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We utilized pulsed-field gel electrophoresis (PFGE) to purify high-molecular-weight DNA from HIV-infected cells. This purification, in combination with our previously described droplet digital PCR (ddPCR) assay, was used to develop a method to quantify proviral integrated HIV DNA free of lower-molecular-weight species of HIV DNA. Episomal 2-long-terminal-repeat (2-LTR) circles were completely cleared from HIV DNA samples. Technical replicates of the complete assay, starting with the same specimens, resulted in no statistical differences in quantification of integrated HIV gag sequences in cellular DNA from cells from HIV-infected subjects after prolonged treatment with antiretroviral therapy (ART). The PFGE ddPCR assay was compared to the Alu-gag quantitative PCR (qPCR) assay, the most widely used assay to measure proviral integrated HIV DNA. Spearman's rho nonparametric correlation determined PFGE ddPCR results to be positively correlated with Alu-gag qPCR results (r = 0.7052; P = 0.0273). In summary, PFGE ddPCR is a sensitive, reproducible, and robust method to measure proviral integrated HIV DNA and is theoretically more accurate than previously described assays, because it is a direct measure of integrated HIV DNA.

HIV-1 can downregulate HLA-C on infected cells, using the viral protein Vpu, and the magnitude of this downregulation varies widely between primary HIV-1 variants. The selection pressures that result in viral downregulation of HLA-C in some individuals, but preservation of surface HLA-C in others are not clear. To better understand viral immune evasion targeting HLA-C, we have characterized HLA-C downregulation by a range of primary HIV-1 viruses. 128 replication competent viral isolates from 19 individuals with effective anti-retroviral therapy, show that a substantial minority of individuals harbor latent reservoir virus which strongly downregulates HLA-C. Untreated infections display no change in HLA-C downregulation during the first 6 months of infection, but variation between viral quasispecies can be detected in chronic infection. Vpu molecules cloned from plasma of 195 treatment naïve individuals in chronic infection demonstrate that downregulation of HLA-C adapts to host HLA genotype. HLA-C alleles differ in the pressure they exert for downregulation, and individuals with higher levels of HLA-C expression favor greater viral downregulation of HLA-C. Studies of primary and mutant molecules identify 5 residues in the transmembrane region of Vpu, and 4 residues in the transmembrane domain of HLA-C, which determine interactions between Vpu and HLA. The observed adaptation of Vpu-mediated downregulation to host genotype indicates that HLA-C alleles differ in likelihood of mediating a CTL response that is subverted by viral downregulation, and that preservation of HLA-C expression is favored in the absence of these responses. Finding that latent reservoir viruses can downregulate HLA-C could have implications for HIV-1 cure therapy approaches in some individuals.

Analytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group ATI studies were assayed for integrated HIV DNA levels. Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV integrated DNA size-exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu-gag assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in the levels of integrated HIV DNA between the pre- and post-ATI time points, with a median ratio of post- to pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI, followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on the levels of integrated proviral DNA in the peripheral blood.IMPORTANCE Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated HIV DNA and is less sample- and labor-intensive than current assays. Using stored samples from AIDS Clinical Trials Group studies, we found that short-term ART discontinuation had minimal impact on integrated HIV DNA levels after ART resumption, providing reassurance about the reservoir effects of short-term treatment interruption trials.

The dynamics of HIV reservoir accumulation off antiretroviral therapy (ART) is underexplored. Levels of integrated HIV DNA in peripheral blood mononuclear cells (PBMCs) were longitudinally monitored before and after antiviral therapy. HIV integration increased over time in both elite controllers (ECs; n = 8) and noncontrollers (NCs; n = 6) before ART, whereas integration remained stable in patients on ART (n = 4). The median annual fold change was higher in NCs than in ECs and negatively correlated with CD4/CD8 T-cell ratio. Cytotoxic T lymphocyte (CTL) function as assessed by infected CD4 T-cell elimination (ICE) and granzyme B activity did not significantly change over time in ECs, suggesting that the gradual increase in integrated HIV DNA observed in ECs was not a result of progressive loss of immune-mediated control. Also, acutely infected (n = 7) but not chronically infected (n = 6) patients exhibited a significant drop in integrated HIV DNA 12 months after ART initiation. In conclusion, in the absence of ART, integrated HIV accumulates over time both in NCs and in ECs, at variable individual rates. Starting ART early in infection leads to a greater drop in integrated HIV DNA than does initiating treatment after years of infection. The increase in integrated HIV DNA over time suggests that early treatment may be of benefit in limiting HIV reservoirs.ImportanceThe establishment of a latent reservoir represents a barrier to cure among HIV-infected individuals. The dynamics of HIV reservoir accumulation over time in patients before antiviral therapy is underexplored, in large part because it is difficult to accurately and reproducibly measure the size of HIV reservoir in this setting. In our study, we compared the dynamics of integrated HIV DNA over time in ECs and NCs before and after ART was initiated. We found that integrated HIV DNA levels progressively increase over time in the absence of ART, but with a higher, albeit variable, rate in NCs compared to ECs. In addition, integrated HIV DNA declines more dramatically when ART is initiated in acute rather than chronic HIV infection, suggesting important differences between acute and chronic infection. Our study highlights the role of HIV replication and CTL control in reservoir accumulation in sanctuary sites and why ART appears to be more effective in acute infection.

