The Alphabet Soup of HIV Reservoir Markers
Purpose of Review
Despite the success of antiretroviral therapy in suppressing HIV, life-long therapy is required to avoid HIV reactivation from long-lived viral reservoirs. Currently, there is intense interest in searching for therapeutic interventions that can purge the viral reservoir to achieve complete remission in HIV patients off antiretroviral therapy. The evaluation of such interventions relies on our ability to accurately and precisely measure the true size of the viral reservoir. In this review, we assess the most commonly used HIV reservoir assays, as a clear understanding of the strengths and weaknesses of each is vital for the accurate interpretation of results and for the development of improved assays.
The quantification of intracellular or plasma HIV RNA or DNA levels remains the most commonly used tests for the characterization of the viral reservoir. While cost-effective and high-throughput, these assays are not able to differentiate between replication-competent or defective fractions or quantify the number of infected cells. Viral outgrowth assays provide a lower bound for the fraction of cells that can produce infectious virus, but these assays are laborious, expensive and substantially underestimate the potential reservoir of replication-competent provirus. Newer assays are now available that seek to overcome some of these problems, including full-length proviral sequencing, inducible HIV RNA assays, ultrasensitive p24 assays and murine adoptive transfer techniques.
The development and evaluation of strategies for HIV remission rely upon our ability to accurately and precisely quantify the size of the remaining viral reservoir. At this time, all current HIV reservoir assays have drawbacks such that combinations of assays are generally needed to gain a more comprehensive view of the viral reservoir. The development of novel, rapid, high-throughput assays that can sensitively quantify the levels of the replication-competent HIV reservoir is still needed.
KeywordsHIV Reservoir Assays QVOA Replication-competent Cure
Compliance with Ethical Standards
Conflict of Interest
JL has received research funding and consulted for Gilead and Merck. JL was funded in part by a grant from NIH/NIAID (125109).
Human and Animal Rights and Informed Consent
There were no human or animal experiments performed for the purpose of this review.
Papers of particular interest have been highlighted as: •• Of major importance
- 5.Sáez-Cirión A, Bacchus C, Hocqueloux L, Avettand-Fenoel V, Girault I, Lecuroux C, et al. Post-treatment HIV-1 controllers with a long-term virological remission after the interruption of early initiated antiretroviral therapy ANRS VISCONTI Study. PLoS Pathog. 2013;9:e1003211.CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Søgaard OS, Graversen ME, Leth S, Olesen R, Brinkmann CR, Nissen SK, et al. The depsipeptide romidepsin reverses HIV-1 latency in vivo. PLoS Pathog. 2015;11:e1005142.Google Scholar
- 8.Rasmussen TA, Tolstrup M, Brinkmann CR, Olesen R, Erikstrup C, Solomon A, et al. Panobinostat, a histone deacetylase inhibitor, for latent virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial. Lancet HIV. 2014;1:e13–21.CrossRefPubMedGoogle Scholar
- 13.Pasternak AO, Jurriaans S, Bakker M, Prins JM, Berkhout B, Lukashov V V. Cellular levels of HIV unspliced RNA from patients on combination antiretroviral therapy with undetectable plasma viremia predict the therapy outcome. PLoS One. 2009;4:e8490.Google Scholar
- 18.Klatt NR, Bosinger SE, Peck M, Richert-Spuhler LE, Heigele A, Gile JP, et al. Limited HIV infection of central memory and stem cell memory CD4+ T cells is associated with lack of progression in viremic individuals. PLoS Pathog. 2014;10:e1004345.Google Scholar
- 19.Nottet HSLM, van Dijk SJ, Fanoy EB, Goedegebuure IW, de Jong D, Vrisekoop N, et al. HIV-1 can persist in aged memory CD4+ T lymphocytes with minimal signs of evolution after 8.3 years of effective highly active antiretroviral therapy. J Acquir Immune Defic Syndr. 2009;50:345–53.CrossRefPubMedGoogle Scholar
