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1. Prior flavivirus immunity skews the yellow fever vaccine response to cross-reactive antibodies with potential to enhance dengue virus infection

Author

Antonio Santos-Peral, Fabian Luppa, Sebastian Goresch, Elena Nikolova, Magdalena Zaucha, Lisa Lehmann, Frank Dahlstroem, Hadi Karimzadeh, Julia Thorn-Seshold, Elena Winheim, Ev-Marie Schuster, Gerhard Dobler, Michael Hoelscher, Beate M. Kümmerer, Stefan Endres, Kilian Schober, Anne B. Krug, Michael Pritsch, Giovanna Barba-Spaeth, and Simon Rothenfusser

Subjects
Science
Abstract

Abstract The yellow fever 17D vaccine (YF17D) is highly effective but is frequently administered to individuals with pre-existing cross-reactive immunity, potentially impacting their immune responses. Here, we investigate the impact of pre-existing flavivirus immunity induced by the tick-borne encephalitis virus (TBEV) vaccine on the response to YF17D vaccination in 250 individuals up to 28 days post-vaccination (pv) and 22 individuals sampled one-year pv. Our findings indicate that previous TBEV vaccination does not affect the early IgM-driven neutralizing response to YF17D. However, pre-vaccination sera enhance YF17D virus infection in vitro via antibody-dependent enhancement (ADE). Following YF17D vaccination, TBEV-pre-vaccinated individuals develop high amounts of cross-reactive IgG antibodies with poor neutralizing capacity. In contrast, TBEV-unvaccinated individuals elicit a non-cross-reacting neutralizing response. Using YF17D envelope protein mutants displaying different epitopes, we identify quaternary dimeric epitopes as the primary target of neutralizing antibodies. Additionally, TBEV-pre-vaccination skews the IgG response towards the pan-flavivirus fusion loop epitope (FLE), capable of mediating ADE of dengue and Zika virus infections in vitro. Together, we propose that YF17D vaccination conceals the FLE in individuals without prior flavivirus exposure but favors a cross-reactive IgG response in TBEV-pre-vaccinated recipients directed to the FLE with potential to enhance dengue virus infection.

Published
2024
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2. A specific molecular signature in SARS-CoV-2–infected kidney biopsies

Author

Pierre Isnard, Paul Vergnaud, Serge Garbay, Matthieu Jamme, Maeva Eloudzeri, Alexandre Karras, Dany Anglicheau, Valérie Galantine, Arwa Jalal Eddine, Clément Gosset, Franck Pourcine, Mohammed Zarhrate, Jean-Baptiste Gibier, Elena Rensen, Stefano Pietropaoli, Giovanna Barba-Spaeth, Jean-Paul Duong-Van-Huyen, Thierry J. Molina, Florian Mueller, Christophe Zimmer, Marco Pontoglio, Fabiola Terzi, and Marion Rabant

Subjects
COVID-19, Nephrology, Medicine
Abstract

Acute kidney injury is one of the most important complications in patients with COVID-19 and is considered a negative prognostic factor with respect to patient survival. The occurrence of direct infection of the kidney by SARS-CoV-2, and its contribution to the renal deterioration process, remain controversial issues. By studying 32 renal biopsies from patients with COVID-19, we verified that the major pathological feature of COVID-19 is acute tubular injury (ATI). Using single-molecule fluorescence in situ hybridization, we showed that SARS-CoV-2 infected living renal cells and that infection, which paralleled renal angiotensin-converting enzyme 2 expression levels, was associated with increased death. Mechanistically, a transcriptomic analysis uncovered specific molecular signatures in SARS-CoV-2–infected kidneys as compared with healthy kidneys and non–COVID-19 ATI kidneys. On the other hand, we demonstrated that SARS-CoV-2 and hantavirus, 2 RNA viruses, activated different genetic networks despite triggering the same pathological lesions. Finally, we identified X-linked inhibitor of apoptosis-associated factor 1 as a critical target of SARS-CoV-2 infection. In conclusion, this study demonstrated that SARS-CoV-2 can directly infect living renal cells and identified specific druggable molecular targets that can potentially aid in the design of novel therapeutic strategies to preserve renal function in patients with COVID-19.

