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BACKGROUND: The human immunodeficiency virus (HIV) remains a major worldwide health problem. Nowadays, many methods have been developed for quantitative detecting human immunodeficiency virus DNA (HIV-DNA), such as fluorescence and colorimetry. However, these methods still have the disadvantages of being expensive and requiring professional technicians. Early diagnosis of pathogens is increasingly dependent on portable instruments and simple point-of-care testing (POCT). Therefore, it is meaningful and necessary to develop portable and cheap methods for detecting disease markers. RESULTS: In this work, a label-free chemiluminescence (CL) method was developed for detecting HIV-DNA via a handheld luminometer. To achieve label-free target detection, the CL catalyst, G-triplex-hemin DNAzyme (G3-hemin DNAzyme), was in-situ assembled in the presence of HIV-DNA. For improving sensitivity, HIV-DNA induced the cyclic strand displacement reaction (SDR), which can form three G3-hemin DNAzymes in one cycle. So, the chemiluminescence reaction between luminol and H2O2 was highly effectively catalyzed, and the CL intensity was linearly related with the concentration of HIV-DNA in the range of 0.05-10 nM with a detection limit of 29.0 pM. Due to the high specificity of hairpin DNA, single-base mismatch can be discriminated, which ensured the specific detection of HIV-DNA. SIGNIFICANCE: In-situ formation of G3-hemin DNAzyme led to label-free and selective detection without complex synthesis and functionalization. Therefore, it offers a cheap, selective, sensitive and portable method for detecting disease-related genes, which is promising for POCT of clinical diagnosis in resource-limited settings.

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The persistence of latent viral genomes in people receiving antiretroviral therapy (ART) is the main obstacle to a cure for human immunodeficiency virus (HIV) infection. Viral reservoirs can be defined as cells harboring HIV genomes that have the ability to produce infectious virions. Precise quantification of the cellular reservoirs of HIV is challenging because these cells are rare, heterogeneous, and outnumbered by a larger number of cells carrying defective genomes. In addition, measuring the inducibility of these proviruses requires functional assays and remains technically difficult. The recent development of single-cell and single-viral genome approaches revealed additional layers of complexity: the cell subsets that harbor proviruses are heterogeneous and their ability to be induced is variable. A substantial fraction of intact HIV genomes may be permanently silenced after years of ART, revealing the underappreciated importance of induction assays. As such, a simple approach that would assess simultaneously the genetic intactness and the inducibility of the reservoir is still lacking. In this study, we review recent advances in the development of methods to quantify and characterize persistently infected cells, and we discuss how these findings can inform the design of future assays aimed at measuring the size of the intact and inducible HIV reservoir.

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OBJECTIVE: HIV DNA sequencing is now routinely used for HIV-infected individuals on antiretroviral therapy (ART) with or without partial genotypic history. Successful amplification of HIV pol gene has yet to be correlated with HIV DNA levels. Here, we assessed the relationship between HIV DNA load and sequencing results. METHODS: We analyzed three different qPCR measurements of total (LTR and LTR-gag) and integrated (Alu-LTR) HIV DNA in blood samples collected from viremic as well as virally suppressed HIV-infected individuals on ART. HIV DNA levels were compared to HIV DNA Sanger sequencing and clinical and therapeutic parameters. RESULTS: Among the 135 individuals analyzed for HIV DNA measurements and sequencing, all three HIV DNA measurements were associated with HIV DNA Sanger sequencing results. A threshold of around 2 and 1.5 log copies/million leukocytes of total HIV DNA was identified for LTR and LTR-gag qPCRs, respectively. Integrated HIV DNA positivity was also associated with successful sequencing. We further compared HIV DNA measurement techniques in an extended cohort of 312 individuals and showed that all measurements correlated between the different techniques, regardless of the HIV-1 subtypes analyzed. However, higher detection rates were observed with LTR (96%) compared to LTR-gag (86%) and Alu-LTR (59%) qPCRs. Duration of virological control on ART and CD4 nadir were the main determinants of HIV reservoir size. CONCLUSIONS: HIV DNA measurement is associated with Sanger sequencing success, regardless of the technique used. In a clinical setting, Application of HIV DNA quantification before sequencing should be further evaluated.

