were in virological failure (≥1000 HIV-1 copies/ml), while median of CD4 T cells were 494 [360-793] cell/mm3 with 48.8% (39/80) immunocompromised. No significant variation in HIV-1 RNA viremia and CD4 T cell count was observed between the three time-points, and 13.7% (11/80) adolescents remained aviremic and immune-competent throughout (stable adolescents). A positive and moderate correlation (r = 0.59; p < 0.001) was found between HIV-1 DNA levels and HIV- 1 RNA viremia. Regarding the CD4 T cell count, a negative and weak correlation (r = -0.28; p = 0.014) was found with HIV-1 DNA loads only among adolescents with viremia. Starting ART within the first year of life, ART for over 9 years and aviremia appear as predictors of low HIV-1 DNA loads. Conclusion: Among ALPHIV, high HIV-1 RNA indicates an elevated viral reservoir size, representing a drawback to cure research. Interestingly, early ART initiation and longer ARTduration lead to sustained viral control and limited HIV-1 reservoir size. As limited size of viral reservoir appears consistent with viral control and immune competence, adolescents with sustained viral control (about 14% of this target population) would be candidates for analytical ART interruptions toward establishing paediatric post-treatment controllers in SSA.

Reflex Human Immunodeficiency Virus (HIV) Type 1 RNA Testing Enables Timely Differentiation of False-Positive Results From Acute HIV Infection.

Accurate, timely human immunodeficiency virus (HIV) diagnosis is critical. Routine HIV screening program data were examined before and after reflex HIV type 1 RNA testing. Reflex testing facilitated confirmation of reactive HIV screening assays (as true or false positives) (odds ratio, 23.7 [95% confidence interval, 6.7-83.4]; P < .0001), improving detection of acute HIV and reducing unconfirmed discordant results.

Effects of anti-retroviral therapy on baseline serum interleukin-18 levels in HIV-I infected patients relative to viral suppression and CD4+ gain: A prospective pilot study.

Background: In HIV infection, dysregulation of cytokines, including interleukin 18 (IL-18), has been linked to poor clinical outcomes in studies mainly conducted in resource-rich countries. This phenomenon has not been well-studied in resource-limited settings where outcomes could be confounded by exposure to endemic infections and genetic factors. Objectives: Therefore, the influence of immunological and virological status of HIV-infected, antiretroviral therapy (ART)-naïve patients on serum IL-18 levels at baseline (pretreatment) and 24 weeks following initiation of combination ART (cART24) in a resource-limited setting was investigated. Methods: Using the cross-sectional and longitudinal mixed method design, a total of Forty-four (44) newly diagnosed consenting HIV patients were consecutively recruited during routine clinic visits at the Nasara Treatment & Care Centre of the Ahmadu Bello University Teaching Hospital (ABUTH), Zaria, Nigeria between December 2016 to January 2018, and followed up for 24 weeks on initiation of first-line cART. Results: Serum IL-18 concentrations, CD4+ T-cell counts (CD4+) counts, and HIV1 RNA levels were determined at baseline and cART24. There was little CD4+ count gain in both <200 and ≥ 200 cell/mm3subgroups despite the high proportion of subjects having virological suppression (n = 35, [80%]) at cART24. However, at cART24 there was a more than a threefold decrease in the level of IL-18 concentration compared to baseline in patients with <200 cells/mm3 and a significant decrease in the median plasma IL-18 concentration in patients with HIV1 RNA <1000 cp/mL at cART24. A multivariate logistic regression model shows IL-18 intermediate quartile to be more related to immunological poor gain as compared to the highest quartile. Conclusion: Our study found high baseline and significantly low levels of IL-18 at cART24 in virologically suppressed subjects but not among virological non-suppressed responders despite comparable IL-18 levels by CD4+ T cell count strata at cART24. These findings have implications for risk stratification and treatment outcomes in HIV-positive persons.

Systems crosstalk between antiviral response and cancerous pathways via extracellular vesicles in HIV-1-associated colorectal cancer.