Published
2023
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3. Defective viral genomes as therapeutic interfering particles against flavivirus infection in mammalian and mosquito hosts

Author

Veronica V. Rezelj, Lucía Carrau, Fernando Merwaiss, Laura I. Levi, Diana Erazo, Quang Dinh Tran, Annabelle Henrion-Lacritick, Valérie Gausson, Yasutsugu Suzuki, Djoshkun Shengjuler, Bjoern Meyer, Thomas Vallet, James Weger-Lucarelli, Veronika Bernhauerová, Avi Titievsky, Vadim Sharov, Stefano Pietropaoli, Marco A. Diaz-Salinas, Vincent Legros, Nathalie Pardigon, Giovanna Barba-Spaeth, Leonid Brodsky, Maria-Carla Saleh, and Marco Vignuzzi

Subjects
Science
Abstract

Defective viral genomes (DVGs) can interfere with virus replication and provide a potential approach to control infection. Here, Rezelj et al. use a combined experimental evolution and computational approach to identify DVG sequences that optimally interfere with Zika virus infection and show antiviral activity in mice and mosquitoes.

Published
2021
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4. FluoRNT: A robust, efficient assay for the detection of neutralising antibodies against yellow fever virus 17D

Author

Magdalena K. Scheck, Lisa Lehmann, Magdalena Zaucha, Paul Schwarzlmueller, Kristina Huber, Michael Pritsch, Giovanna Barba-Spaeth, Oliver Thorn-Seshold, Anne B. Krug, Stefan Endres, Simon Rothenfusser, and Julia Thorn-Seshold

Subjects
Medicine, Science
Abstract

There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016–2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.

Published
2022

5. Dynamic changes in circulating T follicular helper cell composition predict neutralising antibody responses after yellow fever vaccination

Author

Johanna E Huber, Julia Ahlfeld, Magdalena K Scheck, Magdalena Zaucha, Klaus Witter, Lisa Lehmann, Hadi Karimzadeh, Michael Pritsch, Michael Hoelscher, Frank vonSonnenburg, Andrea Dick, Giovanna Barba‐Spaeth, Anne B Krug, Simon Rothenfußer, and Dirk Baumjohann

Subjects
neutralising antibodies, T follicular helper (Tfh) cells, vaccination, viral infection, yellow fever, YF‐17D, Immunologic diseases. Allergy, RC581-607
Abstract

Abstract Objectives T follicular helper (Tfh) cells are the principal T helper cell subset that provides help to B cells for potent antibody responses against various pathogens. In this study, we took advantage of the live‐attenuated yellow fever virus (YFV) vaccine strain, YF‐17D, as a model system for studying human antiviral immune responses in vivo following exposure to an acute primary virus challenge under safe and highly controlled conditions, to comprehensively analyse the dynamics of circulating Tfh (cTfh) cells. Methods We tracked and analysed the response of cTfh and other T and B cell subsets in peripheral blood of healthy volunteers by flow cytometry over the course of 4 weeks after YF‐17D vaccination. Results Using surface staining of cell activation markers to track YFV‐specific T cells, we found increasing cTfh cell frequencies starting at day 3 and peaking around 2 weeks after YF‐17D vaccination. This kinetic was confirmed in a subgroup of donors using MHC multimer staining for four known MHC class II epitopes of YF‐17D. The subset composition of cTfh cells changed dynamically during the course of the immune response and was dominated by the cTfh1‐polarised subpopulation. Importantly, frequencies of cTfh1 cells correlated with the strength of the neutralising antibody response, whereas frequencies of cTfh17 cells were inversely correlated. Conclusion In summary, we describe detailed cTfh kinetics during YF‐17D vaccination. Our results suggest that cTfh expansion and polarisation can serve as a prognostic marker for vaccine success. These insights may be leveraged in the future to improve current vaccine design and strategies.