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As the introduction of antiretroviral therapy (ART) during primary HIV-1 infection (PHI) could restrict the establishment of HIV reservoirs, we aimed to assess the effect of three different ART regimens on HIV-DNA load in people living with HIV (PLWH), who started ART in PHI. Randomized, open-label, multicentric study, including subjects in PHI (defined as an incomplete HIV-1 Western blot and detectable plasma HIV-RNA) in the Italian Network of Acute HIV Infection cohort. Participants were randomly assigned (10:10:8) to a fixed-dose combination of tenofovir alafenamide fumarate (TAF) 10 mg plus emtricitabine (FTC) 200 mg, darunavir 800 mg, and cobicistat 150 mg once daily (group A), or TAF 25 mg plus FTC 200 mg, dolutegravir 50 mg once daily (group B), or an intensified four-drug regimen (TAF 10 mg plus FTC 200 mg, dolutegravir 50 mg, darunavir 800 mg, and cobicistat 150 mg once daily) (group C). The primary endpoint was the decrease of HIV-DNA copies/106 peripheral blood mononuclear cells (PBMCs) at weeks (W) 12 and 48. Secondary endpoints were increased in CD4+ cells and in CD4+/CD8+ ratio and percentage of PLWH reaching undetectable HIV-RNA. HIV-DNA was quantified by Droplet Digital PCR (Biorad QX100) and normalized to RPP30 reference gene. This study was registered in ClinicalTrials.gov (number NCT04225325). Among 78 participants enrolled, 30 were randomized to group 1, 28 to group 2, and 20 to group 3. At baseline, median CD4+ count was 658/µL (476-790), HIV-RNA 5.37 (4.38, 6.12) log10 copies/mL, without statistical difference in their change among groups at weeks 12 and 48 (p = 0.432 and 0.234, respectively). The trial was prematurely discontinued for slow accrual and for COVID-19 pandemic-associated restrictions. In the per-protocol analysis, PLWH (n = 72) with undetectable viral load was 54.3% at W12 and 86.4% at W48. Interestingly, the CD4/CD8 ratio progressively increased over time, up to normalization in almost half of the cohort by week 48, despite a deflection in group 3; no difference was observed by the Fiebig stage (I-III vs. IV-VI). HIV-DNA decreased from 4.46 (4.08, 4.81) log10 copies/106 PBMCs to 4.22 (3.79, 4.49) at week 12, and 3.87 (3.46, 4.34) at week 48, without difference among groups. At multivariable analysis, HIV-DNA delta at W48 was associated only with the increase of CD4+ count by 100 cells/mm3 but not with the Fiebig stage, the CD4+/CD8+ ratio, and treatment arm, despite a higher decrease in group 3. Six adverse events were recorded during our study, which did not cause any withdrawal from the study. We observed a decrease in HIV-DNA from baseline to W48 in PLWH treated during PHI, associated with an increase in CD4+ count, unrelated to the treatment arm.

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BACKGROUND: We aimed at evaluating the temporal trend of drug-resistance and APOBEC editing from HIV-DNA genotypic resistance tests (GRT) in virologically suppressed individuals. MATERIAL AND METHODS: Major resistance mutations (MRM), genotypic susceptibility score (GSS) for the current regimen and APOBEC-related mutations (APO-M) were evaluated. Potential changes in trends of MRM and APO-M over-time were assessed and predictors of MRM detection or sub-optimal GSS (GSS<2) at HIV-DNA-GRT were estimated through logistic regression analyses. RESULTS: Among the 1126 individuals included, 396 (35.2%) harboured at least one MRM (23.4% to NRTI, 18.8% to NNRTI, 7.7% to PI and 1.4% to INSTI [N=724]); 132 (12.3%) individuals showed a GSS <2. APO-M and stop codons were found in 229 (20.3%) and 105 (9.3%) individuals, respectively. APO-DRMs were found in 16.8% of individuals and were more likely observed in those individuals with stop codons (40.0%) compared to those without (14.4%, P<0.001). From 2010 to 2021 no significant changes of resistance or APO-M were found. Positive predictors of MRM detection at HIV-DNA GRT were drug abuse, subtype B infection, and a prolonged and complex treatment history. Perinatal infection and having at least 2 stop codons were associated with a current suboptimal regimen. CONCLUSIONS: In virologically suppressed individuals, resistance in HIV-DNA and the extent of APOBEC editing were generally stable in the last decade. A careful evaluation of APOBEC editing might be helpful to improve the reliability of HIV-DNA GRT. Further investigations are required to understand how to apply the estimation of APOBEC editing in refining genotypic evaluation.

