Tools for Visualizing HIV in Cure Research
Purpose of Review
The long-lived HIV reservoir remains a major obstacle for an HIV cure. Current techniques to analyze this reservoir are generally population-based. We highlight recent developments in methods visualizing HIV, which offer a different, complementary view, and provide indispensable information for cure strategy development.
Recent advances in fluorescence in situ hybridization techniques enabled key developments in reservoir visualization. Flow cytometric detection of HIV mRNAs, concurrently with proteins, provides a high-throughput approach to study the reservoir on a single-cell level. On a tissue level, key spatial information can be obtained detecting viral RNA and DNA in situ by fluorescence microscopy. At total-body level, advancements in non-invasive immuno-positron emission tomography (PET) detection of HIV proteins may allow an encompassing view of HIV reservoir sites.
HIV imaging approaches provide important, complementary information regarding the size, phenotype, and localization of the HIV reservoir. Visualizing the reservoir may contribute to the design, assessment, and monitoring of HIV cure strategies in vitro and in vivo.
KeywordsHIV cure In situ hybridization (ISH) Flow cytometry Microscopy RNA flow Viral reservoir Positron emission tomography (PET)
This work was supported by National Institutes of Health (NIH) grants RO1 HL-092565 (D.E.K); NIAID UM1AI100663 (CHAVI-ID) (D.E.K; Dennis Burton, principal investigator), and the Canadian Institutes for Health Research (project grant # 377124: D.E.K). AEB is the recipient of a CIHR Postdoctoral Fellowship (award no. 152536); DEK is supported by a FRQS Senior Research Scholar Award (award no. 31035).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no competing interests.
Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance
- 1.Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, et al. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science. 1997;278(5341):1295–300. https://doi.org/10.1126/science.278.5341.1295.
- 2.Chun TW, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature. 1997;387(6629):183–8. https://doi.org/10.1038/387183a0.
- 3.Finzi D, Blankson J, Siliciano JD, Margolick JB, Chadwick K, Pierson T, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med. 1999;5(5):512–7. https://doi.org/10.1038/8394.
- 7.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(2):e1003174. https://doi.org/10.1371/journal.ppat.1003174.
- 8.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. 2013;155(3):540–51. https://doi.org/10.1016/j.cell.2013.09.020.
- 9.Hiener B, Horsburgh BA, Eden J-S, Barton K, Schlub TE, Lee E, et al. Identification of genetically intact HIV-1 proviruses in specific CD4(+) T cells from effectively treated participants. Cell Rep. 2017;21(3):813–22. https://doi.org/10.1016/j.celrep.2017.09.081.
- 10.O’Doherty U, Swiggard WJ, Jeyakumar D, McGain D, Malim MH. A sensitive, quantitative assay for human immunodeficiency virus type 1 integration. J Virol. 2002;76(21):10942–50. https://doi.org/10.1128/JVI.76.21.10942-10950.2002.CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Brady T, Kelly BJ, Male F, Roth S, Bailey A, Malani N, et al. Quantitation of HIV DNA integration: effects of differential integration site distributions on Alu-PCR assays. J Virol Methods. 2013;189(1):53–7. https://doi.org/10.1016/j.jviromet.2013.01.004.
- 13.Vandergeeten C, Fromentin R, Merlini E, Lawani MB, DaFonseca S, Bakeman W, et al. Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies. J Virol. 2014;88(21):12385–96. https://doi.org/10.1128/JVI.00609-14.
- 14.Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, Yassine-Diab B, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med. 2009;15(8):893–900. https://doi.org/10.1038/nm.1972.
- 15.Morón-López S, Puertas MC, Gálvez C, Navarro J, Carrasco A, Esteve M, et al. Sensitive quantification of the HIV-1 reservoir in gut-associated lymphoid tissue. PLoS One. 2017;12(4):e0175899. https://doi.org/10.1371/journal.pone.0175899.
- 16.Soriano-Sarabia N, Bateson RE, Dahl NP, Crooks AM, Kuruc JD, Margolis DM, et al. Quantitation of replication-competent HIV-1 in populations of resting CD4 T cells. J Virol. 2014;88(24):14070–7. https://doi.org/10.1128/JVI.01900-14.
- 18.• De Master LK, Liu X, Van Belzen DJ, Trinité B, Zheng L, Agosto LM, et al. A subset of CD4/CD8 double-negative T cells expresses HIV proteins in patients on antiretroviral therapy. J Virol. 2015;90:2165–79. This study combined classical detection of HIV Gag protein with a cellular hallmark of HIV infection, CD4 downregulation, to reduce the false-positive rate associated with Gag staining alone. This enabled the identification of rare cells expressing HIV Gag directly ex vivo in samples from subjects on ART. The use of FAST (Fiber-optic Array Scanning Technology), enabled a relatively high-throughput for a microscopy-based technique. CrossRefGoogle Scholar
- 19.•• Baxter AE, Niessl J, Fromentin R, Richard J, Porichis F, Charlebois R, et al. Single-cell characterization of viral translation-competent reservoirs in HIV-infected individuals. Cell Host Microbe. 2016;20(3):368–80. This study provided the initial demonstration of single-cell detection of translation-competent cellular HIV reservoirs by RNA flow cytometry, directly in samples from HIV-infected individuals. The RNA flow FISH approach used combines the detection of Gag protein with GagPol mRNA and enabled substantial advances in specificity. Importantly, the authors demonstrate that this technique enables the size and cellular distribution of the latent, translation-competent reservoir after latency reversal to be studied at the single-cell level. https://doi.org/10.1016/j.chom.2016.07.015.