Published
2020
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6. Covalently linked dengue virus envelope glycoprotein dimers reduce exposure of the immunodominant fusion loop epitope

Author

Alexander Rouvinski, Wanwisa Dejnirattisai, Pablo Guardado-Calvo, Marie-Christine Vaney, Arvind Sharma, Stéphane Duquerroy, Piyada Supasa, Wiyada Wongwiwat, Ahmed Haouz, Giovanna Barba-Spaeth, Juthathip Mongkolsapaya, Félix A. Rey, and Gavin R. Screaton

Subjects
Science
Abstract

The immunodominant epitope of dengue virus envelope protein (E) induces poorly neutralizing antibodies, which poses a problem for vaccine development. Here, the authors engineer covalently locked E dimers exposing an epitope that has been shown to induce potent and broadly neutralizing antibodies.

Published
2017
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7. Prior flavivirus immunity skews the yellow fever vaccine response to expand cross-reactive antibodies with increased risk of antibody dependent enhancement of Zika and dengue virus infection

Author

Antonio Santos-Peral, Fabian Luppa, Sebastian Goresch, Elena Nikolova, Magdalena Zaucha, Lisa Lehmann, Frank Dahlstroem, Hadi Karimzadeh, Beate M Kummerer, Julia Thorn-Seshold, Elena Winheim, Gerhard Dobler, Michael Hoelscher, Stefan Endres, Anne B Krug, Michael Pritsch, Giovanna Barba-Spaeth, Simon Rothenfusser, Ludwig-Maximilians-Universität München (LMU), Rheinische Friedrich-Wilhelms-Universität Bonn, German Centre for Infection Research (DZIF), Bundeswehr Institute of Microbiology, Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Helmholtz Zentrum München (HMGU), Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was supported by FlavImmunity a combined grant of the German Research foundation (DFG) project number 391217598 to SR and ABK and the French National Research Agency (ANR) project number ANR-17-CE15-0031-01 to GBS, by DFG TRR237 grant project number 369799452 to SR and ABK (TRR237 TPB14) and BMK (TRR237 TPA04), by grants of the iMed consortium of the German Helmholtz Societies to SR, by the Einheit für Klinische Pharmakologie (EKLIP), Helmholtz Zentrum München, Neuherberg, Germany to SR and SE, a Stipend (TI 07.003) by the German Center for Infection Research (DZIF) to FL, grants by the Friedrich Baur Foundation (FBS) to JTS, HK and MP, a Metiphys fellowship of the Medical Faculty of the LMU Munich to MP, by the FöFoLe Program of the Medical Faculty of the LMU Munich to SG, EN, LL, FL, the international doctoral program 'iTarget: Immunotargeting of cancer'funded by the Elite Network of Bavaria to ASP, MZ, SG, LL, EN., and ANR-17-CE15-0031,FLAVIMMUNITY,Comprendre les mécanismes de l'efficacité du vaccin de la fièvre jaune 17D: Une approche immuno-structurelle vers la conception d'un vaccin Pan-flavivirus(2017)

Subjects
vaccine immunogenicity, immunodominance, cross-reactivity, [SDV]Life Sciences [q-bio], pre-existing flavivirus immunity, yellow fever virus
Abstract

Human pathogenic flaviviruses pose a significant health concern and vaccination is the most effective instrument to control their circulation. How pre-existing immunity to antigenically related viruses modulates immunization outcome remains poorly understood. In this study, we evaluated the effect of vaccination against tick-borne encephalitis virus (TBEV) on the epitope immunodominance and immunogenicity of the yellow fever 17D vaccine (YF17D) in a cohort of 250 human vaccinees.Following YF17D vaccination, all study participants seroconverted and generated protective neutralizing antibody titers. At day 28, TBEV pre-immunity did not affect the polyclonal neutralizing response which largely depended on the IgM fraction. We found that sera from TBEV-immunized individuals enhanced YF17D vaccine virus infection via antibody-dependent enhancement (ADE). Upon vaccination, individuals with TBEV pre-immunity had higher concentrations of cross-reactive IgG antibodies with limited neutralizing capacity against YF17D whereas vaccinees without prior flavivirus exposure showed a non-cross-reacting response. Using a set of recombinant YF17D envelope protein mutants displaying different epitopes, we identified quaternary epitopes as the primary target of neutralizing antibodies. Sequential immunizations redirected the IgG response towards the pan-flavivirus fusion loop epitope (FLE) with the potential to mediate enhancement of dengue and Zika virus infections whereas TBEV naïve individuals elicited an IgG response directed towards neutralizing epitopes without an enhancing effect.We propose that the YF17D vaccine effectively conceals the FLE and primes a neutralizing IgG response in individuals with no prior flavivirus exposure. In contrast, the response in TBEV-experienced recipients favors weakly-neutralizing, cross-reactive epitopes potentially increasing the risk of severe dengue and Zika disease due to ADE.