HIV-1 associated colorectal cancer (HA-CRC) is one of the most understudied non-AIDS-defining cancers. In this study, we analyzed the proteome of HA-CRC and the paired remote tissues (HA-RT) through data-independent acquisition mass spectrometry (MS). The quantified proteins could differentiate the HA-CRC and HA-RT groups per PCA or cluster analyses. As a background comparison, we reanalyzed the MS data of non-HIV-1 infected CRC (non-HA-CRC) published by CPTAC. According to the GSEA results, we found that HA-CRC and non-HA-CRC shared similarly over-represented KEGG pathways. Hallmark analysis suggested that terms of antiviral response were only significantly enriched in HA-CRC. The network and molecular system analysis centered the crosstalk of IFN-associated antiviral response and cancerous pathways, which was favored by significant up-regulation of ISGylated proteins as detected in the HA-CRC tissues. We further proved that defective HIV-1 reservoir cells as represented by the 8E5 cells could activate the IFN pathway in human macrophages via horizonal transfer of cell-associated HIV-1 RNA (CA-HIV RNA) carried by extracellular vesicles (EVs). In conclusion, HIV-1 reservoir cells secreted and CA-HIV RNA-containing EVs can induce IFN pathway activation in macrophages that contributes to one of the mechanistic explanations of the systems crosstalk between antiviral response and cancerous pathways in HA-CRC.

Elevation of cell-associated HIV-1 transcripts in CSF CD4+ T cells, despite effective antiretroviral therapy, is linked to brain injury.

Antiretroviral therapy (ART) can attain prolonged undetectable HIV-1 in plasma and cerebrospinal fluid (CSF), but brain injury remains prevalent in people living with HIV-1 infection (PLHIV). We investigated cell-associated (CA)-HIV-1 RNA transcripts in cells in CSF and blood, using the highly sensitive Double-R assay, together with proton Magnetic Resonance Spectroscopy (1H MRS) of major brain metabolites, in sixteen PLHIV. 14/16 CSF cell samples had quantifiable CA-HIV-1 RNA, at levels significantly higher than in their PBMCs (median 9,266 vs 185 copies /106 CD4+ T-cells; p<0.0001). In individual PLHIV, higher levels of HIV-1 transcripts in CSF cells were associated with greater brain injury in the frontal white matter (Std ß=-0.73; p=0.007) and posterior cingulate (Std ß=-0.61; p=0.03). 18-colour flow cytometry revealed that the CSF cells were 91% memory T-cells, equally CD4+ and CD8+ T-cells, but fewer B cells (0.4 %), and monocytes (3.1%). CXCR3+CD49d+integrin ß7-, CCR5+CD4+ T-cells were highly enriched in CSF, compared with PBMC (p <0.001). However, CA-HIV-1 RNA could not be detected in 10/16 preparations of highly purified monocytes from PBMC, and was extremely low in the other six. Our data show that elevated HIV-1 transcripts in CSF cells were associated with brain injury, despite suppressive ART. The cellular source is most likely memory CD4+ T cells from blood, rather than trafficking monocytes. Future research should focus on inhibitors of this transcription to reduce local production of potentially neurotoxic and inflammatory viral products.

Evaluating the Dual-Target Aptima HIV-1 Quant Dx Assay: Comparison between Viral Loads Measured with pol and LTR Targets in the Same Samples.

For effective management of HIV-1 patients, accurate measurement of HIV-1-RNA viral load (VL) is fundamental. The latest generation molecular assays for monitoring VL perform simultaneous detection of two regions of the viral genome, but without specifying the target used for VL quantitation. By using the "open" software (research use only [RUO]) of Aptima HIV-1 Quant Dx Assay (Aptima) which provides both results obtained with the pol and LTR targets, we were able to compare n = 500 plasma samples results from chronically HIV-1-infected patients under antiretroviral therapy (ART). Correlation and concordance were analyzed. By stratifying VL into two groups (<30 and ≥30 copies/mL HIV-1-RNA) according to pol-based results and matching them with their respective LTR values, concordance was substantial (κ = 0.635; 95%CI = 0.569 to 0.700) as expected. Considering the specimens (n = 224) with VL exactly quantified (i.e., ≥30 copies/mL) with both targets, an optimal correlation subsisted (r = 0.8882; P < 0.0001) and Bland-Altman plot showed no significant mean difference between them. However, by stratifying all these data in three ranges (30 to 200, 201 to 1,000, and >1,000 copies/mL) according to pol-based results, concordance analysis showed fair agreement (κ = 0.344; 95%CI = 0.257 to 0.432). Indeed, after excluding mutually concordant VL values in each range (n = 134), the remaining discordant samples (n = 90; 40.1%) showed significant (P < 0.05) difference between VL measured with the two targets. With the Aptima "open" software, samples with pol-based VL <1,000 copies (cp)/mL HIV-1-RNA, the corresponding LTR values were on average 0.5 log10 cp/mL higher. Further studies on these discrepancies and the nature of viral RNA elements detected only with the LTR despite efficient ART are in progress. IMPORTANCE The last generation dual-target platforms for quantification of HIV-1 RNA return a single value of viral load (VL) derived from a combined reading of two HIV-1 genome targets. By using a modified version of Aptima software, providing both the VL results obtained from pol and LTR amplification separately, we observed discordant VL results in some samples from HIV-1-infected patients on antiretroviral therapy. In particular, some samples with pol-based quantified <1,000 copies/mL VL showed the LTR-based value on average 0.5 log10 copies/mL higher, and other samples, always by treated patients, showed VL exclusively quantified with LTR target while the corresponding pol-based VL results were completely undetected. Standard software of double-target based diagnostic systems does not allow recognizing discrepant VL values in these particular, but not rare, clinical specimens. This issue could have implications for clinical management by leading physicians to consider changing antiretroviral regimen based on presumed failure of antiretroviral therapy.

