Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress 2023 EHA Congress 2023 ASCO Annual Meeting
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on sleep in brain health

LIVE: Thursday 2nd May 2024, 18:00-19:30 (CEST)

Quality sleep is essential for health. But what happens to our brains when sleep patterns are disturbed? Join our experts to explore the interplay between sleep disruption and neurological diseases, and the questions that you need to be asking your patients to help you prevent the harmful effects of sleep deprivation.
ADVANCED SEARCH
Log in
Top
AIDS Research and Therapy
Published in: AIDS Research and Therapy 1/2017

Open Access 01-12-2017 | Review

RNA flow cytometric FISH for investigations into HIV immunology, vaccination and cure strategies

Authors: Amy E. Baxter, Julia Niessl, Antigoni Morou, Daniel E. Kaufmann

Published in: AIDS Research and Therapy | Issue 1/2017

Login to get access

Abstract

Despite the tremendous success of anti-retroviral therapy (ART) no current treatment can eradicate latent HIV reservoirs from HIV-infected individuals or generate, effective HIV-specific immunity. Technological limitations have hampered the identification and characterization of both HIV-infected cells and HIV-specific responses in clinical samples directly ex vivo. RNA-flow cytometric fluorescence in situ hybridisation (RNA Flow-FISH) is a powerful technique, which enables detection of mRNAs in conjunction with proteins at a single-cell level. Here, we describe how we are using this technology to address some of the major questions remaining in the HIV field in the era of ART. We discuss how CD4 T cell responses to HIV antigens, both following vaccination and HIV infection, can be characterized by measurement of cytokine mRNAs. We describe how our development of a dual HIV mRNA/protein assay (HIVRNA/Gag assay) enables high sensitivity detection of very rare HIV-infected cells and aids investigations into the translation-competent latent reservoir in the context of HIV cure.
Literature
1.
Margolis DM, Garcia JV, Hazuda DJ, Haynes BF. Latency reversal and viral clearance to cure HIV-1. Science. 2016;353:6517.CrossRef
2.
3.
Swain SL, McKinstry KK, Strutt TM. Expanding roles for CD4+ T cells in immunity to viruses. Nat Rev Immunol. 2012;12:136–48.PubMedPubMedCentral
4.
5.
6.
Havenar-Daughton C, Lee JH, Crotty S. Tfh cells and HIV bnAbs, an immunodominance model of the HIV neutralizing antibody generation problem. Immunol Rev. 2017;275:49–61.CrossRefPubMed
7.
Porichis F, Hart MG, Griesbeck M, Everett HL, Hassan M, Baxter AE, Lindqvist M, Miller SM, Soghoian DZ, Kavanagh DG, Reynolds S, Norris B, Mordecai SK, Nguyen Q, Lai C, Kaufmann DE. High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat Commn. 2014;5:5641.CrossRef
8.
Baxter AE, Niessl J, Fromentin R, Richard J, Porichis F, Charlebois R, Massanella M, Brassard N, Alsahafi N, Delgado G-G, Routy J-P, Walker BD, Finzi A, Chomont N, Kaufmann DE. Single-cell characterization of viral translation-competent reservoirs in HIV-infected individuals. Cell Host Microbe. 2016;20:368–80.CrossRefPubMed
9.
Baxter AE, Niessl J, Fromentin R, Richard J, Porichis F, Massanella M, Brassard N, Alsahafi N, Routy J-P, Finzi A, Chomont N, Kaufmann DE. Multi-parametric characterization of rare HIV-infected cells using an RNA-flow FISH technique. Nat Protoc. In Press.
10.
Haynes BF, Gilbert PB, Mcelrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, Evans DT, Montefiori DC, Karnasuta C, Sutthent R, Liao H-X, Devico AL, Lewis GK, Williams C, Pinter A, Fong Y, Janes H, DeCamp A, Huang Y, Rao M, Billings EM, Karasavvas N, Robb ML, Ngauy V, de Souza MS, Paris R, Ferrari G, Bailer RT, Soderberg KA, Andrews C, et al. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med. 2012;366:1275–86.CrossRefPubMedPubMedCentral
11.
Porichis F, Hart MG, Zupkosky J, Barblu L, Kwon DS, McMullen A, Brennan T, Ahmed R, Freeman GJ, Kavanagh DG, Kaufmann DE. Differential impact of PD-1 and/or interleukin-10 blockade on HIV-1-specific CD4 T cell and antigen-presenting cell functions. J Virol. 2014;88:2508–18.CrossRefPubMedPubMedCentral
12.
Havenar-Daughton C, Reiss SM, Carnathan DG, Wu JE, Kendric K, de la Peña AT, Kasturi SP, Dan JM, Bothwell M, Sanders RW, Pulendran B, Silvestri G, Crotty S. Cytokine-independent detection of antigen-specific germinal center T follicular helper cells in immunized nonhuman primates using a live cell activation-induced marker technique. J Immunol. 2016;197:994–1002.CrossRefPubMedPubMedCentral
13.
Burton DR, Ahmed R, Barouch DH, Butera ST, Crotty S, Godzik A, Kaufmann DE, Mcelrath MJ, Nussenzweig MC, Pulendran B, Scanlan CN, Schief WR, Silvestri G, Streeck H, Walker BD, Walker LM, Ward AB, Wilson IA, Wyatt R. A blueprint for HIV vaccine discovery. Cell Host Microbe. 2012;12:396–407.CrossRefPubMedPubMedCentral
14.