Published
2023

8. Environmental and genetic drivers of population differences in SARS-CoV-2 immune responses

Author

Yann Aquino, Aurélie Bisiaux, Zhi Li, Mary O’Neill, Javier Mendoza-Revilla, Sarah Hélène Merkling, Gaspard Kerner, Milena Hasan, Valentina Libri, Vincent Bondet, Nikaïa Smith, Camille de Cevins, Mickaël Ménager, Francesca Luca, Roger Pique-Regi, Giovanna Barba-Spaeth, Stefano Pietropaoli, Olivier Schwartz, Geert Leroux-Roels, Cheuk-Kwong Lee, Kathy Leung, Joseph T.K. Wu, Malik Peiris, Roberto Bruzzone, Laurent Abel, Jean-Laurent Casanova, Sophie A. Valkenburg, Darragh Duffy, Etienne Patin, Maxime Rotival, Lluis Quintana-Murci, Génétique Evolutive Humaine - Human Evolutionary Genetics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Collège Doctoral, Sorbonne Université (SU), Interactions Virus-Insectes - Insect-Virus Interactions (IVI), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Immunologie Translationnelle - Translational Immunology lab, Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Inflammatory Responses and Transcriptomic Networks in diseases (Equipe Inserm U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Wayne State University [Detroit], University of Rome 'Tor Vergeta', Università degli Studi di Roma Tor Vergata [Roma], Virologie Structurale - Structural Virology, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Universiteit Gent = Ghent University (UGENT), Ghent University Hospital, Hong Kong Red Cross Blood Transfusion Service, Hospital Authority, The University of Hong Kong (HKU), Rockefeller University [New York], CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Human genetics of infectious diseases: Complex predisposition (Equipe Inserm U1163), Howard Hughes Medical Institute (HHMI), University of Melbourne, Hong Kong Science and Technology Parks Corporation (HKSTP), Collège de France - Chaire Génomique humaine et évolution, Collège de France (CdF (institution)), The Human Evolutionary Genetics Laboratory is supported by the Institut Pasteur, the Collège de France, the Centre Nationale de la Recherche Scientifique (CNRS), the Agence Nationale de la Recherche (ANR) grants COVID-19-POPCELL (ANR-21-CO14-0003-01), POPCELL-REG (ANR-22-CE12-0030-01) and COVIFERON (ANR-21-RHUS-0008), the EU HORIZON-HLTH-2021-DISEASE-04-07 grant UNDINE (no. 101057100), the French Government’s Investissement d’Avenir program, Laboratoires d’Excellence 'Integrative Biology of Emerging Infectious Diseases' (ANR-10- LABX-62-IBEID) and 'Milieu Intérieur' (ANR-10-LABX-69-01), the Fondation pour la Recherche Médicale (Equipe FRM DEQ20180339214), the Fondation Allianz-Institut de France, and the Fondation de France (no. 00106080). K.L., J.T.K.W. and M.P. are supported by the Health and Medical Research Fund Commissioned Research on the Novel Coronavirus Disease, Hong Kong SAR (COVID190126), S.A.V by the Theme-based Research Scheme of the Research Grants Council of the Hong Kong SAR (T11-705/21-N, SAV: T11-712/19-N), and M.P., R.B. and D.D. by InnoHK, an initiative of the Innovation and Technology Commission, the Government of the Hong Kong SAR., ANR-21-CO14-0003,COVID-19-POPCELL,Facteurs génétiques et infectieux à l'origine de la variabilité populationnelle de la réponse immunitaire à l'infection par le SARS-CoV-2(2021), ANR-22-CE12-0030,popCell-REG,Variation populationnelle des profils d'accessibilité de la chromatine à l'échelle unicellulaire(2022), ANR-21-RHUS-0008,COVIFERON,Covid-19 and interferons: from discovery to therapy(2021), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010)