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In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART ('HIV reservoir'). One such technique for HIV RNA quantitation, 'HIV transcription profiling', developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the 'HIV transcription profiling' technique to Qiagen's dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

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BACKGROUND: Elite controllers are able to control viral replication without antiretroviral therapy. Exceptional elite controllers do not show disease progression for more than 25 years. Different mechanisms have been proposed and several elements of both innate and adaptive immunity are implicated. Vaccines are immune stimulating agents that can promote HIV-RNA transcription; transient plasma HIV-RNA detectability has been described within 7-14 days after different vaccinations. The most reliable mechanism involved in virosuppressed people living with HIV is a generalized inflammatory response that activates bystander cells harboring latent HIV. So far no data about viral load increase in elite controllers after SARS-CoV-2 vaccination are reported in literature. CASE PRESENTATION: We report the case of a 65-year-old woman of European ancestry, diagnosed with HIV-1/HCV co-infection more than 25 years ago. Since then, HIV-RNA remained undetectable and she never received ARV therapy. In 2021 she was vaccinated with mRNA-BNT162b2 vaccine (Pfizer-BioNTech®). She was administered with three doses in June, July and October 2021, respectively. The last available viral load was undetectable in March 2021. We observed an increase of VL at 32 cp/ml and 124 cp/mL, two and seven months after the second vaccine dose, respectively. During monthly follow-up, HIV-RNA gradually and spontaneously dropped becoming undetectable without ARV intervention. COVID-19 serology was positive with IgG 535 BAU/mL, showing response to vaccination. We measured total HIV-DNA at different time-points and we found it detectable both at the time of the higher plasma HIV-RNA (30 cp/10^6 PBMCs) and when it was undetectable (13 cp/10^6 PBMCs), in reduction. CONCLUSIONS: This case is the first report, to our knowledge, describing a rebound of plasma HIV-RNA in an elite controller after three doses of mRNA-BNT162b2 vaccine for SARS-CoV-2. Concomitantly with a spontaneous reduction of plasma HIV-RNA ten months after the third dose of mRNA-BNT162b2 vaccine (Pfizer-BioNTech®) without antiretroviral therapy intervention, we observed a reduction of total HIV-DNA in peripheral mononuclear cells. The potential role of vaccinations in altering HIV reservoir, even in elite controllers when plasma HIV-RNA is undetectable, could be a valuable aspect to take into account for the future HIV eradication interventions.

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BACKGROUND: HIV DNA mirrors the number of infected cells and the size of the HIV viral reservoir. The aim of this study was to evaluate the effect of pre-cART HIV DNA levels as a predictive marker of immune reconstitution and on the post-cART CD4 counts trends. METHODS: HIV DNA was isolated from PBMCs and quantified by real-time PCR. Immune reconstitution was assessed up to four years. Piecewise-linear mixed models were used to describe CD4 count changes. RESULTS: 148 people living with HIV (PLWH) were included. The highest rate of immune reconstitution was observed during the first trimester. There was a trend showing that high HIV RNA level resulted in greater increase in CD4 count, especially during the first trimester of cART (difference above vs. below median 15.1 cells/µL/month; 95% CI -1.4-31.5; p = 0.073). Likewise, higher HIV DNA level would predict greater CD4 increases, especially after the first trimester (difference above vs. below median 1.2 cells/µL/month; 95% CI -0.1-2.6; p = 0.071). Higher DNA and RNA levels combined were significantly associated with greater CD4 increase past the first trimester (difference high/high vs. low/low 2.1 cells/µL/month; 95% CI 0.3-4.0; p = 0.024). In multivariable analysis, lower baseline CD4 counts predicted a greater CD4 rise. CONCLUSIONS: In successfully treated PLWH, pre-cART HIV DNA and HIV RNA levels are predictors of immune reconstitution.

