HIV-1 DNA Quantification Research Articles

Comparison of HIV-1 DNA load measurements in blood and in relation to successful proviral sequencing

ObjectiveHIV 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. MethodsWe 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. ResultsAmong 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. ConclusionsHIV 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.

A label-free multicolor colorimetric and fluorescence dual mode biosensing of HIV-1 DNA based on the bifunctional NiFe2O4@UiO-66 nanozyme

Finding the DNA of the human immune deficiency virus (HIV) with simple and sensitive detection is the main challenge in early diagnosis of AIDS. Herein, two-point separation strategies based on the colorimetric and fluorescence are introduced. The naked-eye qualitative and semiquantitative colorimetric, and also accuracy fluorescence quantification of HIV-1 DNA were applied using label-free NiFe2O4@UiO-66 nanozyme with both functions of peroxidase-mimetic like and emitting fluorescence. The DNA probe-conjugated nanozyme is employed to hybridize a sequence of HIV-1. NiFe2O4@UiO-66 nanozymes catalyze the decomposition of H2O2 to •OH which can produce a remarkable fluorescent product 2-hydroxyterephthalic acid (TAOH) by the oxidation of the bridging ligand of weakly fluorescent terephthalic acid (TA). The accessibility of H2O2 toward confined-NiFe2O4 MNPs was reduced by increasing the HIV-1 target DNA concentration, resulting in the fluorescence intensity of TAOH being decreased. Meanwhile, remaining the unreacted H2O2 was transferred an acidic colorimetric solution containing FeSO4 and gold nanorods (AuNRs). Increasing the amount of H2O2 available for longitudinal etching of AuNRs due to •OH-generating Fe+2-catalyzed H2O2 is reponsible for different colors from brownish to colorless depending on the HIV-1 target DNA concentration. The fluorescence intensity and obtained colors have offered the sensitive biosensing methods with a linear range from 0.05 to 300 and 1–200 pM, respectively with a detection limit as low as 1 fM. Our study revealed that the applied sensing assay provides a cost-effective and straightforward qualitative, semiquantitative, and sensitive quantitation visible monitoring without the necessity of high-end instruments for HIV-1 detection in a human blood plasma/serum samples.

Assessing the Variability of Cell-Associated HIV DNA Quantification through a Multicenter Collaborative Study.

ABSTRACTReliable 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.

Accuracy of real-time PCR and digital PCR for the monitoring of total HIV DNA under prolonged antiretroviral therapy

Total HIV DNA is a standard marker to monitor the HIV reservoir in people living with HIV. We investigated HIV DNA quantification accuracy by a real-time PCR kit (qPCR) and digital PCR (dPCR) method within the same set of primers and probes. Among 48 aviremic patients followed for up to 7 years with qPCR, the mean coefficient of variation of total HIV DNA between two successive measurements was 77% (± 0.42log10 HIVDNA copies/106 PBMC). The total HIV DNA quantified by the two PCR methods has a high correlation (0.99 and 0.83, for 8E5 and PLHIV samples, respectively), but we observed better repeatability and reproducibility of the dPCR compared to the qPCR (CV of 11.9% vs. 24.7% for qPCR, p-value = 0.024). Furthermore, we highlighted a decay of the number of HIV copies in the 8E5 cell line qPCR standard over time (from 0.73 to 0.43 copies per cell), contributing to variations of HIV DNA results in patients whose HIV reservoir should be theoretically stabilized. Our study highlighted that absolute quantification of total HIV DNA by dPCR allows more accurate monitoring of the HIV reservoir than qPCR in patients under prolonged antiretroviral therapy.

