title: A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals.
abstract: BACKGROUND
  Quantifying 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  Our 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.
  
  
  BACKGROUND
  In 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.
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.
authors.forename: Francesco Andrea
authors.initials: FA
authors.lastname: Procopio
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.
authors.forename: Rémi
authors.initials: R
authors.lastname: Fromentin
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.
authors.forename: Deanna A
authors.initials: DA
authors.lastname: Kulpa
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Brehm
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.
authors.forename: Anne-Gaelle
authors.initials: AG
authors.lastname: Bebin
authors.affiliation: University of California San Diego, La Jolla, California and Veterans Affairs San Diego Healthcare System, San Diego, CA, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Strain
authors.affiliation: University of California San Diego, La Jolla, California and Veterans Affairs San Diego Healthcare System, San Diego, CA, USA.
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Richman
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors.forename: Una
authors.initials: U
authors.lastname: O'Doherty
authors.affiliation: Centre for Virus Research, Westmead Millennium Institute, Westmead, Australia ; Sydney Medical School, University of Sydney, Sydney, Australia.
authors.forename: Sarah
authors.initials: S
authors.lastname: Palmer
authors.affiliation: Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Frederick M
authors.initials: FM
authors.lastname: Hecht
authors.affiliation: Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Hoh
authors.affiliation: Infectious Disease, Merck Research Laboratories, West Point, PA, USA.
authors.forename: Richard J O
authors.initials: RJ
authors.lastname: Barnard
authors.affiliation: Infectious Disease, Merck Research Laboratories, West Point, PA, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Miller
authors.affiliation: Infectious Disease, Merck Research Laboratories, West Point, PA, USA.
authors.forename: Daria J
authors.initials: DJ
authors.lastname: Hazuda
authors.affiliation: Department of Medicine, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Deeks
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.
authors.forename: Rafick-Pierre
authors.initials: RP
authors.lastname: Sékaly
authors.affiliation: Vaccine and Gene Therapy Institute Florida, Port St. Lucie, FL, USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Chomont
mesh_terms: D000328:Adult; D044966:Anti-Retroviral Agents / Q000008:administration & dosage; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D004279:DNA, Viral / Q000097:blood; D005260:Female; D015658:HIV Infections / Q000097:blood* / Q000188:drug therapy; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D012367:RNA, Viral / Q000097:blood*; D017735:Virus Latency
chemical_list: D044966:Anti-Retroviral Agents; D004279:DNA, Viral; D012367:RNA, Viral
keywords: Eradication; HIV; Inducible virus; Latency; Multiply spliced RNA; Reservoir; TILDA