HibeRNAtion: HIV-1 RNA Metabolism and Viral Latency.

HIV-1 infection remains non-curative due to the latent reservoir, primarily a small pool of resting memory CD4+ T cells bearing replication-competent provirus. Pharmacological reversal of HIV-1 latency followed by intrinsic or extrinsic cell killing has been proposed as a promising strategy to target and eliminate HIV-1 viral reservoirs. Latency reversing agents have been extensively studied for their role in reactivating HIV-1 transcription in vivo, although no permanent reduction of the viral reservoir has been observed thus far. This is partly due to the complex nature of latency, which involves strict intrinsic regulation at multiple levels at transcription and RNA processing. Still, the molecular mechanisms that control HIV-1 latency establishment and maintenance have been almost exclusively studied in the context of chromatin remodeling, transcription initiation and elongation and most known LRAs target LTR-driven transcription by manipulating these. RNA metabolism is a largely understudies but critical mechanistic step in HIV-1 gene expression and latency. In this review we provide an update on current knowledge on the role of RNA processing mechanisms in viral gene expression and latency and speculate on the possible manipulation of these pathways as a therapeutic target for future cure studies.

Association of High Ratio of CSF/Plasma HIV-1 RNA with Central Nervous System Co-Infection in HIV-1-Positive Treatment-Naive Patients.

Cerebrospinal fluid (CSF) human immunodeficiency virus-1 (HIV-1) ribonucleic acid (RNA) at higher levels than in plasma has been observed in HIV-1-positive patients and defined as CSF/plasma discordance or CSF escape. Discordance is particularly seen in untreated patients with antiretroviral agents. Quantitative data regarding its association with blood−brain barrier (BBB) damage and intracranial co-infection with other pathogens are limited. Therefore, we used the CSF to plasma HIV-1 RNA ratio (HRR) to determine its relation to central nervous system (CNS) co-infection in HIV-1-positive treatment-naïve individuals. We retrospectively recruited the subjects with HIV-1-positive and potential neurological deficits. A lumbar puncture was performed before the antiretroviral therapy. The paired CSF/plasma HIV-1 RNA samples were analyzed. Univariate and multivariate logistic regression models and multiple spine regression analyses were performed to assess the association between the HRR and CNS co-infection. A total of 195 patients with 78% males (median age: 49 years) were included in this study, of whom 98 (50.2%) had CNS co-infection with other pathogens. The receiver-operating characteristic curve analysis showed that the optimal cutoff value for the HRR to predict the CNS co-infection was 1.00. Higher HRR (≥1) was significantly associated with tuberculous meningitis (OR 6.50, 95% CI 2.08−20.25, p = 0.001), cryptococcus meningitis (OR 7.58, 95% CI 2.10−27.32, p = 0.001), and multiple co-infection (OR 4.04, 95% CI 1.02−16.04, p = 0.047). Higher HRR (≥1) (OR 3.01, 95% CI 1.09−8.73, p = 0.032) was independently associated with the CNS co-infection after adjusting for covariates. No significant nonlinear association was found between the HRR and CNS co-infection in the multivariate spline regression (p > 0.05) and a positive relationship was found between the HRR and CNS co-infection when the HRR was ≥0.78. Higher HRR was associated with an increased risk of CNS co-infection in HIV-1-positive patients. The relationship between the HRR and CNS co-infection may be related to the BBB disturbance and warrants further investigation with a large, longitudinal cohort.