External quality assessment of HIV-1 DNA quantification assays used in the clinical setting in Italy

Total cell-associated HIV-1 DNA is a surrogate marker of the HIV-1 reservoir, however, certified systems for its quantification are not available. The Italian HIV DNA Network was launched to validate HIV-1 DNA quantification methods in use at University and Hospital labs. A quality control panel including HIV-1 DNA standards, reconstructed blood samples (RBSs) and DNA from different HIV-1 subtypes was blindly tested by 12 participating labs by quantitative real-time PCR (n = 6), droplet digital PCR (n = 3) or both (n = 3). The median 95% hit rate was 4.6 (3.7–5.5) copies per test and linearity in the tested range was excellent (R2 = 1.000 [1.000–1.000]). The median values obtained across labs were 3,370 (2,287–4,245), 445 (299–498), 59 (40–81) and 7 (6–11) HIV-1 DNA copies, for the 3,584, 448, 56 and 7-copy standards, respectively. With RBSs, measured values were within twofold with respect to the median in two thirds of cases. HIV-1 subtypes were missed (CRF01_AE by 3 labs) or underestimated by > 1 log (subtypes A, C, D, F by one lab; CRF01_AE by one lab; CRF02_AG by one lab). The overall performance was excellent with HIV-1 DNA standards, however detection of different HIV-1 subtypes must be improved.

Assessment of intra-sample variability in HIV-1 DNA drug resistance genotyping.

HIV-1 drug resistance testing can be performed in proviral DNA. The non-homogenous distribution of viral variants in cells can impact the performance of this method. We assessed the variability of HIV-1 DNA genotyping results in the same blood sample using a next-generation sequencing (NGS) method. For each included patient, a blood sample from a single venipuncture was split into five 1 mL aliquots, which were independently tested in the same run. HIV-1 DNA was quantified in blood samples using real-time PCR, and NGS was performed with the Sentosa platform combined with the Sentosa SQ HIV genotyping Assay. A total of 60 aliquots from 12 samples (12 patients) were tested. The median age was 45.50 years old, and all patients were treated with antiretrovirals. A significant variability can sometimes be observed in HIV-1 DNA quantification between aliquots from the same sample, with a coefficient of variation ranging from 23% to 89%. The analysis of resistance-associated mutations (RAMs) with a 20% cut-off found some discordances in RAMs profile between aliquots from the same sample for 5, 3 and 3 patients in the reverse transcriptase, protease and integrase genes, respectively. The analysis with a lower cut-off (10%) showed additional mutations, but did not improve the intra-sample concordance. There is an intra-sample variability in HIV-1 DNA resistance test results, and repetition may sometimes bring additional information, but the extent of its clinical impact still requires further investigation.

T cell immune discriminants of HIV reservoir size in a pediatric cohort of perinatally infected individuals.

The size of the latent HIV reservoir is associated with the timing of therapeutic interventions and overall health of the immune system. Here, we demonstrate that T cell phenotypic signatures associate with viral reservoir size in a cohort of HIV vertically infected children and young adults under durable viral control, and who initiated anti-retroviral therapy (ART) <2 years old. Flow cytometry was used to measure expression of immune activation (IA), immune checkpoint (ICP) markers, and intracellular cytokine production after stimulation with GAG peptides in CD4 and CD8 T cells from cross-sectional peripheral blood samples. We also evaluated the expression of 96 genes in sort-purified total CD4 and CD8 T cells along with HIV-specific CD4 and CD8 T cells using a multiplexed RT-PCR approach. As a measure of HIV reservoir, total HIV-DNA quantification by real-time PCR was performed. Poisson regression modeling for predicting reservoir size using phenotypic markers revealed a signature that featured frequencies of PD-1+CD4 T cells, TIGIT+CD4 T cells and HIV-specific (CD40L+) CD4 T cells as important predictors and it also shows that time of ART initiation strongly affects their association with HIV-DNA. Further, gene expression analysis showed that the frequencies of PD-1+CD4 T cells associated with a CD4 T cell molecular profile skewed toward an exhausted Th1 profile. Our data provide a link between immune checkpoint molecules and HIV persistence in a pediatric cohort as has been demonstrated in adults. Frequencies of PD-1+ and TIGIT+CD4 T cells along with the frequency of HIV-specific CD4 T cells could be associated with the mechanism of viral persistence and may provide insight into potential targets for therapeutic intervention.