- 20.• Baxter AE, Niessl J, Fromentin R, Richard J, Porichis F, Massanella M, et al. Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique. Nat Protoc. 2017;12(10):2029–49. This paper provides a detailed protocol for the detection and characterization of translation-competent reservoir cells, identified by the concurrent expression of HIV Gag protein and vRNAs using the RNA flow FISH technique. https://doi.org/10.1038/nprot.2017.079.
- 21.• 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(20):9018–28. This study provided the demonstration that the RNA flow cytometry approach could be applied to in vitro models of HIV latency to detect and characterize cells expressing HIV mRNA and/or HIV protein. https://doi.org/10.1128/JVI.01448-16.
- 22.•• Grau-Expósito J, Serra-Peinado C, Miguel L, Navarro J, Curran A, Burgos J, et al. A novel single-cell FISH-flow assay identifies effector memory CD4 T cells as a major niche for HIV-1 transcription in HIV-infected patients. MBio. 2017;8:e00876–17. This work describes a key advance for the RNA flow FISH technique; the identification of GagPol vRNA+ cells directly in samples from HIV-infected individuals, without the requirement for concurrent Gag protein detection, thus identifying the transcription-competent reservoir. The authors demonstrate that this approach enables the identification of the transcription-competent reservoir after latency reversal. CrossRefPubMedPubMedCentralGoogle Scholar
- 23.Cavert W, Notermans DW, Staskus K, Wietgrefe SW, Zupancic M, Gebhard K, et al. Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection. Science. 1997;276(5314):960–4. https://doi.org/10.1126/science.276.5314.960.
- 24.Fletcher CV, Staskus K, Wietgrefe SW, Rothenberger M, Reilly C, Chipman JG, et al. Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues. Proc Natl Acad Sci U S A. 2014;111(6):2307–12. https://doi.org/10.1073/pnas.1318249111.
- 25.•• Deleage C, Wietgrefe SW, Del Prete G, Morcock DR, Hao XP, Piatak M Jr, et al. Defining HIV and SIV reservoirs in lymphoid tissues. Pathog Immun. 2016;1(1):68–106. This study provided an initial demonstration of the in situ-hybridization Scope approach for the detection and localization of vRNA and vDNA in lymphoid tissues from SIV-infected non-human primates or HIV-infected subjects. Importantly, this assay shows greater speed compared to previous hybridization techniques and allows the concurrent detection of vRNA and vDNA on the same slide. https://doi.org/10.20411/pai.v1i1.100.CrossRefPubMedPubMedCentralGoogle Scholar
- 26.•• Estes JD, Kityo C, Ssali F, Swainson L, Makamdop KN, Del Prete GQ, et al. Defining total-body AIDS-virus burden with implications for curative strategies. Nat. Med. [Internet]. 2017; https://doi.org/10.1038/nm.4411. This study was the first to investigate how the in situ-hybridization DNA/RNAScope technique could be used to evaluate the contribution of various tissues to the SIV reservoir in ART-treated SIV-infected non-human primates. Based on these results, the authors use tissue biopsies from ART-treated HIV-infected subjects to estimate the total-body size of the replication-competent HIV reservoir.
- 27.•• Santangelo PJ, Rogers KA, Zurla C, Blanchard EL, Gumber S, Strait K, et al. Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy-treated macaques. Nat Methods. 2015;12(5):427–32. This key method paper details the use of a non-invasive immuno-PET/CT approach to analyze the anatomic localization of viral protein-expressing cells throughout the whole body in untreated and ART-treated SIV-infected non-human primates. https://doi.org/10.1038/nmeth.3320.
- 28.Graf EH, Pace MJ, Peterson BA, Lynch LJ, Chukwulebe SB, Mexas AM, et al. Gag-positive reservoir cells are susceptible to HIV-specific cytotoxic T lymphocyte mediated clearance in vitro and can be detected in vivo. PLoS One. 2013;8(8):e71879. https://doi.org/10.1371/journal.pone.0071879.
- 29.Krivacic RT, Ladanyi A, Curry DN, Hsieh HB, Kuhn P, Bergsrud DE, et al. A rare-cell detector for cancer. Proc Natl Acad Sci U S A. 2004;101(29):10501–4. https://doi.org/10.1073/pnas.0404036101.