Locci M, Havenar-Daughton C, Landais E, Wu J, Kroenke MA, Arlehamn CL, Su LF, Cubas R, Davis MM, Sette A, Haddad EK, International AIDS Vaccine Initiative Protocol C Principal Investigators, Poignard P, Crotty S. Human circulating PD-1(+) CXCR3(-) CXCR5(+) memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses. Immunity. 2013;39(4):758–69.CrossRefPubMedPubMedCentral
15.
Gaublomme JT, Yosef N, Lee Y, Gertner RS, Yang LV, Wu C, Pandolfi PP, Mak T, Satija R, Shalek AK, Kuchroo VK, Park H, Regev A. Single-cell genomics unveils critical regulators of Th17 cell pathogenicity. Cell. 2015;163:1400–12.CrossRefPubMedPubMedCentral
16.
Shalapour S, Font-Burgada J, Di Caro G, Zhong Z, Sanchez-Lopez E, Dhar D, Willimsky G, Ammirante M, Strasner A, Hansel DE, Jamieson C, Kane CJ, Klatte T, Birner P, Kenner L, Karin M. Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy. Nature. 2015;521:94–8.CrossRefPubMedPubMedCentral
17.
Chun TW, Carruth L, Finzi D, Shen X, DiGiuseppe JA, Taylor H, Hermankova M, Chadwick K, Margolick J, Quinn TC, Kuo YH, Brookmeyer R, Zeiger MA, Barditch-Crovo P, Siliciano RF. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature. 1997;387:183–8.CrossRefPubMed
18.
Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, Lloyd AL, Nowak MA, Fauci AS. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci. 1997;94:13193–7.CrossRefPubMedPubMedCentral
19.
Vandergeeten C, Fromentin R, Merlini E, Lawani MB, DaFonseca S, Bakeman W, McNulty A, Ramgopal M, Michael N, Kim JH, Ananworanich J, Chomont N. Cross-clade ultrasensitive PCR-based assays to measure HIV persistence in large-cohort studies. J Virol. 2014;88:12385–96.CrossRefPubMedPubMedCentral
20.
Finzi D, Hermankova M, Pierson T, Carruth LM, Buck C, Chaisson RE, Quinn TC, Chadwick K, Margolick J, Brookmeyer R, Gallant J, Markowitz M, Ho DD, Richman DD, Siliciano RF. Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. Science. 1997;278:1295–300.CrossRefPubMed
21.
Laird GM, Eisele EE, Rabi SA, Lai J, Chioma S, Blankson JN, Siliciano JD, Siliciano RF. Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay. PLoS Pathog. 2013;9:e1003398.CrossRefPubMedPubMedCentral
22.
Procopio FA, Fromentin R, Kulpa DA, Brehm JH, Bebin A-G, Strain MC, Richman DD, O’Doherty U, Palmer S, Hecht FM, Hoh R, Barnard RJO, Miller MD, Hazuda DJ, Deeks SG, Sekaly R-P, Chomont N. A Novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals. EBioMedicine. 2015;2:872–81.CrossRef
23.
Ho Y-C, Shan L, Hosmane NN, Wang J, Laskey SB, Rosenbloom DIS, Lai J, Blankson JN, Siliciano JD, Siliciano RF. Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure. Cell. 2013;155:540–51.CrossRefPubMedPubMedCentral
24.
25.
Chomont N, El-Far M, Ancuta P, Trautmann L, Procopio FA, Yassine-Diab B, Boucher G, Boulassel M-R, Ghattas G, Brenchley JM, Schacker TW, Hill BJ, Douek DC, Routy J-P, Haddad EK, Sekaly R-P. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med. 2009;15:893–900.CrossRefPubMedPubMedCentral
26.
Perreau M, Savoye A-L, De Crignis E, Corpataux J-M, Cubas R, Haddad EK, De Leval L, Graziosi C, Pantaleo G. Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production. J Exp Med. 2013;210:143–56.CrossRefPubMedPubMedCentral
27.
Fukazawa Y, Lum R, Okoye AA, Park H, Matsuda K. B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers. Nat Med. 2015;21(2):132–9.CrossRefPubMedPubMedCentral
28.
Lorenzo-Redondo R, Fryer HR, Bedford T, Kim E-Y, Archer J, Pond SLK, Chung Y-S, Penugonda S, Chipman JG, Fletcher CV, Schacker TW, Malim MH, Rambaut A, Haase AT, McLean AR, Wolinsky SM. Persistent HIV-1 replication maintains the tissue reservoir during therapy. Nature. 2016;530:51–6.CrossRefPubMedPubMedCentral
29.
30.
Kearney MF, Spindler J, Shao W, Yu S, Anderson EM, O’Shea A, Rehm C, Poethke C, Kovacs N, Mellors JW, Coffin JM, Maldarelli F. Lack of detectable HIV-1 molecular evolution during suppressive antiretroviral therapy. PLoS Pathog. 2014;10:e1004010.CrossRefPubMedPubMedCentral
31.
Spina CA, Anderson J, Archin NM, Bosque A, Chan J, Famiglietti M, Greene WC, Kashuba A, Lewin SR, Margolis DM, Mau M, Ruelas D, Saleh S, Shirakawa K, Siliciano RF, Singhania A, Soto PC, Terry VH, Verdin E, Woelk C, Wooden S, Xing S, Planelles V. An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+T Cells from aviremic patients. PLoS Pathog. 2013;9:e1003834.CrossRefPubMedPubMedCentral
32.
Rasmussen TA, Lewin SR. Shocking HIV out of hiding: where are we with clinical trials of latency reversing agents? Curr Opin HIV AIDS. 2016;11:394–401.CrossRefPubMed
Metadata
Title
RNA flow cytometric FISH for investigations into HIV immunology, vaccination and cure strategies
Authors
Amy E. Baxter
Julia Niessl
Antigoni Morou
Daniel E. Kaufmann
Publication date
01-12-2017
Publisher
BioMed Central
Published in
AIDS Research and Therapy / Issue 1/2017
Electronic ISSN: 1742-6405
DOI
https://doi.org/10.1186/s12981-017-0171-x