Quantification of the HIV-1 total reservoir in the peripheral blood of naïve and treated patients by a standardised method derived from a commercial HIV-1 RNA quantification assay.

HIV-1 DNA can persist in host cells, establishing a latent reservoir. This study was aimed to develop an extraction and amplification protocol for HIV-1 DNA quantification by modifying a quantitative commercial assay. HIV-1 DNA was extracted on an Abbott m2000sp instrument, using an open-mode protocol. Two calibrators, spiked with a plasmid containing HIV-1 genome (103 and 105 cps/mL), were extracted and amplified to generate a master calibration curve. Precision, accuracy, linear dynamic range, limit of quantification (LOQ) and limit of detection (LOD) were determined. A cohort of patients, naïve or chronically infected, was analysed. Calibration curve was obtained from 42 replicates of standards (stds); precision was calculated (coefficients of variability [CVs] below 10%); accuracy was higher than 90%. Linearity covered the entire range tested (10-104 copies per reaction), and LOD (95%) was 12 copies per reaction. HIV-1 DNA was significantly higher (p < 0.0001) in drug-naïve (62) than in chronically treated patients (50), and proviral loads correlated with lymphocytes (p = 0.0002) and CD4+ (p < 0.0001) counts only in naïve patients. Both groups displayed a significant inverse correlation between CD4+ nadir and proviral loads. A significant correlation (p < 0.0001) between viraemia and HIV-1 reservoir was disclosed. No significant difference was obtained from the comparison between proviral loads on whole blood and peripheral blood mononuclear cells (PBMCs) from the same patient. The novelty of our approach relies on the selection of appropriate reference standard extracted and amplified as clinical specimens avoiding any underestimation of the reservoir. Results confirm HIV-1 DNA as a marker of disease progression, supporting the relationship between the width of latent reservoir and the immunological status of the patient.

Comparison of Ultrabio HIV DNA PCR and Gag Real-Time PCR Assays for Total Hiv-1 DNA Quantification

Total HIV-1 DNA has been shown to reflect reservoir size in HIV-1 infected individuals and correlate with viral rebound time after therapy interruption. Quantification of total HIV-1 DNA level may be used to study and evaluate reservoir elimination therapies. There is currently no FDA approved HIV- 1 DNA quantification assay available. The performance of a research uses only kit, Ultrabio HIV DNA quantification kit, was compared to a commonly used laboratory qPCR method targeting the gag region (GAG PCR). The limit of detection (LOD) and precision was evaluated with serially diluted 8E5/LAV DNA. LOD of Ultrabio Kit and GAG PCR was determined to be 4 and 25 copies/reaction, respectively. The Ultrabio Kit showed less variation when HIV-1 DNA concentration was lower than 20 copies/reaction. Twentysix HIV-1 infected patients’ PBMCs samples were quantified by both methods and the Ultrabio kit showed higher sensitivity. GAG PCR was unable to detect 2 of the samples while Ultrabio Kit was able to detect all. Both 2 samples had undetectable viral load. The quantified HIV DNA copies/million cells of the 24 detected samples by both methods showed good correlation, r=0.91. The difference was within one log.

Abundant HIV-infected cells in blood and tissues are rapidly cleared upon ART initiation during acute HIV infection.

The timing and location of the establishment of the viral reservoir during acute HIV infection remain unclear. Using longitudinal blood and tissue samples obtained from HIV-infected individuals at the earliest stage of infection, we demonstrate that frequencies of infected cells reach maximal values in gut-associated lymphoid tissue and lymph nodes as early as Fiebig stage II, before seroconversion. Both tissues displayed higher frequencies of infected cells than blood until Fiebig stage III, after which infected cells were equally distributed in all compartments examined. Initiation of antiretroviral therapy (ART) at Fiebig stages I to III led to a profound decrease in the frequency of infected cells to nearly undetectable level in all compartments. The rare infected cells that persisted were preferentially found in the lymphoid tissues. Initiation of ART at later stages (Fiebig stages IV/V and chronic infection) induced only a modest reduction in the frequency of infected cells. Quantification of HIV DNA in memory CD4+ T cell subsets confirmed the unstable nature of most of the infected cells at Fiebig stages I to III and the emergence of persistently infected cells during the transition to Fiebig stage IV. Our results indicate that although a large pool of cells is infected during acute HIV infection, most of these early targets are rapidly cleared upon ART initiation. Therefore, infected cells present after peak viremia have a greater ability to persist.

