Detecting Sources of Immune Activation and Viral Rebound in HIV Infection

Stephen W. Wietgrefe, Lijie Duan, Jodi Anderson, Guillermo Marques, Mark Sanders, Nathan W. Cummins, Andrew D. Badley, Curtis Dobrowolski, Jonathan Karn, Amelie Pagliuzza, Nicolas Chomont, Geremy Sannier, Mathieu Dube, Daniel E. Kaufmann, Paul Zuck, Guoxin Wu, Bonnie J. Howell, Cavan Reilly, Alon Herschhorn, Timothy W. SchackerAshley T. Haase

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA +/p24 + cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

Original languageEnglish (US)
JournalJournal of virology
Issue number15
StatePublished - Aug 2022

Bibliographical note

Funding Information:
We thank the U.S. National Institutes of Health and the Canadian Institutes of Health Research for support (NIH R01 AI110173, R01 AI120698, R21 AI136731, R01 AI134406, and R56 AI145407; CIHR 152977 and 364408) as well as Colleen O’Neill for assistance with the preparation of the manuscript. D.E.K is a FRQS Merit Research Scholar. N.C. is supported by Research Scholar Career Awards of the Quebec Health Research Fund (FRQS no. 253292).

Publisher Copyright:
© 2022 American Society for Microbiology. All rights reserved.


  • HIV
  • immune activation
  • viral rebound
  • Immunoassay
  • Reproducibility of Results
  • HIV Core Protein p24/genetics
  • Antigens, Viral/analysis
  • Humans
  • In Situ Hybridization, Fluorescence
  • HIV Infections/immunology
  • Anti-HIV Agents/administration & dosage
  • HIV-1/genetics
  • Sensitivity and Specificity
  • env Gene Products, Human Immunodeficiency Virus/genetics
  • RNA, Messenger/analysis
  • CD4-Positive T-Lymphocytes
  • Viral Tropism
  • RNA, Viral/analysis
  • Virus Activation

PubMed: MeSH publication types

  • Journal Article


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