Performance assessment of the new Xpert® HIV-1 viral load XC assay for quantification of HIV-1 viral loads.

BACKGROUND: HIV-1 RNA quantification is a key component of treatment monitoring. OBJECTIVES: To assess the performance of a redesigned HIV-1 RNA quantitative assay that uses a dual-target approach: Xpert® HIV-1 Viral Load (VL) XC. STUDY DESIGN: Fresh and frozen samples (N = 533) from HIV-1 positive patients tested with Abbott HIV-1 assays (Alinity m and RealTime [m2000]) were retested using the new Xpert XC assay. Three samples with known underquantification using the previous single-target Xpert assay were retested. RESULTS: The Xpert XC assay yielded valid results in 98.5% (N = 528/536) of cases and showed high sensitivity in 80 fresh samples that had undetectable VLs or ≤1.7 log copies/mL with Alinity m. Linear regression and Bland-Altman analyses showed high concordance with the Abbott tests for quantified samples over a wide VL range (1.6-6.9 copies/mL), including non-B subtypes (mean difference=-0.1±0.23 log copies/mL). Mutations associated with integrase resistance did not impact the results. Very good linearity and reproducibility was shown for the tested subtypes B, CRF06_cpx, and CRF02_AG. Xpert XC VLs in samples that were previously underquantified using the original single-target Xpert assay were similar to those detected by the Abbott assays (±0.11 log copies/mL). CONCLUSIONS: The Xpert XC assay showed excellent correlation with the Abbott assays for all tested HIV-1 subtypes. Sensitivity, linearity and accuracy were high in the therapeutically relevant VL range. With a time to result of only 90 min, this on-demand decentralized assay is a safe, reliable and fast option for VL monitoring in HIV-1-infected patients.

Phase IIa Proof-of-Concept Evaluation of the Antiviral Efficacy, Safety, Tolerability, and Pharmacokinetics of the Next-Generation Maturation Inhibitor GSK3640254.

BACKGROUND: GSK3640254 (GSK'254) is a next-generation human immunodeficiency virus type 1 (HIV-1) maturation inhibitor with pharmacokinetics (PK) supporting once-daily therapy. METHODS: This phase IIa double-blind (sponsor-unblinded), randomized, placebo-controlled, adaptive study evaluated antiviral effect, safety, tolerability, and PK of once-daily GSK'254 monotherapy administered with food (moderate-fat meal) in HIV-1-positive, treatment-naive adults. In part 1, participants received GSK'254 10 or 200 mg for 10 days. In part 2, participants received GSK'254 40, 80, or 140 mg for 7 days, modified from 10 days by a protocol amendment to decrease potential for resistance-associated mutations (RAMs). The primary endpoint was maximum change from baseline in HIV-1 RNA. RESULTS: Maximum changes in HIV-1 RNA of -0.4, -1.2, -1.0, -1.5, and -2.0 log10 occurred with GSK'254 10, 40, 80, 140, and 200 mg, respectively. Regardless of dosing duration, doses ≥40 mg resulted in ≥1-log10 declines in HIV-1 RNA. Plasma PK was generally dose proportional to 140 mg but non-proportional between 140 and 200 mg. Four participants in the 200-mg group developed RAMs on day 11 in part 1, 1 with phenotypic resistance. No RAMs occurred in part 2. Adverse events (AEs) were reported by 22 (65%) participants; headache was the most common (n = 4). Two non-drug-related serious AEs occurred. All AEs were of mild-to-moderate intensity, except for 2 grade 3 non-drug-related AEs in 1 participant. CONCLUSIONS: This monotherapy study established a dose-antiviral response relationship for GSK'254. No safety or tolerability concerns were noted. These results supported dose selection for the ongoing phase IIb study (ClinicalTrials.gov: NCT04493216). CLINICAL TRIALS REGISTRATION: NCT03784079.

ABX464 Decreases the Total Human Immunodeficiency Virus (HIV) Reservoir and HIV Transcription Initiation in CD4+ T Cells From Antiretroviral Therapy-Suppressed Individuals Living With HIV.