Virological Outcome After Choice of Antiretroviral Regimen Guided by Proviral HIV-1 DNA Genotyping in a Real-Life Cohort of HIV-Infected Patients.

Issues have been raised concerning clinical relevance of HIV-1 proviral DNA genotypic resistance test (DNA GRT). To assess impact of DNA GRT on choice of antiretroviral therapy (ART) and subsequent virological outcome, we retrospectively reviewed decision-making and viral load (VL) evolution following DNA GRT performed in our center between January 2012 and December 2017, except those prescribed within the framework of a clinical trial. A total of 304 DNA GRTs were included, 185 (62%) performed in a context of virological success. Only 34% of tests were followed by ART change, more frequently in situation of virological success (39% vs. 26%, p = 0.02). In this situation, ART change guided by DNA GRT led to VL >20 copies/mL after 6 months in 5% of cases. In multivariate analysis, higher HIV DNA quantification (p = 0.01) was associated with occurrence of viremia. A higher nadir of CD4 count (p = 0.04) and a longer time with VL <20 copies/mL (p = 0.04) were independently associated with a lower risk of viremia. In situation of low-level viremia, ART change guided by DNA GRT led to VL <20 copies/mL after 6 months in 52% of cases, while decision to maintain the same treatment led to VL <20 copies/mL in 74% of cases. In multivariate analysis, longer time with VL >20 copies/mL (p = 0.02) was associated with persistence of virological replication. In conclusion, in situation of virological success, use of DNA GRT in addition to analysis of historical RNA GRT to guide ART optimization appears safe. Its prescription framework in situation of low-level viremia deserves to be better defined.

Proliferative memory SAMHD1low CD4+ T cells harbour high levels of HIV-1 with compartmentalized viral populations.

We previously reported the presence of memory CD4+ T cells that express low levels of SAMHD1 (SAMHD1low) in peripheral blood and lymph nodes from both HIV-1 infected and uninfected individuals. These cells are enriched in Th17 and Tfh subsets, two populations known to be preferentially targeted by HIV-1. Here we investigated whether SAMHD1low CD4+ T-cells harbour replication-competent virus and compartimentalized HIV-1 genomes. We sorted memory CD4+CD45RO+SAMHD1low, CD4+CD45RO+SAMHD1+ and naive CD4+CD45RO-SAMHD1+ cells from HIV-1-infected patients on anti-retroviral therapy (c-ART) and performed HIV-1 DNA quantification, ultra-deep-sequencing of partial env (C2/V3) sequences and phenotypic characterization of the cells. We show that SAMHD1low cells include novel Th17 CCR6+ subsets that lack CXCR3 and CCR4 (CCR6+DN). There is a decrease of the % of Th17 in SAMHD1low compartment in infected compared to uninfected individuals (41% vs 55%, p<0.05), whereas the % of CCR6+DN increases (7.95% vs 3.8%, p<0.05). Moreover, in HIV-1 infected patients, memory SAMHD1low cells harbour high levels of HIV-1 DNA compared to memory SAMHD1+ cells (4.5 vs 3.8 log/106cells, respectively, p<0.001), while naïve SAMHD1+ showed significantly lower levels (3.1 log/106cells, p<0.0001). Importantly, we show that SAMHD1low cells contain p24-producing cells. Moreover, phylogenetic analyses revealed well-segregated HIV-1 DNA populations with compartmentalization between SAMHD1low and SAMHD1+ memory cells, and limited viral exchange. As expected, the % of Ki67+ cells was significantly higher in SAMHD1low compared to SAMHD1+ cells. There was positive association between levels of HIV-1 DNA and Ki67+ in memory SAMHD1low cells, but not in memory and naïve SAMHD1+ CD4+ T-cells. Altogether, these data suggest that proliferative memory SAMHD1low cells contribute to viral persistence.