- 30.Das M, Riess JW, Frankel P, Schwartz E, Bennis R, Hsieh HB, et al. ERCC1 expression in circulating tumor cells (CTCs) using a novel detection platform correlates with progression-free survival (PFS) in patients with metastatic non-small-cell lung cancer (NSCLC) receiving platinum chemotherapy. Lung Cancer. 2012;77(2):421–6. https://doi.org/10.1016/j.lungcan.2012.04.005.
- 34.Geleziunas R, Bour S, Wainberg MA. Correlation between high level gp160 expression and reduced CD4 biosynthesis in clonal derivatives of human immunodeficiency virus type 1-infected U-937 cells. J Gen Virol. 1994;75(Pt 4):857–65. https://doi.org/10.1099/0022-1317-75-4-857.CrossRefPubMedGoogle Scholar
- 36.Wildum S, Schindler M, Munch J, Kirchhoff F. Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection. J Virol. 2006;80(16):8047–59. https://doi.org/10.1128/JVI.00252-06.CrossRefPubMedPubMedCentralGoogle Scholar
- 38.Patterson BK, Mosiman VL, Cantarero L, Furtado M, Bhattacharya M, Goolsby C. Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype. Cytometry. 1998;31(4):265–74. https://doi.org/10.1002/(SICI)1097-0320(19980401)31:4<265::AID-CYTO6>3.0.CO;2-I.CrossRefPubMedGoogle Scholar
- 41.Wang F, Flanagan J, Su N, Wang L-C, Bui S, Nielson A, et al. RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 2012;14(1):22–9. https://doi.org/10.1016/j.jmoldx.2011.08.002.
- 42.• Porichis F, Hart MG, Griesbeck M, Everett HL, Hassan M, Baxter AE, et al. High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat Commun. 2014;5:5641. This paper provides the initial description of the commerical RNA flow cytometry approach discussed in this review. The authors demonstrated the high-throughput detection of cellular mRNAs using the RNA flow FISH assay based on a branched DNA labeling technique originally developed for microscopy. This study provided a framework for many of the RNA flow studies discussed here. https://doi.org/10.1038/ncomms6641.
- 43.Bagnarelli P, Valenza A, Menzo S, Sampaolesi R, Varaldo PE, Butini L, et al. Dynamics and modulation of human immunodeficiency virus type 1 transcripts in vitro and in vivo. J Virol. 1996;70(11):7603–13.Google Scholar
- 44.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(4):e1004071. https://doi.org/10.1371/journal.ppat.1004071.
- 45.Jiang G, Mendes EA, Kaiser P, Sankaran-Walters S, Tang Y, Weber MG, et al. Reactivation of HIV latency by a newly modified Ingenol derivative via protein kinase Cδ-NF-κB signaling. AIDS. 2014;28(11):1555–66. https://doi.org/10.1097/QAD.0000000000000289.
- 48.Embretson J, Zupancic M, Ribas JL, Burke A, Racz P, Tenner-Racz K, et al. Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature. 1993;362(6418):359–62. https://doi.org/10.1038/362359a0.
- 50.Miller CJ, Li Q, Abel K, Kim E-Y, Ma Z-M, Wietgrefe S, et al. Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J Virol. 2005;79(14):9217–27. https://doi.org/10.1128/JVI.79.14.9217-9227.2005.
- 51.Li Q, Duan L, Estes JD, Ma Z-M, Rourke T, Wang Y, et al. Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature. 2005;434(7037):1148–52. https://doi.org/10.1038/nature03513.
- 52.Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, et al. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med. 2004;200(6):761–70. https://doi.org/10.1084/jem.20041196.
- 56.Fukazawa Y, Lum R, Okoye AA, Park H, Matsuda K, Bae JY, et al. B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers. Nat Med. 2015;21(2):132–9. https://doi.org/10.1038/nm.3781.
- 57.Li Q, Skinner PJ, Ha S-J, Duan L, Mattila TL, Hage A, et al. Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection. Science. 2009;323(5922):1726–9. https://doi.org/10.1126/science.1168676.
- 58.Gerner MY, Kastenmuller W, Ifrim I, Kabat J, Germain RN. Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes. Immunity. 2012;37(2):364–76. https://doi.org/10.1016/j.immuni.2012.07.011.CrossRefPubMedPubMedCentralGoogle Scholar
- 60.Mestel R. Cancer: imaging with antibodies. Nature 2017;543:743–746, 7647, DOI: https://doi.org/10.1038/543743a.
- 61.Marban C, Forouzanfar F, Ait-Ammar A, Fahmi F, El Mekdad H, Daouad F, et al. Targeting the brain reservoirs: toward an HIV cure. Front. Immunol. [Internet]. 2016;7. Available from: https://doi.org/10.3389/fimmu.2016.00397
- 63.Arakelyan A, Fitzgerald W, King DF, Rogers P, Cheeseman HM, Grivel J-C, et al. Flow virometry analysis of envelope glycoprotein conformations on individual HIV virions. Sci Rep. 2017;7(1):948. https://doi.org/10.1038/s41598-017-00935-w.