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PURPOSEOF REVIEW: In the current quest for a complete cure for HIV/AIDS, the persistence of a long-lived reservoir of cells carrying replication-competent proviruses is the major challenge. Here, we describe the main elements and characteristics of several widely used assays of HIV latent reservoir detection. RECENT FINDINGS: To date, researchers have developed several different HIV latent reservoir detection assays. Among them, the in vitro quantitative viral outgrowth assay (QVOA) has been the gold standard for assessing latent HIV-1 viral load. The intact proviral DNA assay (IPDA) based on PCR also demonstrated the predominance of defective viruses. However, these assays all have some drawbacks and may still be inadequate in detecting the presence of ultralow levels of latent virus in many patients who were initially thought to have been cured, but eventually showed viral rebound. An accurate and precise measurement of the HIV reservoir is therefore needed to evaluate curative strategies, aimed to functional cure or sterilizing cure.

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OBJECTIVES: To investigate HIV-DNA and residual viremia (RV) levels over 96 weeks (W96) in virologically-suppressed HIV-1-infected individuals enrolled in the Be-OnE Study. Individuals were randomised to continue a two-drug regimen with dolutegravir (DTG) plus one reverse transcriptase inhibitor (RTI) or to switch to elvitegravir/cobicistat/emtricitabine/tenofovir-alafenamide (E/C/F/TAF). STUDY DESIGN: Total HIV-DNA and RV were evaluated at baseline, W48 and W96 using droplet digital polymerase chain reaction (ddPCR) technique. Potential relationships between viro-immunological parameters and between/within arms were also assessed. RESULTS: Median (interquartile range [IQR]) HIV-DNA was 2247 (767-4268), 1587 (556-3543) and 1076 (512-2345) copies/106 CD4+T-cells at baseline, W48 and at W96, respectively; RV was 3 (1-5), 4 (1-9) and 2 (2-4) copies/mL, respectively, with no significant differences between arms. A significant reduction in HIV-DNA and RV from baseline to W96 was observed in the E/C/F/TAF arm (HIV-DNA: -285 [-2257; -45], P=0.010; RV: -1 [-3;0], P=0.007). In the DTG + 1 RTI arm, HIV-DNA and RV levels remained stable (HIV-DNA: -549 [-2269;+307], P=0.182; RV: -1 [-3;+1], P=0.280). For both HIV-DNA and RV, there were no significant changes over time between the arms. A positive correlation was found between baseline HIV-DNA and HIV-DNA at W96 (E/C/F/TAF: Spearman correlation coefficient (rs)=0.726, P=0.0004; DTG + 1 RTI: rs=0.589, P=0.010). In general, no significant correlations were found between HIV-DNA, RV and immunological parameters over time. CONCLUSIONS: In virologically-suppressed individuals, there was a small reduction in HIV-DNA and HIV-RNA levels from baseline to W96 in individuals who switched to the E/C/F/TAF arm compared with those who remained on DTG + 1 RTI. However, there were no significant differences between the two arms in the changes in HIV-DNA and HIV-RNA over time.