BACKGROUND: Antiretroviral therapy (ART) intensification and disruption of latency have been suggested as strategies to eradicate HIV. ABX464 is a novel antiviral that inhibits HIV RNA biogenesis. We investigated its effect on HIV transcription and total and intact HIV DNA in CD4+ T cells from ART-suppressed participants enrolled in the ABIVAX-005 clinical trial (NCT02990325). METHODS: Peripheral CD4+ T cells were available for analysis from 9 ART-suppressed participants who were treated daily with 150 mg of ABX464 for 4 weeks. Total and intact HIV DNA and initiated, 5'elongated, unspliced, polyadenylated, and multiply-spliced HIV transcripts were quantified at weeks 0, 4, and 8 using ddPCR. RESULTS: We observed a significant decrease in total HIV DNA (P = .008, median fold change (mfc) = 0.8) and a lower median level of intact HIV DNA (P = not significant [n.s.], mfc = 0.8) after ABX464 treatment. Moreover, we observed a decrease in initiated HIV RNA per million CD4+ T cells and per provirus (P = .05, mfc = 0.7; P = .004, mfc = 0.5, respectively), a trend toward a decrease in the 5'elongated HIV RNA per provirus (P = .07, mfc = 0.5), and a lower median level of unspliced HIV RNA (P = n.s., mfc = 0.6), but no decrease in polyadenylated or multiply-spliced HIV RNA. CONCLUSIONS: In this substudy, ABX464 had a dual effect of decreasing total HIV DNA (and possibly intact proviruses) and HIV transcription per provirus. To further characterize its specific mechanism of action, long-term administration of ABX464 should be studied in a larger cohort. CLINICAL TRIALS REGISTRATION: NCT02990325.

Atlas of the HIV-1 Reservoir in Peripheral CD4 T Cells of Individuals on Successful Antiretroviral Therapy.

Knowing the mechanisms that govern the persistence of infected CD4+ subpopulations could help us to design new therapies to cure HIV-1 infection. We evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ subpopulations from 14 HIV-1-infected individuals on antiretroviral therapy to analyze its relationship with HIV-1 transcription, immune activation, and cell proliferation. A unique large blood donation was used to isolate CD4, CD4 resting (CD4r), CD4 activated (CD4a), T naive (TN), T stem cell memory (TSCM), T central memory (TCM), T transitional memory (TTM), T effector memory (TEM), circulating T follicular helper (cTFH), TCD20, TCD32, and resting memory TCD2high (rmTCD2high) cells. HIV-1 DNA measured by droplet digital PCR ranged from 3,636 copies/106 in TTM to 244 in peripheral blood mononuclear cells (PBMCs), with no subpopulation standing out for provirus enrichment. Importantly, all the subpopulations harbored intact provirus by intact provirus DNA assay (IPDA). TCD32, cTFH, and TTM had the highest levels of HIV-1 transcription measured by fluorescent in situ hybridization with flow cytometry (FISH/flow), but without reaching statistical differences. The subpopulations more enriched in provirus had a memory phenotype, were less activated (measured by CD38+/HLA-DR+), and expressed more programmed cell death 1 (PD-1). Conversely, subpopulations transcribing more HIV-1 RNA were not necessarily enriched in provirus and were more activated (measured by CD38+/HLA-DR+) and more proliferative (measured by Ki-67). In conclusion, the HIV reservoir is composed of a mosaic of subpopulations contributing to the HIV-1 persistence through different mechanisms such as susceptibility to infection, provirus intactness, or transcriptional status. The narrow range of reservoir differences between the different blood cell subsets tested suggests limited efficacy in targeting only specific cell subpopulations during HIV-1 cure strategies. IMPORTANCE The main barrier for HIV-1 cure is the presence of latently infected CD4+ T cells. Although various cell subpopulations have been identified as major HIV-1 reservoir cells, the relative contribution of infected CD4 subpopulations in the HIV-1 reservoir remains largely unknown. Here, we evaluated the simultaneous distribution of the HIV-1 reservoir in 13 CD4+ T-cell subpopulations in peripheral blood from HIV-1-infected individuals under suppressive antiretroviral therapy. We found that the HIV-1 reservoir is composed of a mosaic of cell subpopulations, with heterogeneous proviral DNA, HIV-1 transcription, and activation status. Hence, each cell subpopulation contributes to the HIV-1 persistence through different mechanisms such as susceptibility to infection, rates of intact provirus, transcriptional status or half-life. This research provides new insights into the composition of the HIV-1 reservoir, suggesting that, to be effective, eradication strategies must simultaneously target multiple cell subpopulations.