- 28.•• Bruner KM, Murray AJ, Pollack RA, Soliman MG, Laskey SB, Capoferri AA, et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat Med. 2016;22:1043–9. Study comparing the proportion of intact and defective proviruses in HIV patients treated early vs. late after infection. Compared to the intact proviral assay, CA-DNA assays significantly overestimated the intact reservoir size while QVOA significantly underestimated the intact HIV reservoir size. CrossRefPubMedPubMedCentralGoogle Scholar
- 32.Iyer SR, Yu D, Biancotto A, Margolis LB, Wu Y. Measurement of human immunodeficiency virus type 1 preintegration transcription by using Rev-dependent Rev-CEM cells reveals a sizable transcribing DNA population comparable to that from proviral templates. J Virol. 2009;83:8662–73.CrossRefPubMedPubMedCentralGoogle Scholar
- 44.•• Deleage C, Wietgrefe SW, Del Prete G, Morcock DR, Hao XP, Piatak M, et al. Defining HIV and SIV reservoirs in lymphoid tissues. Pathog Immun. 2016;1:68–106. Study demonstrating the use of RNAscope and DNAscope to detect latently infected cells (vDNA+ vRNA-) and actively infected cells (vDNA+ vRNA+). CrossRefPubMedPubMedCentralGoogle Scholar
- 46.Ho Y-C, Shan L, Hosmane NN, Wang J, Laskey SB, Rosenbloom DIS, et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell Elsevier Inc. 2013;155:540–51.Google Scholar
- 48.Pasternak AO, Adema KW, Bakker M, Jurriaans S, Berkhout B, Cornelissen M, et al. Highly sensitive methods based on seminested real-time reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 unspliced and multiply spliced RNA and proviral DNA. J Clin Microbiol. 2008;46:2206–11.CrossRefPubMedPubMedCentralGoogle Scholar
- 52.Kearney MF, Wiegand A, Shao W, Coffin JM, Mellors JW, Lederman M, et al. Origin of rebound plasma HIV includes cells with identical proviruses that are transcriptionally active before stopping of antiretroviral therapy. J Virol. 2016; 90:1369–1376.Google Scholar
- 53.Martrus G, Niehrs A, Cornelis R, Rechtien A, García-Beltran W, Lütgehetmann M, et al. Kinetics of HIV-1 latency reversal quantified on the single cell level using a novel flow-based technique. J. Virol. 2016;90:9018–28.Google Scholar
- 54.Romerio F, Zapata J. Detection and enrichment to near purity of rare HIV-1 infected cells by PrimeFlow RNA. J. Virus Erad. 2015; Suppl 1:1–18 Oral Presentation 3.2.Google Scholar
- 56.Palmer S, Wiegand AP, Maldarelli F, Mican JM, Polis M, Dewar RL, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol. 2003;41:4561–36.Google Scholar
- 60.Leth S, Schleimann MH, Nissen SK, Højen JF, Olesen R, Graversen ME, et al. Combined effect of Vacc-4×, recombinant human granulocyte macrophage colony-stimulating factor vaccination, and romidepsin on the HIV-1 reservoir (REDUC): a single-arm, phase 1B/2A trial. Lancet HIV. 2016;3018:1–10.Google Scholar
- 70.•• Eriksson S, Graf EH, Dahl V, Strain MC, Yukl SA, Lysenko ES, et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog. 2013;9:e1003174.The authors compared 11 different approaches to measure the virus reservoir. Study shows that most assays do not correlate with QVOA results, except for the HIV-1 integrated DNA assay and HIV-1 RNA/DNA ratio in CD4 T cells from rectal tissue. Google Scholar
- 72.Wei DG, Chiang V, Fyne E, Balakrishnan M, Barnes T, Graupe M, et al. Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing. PLoS Pathog. 2014;10:e1004071.Google Scholar
- 77.Howell B, Wu G, Swanson M, Lu M, Graham G, Strizki J, et al. Developing and applying ultrasensitive p24 protein immunoassay for HIV lateny. J. Virus Erad. 2015; Suppl 1:1–18 Oral Presentation 3.1.Google Scholar
- 80.Mothe B, Climent N, Plana M, Rosàs M, Jiménez JL, Muñoz-Fernández MÁ, et al. Safety and immunogenicity of a modified vaccinia Ankara-based HIV-1 vaccine (MVA-B) in HIV-1-infected patients alone or in combination with a drug to reactivate latent HIV-1. J Antimicrob Chemother. 2015;70:1833–42.PubMedGoogle Scholar