skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA
Title: Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA
A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1 + adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/10 6 CD4 + T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/10 6 CD4 + T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.  more » « less
Award ID(s):
1738428
PAR ID:
10440064
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Proceedings of the National Academy of Sciences
Volume:
117
Issue:
31
ISSN:
0027-8424
Page Range / eLocation ID:
18692 to 18700
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, Nature Communications)
    Abstract The HIV reservoir consists of infected cells in which the HIV-1 genome persists as provirus despite effective antiretroviral therapy (ART). Studies exploring HIV cure therapies often measure intact proviral DNA levels, time to rebound after ART interruption, or ex vivo stimulation assays of latently infected cells. This study utilizes barcoded HIV to analyze the reservoir in humanized mice. Using bulk PCR and deep sequencing methodologies, we retrieve 890 viral RNA barcodes and 504 proviral barcodes linked to 15,305 integration sites at the single RNA or DNA molecule in vivo. We track viral genetic diversity throughout early infection, ART, and rebound. The proviral reservoir retains genetic diversity despite cellular clonal proliferation and viral seeding by rebounding virus. Non-proliferated cell clones are likely the result of elimination of proviruses associated with transcriptional activation and viremia. Elimination of proviruses associated with viremia is less prominent among proliferated cell clones. Proliferated, but not massively expanded, cell clones contribute to proviral expansion and viremia, suggesting they fuel viral persistence. This approach enables comprehensive assessment of viral levels, lineages, integration sites, clonal proliferation and proviral epigenetic patterns in vivo. These findings highlight complex reservoir dynamics and the role of proliferated cell clones in viral persistence. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; ; et al (, The Journal of Infectious Diseases)
    Abstract Background HIV-1 proviruses persist in people on antiretroviral therapy (ART) but most are defective and do not constitute a replication-competent reservoir. The decay of infected cells carrying intact compared with defective HIV-1 proviruses has not been well defined in people on ART. Methods We separately quantified intact and defective proviruses, residual plasma viremia, and markers of inflammation and activation in people on long-term ART. Results Among 40 participants tested longitudinally from a median of 7.1 years to 12 years after ART initiation, intact provirus levels declined significantly over time (median half-life, 7.1 years; 95% confidence interval [CI], 3.9–18), whereas defective provirus levels did not decrease. The median half-life of total HIV-1 DNA was 41.6 years (95% CI, 13.6–75). The proportion of all proviruses that were intact diminished over time on ART, from about 10% at the first on-ART time point to about 5% at the last. Intact provirus levels on ART correlated with total HIV-1 DNA and residual plasma viremia, but there was no evidence for associations between intact provirus levels and inflammation or immune activation. Conclusions Cells containing intact, replication-competent proviruses are selectively lost during suppressive ART. Defining the mechanisms involved should inform strategies to accelerate HIV-1 reservoir depletion. 
    more » « less
  3. ; ; (, Biosensors)
    The development of point-of-care, cost-effective, and easy-to-use assays for the accurate counting of CD4+ T cells remains an important focus for HIV-1 disease management. The CD4+ T cell count provides an indication regarding the overall success of HIV-1 treatments. The CD4+ T count information is equally important for both resource-constrained regions and areas with extensive resources. Hospitals and other allied facilities may be overwhelmed by epidemics or other disasters. An assay for a physician’s office or other home-based setting is becoming increasingly popular. We have developed a technology for the rapid quantification of CD4+ T cells. A double antibody selection process, utilizing anti-CD4 and anti-CD3 antibodies, is tested and provides a high specificity. The assay utilizes a microfluidic chip coated with the anti-CD3 antibody, having an improved antibody avidity. As a result of enhanced binding, a higher flow rate can be applied that enables an improved channel washing to reduce non-specific bindings. A wide-field optical imaging system is also developed that provides the rapid quantification of cells. The designed optical setup is portable and low-cost. An ImageJ-based program is developed for the automatic counting of CD4+ T cells. We have successfully isolated and counted CD4+ T cells with high specificity and efficiency greater than 90%. 
    more » « less
  4. ; (, Journal of life sciences)
    null (Ed.)
    Human immunodeficiency virus (HIV) preferentially infects T-lymphocytes by integrating into host DNA and forming a latent transcriptionally silent provirus. As previously shown, HIV-1 alters migration modes of T-lymphocytes by co-regulating viral gene expression with human C-X-C chemokine receptor-4 (CXCR4). Here, we show that motility of infected T-lymphocytes is cell size dependent. In cell migration assays, migrating cells are consistently larger than non-migrating cells. This effect is drug-treatment independent. The cell size dependent motility observed in a previously generated Jurkat latency model correlates with the motility of primary human CD4+ T-cells containing a modified HIV-1 full-length construct JLatd2GFP. In addition, large migrating T-cells, latently infected with HIV, show a slightly decreased rate of reactivation from latency. these results demonstrate that HIV reactivation is cell migration-dependent, where host cell size acts as a catalyst for altered migration velocity. We believe that host cell size controlled migration uncovers an additional mechanism of cellular controlled viral fate determination important for virus dissemination and reactivation from latency. This observation may provide more insights into viral-host interactions regulating cell migration and reactivation from latency and helps in the design and implementation of novel therapeutic strategies. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, Nature Communications)
    Abstract Despite major advances in HIV testing, ultrasensitive detection of early infection remains challenging, especially for the viral capsid protein p24, which is an early virological biomarker of HIV-1 infection. Here, To improve p24 detection in patients missed by immunological tests that dominate the diagnostics market, we show a click chemistry amplified nanopore (CAN) assay for ultrasensitive quantitative detection. This strategy achieves a 20.8 fM (0.5 pg/ml) limit of detection for HIV-1 p24 antigen in human serum, demonstrating 20~100-fold higher analytical sensitivity than nanocluster-based immunoassays and clinically used enzyme-linked immunosorbent assay, respectively. Clinical validation of the CAN assay in a pilot cohort shows p24 quantification at ultra-low concentration range and correlation with CD4 count and viral load. We believe that this strategy can improve the utility of p24 antigen in detecting early infection and monitoring HIV progression and treatment efficacy, and also can be readily modified to detect other infectious diseases. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email