IRF7 and RNH1 are modifying factors of HIV-1 reservoirs: a genome-wide association analysis.

BACKGROUND: Combination antiretroviral treatment (cART) cannot eradicate HIV-1 from the body due to the establishment of persisting viral reservoirs which are not affected by therapy and reinitiate new rounds of HIV-1 replication after treatment interruption. These HIV-1 reservoirs mainly comprise long-lived resting memory CD4+ T cells and are established early after infection. There is a high variation in the size of these viral reservoirs among virally suppressed individuals. Identification of host factors that contribute to or can explain this observed variation could open avenues for new HIV-1 treatment strategies. METHODS: In this study, we conducted a genome-wide quantitative trait locus (QTL) analysis to probe functionally relevant genetic variants linked to levels of cell-associated (CA) HIV-1 DNA, CA HIV-1 RNA, and RNA:DNA ratio in CD4+ T cells isolated from blood from a cohort of 207 (Caucasian) people living with HIV-1 (PLHIV) on long-term suppressive antiretroviral treatment (median = 6.6 years). CA HIV-1 DNA and CA HIV-1 RNA levels were measured with corresponding droplet digital PCR (ddPCR) assays, and genotype information of 522,455 single-nucleotide variants was retrieved via the Infinium Global Screening array platform. RESULTS: The analysis resulted in one significant association with CA HIV-1 DNA (rs2613996, P < 5 × 10-8) and two suggestive associations with RNA:DNA ratio (rs7113204 and rs7817589, P < 5 × 10-7). Then, we prioritized PTDSS2, IRF7, RNH1, and DEAF1 as potential HIV-1 reservoir modifiers and validated that higher expressions of IRF7 and RNH1 were accompanied by rs7113204-G. Moreover, RNA:DNA ratio, indicating relative HIV-1 transcription activity, was lower in PLHIV carrying this variant. CONCLUSIONS: The presented data suggests that the amount of CA HIV-1 DNA and RNA:DNA ratio can be influenced through PTDSS2, RNH1, and IRF7 that were anchored by our genome-wide association analysis. Further, these observations reveal potential host genetic factors affecting the size and transcriptional activity of HIV-1 reservoirs and could indicate new targets for HIV-1 therapeutic strategies.

The Clonal Expansion Dynamics of the HIV-1 Reservoir: Mechanisms of Integration Site-Dependent Proliferation and HIV-1 Persistence.

More than 50% of the HIV-1 latent reservoir is maintained by clonal expansion. The clonally expanded HIV-1-infected cells can contribute to persistent nonsuppressible low-level viremia and viral rebound. HIV-1 integration site and proviral genome landscape profiling reveals the clonal expansion dynamics of HIV-1-infected cells. In individuals under long-term suppressive antiretroviral therapy (ART), HIV-1 integration sites are enriched in specific locations in certain cancer-related genes in the same orientation as the host transcription unit. Single-cell transcriptome analysis revealed that HIV-1 drives aberrant cancer-related gene expression through HIV-1-to-host RNA splicing. Furthermore, the HIV-1 promoter dominates over the host gene promoter and drives high levels of cancer-related gene expression. When HIV-1 integrates into cancer-related genes and causes gain of function of oncogenes or loss of function of tumor suppressor genes, HIV-1 insertional mutagenesis drives the proliferation of HIV-1-infected cells and may cause cancer in rare cases. HIV-1-driven aberrant cancer-related gene expression at the integration site can be suppressed by CRISPR-mediated inhibition of the HIV-1 promoter or by HIV-1 suppressing agents. Given that ART does not suppress HIV-1 promoter activity, therapeutic agents that suppress HIV-1 transcription and halt the clonal expansion of HIV-1-infected cells should be explored to block the clonal expansion of the HIV-1 latent reservoir.

Variations in the Abortive HIV-1 RNA Hairpin Do Not Impede Viral Sensing and Innate Immune Responses.

