Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth (original) (raw)
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HIV-1 DNA predicts disease progression and post-treatment virological control
eLife, 2014
In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.
Rapid Quantification of the Latent Reservoir for HIV-1 Using a Viral Outgrowth Assay
HIV-1 persists in infected individuals in a stable pool of resting CD4 + T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4 + T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4 + T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and costeffective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.
Cellular HIV-1 DNA quantification and short-term and long-term response to antiretroviral therapy
Journal of Antimicrobial Chemotherapy, 2011
The aim of our study was to determine whether HIV-1 DNA level before antiretroviral therapy (ART) was associated with short-and long-term virological and immunological responses. Methods: Patients starting first-line protease inhibitor-containing regimens were enrolled in a prospective multicentre cohort in 1998-99. HIV-1 DNA was quantified using real-time PCR at baseline and after 1 year of ART. The association between HIV-1 DNA and virological and immunological responses after 1 and 7 years on ART was studied in multivariate regression models along with other biological and clinical variables. Virological failure (VF) at month 12 (M12) was defined as a plasma HIV-1 RNA .500 copies/mL. Time to death or two plasma HIV-1 RNA .500 copies/mL between M12 and M84 was studied for long-term VF. Results: HIV-1 DNA levels were measured in 148 patients. The median baseline peripheral blood mononuclear cell (PBMC) HIV-1 DNA was 3.7 log 10 copies/10 6 PBMCs. At M12, the median PBMC HIV-1 DNA was 2.99 log 10 copies/10 6 PBMCs. The median decrease in PBMC HIV-1 DNA between M0 and M12 was 20.7 log 10 copies/ 10 6 PBMCs. Higher baseline PBMC HIV-1 DNA and plasma HIV-1 RNA were independently associated with a higher risk of VF at M12. Only the baseline plasma HIV-1 RNA was independently associated with long-term virological response. The baseline CD4 cell count was the only parameter associated with short-and longterm immunological responses. Conclusions: HIV-1 DNA impacted the virological response in our cohort. Further research is warranted to study the impact of HIV-1 DNA with currently recommended first-line cART.
Journal of Clinical Investigation
Proliferation of CD4 + T cells harboring HIV-1 proviruses is a major contributor to viral persistence in people on antiretroviral therapy (ART). To determine whether differential rates of clonal proliferation or HIV-1-specific CTL pressure shape the provirus landscape, we performed the intact proviral DNA assay (IPDA) and obtained 661 nearfull length provirus sequences from eight individuals with suppressed viral loads on ART at time points seven years apart. We observed slow decay of intact proviruses but no changes in the proportions of various types of defective proviruses. The proportion of intact proviruses in expanded clones was similar to that of defective proviruses in clones. Intact proviruses observed in clones did not have more escaped CTL epitopes than intact proviruses observed as singlets. Concordantly, total proviruses at later timepoints or observed in clones were not enriched in escaped or unrecognized epitopes. Three individuals with natural control of HIV-1 infection (controllers) on ART, included because controllers have strong HIV-1-specific CTL responses, had a smaller proportion of intact proviruses but a similar distribution of defective provirus types and escaped or unrecognized epitopes as the other individuals. This work suggests that CTL selection does not significantly check clonal proliferation of infected cells or greatly alter the provirus landscape in people on ART.