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Objective: To investigate the relationship between abnormal activation of T cell subsets in peripheral whole blood and the recovery of immune function in persons infected with HIV-1, and to examine the relationship between the size of the viral reservoir of HIV-1 DNA and T cell subsets. Methods: HIV-1-infected persons who underwent routine testing between July 2019 and May 2020 were the target population of the study. According to whether, at the time of enrollment, their CD4+ T cells reached 500 cells/µL after antiretroviral therapy (ART), HIV-1-infected persons were divided into two groups, 76 in the deficiency group and 61 in the immune recovery group. In addition, 22 people who were not exposed to HIV-1, and who were tested negative for HIV-1 antibody were selected as the control group. For the three groups of subjects, tests of the T cell subsets were conducted. A total of 77 HIV-1-infected persons, with 44 from the deficiency group and 33 from the recovery group, were examined for HIV-1 DNA reservoir. The deficiency group and the recovery group were followed up 6 months later and the CD4+ T cell test results of 133 blood samples were collected, with 74 from the deficiency group and 59 from the recovery group. Results: The proportions of activated CD4+ and CD8+ T cells of the deficiency group were higher than those of the recovery group and the control group. The proportions of senescent CD4+ and CD8+ T cells in the deficiency group were comparable to those of the recovery group, which were higher than those of the control group, showing significant differences only in senescent CD8+ T cells, and no significant difference in senescent CD4+ T cells. The deficiency group expressed higher levels of effector memory CD4+ T and CD8+ T cells than the control group did, and the recovery group only expressed a higher level of effect memory CD8+ T cells. Both the deficiency group and the recovery group showed lower levels of central memory CD4+ T and CD8+ T cells than the control group did, and the recovery group had an even lower level of central memory CD4+ T cells than the deficiency group did. The recovery group showed a higher expression level of naïve CD4+ T cells, and the deficiency group and the recovery group had lower expression levels of naïve CD8+ T cells than the control group did. There was no correlation between the size of the viral reservoir of HIV-1 DNA and CD4+ T cell count or the T cell subsets. Activated CD4+ T cells, activated CD8+ T cells, and central memory CD4+ T cells were negatively correlated with the follow-up findings for CD4+ T cells, with r at －0.378, －0.334, and －0.322, respectively ( P<0.05). Naïve CD4+ T cells and naïve CD8+ T cells were positively correlated with the follow-up findings for CD4+ T cell subset, with r at 0.350 and 0.267, respectively ( P<0.05). Conclusion: HIV-1 infected persons have varying degrees of abnormal immune activation of T cell subsets. The abnormal activation of some T-cell subsets is partly associated with the subsequent recovery of immune functions and the size of the viral reservoir of HIV-1 DNA was not associated with the T cell subsets.

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BACKGROUND: Understanding immune responses after HBV vaccination is important to prevent HBV infection in PLWH and to achieve successful treatment. METHODS: Thirty-two PLWHs with CD4+ cell count > 350 cells/µL and HIV RNA < 200 copies/mL were vaccinated with 20 µg of HBV vaccine at weeks 0, 4, and 24 in this prospective study. We measured total HIV DNA levels, HBsAb titers and HBsAg-specific T-cell responses during follow-up time. RESULTS: All patients achieved protective HBsAb titer after immunization. The magnitude of the IFN-r and TNF-a response to HBsAg was 22.0 (IQR: 6.5-65.0) and 106.50 (IQR: 58.5-203.0) spot-forming cells (SFC)/105 PBMC, respectively at week 0. The level of IFN-r secreted at weeks 12 and weeks 36 to 48 was comparable with that at week 0. However, IFN-r response was higher at weeks 12 than that at weeks 36 to 48 (p = 0.02). The level of TNF-a secreted at weeks 12 was higher than that at week 0 (p < 0.001). Total HIV DNA levels were 2.76 (IQR: 2.47-3.07), 2.77 (IQR: 2.50-3.09), 2.77 (IQR: 2.41-2.89) log10 copies/106 PBMCs at weeks 0, 12, 36 to 48, respectively. No correlation was observed between IFN-r and TNF-a levels and HBsAb titer as well as total HIV DNA levels after immunization. CONCLUSION: Humoral immunity was satisfactory, but cellular immunity and decline in HIV reservoir were not optimal after HBV vaccine immunization in these patients.

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We report that people with human immunodeficiency virus (HIV) diagnosed with coronary artery atherosclerotic plaques display higher levels of HIV DNA compared with those without atherosclerotic plaques. In a multivariable prediction model that included 27 traditional and HIV-related risk factors, measures of HIV DNA were among the most important predictors of atherosclerotic plaque formation.

