Una O’Doherty

Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR.

In this review, we compare four assays that are currently used to measure HIV integration and discuss their strengths and weaknesses. We then outline advances that have been made toward development of a more robust, more sensitive, quantitative HIV integration assay suitable for clinical use. The assay that we have developed uses repetitive-sampling Alu-gag PCR. The detailed protocol describes our assay step-by-step, the creation of an integration standard cell line and accompanying standard curve, as well as the quantitation of integration and calculation of associated error estimates. Finally, we speculate on fundamental, unresolved issues in HIV latency that can be addressed by measuring HIV integration.

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Significance The presence of “defective” HIV-1 proviruses in HIV-infected patients has been well documented. The current consensus view of the “defective” proviruses is that these are dead-end products that do not give rise to progeny virus and thus collectively represent a “graveyard” of viruses. We describe the presence of defective HIV-1 proviruses capable of transcribing novel unspliced HIV-RNA species in HIV-infected patients on combination antiretroviral therapy. We propose that the proviruses persistently present in combination antiretroviral therapy-treated patients are not defective in a conventional sense, but rather represent incomplete forms of proviruses encoding translationally competent HIV-RNA transcripts. Strategies directed toward curing HIV-1 infection and eliminating the state of persistent immune activation need to include approaches designed to eliminate cells harboring such proviruses. 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.

HIV latency and integration site placement in five cell-based models

BackgroundHIV infection can be treated effectively with antiretroviral agents, but the persistence of a latent reservoir of integrated proviruses prevents eradication of HIV from infected individuals. The chromosomal environment of integrated proviruses has been proposed to influence HIV latency, but the determinants of transcriptional repression have not been fully clarified, and it is unclear whether the same molecular mechanisms drive latency in different cell culture models.ResultsHere we compare data from five different in vitro models of latency based on primary human T cells or a T cell line. Cells were infected in vitro and separated into fractions containing proviruses that were either expressed or silent/inducible, and integration site populations sequenced from each. We compared the locations of 6,252 expressed proviruses to those of 6,184 silent/inducible proviruses with respect to 140 forms of genomic annotation, many analyzed over chromosomal intervals of multiple lengths. A regularized logistic regression model linking proviral expression status to genomic features revealed no predictors of latency that performed better than chance, though several genomic features were significantly associated with proviral expression in individual models. Proviruses in the same chromosomal region did tend to share the same expressed or silent/inducible status if they were from the same cell culture model, but not if they were from different models.ConclusionsThe silent/inducible phenotype appears to be associated with chromosomal position, but the molecular basis is not fully clarified and may differ among in vitro models of latency.

Concurrent Measures Of Total And Integrated HIV DNA Monitor Reservoirs And Ongoing Replication In Eradication Trials

Objectives:Interest in targeting HIV reservoirs is fueling trials that may decrease reservoir size and/or induce viral replication. Therefore, we aimed to develop strategies to sensitively measure changes in these parameters in patients on and off antiretroviral therapy (ART). Achieving these goals may help evaluate the effects of future clinical trials. Design:To determine the relationship between measurements of total and integrated HIV DNA and their role as markers of reservoir size and ongoing replication, these parameters were measured during the first year of ART, during long-term effective ART, and during a clinical trial aimed at targeting reservoirs. Methods:Total and integrated HIV DNA were measured in patient samples using quantitative PCR techniques. CD4+T cell counts and plasma viremia were also monitored. Results:Unintegrated HIV DNA became undetectable during the first year of ART. Total and integrated HIV DNA levels were generally equal in well controlled patients on ART, and low-level plasma viremia correlated best with integration measures. Finally, patients who controlled plasma viremia (<400 copies/ml) during interferon-&agr; monotherapy exhibited a decrease in the level of integrated but not total HIV DNA and a rise in the ratio of total to integrated HIV DNA over time. Conclusion:Our findings suggest that appearance of unintegrated HIV DNA reflects residual HIV expression and de-novo reverse transcription, providing insight into the mechanism by which interferon-&agr; reduces the HIV reservoir. We conclude that concurrent measurements of total and integrated HIV DNA provide information regarding reservoir size and ongoing replication in trials targeting HIV.

Gag-Positive Reservoir Cells Are Susceptible to HIV-Specific Cytotoxic T Lymphocyte Mediated Clearance In Vitro and Can Be Detected In Vivo

Resting CD4+ T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+ T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+ T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+ T cells expressed HIV Gag and were cleared by autologous CD8+ T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+ T cells exists in vivo in patients well controlled on therapy.

Anti-HIV Antibody Responses and the HIV Reservoir Size during Antiretroviral Therapy

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

Clinical use of lentiviral vectors

Viral vectors provide an efficient means for modification of eukaryotic cells, and their use is now commonplace in academic laboratories and industry for both research and clinical gene therapy applications. Lentiviral vectors, derived from the human immunodeficiency virus, have been extensively investigated and optimized over the past two decades. Third-generation, self-inactivating lentiviral vectors have recently been used in multiple clinical trials to introduce genes into hematopoietic stem cells to correct primary immunodeficiencies and hemoglobinopathies. These vectors have also been used to introduce genes into mature T cells to generate immunity to cancer through the delivery of chimeric antigen receptors (CARs) or cloned T-cell receptors. CAR T-cell therapies engineered using lentiviral vectors have demonstrated noteworthy clinical success in patients with B-cell malignancies leading to regulatory approval of the first genetically engineered cellular therapy using lentiviral vectors. In this review, we discuss several aspects of lentiviral vectors that will be of interest to clinicians, including an overview of lentiviral vector development, the current uses of viral vectors as therapy for primary immunodeficiencies and cancers, large-scale manufacturing of lentiviral vectors, and long-term follow-up of patients treated with gene therapy products.

Measuring integrated HIV DNA ex vivo and in vitro provides insights about how reservoirs are formed and maintained

The identification of the most appropriate marker to measure reservoir size has been a great challenge for the HIV field. Quantitative viral outgrowth assay (QVOA), the reference standard to quantify the amount of replication-competent virus, has several limitations, as it is laborious, expensive, and unable to robustly reactivate every single integrated provirus. PCR-based assays have been developed as an easier, cheaper and less error-prone alternative to QVOA, but also have limitations. Historically, measuring integrated HIV DNA has provided insights about how reservoirs are formed and maintained. In the 1990s, measuring integrated HIV DNA was instrumental in understanding that a subset of resting CD4 T cells containing integrated HIV DNA were the major source of replication-competent virus. Follow-up studies have further characterized the phenotype of these cells containing integrated HIV DNA, as well as shown the correlation between the integration levels and clinical parameters, such as duration of infection, CD4 count and viral load. Integrated HIV DNA correlates with total HIV measures and with QVOA. The integration assay has several limitations. First, it largely overestimates the reservoir size, as both defective and replication-competent proviruses are detected. Since defective proviruses are the majority in patients on ART, it follows that the number of proviruses capable of reactivating and releasing new virions is significantly smaller than the number of integrated proviruses. Second, in patients on ART clonal expansion could theoretically lead to the preferential amplification of proviruses close to an Alu sequence though longitudinal studies have not captured this effect. Proviral sequencing combined with integration measures is probably the best estimate of reservoir size, but it is expensive, time-consuming and requires considerable bioinformatics expertise. All these reasons limit its use on a large scale. Herein, we review the utility of measuring HIV integration and suggest combining it with sequencing and total HIV measurements can provide insights that underlie reservoir maintenance.

HLA-C downregulation by HIV-1 adapts to host HLA genotype

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.