Abstract
Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIVRNA/Gag method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5–1 Gagpol mRNA+/Gag protein+ cells per million CD4 T cells. Uniquely, the HIVRNA/Gag assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.
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References
Margolis, D.M., Garcia, J.V., Hazuda, D.J. & Haynes, B.F. Latency reversal and viral clearance to cure HIV-1. Science 353, aaf6517 (2016).
Stephenson, K.E., D'Couto, H.T. & Barouch, D.H. New concepts in HIV-1 vaccine development. Curr. Opin. Immunol. 41, 39–46 (2016).
Lederman, M.M., Funderburg, N.T., Sekaly, R.-P., Klatt, N.R. & Hunt, P.W. Residual immune dysregulation syndrome in treated HIV infection. Adv. Immunol. 119, 51–83 (2013).
Klatt, N.R., Chomont, N., Douek, D.C. & Deeks, S.G. Immune activation and HIV persistence: implications for curative approaches to HIV infection. Immunol. Rev. 254, 326–342 (2013).
Lederman, M.M. et al. A cure for HIV infection: 'Not in My Lifetime' or 'Just Around the Corner'? Pathog. Immunol. 1, 154–164 (2016).
Deeks, S.G. HIV: shock and kill. Nature 487, 439–440 (2012).
Rasmussen, T.A. & Lewin, S.R. Shocking HIV out of hiding: where are we with clinical trials of latency reversing agents? Curr. Opin. HIV AIDS 11, 394–401 (2016).
Whitney, J.B. et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature 512, 74–77 (2014).
Ananworanich, J. et al. HIV DNA set point is rapidly established in acute HIV infection and dramatically reduced by early ART. EBioMedicine 11, 68–72 (2016).
Siliciano, J.D. et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells. Nat. Med. 9, 727–728 (2003).
Crooks, A.M. et al. Precise quantitation of the latent HIV-1 reservoir: implications for eradication strategies. J. Infect. Dis. 212, 1361–1365 (2015).
Finzi, D. et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278, 1295–1300 (1997).
Chun, T.W. et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. USA 94, 13193–13197 (1997).
Eriksson, S. et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog. 9, e1003174 (2013).
Baxter, A.E. et al. Single-cell characterization of viral translation-competent reservoirs in HIV-infected individuals. Cell Host Microbe 20, 368–380 (2016).
Sattentau, Q.J. & Stevenson, M. Macrophages and HIV-1: an unhealthy constellation. Cell Host Microbe 19, 304–310 (2016).
Chun, T.W. et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387, 183–188 (1997).
Vandergeeten, C. et al. Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies. J. Virol. 88, 12385–12396 (2014).
Laird, G.M. et al. Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay. PLoS Pathog. 9, e1003398 (2013).
Ho, Y.-C. et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 155, 540–551 (2013).
Bruner, K.M. et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat. Med. 22, 1043–1049 (2016).
Bruner, K.M., Hosmane, N.N. & Siliciano, R.F. Towards an HIV-1 cure: measuring the latent reservoir. Trends Microbiol. 23, 192–203 (2015).
Procopio, F.A. et al. A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals. EBioMedicine 2, 872–881 (2015).
Porichis, F. et al. High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat. Commun. 5, 5641 (2014).
DiNapoli, S.R., Hirsch, V.M. & Brenchley, J.M. Macrophages in progressive human immunodeficiency virus/simian immunodeficiency virus infections. J. Virol. 90, 7596–7606 (2016).
Jambo, K.C. et al. Small alveolar macrophages are infected preferentially by HIV and exhibit impaired phagocytic function. Mucosal Immunol. 7, 1116–1126 (2014).
Borzì, R.M. et al. A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA. J. Immunol. Methods 193, 167–176 (1996).
Wilburn, K.M. et al. Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses. Retrovirology 13, 55 (2016).
Chargin, A. et al. Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood. J. Clin. Microbiol. 53, 60–66 (2014).
Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P. & Roederer, M. Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1, 1522–1530 (2006).
Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P.K. & Roederer, M. Quality assurance for polychromatic flow cytometry using a suite of calibration beads. Nat. Protoc. 7, 2067–2079 (2012).
Sacha, J.B. & Watkins, D.I. Synchronous infection of SIV and HIV in vitro for virology, immunology and vaccine-related studies. Nat. Protoc. 5, 239–246 (2010).
Acknowledgements
We thank J. Girouard, the clinical staff at McGill University Health Centre and all study participants; D. Gauchat, the CRCHUM Flow Cytometry Platform, O. Debbeche, the CRCHUM BSL3 Platform, D. Zenklusen and C. Lai for technical assistance; and D. Malayter for technical support. This study was supported by the National Institutes of Health (HL-092565, AI100663 CHAVI-ID, AI113096, AI118544), the Delaney AIDS Research Enterprise (DARE; 1U19AI096109), the Canadian Institutes for Health Research (137694; Canadian HIV Cure Enterprise), a Canada Foundation for Innovation grant, the FRQS AIDS and Infectious Diseases Network and the Foundation for AIDS Research (108928-56-RGRL). D.E.K. and N.C. are supported by FRQS Research Scholar Awards. A.F. is the recipient of a Canada Research Chair. J.-P.R. is the holder of the Louis Lowenstein Chair, McGill University. A.E.B. is the recipient of a CIHR Fellowship (award no. 152536). J.N. is the recipient of a scholarship from the Bavarian Research Alliance (BayFor). J.R. is the recipient of CIHR Fellowship Award no. 135349. N.A. is the recipient of a King Abdullah scholarship from the Saudi government.
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A.E.B., F.P. and D.E.K. conceived and developed the HIVRNA/Gag assay, with input from A.F. and N.C.; A.E.B., J.N., R.F., J.R., N.B., M.M. and N.A. modified the protocol, designed specific experiments and provided reagents; J.-P.R. obtained IRB approval and recruited participants to provide primary samples; D.E.K. provided supervision; A.E.B. and D.E.K. wrote the manuscript and all authors approved the final version.
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Supplementary Figure 1 Example gating strategy.
Cells are gated as lymphocytes (a), single cells (b), live cells (c), exclusion channel negative (d), CD3+ T cells (e). Note that some latency reversing agents may cause downregulation of CD3/CD4, therefore depending on the experimental design the final gate may be excluded. Samples were acquired on a modified 5-laser BD LSRII and analyzed using FlowJo Versions 9 and 10 for Mac.
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Baxter, A., Niessl, J., Fromentin, R. et al. Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique. Nat Protoc 12, 2029–2049 (2017). https://doi.org/10.1038/nprot.2017.079
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DOI: https://doi.org/10.1038/nprot.2017.079
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