Quantification of HIV-DNA and residual viremia in patients starting ART by droplet digital PCR: Their dynamic decay and correlations with immunological parameters and virological success

BackgroundAccurate quantification of total HIV-DNA and residual-viremia by sensitive assays is extremely useful to optimize monitoring of ART-treated patients. ObjectivesTo evaluate the performances of two ddPCR-based assays for HIV-DNA and residual-viremia quantification, and the correlations of pre-ART HIV-DNA with plasma HIV-RNA, CD4 + T, CD4/CD8 and virological-success (VS) during first-line ART. Study designPlasma HIV-RNA, total HIV-DNA, CD4 + T, CD4/CD8 were evaluated at baseline of ART, at VS (viral-load <50copies/ml), and at 6 months after VS (6moVS) in 57 newly-diagnosed HIV-1 infected patients, receiving first-line modern ART. HIV-DNA (log10 copies/106CD4 + T) and residual-viremia (copies/ml) were measured with in-house ddPCR assays. Correlations were assessed by Spearman and Jonckheere-Terpstra tests. ResultsHIV-DNA and residual-viremia assays showed a good linear trend between the expected and obtained values (R2 = 0.9913 and 0.9945); lower limits of detection were 32 copies/106CD4 + T and 2 copies/ml, respectively. At baseline, median (IQR) plasma HIV-RNA and HIV-DNA were 4.88(4.28–5.36)log10 copies/ml and 4.00(3.36–4.51) log10 copies/106CD4 + T cells. Residual-viremia was 8(2–26) and 4(2–12) copies/ml at VS and 6moVS. Pre-ART HIV-DNA positively correlated with plasma HIV-RNA at BL (Rho = 0.708, p < 0.001), and with residual-viremia at VS (Rho:0.383,p = 0.002). Notably, higher HIV-DNA correlated with longer time to achieve VS (median[IQR],weeks: 17.8[12.3–29.0] for HIV-DNA ≥4.5 vs. 7.4[4.1–8.7] for HIV-DNA<4.5, p < 0.001). Furthermore, pre-ART HIV-DNA negatively correlated with CD4 + T and CD4/CD8 at baseline, VS and 6moVS. ConclusionsOur results support the adoption of ddPCR-based assays for both HIV-DNA and residual-viremia quantifications and corroborate that pre-ART HIV-DNA is an excellent indicator in predicting viroimmunological response and VS in patients starting ART.

In-depth validation of total HIV-1 DNA assays for quantification of various HIV-1 subtypes

HIV-1 DNA quantification serves as an important reservoir biomarker in HIV cure trials. However, the high genetic diversity of HIV-1 represented by different subtypes may bring inaccuracy in quantifying HIV-1 DNA and a sensitive and validated assay covering diverse HIV-1 subtypes is lacking. Therefore, we cross-validated total HIV-1 DNA assays described in literature using a three-step comparative analysis. First, a bioinformatics tool was developed in-house to perform an in silico evaluation of 67 HIV-1 DNA assays. Secondly, these selected assays were in vitro validated using a panel of different HIV-1 subtypes and, finally, ex vivo assessed on selected patient samples with different HIV-1 subtypes. Our results show that quantification of HIV-1 DNA substantially differs between assays and we advise five best performing HIV-1 DNA assays for ddPCR and qPCR (Schvachsa_2007, Viard_2004, Heeregrave_2009, Van_der_Sluis_2013, Yu_2008 and Yun_2002). This in-depth analysis of published HIV-1 DNA assays indicates that not all assays guarantee an optimal measurement of HIV-1 DNA, especially when looking across subtypes. Using an in-depth cross-validation, we were able to validate HIV-1 DNA assays that are suitable for quantification of HIV-1 DNA in a wide variety of HIV-1 infected patients.