The highly conserved trans-acting response element (TAR) present in the RNA genome of human immunodeficiency virus 1 (HIV-1) is a stably folded hairpin structure involved in viral replication. However, TAR is also sensed by viral sensors, leading to antiviral immunity. While high variation in the TAR RNA structure renders the virus replication-incompetent, effects on viral sensing remain unclear. Here, we investigated the role of TAR RNA structure and stability on viral sensing. TAR mutants with deletions in the TAR hairpin that enhanced thermodynamic stability increased antiviral responses. Strikingly, TAR mutants with lower stability due to destabilization of the TAR hairpin also increased antiviral responses without affecting pro-inflammatory responses. Moreover, mutations that affected the TAR RNA sequence also enhanced specific antiviral responses. Our data suggest that mutations in TAR of replication-incompetent viruses can still induce immune responses via viral sensors, hereby underscoring the robustness of HIV-1 RNA sensing mechanisms.

Abortive HIV-1 RNA induces pro-IL-1ß maturation via protein kinase PKR and inflammasome activation in humans.

The proinflammatory cytokine IL-1ß mediates high levels of immune activation observed during acute and chronic human immunodeficiency virus 1 (HIV-1) infection. Little is known about the mechanisms that drive IL-1ß activation during HIV-1 infection. Here, we have identified a crucial role for abortive HIV-1 RNAs in inducing IL-1ß in humans. Abortive HIV-1 RNAs were sensed by protein kinase RNA-activated (PKR), which triggered activation of the canonical NLRP3 inflammasome and caspase-1, leading to pro-IL-1ß processing and secretion. PKR activated the inflammasome via ROS generation and MAP kinases ERK1/2, JNK, and p38. Inhibition of PKR during HIV-1 infection blocked IL-1ß production. As abortive HIV-1 RNAs are produced during productive infection and latency, our data strongly suggest that targeting PKR signaling might attenuate immune activation during acute and chronic HIV-1 infection.

Restriction Factors expression decreases in HIV-1 patients after cART.

Activation of interferon (IFN) mediated responses and the consequent expression of restriction factors (RFs) represent an early line of defense against HIV-1 infection. The levels of viral replication and the antiviral are among the determinants influencing RFs' expression pattern. A deeper understanding of the molecular mechanisms regulating RFs activity and their relationship with viral replication factors might lead to new therapeutic strategies based on the enhancement of immune response against the virus. The aim of this study is to perform a longitudinal evaluation of the variations in the levels of a group of selected RFs (APOBEC3G, BST2, TRIM5α, MX2, SAMHD1, SERINC3/5, IFI16 and STING) to determine the impact of cART on their expression in HIV-1 positive patients. Together with RFs expression, immunological and virological parameters (plasma HIV1-RNA load and total HIV1-DNA) were longitudinally evaluated in a cohorts fourteen HIV-1 cART na ve patients, who were evaluated at diagnosis (T0) and followed at 4 (T1) and 8 (T2) months after starting cART. Fourteen long-term treated patients who achieved sustained undetectable viremia for at least 2 years were also included in the study as a reference group. We observed a restoration of immunological conditions during cART, together with a progressive decrease of HIV1-RNA load, which became undetectable at 8 months after starting treatment. On the other hand, despite showing a trend towards decrease, total HIV1-DNA remained detectable after reaching viral suppression, similarly to what observed in long term treated patients. The expression of APOBEC3G, SAMHD1, BST2, IFI16, SERINC3, and SERINC5 was higher at the time of diagnosis and decreased significantly during therapy, reaching levels similar to the ones observed in virally suppressed patients. On the other hand, MX2 and TRIM5a high expression values up to T0, reaching lower levels immediately after the initiation of cART treatment. Correlation analysis showed a positive association between the expression levels of APOBEC3G, IFI16, MX2, SAMHD1, SERINC3 and TRIM5α with the HIV-1 viral load. On the contrary, no significant association was observed for BST2, SERINC5 and STING, even BST2 expression showed a tendency to correlate with viral load. We observed a tendency for a positive association of MX2, SAMHD1 and SERINC5 with the size of viral reservoir and a trend for a negative association for STING. STING appeared also as the only one factor whose expression correlates with the CD4 count and the CD4/CD8 ratio. Our data confirm the correlation between viral replication and expression of RFs, with, the levels of cellular defense proteins decreasing in parallel to the reduction of viral replication.