Other articles of this Issue 1/2017

AIDS Research and Therapy 1/2017 Go to the issue

Research

The role of the glycosyl moiety of myricetin derivatives in anti-HIV-1 activity in vitro

Research

CLINICAL outcomes and loss to follow-up among people living with HIV participating in the NAMWEZA intervention in Dar es Salaam, Tanzania: a prospective cohort study

Short Report

Drug resistance patterns following pharmacy stock shortage in Nigerian Antiretroviral Treatment Program

Research

Hospitalization causes and outcomes in HIV patients in the late antiretroviral era in Colombia

Review

Eradication of HIV-1 latent reservoirs through therapeutic vaccination

Research

Effect of Moringa oleifera Lam. leaf powder on the pharmacokinetics of nevirapine in HIV-infected adults: a one sequence cross-over study

ACC 2024 Congress Coverage

Cardiology Video

Highlights from the ACC 2024 Congress

Watch now

Watch official videos from the ACC congress summarizing key highlights from the last year in heart failure, cardiomyopathies, valvular disease, pulmonary vascular disease, and pediatric cardiology.

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

16-04-2024 Type 2 Diabetes News

Incentivised to exercise

11-04-2024 Lifestyle Modifications News

New intervention for STEMI-related cardiogenic shock

10-04-2024 Myocardial Infarction News

» View more highlights on the ACC 2024 congress hub page

Image Credits