Comparative analysis and generation of a robust HIV-1 DNA quantification assay

HIV-1 infection cannot be cured due to the presence of the latent reservoir (LR). Novel cure or treatment strategies, such as “shock and kill” or therapeutic vaccination, aim to reduce or eradicate the LR. Cure strategies utilise robust DNA quantification assays to measure the change in the LR in low copy scenarios. No standard assay exists, which impedes the reliable comparison of results from different therapy and vaccine trials and HIV-1 total DNA quantification methods have not been previously compared. The HIV-1 long terminal repeat (LTR) has been shown to be the best target for DNA quantification. We have analysed two HIV-1 quantification assays, both able to differentiate between the variant HIV-1 DNA forms via the use of pre-amplification and primers targeting LTR. We identify a strong correlation (r=0.9759, P<0.0001) between assays which is conserved in low copy samples (r=0.8220, P<0.0001) indicating that these assays may be used interchangeably. The RvS assay performed significantly (P=0.0021) better than the CV assay when quantifying HIV-1 total DNA in patient CD4+ T lymphocytes. Sequence analysis demonstrated that viral diversity can limit DNA quantification, however in silico analysis of the primers indicated that within the target region nucleotide miss-matches appear infrequently. Further in silico analysis using up to-date sequence information led to the improvement of primers and enabled us to establish a more broadly specific assay with significantly higher HIV-1 DNA quantification capacity in patient samples (p=0.0057, n=17).

Quantification of HIV DNA Using Droplet Digital PCR Techniques.

HIV persists, despite effective antiretroviral therapy, in long-lived cells, posing a major barrier toward a cure. A key step in the HIV replication cycle and a hallmark of the Retroviridae family is the integration of the viral DNA into the host genome. Once integrated, HIV expression is regulated by host machinery and the provirus persists until the cell dies. A reservoir of cells harboring replication-competent proviruses can survive for years, and mechanisms that maintain that reservoir are under investigation. The majority of integrated proviruses, however, are defective or have large deletions, and the composition of the proviral landscape during therapy remains unknown. Methods to quantify HIV proviruses are useful in investigating HIV persistence. Presented in this unit is a method for total HIV DNA quantification of various HIV genome targets that utilizes the next-generation PCR platform, digital PCR. The abundance of various HIV gene targets reflects the overall proviral composition. In this protocol, total genomic DNA is isolated from patient-derived cells and then used as a template for droplet digital PCR, in which the PCR reaction is partitioned into approximately 20,000 individual droplets, PCR amplified to an end point, and subjected to absolute quantification by counting the number of positive and negative droplets. Copy number is directly calculated using straightforward Poisson correction. Additionally, this methodological approach can be used to obtain absolute quantification of other DNA targets. © 2018 by John Wiley & Sons, Inc.

Development of sensitive ddPCR assays to reliably quantify the proviral DNA reservoir in all common circulating HIV subtypes and recombinant forms.