Exploration of an Efficient Simultaneous Molecular Detection Method of HIV, HCV, and Syphilis from a Single Dried Blood Spot.

OBJECTIVE: The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma. METHOD: A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma. RESULTS: Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log 10 copies/mL). There were two samples (2/94) with undetectable HCV RNA in DBS, while measurable HCV RNA levels were present in plasma (-5 to 5.99 log 10 copies/mL). The correlation between HIV-1 RNA light chain variable region (VL) values obtained from plasma and DBS showed that r = 0.683 ( P < 0.01), n = 27 and r = 0.612 ( P < 0.01), n = 89 in HCV RNA. Bland-Altman analysis revealed that in HIV-1 RNA, the mean (± SD) difference between HIV-1 RNA in plasma and DBS was 1.00 ± 1.01 log 10 copies/mL, and all samples were within ± 1.96 SD (-0.97 to 2.97 log 10 copies/mL) for DBS. The mean difference (± SD) in HCV RNA was 0.15 ± 1.08 log 10 copies/mL, and 94.38% (84/89) were within ± 1.96 SD (-1.96 to 2.67 log 10 copies/mL). Overall, HIV-1 RNA and HCV RNA levels obtained from a DBS were lower than those obtained from plasma. HIV-1 DNA in a DBS showed concordant results with HIV-1 RNA in plasma. HIV-1 DNA RT-PCR using a DBS showed acceptable performance. CONCLUSION: The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

Role of antenatal plasma cytomegalovirus DNA levels on pregnancy outcome and HIV-1 vertical transmission among mothers in the University of Zimbabwe birth cohort study (UZBCS).

INTRODUCTION: Despite being a leading infectious cause of childhood disability globally, testing for cytomegalovirus (CMV) infections in pregnancy is generally not done in Sub-Sahara Africa (SSA), where breastfeeding practice is almost universal. Whilst CMV and human immunodeficiency virus (HIV) are both endemic in SSA, the relationship between antenatal plasma CMV-DNA, HIV-1-RNA levels and HIV-1-mother to child transmission (MTCT) including pregnancy outcomes remains poorly described. METHODS: Pregnant women at least 20 weeks' gestational age at enrolment were recruited from relatively poor high-density suburbs in Harare, Zimbabwe. Mother-infant dyads were followed up until 6 months postpartum. In a case-control study design, we tested antenatal plasma CMV-DNA levels in all 11 HIV-1 transmitting mothers, as well as randomly selected HIV-infected but non-transmitting mothers and HIV-uninfected controls. CMV-DNA was detected and quantified using polymerase chain reaction (PCR) technique. Antenatal plasma HIV-1-RNA load was quantified by reverse transcriptase PCR. Infants' HIV-1 infection was detected using qualitative proviral DNA-PCR. Predictive value of antenatal plasma CMV-DNAemia (CMV-DNA of > 50 copies/mL) for HIV-1-MTCT was analyzed in univariate and multivariate regression analyses. Associations of CMV-DNAemia with HIV-1-RNA levels and pregnancy outcomes were also explored. RESULTS: CMV-DNAemia data were available for 11 HIV-1 transmitting mothers, 120 HIV-infected but non-transmitting controls and 46 HIV-uninfected mothers. In a multivariate logistic regression model, we found a significant association between CMV-DNAemia of > 50 copies/mL and HIV-1 vertical transmission (p = 0.035). There was no difference in frequencies of detectable CMV-DNAemia between HIV-infected and -uninfected pregnant women (p = 0.841). However, CMV-DNA levels were higher in immunosuppressed HIV-infected pregnant women, CD4 < 200 cells/µL (p = 0.018). Non-significant associations of more preterm births (< 37 weeks, p = 0.063), and generally lower birth weights (< 2500 g, p = 0.450) were observed in infants born of HIV-infected mothers with CMV-DNAemia. Furthermore, in a multivariate analysis of HIV-infected but non-transmitting mothers, CMV-DNAemia of > 50 copies/mL correlated significantly with antenatal plasma HIV-1-RNA load (p = 0.002). CONCLUSION: Antenatal plasma CMV-DNA of > 50 copies/mL may be an independent risk factor for HIV-1-MTCT and higher plasma HIV-1-RNA load, raising the possibility that controlling antenatal CMV-DNAemia might improve infant health outcomes. Further studies with larger sample sizes are warranted to confirm our findings.