Ultrasensitive HIV-1 p24 Assay Detects Single Infected Cells and Differences in Reservoir Induction by Latency Reversal Agents

Autor: Olivier Schwartz, Olivier Lambotte, Matthew L. Albert, Valérie Monceaux, Katia Bourdic, Caroline Passaes, Nicolas Noel, Darragh Duffy, Annie David, Michaela Müller-Trutwin, Jérémie Decalf, Mathieu Angin, Timothée Bruel, Asier Sáez-Cirión
Přispěvatelé: HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Institut Pasteur [Paris] (IP), Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Immunobiologie des Cellules dendritiques, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Genentech, Inc., Genentech, Inc. [San Francisco], Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Neuro-Immuno-Virologie, Service de Neurovirologie EMI E-01 (UMR E01), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Centre de Recherche Translationnelle - Center for Translational Science (CRT), This work was supported by grants from MSDAVENIR (http://www.msdavenir.fr/fr/accueil/) and ANRS—France Recherche Nord & Sud Sida-HIV Hépatites (http://anrs.fr/) to A.S.-C. and by Pasteurinnov (http://www.pasteur.fr) to D.D. C.P.B.P. and M.L.A. weresupported by research fellowships from ANRS and Marie Skłodowska-Curie actions (http://ec.europa.eu/research mariecurieactions/). T.B. was supported by funds from the Vaccine Research Institute (http://www.recherchevaccinvih.fr/)., The members of the ANRS RHIVIERA (Remission of HIV Era) Consortium are as follows. The coordinators were Asier Sáez-Cirión (Institut Pasteur, Paris, France) and Christine Rouzioux (CHU Necker, Paris, France). Scientific and clinical partners were Françoise Barré-Sinoussi (Institut Pasteur, Paris, France), Brigitte Autran (CHU Pitié- Salpetriere and Université Pierre et Marie Curie, Paris, France), Monsef Benkirane (Institut de Genetique Humaine, Montpellier, France), Jeremie Guedj (Institut Claude Bernard, Paris, France), Laurent Hocqueloux (CHR Orleans La Source, Orleans, France), Christine Katlama (CHU Pitié Salpetriere, Paris, France), Olivier Lambotte (CHU Kremlin- Bicêtre, Le Kremlin-Bicêtre, France), Roger Legrand (IDMIT, CEA, Fontenay-aux-Roses, France), Laurence Meyer (INSERM, Le Kremlin-Bicêtre, France), Hugo Mouquet (Institut Pasteur, Paris, France), Michaela Müller-Trutwin (Institut Pasteur, Paris, France), Anne- Marie Taburet (CHU Kremlin-Bicêtre, France), and Carine Van Lint (Université Libre de Bruxelles, Brussels, Belgium). Participants from ANRS were Jean François Delfraissy (Director), Livia Pedroza Martins (Head of HIV Basic Research), and Sandrine Couffin- Cadiergues (Head of HIV Clinical Research), HIV, Inflammation et persistance, Institut Pasteur [Paris], Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Vaccine Research Institute (VRI), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Jazyk: angličtina
Rok vydání: 2017
Předmět:
Zdroj: Journal of Virology
Journal of Virology, 2017, 91 (6), pp.e02296-16. ⟨10.1128/JVI.02296-16⟩
Journal of Virology, American Society for Microbiology, 2017, 91 (6), pp.e02296-16. ⟨10.1128/JVI.02296-16⟩
ISSN: 0022-538X
1098-5514
DOI: 10.1128/JVI.02296-16⟩
Popis: The existence of HIV reservoirs in infected individuals under combined antiretroviral therapy (cART) represents a major obstacle toward cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, a method which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which require large numbers of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1,000-fold more sensitive than classical enzyme-linked immunosorbent assays (ELISAs) in the quantification of HIV-1 Gag p24 production in samples from HIV-infected individuals. Results from ultrasensitive p24 assays were compared to those from conventional viral RNA reverse transcription-quantitative PCR (RT-qPCR)-based assays and from outgrowth assay readout by flow cytometry. Using serial dilutions and flow-based single-cell sorting, we show that viral proteins produced by a single infected cell can be detected by the ultrasensitive p24 assay. This unique sensitivity allowed the early (as soon as day 1 in 43% of cases) and more efficient detection and quantification of p24 in phytohemagglutinin-L (PHA)-stimulated CD4 + T cells from individuals under effective cART. When seven different classes of latency reversal agents (LRA) in resting CD4 + T cells from HIV-infected individuals were tested, the ultrasensitive p24 assay revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected CD4 + T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals. IMPORTANCE The persistence of HIV reservoirs in infected individuals under effective antiretroviral treatment represents a major obstacle toward cure. Different methods to estimate HIV reservoirs exist, but there is currently no optimal assay to measure HIV reservoirs in HIV eradication interventions. In the present study, we report an ultrasensitive digital ELISA platform for quantification of the HIV-1 protein p24. This method was employed to assess the early reactivation of infectious virus from reservoirs in HIV-1-infected individuals. We found that viral proteins produced by a single infected cell can be detected by an ultrasensitive p24 assay. This unprecedented resolution showed major advantages in comparison to other techniques currently used to assess viral replication in reactivation studies. In addition, such a highly sensitive assay allows discrimination of drug-induced reactivation of productive HIV based on protein expression. The present study heralds new opportunities to evaluate the HIV reservoir and the efficacy of drugs used to target it.
Databáze: OpenAIRE
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