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【Objective】 To establish a magnetic bead enrichment strategy for the detection of human immunodeficiency virus deoxyribonucleic acid (HIV DNA) in peripheral blood, and to verify the improvement of the sensitivity of this method for the detection of HIV DNA in HIV infected patients after early antiretrovital treatment (ART). 【Methods】 Peripheral whole blood was collected at 4 timepoints in one ART HIV window period (WP) patient. Peripheral blood mononuclear cells (PBMCs) were isolated on a Ficoll gradient. CD4+ T lymphocytes were enriched from total PBMCs by negative sorting. HIV DNA concentration in magnetic beads enriched group and whole blood group was detected by HIV DNA detection kit. 【Results】 CD4+ T cells were isolated by magnetic beads and identified by FCM for purity at (96.4 ± 2.6)%. The viability was (95.9 ± 2.9)%, as demonstrated by trypan blue staining. The person on continued ART treatment in this study had significantly greater reduction in HIV viral load and undetectable HIV plasma RNA at follow up timepoint 4. No HIV DNA was detected in the whole blood group at all 4 timepoints. The quantitative results of HIV DNA in the CD4+ T lymphocyte group of the magnetic bead enrichment group were 73.4, 429.3, 137.1, 449.9 copies/106 CD4+ T cell′s respectively. 【Conclusion】 The magnetic bead enrichment method can be more sensitive in detecting the limit low copy HIV DNA in blood samples, and provide early confirmatory data for HIV WP infection and breakthrough infection after ART treatment.

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Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.

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The extent to which perinatally HIV-infected children, following cART initiation, develop a low proviral reservoir burden over time, as measured by HIV DNA droplet-digital polymerase chain reaction (ddPCR) and the effect on HIV antibody is not well characterized. We measured proviral HIV DNA and plasma RNA virus load (VL) in 37 perinatally HIV-infected children at 6 months of age who initiated stable cART. At 6-11 years of age, HIV proviral DNA, HIV VL (RNA), and HIV antibody by Western Blot (WB) were assessed. CART was initiated before 6 months of age in 13 children and after 6 months in 24. At school age, the HIV DNA levels did not differ by the timing of cART, and the HIV DNA levels were lower in children with negative/indeterminate WB (p = 0.0256). Children with undetectable HIV RNA VL > 50% of the time since cART initiation had lower median DNA VL than children with undetectable VL < 50% of the time (p = 0.07). Long-term viral suppression in perinatally HIV-infected children is associated with a decrease in HIV antibodies and reduced HIV reservoirs.

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Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi+ RRE+), 3' defective (Psi+ RRE-), and 5' defective (Psi- RRE+) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4+ T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/106 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/106 cells; 5.6%) or intact (0.6 copies/106 cells; <1%) HIV RNA. Likewise, the frequency of CD4+ T cells with 3' defective HIV RNA was greater than the frequency with 5' defective or intact HIV RNA. Intact HIV RNA was transcribed by a median of 0.018% of all proviruses and 2.2% of intact proviruses. The vast excess of 3' defective RNA over 5' defective or intact HIV RNA, which was not observed for HIV DNA, suggests that HIV transcription is completely blocked prior to the RRE in most cells with intact proviruses and/or that cells transcribing intact HIV RNA are cleared at very high rates. IMPORTANCE We developed a new assay that can distinguish and quantify intact (potentially infectious) as well as defective HIV RNA. In ART-treated individuals, we found that the vast majority of all HIV RNA is defective at the 3' end, possibly due to incomplete transcriptional processivity. Only a very small percentage of all HIV RNA is intact, and very few total or intact proviruses transcribe intact HIV RNA. Though rare, this intact HIV RNA is tremendously important because it is necessary to serve as the genome of infectious virions that allow transmission and spread, including rebound after stopping ART. Moreover, intact viral RNA may contribute disproportionately to the immune activation, inflammation, and organ damage observed with untreated and treated HIV infection. The intact viral RNA assay can be applied to many future studies aimed at better understanding HIV pathogenesis and barriers to HIV cure.