IntroductionThe latent reservoir is the main barrier on the road to HIV cure, and clinical approaches towards eradication are often evaluated by their effect on proviral DNA. To ensure inclusiveness and representativeness in HIV cure studies, proviral DNA quantification assays that are able to detect all common circulating HIV clades are urgently needed. Here, three HIV DNA assays targeting three different genomic regions were evaluated for their sensitivity and subtype‐tolerance using digital PCR.MethodsA subtype‐B‐specific assay targeting gag (GAG) and two assays targeting conserved sequences in ltr and pol (LTR and JO) were assessed for their sensitivity and subtype‐tolerance in digital PCR (Bio‐Rad QX200), using a panel of serially diluted subtype reference plasmids as well as a panel of clinical isolates. Both panels represent subtypes A, B, C, D, F, G and circulating recombinant forms (CRFs) AE and AG, which together are responsible for 94% of HIV infections worldwide.Results HIV subtype was observed to greatly affect HIV DNA quantification results. Robust regression analysis of the serially diluted plasmid panel showed that the GAG assay was only able to linearly quantify subtype B, D and G isolates (4/13 reference plasmids, average R 2 = 0.99), whereas LTR and JO were able to quantify all tested isolates (13/13 reference plasmids, respective average R 2 = 0.99 and 0.98). In the clinical isolates panel, isolates were considered detectable if all replicates produced a positive result. The GAG assay could detect HIV DNA in four out of five subtype B and one out of two subtype D isolates, whereas the LTR and JO assays detected HIV DNA in all twenty‐nine tested isolates. LTR and JO results were found to be equally precise but more precise than GAG.ConclusionsThe results demonstrate the need for a careful validation of proviral reservoir quantification assays prior to investigations into non‐B subtype reservoirs. The LTR and JO assays can sensitively and reliably quantify HIV DNA in a panel that represents the worldwide most prevalent subtypes and CRFs (A, B, C, D, AE, F, G and AG), justifying their application in future trials aimed at global HIV cure.

Quantification of total HIV-1 DNA in buffy coat cells, feasibility and potential added value for clinical follow-up of HIV-1 infected patients on ART

BackgroundSuccessfully treated HIV-1 infected patients have a sustained undetectable viral RNA load. In these cases the total HIV-1 DNA load may constitute a valuable tool to further follow the overall viral burden. The value of this marker outside of cure research has been rarely studied. ObjectivesTo develop a quantitative (q)PCR for total HIV-1 DNA quantification in buffy coat cells and to evaluate the value of this parameter in clinical follow-up. Study designA qPCR using primers and a probe in the conserved HIV-1 LTR region was adapted for use on DNA extracted from buffy coat cells. Sensitivity, accuracy and reproducibility were evaluated using 8E5 cells and samples from naive and treatment experienced patients. The clinical value of DNA load analysis was assessed by testing 119 longitudinal samples from 9 patients before and after ART initiation and 249 cross sectional samples from therapy-experienced patients. ResultsInter- and intra-assay coefficients of variability were 5.56 and 5.94 (%CV). HIV-1 DNA was detected in 249 of the 263 (94.7%) patients on ART for at least 5 months (median: 53 months; IQR: 28–84 months). The HIV-1 DNA load varied between 0.60 and 3.37 copies/106 blood cells and showed significant correlation with the pre-ART CD4+ T-cell count nadir and peak viral RNA load. ART initiation resulted in a slow and limited decline of the total HIV-1 DNA concentration. ConclusionsQuantification of total HIV-1 DNA from buffy coat cells is feasible, sensitive and reliable. Although determination of the on-therapy HIV-1 DNA load may be informative, regular testing has limited clinical value because of the very slow evolution.

Exploring viral reservoir: The combining approach of cell sorting and droplet digital PCR.

Combined antiretroviral therapy (cART) blocks different steps of HIV replication and maintains plasma viral RNA at undetectable levels. The virus can remain in long-living cells and create a reservoir where HIV can restart replicating after cART discontinuation. A persistent viral production triggers and maintains a persistent immune activation, which is a well-known feature of chronic HIV infection, and contributes either to precocious aging, or to the increased incidence of morbidity and mortality of HIV positive patients. The new frontier of the treatment of HIV infection is nowadays eradication of the virus from all host cells and tissues. For this reason, it is crucial to have a clear and precise idea of where the virus hides, and which are the cells that keep it silent. Important efforts have been made to improve the detection of viral reservoirs, and new techniques are now giving the opportunity to characterize viral reservoirs. Among these techniques, a strategic approach based upon cell sorting and droplet digital PCR (ddPCR) is opening new horizons and opportunities of research. This review provides an overview of the methods that combine cell sorting and ddPCR for the quantification of HIV DNA in different cell types, and for the detection of its maintenance.

Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.

The most immediate and evident effect of mucosal exposure to semen in vivo is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and IL1A, CSF2, IL7, PTGS2, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site.