BackgroundA major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir"). We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART).MethodsWe evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS) assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR); integrated DNA (Alu PCR); unspliced RNA (rtPCR), multiply-spliced RNA (TILDA), residual plasma HIV RNA (single copy PCR), and replication-competent virus (outgrowth assay). We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR). Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables.ResultsAmong 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years), the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004) and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003). However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P

BackgroundQuantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging.MethodsWe developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.FindingsIn suppressed individuals on ART, we found the median frequency of latently infected CD4 + T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4 + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART.InterpretationsOur results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation.Research in contextIn this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

Since the first HIV-cured person was reported in 2009, a strong interest in developing highly sensitive HIV and SIV reservoir assays has emerged. In particular, the question arose about the comparative value of state-of-the-art assays to measure and characterize the HIV reservoir, and how these assays can be applied to accurately detect changes in the reservoir during efforts to develop a cure for HIV infection. Second, it is important to consider the impact on the outcome of clinical trials if these relatively new HIV reservoir assays are incorporated into clinical trial endpoints and/or used for clinical decision-making. To understand the advantages and limitations and the regulatory implications of HIV reservoir assays, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored and convened a meeting on September 16, 2022, to discuss the state of knowledge concerning these questions and best practices for selecting HIV reservoir assays for a particular research question or clinical trial protocol. [ABSTRACT FROM AUTHOR]

Background: Rapid analytics to predict circulating hematopoietic stem cells are valuable for optimal management of mobilization, particularly for the use of newer and costly mobilization agents such as plerixafor.Study Design and Methods: We used stepwise, linear multiple regression modeling applied to cell population data collected by routine hematology analyzers (Beckman Coulter DxH 800) on patients undergoing autologous stem cell collection (n = 131). Beta coefficients were used to derive a formula for a stem cell index (SCI). We then tested the correlation of SCI with stem cell counts and performance of the SCI as a predictor of poor mobilization with external validation in a separate cohort (n = 183).Results: The SCI correlated strongly with CD34 counts by flow cytometry (r = 0.8372 in the development cohort, r = 0.8332 in the external validation cohort) and compares favorably with other rapid stem cell enumerating technologies. In the external validation cohort, the SCI performed well as a predictor (receiver operating characteristic area under the curve, 0.9336) of poor mobilization (CD34 count < 10), with a sensitivity of 72% and a specificity of 93%. When prevalence of poor mobilization was 33%, this resulted in a positive predictive value of 83% and a negative predictive value of 87%. The SCI also showed promise in tracking responses to plerixafor administration.Conclusion: The findings demonstrate the utility of the cell population data collected by hematology analyzers to provide rapid data beyond standard complete blood counts, particularly for stem cell count prediction, requiring no additional reagents, specimen, or instrumentation. [ABSTRACT FROM AUTHOR]

Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5'LTR-to-3'LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of "defective" proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not "silent," but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins. [ABSTRACT FROM AUTHOR]

The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1) in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4+ and CD8+ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies. [ABSTRACT FROM AUTHOR]

Next-generation sequencing (NGS) represents a powerful tool to unravel the genetic make-up of the HIV reservoir but limited data exist on its use in vitro. Moreover, most NGS studies do not separate integrated from unintegrated DNA even though selection pressures on these two forms should be distinct. We reasoned we could use NGS to compare infection of resting and activated CD4 T cells in vitro to address how the metabolic state affects reservoir formation and dynamics. To address these questions we obtained HIV sequences at 2, 4 and 8 days after NL4-3 infection of metabolically activated and quiescent CD4 T cells (cultured with 2 ng/mL IL-7). We compared the composition of integrated and total HIV DNA by isolating integrated HIV DNA using pulsed-field electrophoresis before performing sequencing. After a single-round infection the majority of integrated HIV DNA was intact in both resting and activated T cells. The decay of integrated intact proviruses was rapid and similar in both quiescent and activated T cells. Defective forms accumulated relative to intact ones analogously to what is observed in vivo. Massively deleted viral sequences formed more frequently in resting cells likely due to lower deoxynucleoside triphosphate (dNTP) levels and the presence of multiple restriction factors. To our surprise, the majority of these deleted sequences did not integrate into the human genome. The use of NGS to study reservoir dynamics in vitro provides a model that recapitulates important aspects of reservoir dynamics. Moreover, separating integrated from unintegrated HIV DNA is important in some clinical settings to properly study selection pressures. [ABSTRACT FROM AUTHOR]