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Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique

Nature Protocols volume 12, pages 2029–2049 (2017)Cite this article

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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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Figure 1: Impact of Poisson distribution on the detection of rare HIVRNA+/Gag+ events.
Figure 2: Part I of the protocol.
Figure 3: Part IIA of the protocol.
Figure 4: Part IIB of the protocol.
Figure 5: Representative staining of mRNA-positive controls processed as described in the complete protocol, demonstrating the expected distribution of fluorescence intensities and frequencies of mRNA+ cells.
Figure 6: Example flow cytometry plots from Steps 104 to 109 showing primary CD4 T-cell samples processed with the HIVRNA/Gag assay, demonstrating expected staining patterns and frequencies of HIVRNA+/Gag+ cells.
Figure 7: Comparison of single mRNA, dual mRNA and protein staining on subject samples.
Figure 8: Example HIVRNA/Gag assay staining and concurrent phenotyping of the translation-competent latent viral reservoir following LRA-induced reactivation for a virally suppressed, ART-treated individual.
Figure 9: The HIVRNA/Gag assay enables the microscopy analysis of rare populations of HIV-infected CD4 T cells, by sorting the rare populations of interest before microscopy.

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References

  1. 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).

    Article  Google Scholar 

  2. Stephenson, K.E., D'Couto, H.T. & Barouch, D.H. New concepts in HIV-1 vaccine development. Curr. Opin. Immunol. 41, 39–46 (2016).

    Article  CAS  Google Scholar 

  3. 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).

    Article  CAS  Google Scholar 

  4. 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).

    Article  Google Scholar 

  5. 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).

    Article  Google Scholar 

  6. Deeks, S.G. HIV: shock and kill. Nature 487, 439–440 (2012).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

  8. Whitney, J.B. et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys. Nature 512, 74–77 (2014).

    Article  CAS  Google Scholar 

  9. 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).

    Article  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. Crooks, A.M. et al. Precise quantitation of the latent HIV-1 reservoir: implications for eradication strategies. J. Infect. Dis. 212, 1361–1365 (2015).

    Article  CAS  Google Scholar 

  12. Finzi, D. et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science 278, 1295–1300 (1997).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Eriksson, S. et al. Comparative analysis of measures of viral reservoirs in HIV-1 eradication studies. PLoS Pathog. 9, e1003174 (2013).

    Article  CAS  Google Scholar 

  15. Baxter, A.E. et al. Single-cell characterization of viral translation-competent reservoirs in HIV-infected individuals. Cell Host Microbe 20, 368–380 (2016).

    Article  CAS  Google Scholar 

  16. Sattentau, Q.J. & Stevenson, M. Macrophages and HIV-1: an unhealthy constellation. Cell Host Microbe 19, 304–310 (2016).

    Article  CAS  Google Scholar 

  17. Chun, T.W. et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 387, 183–188 (1997).

    Article  CAS  Google Scholar 

  18. Vandergeeten, C. et al. Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies. J. Virol. 88, 12385–12396 (2014).

    Article  Google Scholar 

  19. Laird, G.M. et al. Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay. PLoS Pathog. 9, e1003398 (2013).

    Article  CAS  Google Scholar 

  20. Ho, Y.-C. et al. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell 155, 540–551 (2013).

    Article  CAS  Google Scholar 

  21. Bruner, K.M. et al. Defective proviruses rapidly accumulate during acute HIV-1 infection. Nat. Med. 22, 1043–1049 (2016).

    Article  CAS  Google Scholar 

  22. Bruner, K.M., Hosmane, N.N. & Siliciano, R.F. Towards an HIV-1 cure: measuring the latent reservoir. Trends Microbiol. 23, 192–203 (2015).

    Article  CAS  Google Scholar 

  23. 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).

    Article  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. Jambo, K.C. et al. Small alveolar macrophages are infected preferentially by HIV and exhibit impaired phagocytic function. Mucosal Immunol. 7, 1116–1126 (2014).

    Article  CAS  Google Scholar 

  27. 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).

    Article  Google Scholar 

  28. 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).

    Article  Google Scholar 

  29. Chargin, A. et al. Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood. J. Clin. Microbiol. 53, 60–66 (2014).

    Article  Google Scholar 

  30. Perfetto, S.P., Ambrozak, D., Nguyen, R., Chattopadhyay, P. & Roederer, M. Quality assurance for polychromatic flow cytometry. Nat. Protoc. 1, 1522–1530 (2006).

    Article  CAS  Google Scholar 

  31. 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).

    Article  CAS  Google Scholar 

  32. 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).

    Article  CAS  Google Scholar 

Download references

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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Author notes

  1. Filippos Porichis

    Present address: Current address: EMD Serono-Merck, Billerica, Massachusetts, USA.,

Authors and Affiliations

  1. Research Centre of the Centre Hospitalier de l'Université de Montréal (CRCHUM) and Université de Montréal, Montreal, Quebec, Canada

    Amy E Baxter, Julia Niessl, Rémi Fromentin, Jonathan Richard, Marta Massanella, Nathalie Brassard, Nirmin Alsahafi, Andrés Finzi, Nicolas Chomont & Daniel E Kaufmann

  2. Center for HIV/AIDS Vaccine Immunology and,

    Amy E Baxter, Julia Niessl & Daniel E Kaufmann

  3. Immunogen Discovery (CHAVI-ID), La Jolla, California, USA

    Amy E Baxter, Julia Niessl & Daniel E Kaufmann

  4. Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA

    Filippos Porichis

  5. Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada

    Nirmin Alsahafi & Andrés Finzi

  6. Chronic Viral Illnesses Service and Division of Hematology, McGill University Health Centre, Montreal, Quebec, Canada

    Jean-Pierre Routy

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Contributions

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.

Corresponding author

Correspondence to Daniel E Kaufmann.

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The authors declare no competing financial interests.

Integrated supplementary information

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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Supplementary Figure 1. (PDF 218 kb)

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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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