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INTRODUCTION: Proviral HIV DNA integrated within CD4 T-cells maintains an archive of viral variants that replicate during the course of the infection, including variants with reduced drug susceptibility. We considered studies that investigated archived drug resistance, with a focus on virologically suppressed patients and highlighted interpretative caveats and gaps in knowledge. RESULTS: Either Sanger or deep sequencing can be used to investigate resistance-associated mutations (RAMs) in HIV DNA recovered from peripheral blood. Neither technique is free of limitations. Furthermore, evidence regarding the establishment, maintenance, expression and clinical significance of archived drug-resistant variants is conflicting. This in part reflects the complexity of the HIV proviral landscape and its dynamics during therapy. Clinically, detection of RAMs in cellular HIV DNA has a variable impact on treatment outcomes, modulated by the drugs affected, treatment duration and additional determinants of virological failure, including those leading to suboptimal drug exposure. CONCLUSIONS: Sequencing cellular HIV DNA can provide helpful complementary information in treatment-experienced patients with suppressed plasma HIV RNA who require a change of regimen. However, care should be taken when interpreting the results. Presence of RAMs is not necessarily a barrier to treatment success. Conversely, even the most sensitive sequencing techniques will fail to provide a comprehensive view of the HIV DNA archive. To inform treatment decisions appropriately, the overall clinical and treatment history of a patient must always be considered alongside the results of resistance testing. Prospective controlled studies are needed to validate the utility of drug resistance testing using cellular HIV DNA.

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Reliable and accurate quantification of cell-associated HIV DNA (CA HIV DNA) is critical for early infant diagnosis, clinical management of patients under therapy, and to inform new therapeutics efficacy. The present study assessed the variability of CA HIV DNA quantification obtained from various assays and the value of using reference materials to help harmonize the measurements. Using a common set of reagents, our multicenter collaborative study highlights significant variability of CA HIV DNA quantification and lower limit of quantification across assays. The quantification of CA HIV DNA from a panel of infected PBMCs can be harmonized through cross-subtype normalization but assay calibration with the commonly used 8E5 cell line failed to reduce quantification variability between assays, demonstrating the requirement to thoroughly evaluate reference material candidates to help improve the comparability of CA HIV DNA diagnostic assay performance. IMPORTANCE Despite a global effort, HIV remains a major public health burden with an estimated 1.5 million new infections occurring in 2020. HIV DNA is an important viral marker, and its monitoring plays a critical role in the fight against HIV: supporting diagnosis in infants and underpinning clinical management of patients under therapy. Our study demonstrates that HIV DNA measurement of the same samples can vary significantly from one laboratory to another, due to heterogeneity in the assay, protocol, and reagents used. We show that when carefully selected, reference materials can reduce measurement variability and harmonize HIV DNA quantification across laboratories, which will help contribute to improved diagnosis and clinical management of patients living with HIV.

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The quantification of proviral DNA is raising interest in view of clinical management and functional HIV eradication. Measures of all unintegrated HIV DNA (uDNA) forms in infected reservoir cells provides information on recent replication events that is not found from other proviral DNA assays. To evaluate its actual relevance in a cohort of perinatally-infected adult HIV patients (PHIV), we studied how peripheral blood mononuclear cell uDNA levels correlated with total HIV DNA (tDNA) and with overall replication or innate immune control parameters including NK cell activation/exhaustion and lymphoid turnover. Twenty-two PHIV were included, with successfully controlled HIV (HIV RNA <50 copies/mL) on combined antiretroviral therapy for mean of 8.7 ± 3.9 years. uDNA accounted for 16 [5.2-83.5] copies/µg and was strongly correlated with tDNA (ρ=0.700, p=0.001). Flow cytometric analysis of peripheral NK cells showed that CD69 expression was directly correlated uDNA (p=0.0412), but not with tDNA. Interestingly, CD56-CD16+NK cells which include newly described inflammatory precursors and terminally differentiated cells were directly correlated with uDNA levels (p<0.001), but not with tDNA, and an inverse association was observed between the proportion of NKG2D+ NK cells and uDNA (ρ=-0.548, p=0.015). In addition, CD34+DNAM-1brightCXCR4+ inflammatory precursor frequency correlated directly with uDNA levels (ρ=0.579, p=0.0075). The frequencies of CD56-CD16+ and CD34+DNAM-1brightCXCR4+ cells maintained association with uDNA levels in a multivariable analysis (p=0.045 and p=0.168, respectively). Thus, control of HIV-1 reservoir in aviremic patients on ART is an active process associated with continuous NK cell intervention and turnover, even after many years of treatment. Quantification of linear and circular uDNA provides relevant information on the requirement for ongoing innate immune control in addition to ART, on recent replication history and may help stratify patients for functional HIV eradication protocols with targeted options.

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