Subjects
[SDV]Life Sciences [q-bio]
Abstract

The RNA sequencing data generated and analyzed in this study have been deposited in the Institut Pasteur data repository, OWEY, which can be accessed via the following link: https://doi.org/XXXX. The genome-wide genotyping data generated or used in this study have been deposited in OWEY and can be accessed at the following URL: https://doi.org/XXXX. Data access and use is restricted to academic research related to the variability of the human immune response.; Humans display vast clinical variability upon SARS-CoV-2 infection 1–3 , partly due to genetic and immunological factors 4 . However, the magnitude of population differences in immune responses to SARS-CoV-2 and the mechanisms underlying such variation remain unknown. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells from 222 healthy donors of various ancestries stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces a weaker, but more heterogeneous interferon-stimulated gene activity than influenza A virus, and a unique pro-inflammatory signature in myeloid cells. We observe marked population differences in transcriptional responses to viral exposure that reflect environmentally induced cellular heterogeneity, as illustrated by higher rates of cytomegalovirus infection, affecting lymphoid cells, in African-descent individuals. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell proportions on population differences in immune responses, with genetic variants having a narrower but stronger effect on specific loci. Additionally, natural selection has increased immune response differentiation across populations, particularly for variants associated with SARS-CoV-2 responses in East Asians. We document the cellular and molecular mechanisms through which Neanderthal introgression has altered immune functions, such as its impact on the myeloid response in Europeans. Finally, colocalization analyses reveal an overlap between the genetic architecture of immune responses to SARS-CoV-2 and COVID-19 severity. Collectively, these findings suggest that adaptive evolution targeting immunity has also contributed to current disparities in COVID-19 risk.

Published
2023

9. Human type I IFN deficiency does not impair B cell response to SARS-CoV-2 mRNA vaccination

Author

Aurélien Sokal, Paul Bastard, Pascal Chappert, Giovanna Barba-Spaeth, Slim Fourati, Alexis Vanderberghe, Pauline Lagouge-Roussey, Isabelle Meyts, Adrian Gervais, Magali Bouvier-Alias, Imane Azzaoui, Ignacio Fernández, Andréa de la Selle, Qian Zhang, Lucy Bizien, Isabelle Pellier, Agnès Linglart, Anya Rothenbuhler, Estelle Marcoux, Raphael Anxionnat, Nathalie Cheikh, Juliane Léger, Blanca Amador-Borrero, Fanny Fouyssac, Vanessa Menut, Jean-Christophe Goffard, Caroline Storey, Caroline Demily, Coralie Mallebranche, Jesus Troya, Aurora Pujol, Marie Zins, Pierre Tiberghien, Paul E. Gray, Peter McNaughton, Anna Sullivan, Jane Peake, Romain Levy, Laetitia Languille, Carlos Rodiguez-Gallego, Bertrand Boisson, Sébastien Gallien, Bénédicte Neven, Marc Michel, Bertrand Godeau, Laurent Abel, Felix A. Rey, Jean-Claude Weill, Claude-Agnès Reynaud, Stuart G. Tangye, Jean-Laurent Casanova, Matthieu Mahévas, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Rockefeller University [New York], Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), CHU Henri Mondor [Créteil], Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Centre Hospitalier Universitaire d'Angers (CHU Angers), PRES Université Nantes Angers Le Mans (UNAM), Université d'Angers (UA), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Université Paris-Saclay, Hôpital Nord Franche-Comté [Hôpital de Trévenans] (HNFC), Service de pédiatrie [CHRU Besançon], Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), AP-HP. Nord - Université Paris Cité, Université Paris Cité (UPCité), Hôpital Lariboisière-Fernand-Widal [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Centre Hospitalier Universitaire de Nancy (CHU Nancy), Hôpital Mère-Enfant [Hôpital Hôtel Dieu, Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Université libre de Bruxelles (ULB), Hôpital Robert Debré, Centre Hospitalier le Vinatier [Bron], Hospital Universitario Infanta Leonor [Madrid], Institut d'Investigació Biomèdica de Bellvitge [Barcelone] (IDIBELL), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Etablissement Français du Sang [La Plaine Saint-Denis] (EFS), Sydney Children's hospital, University of New South Wales [Sydney] (UNSW), Children’s Health Queensland [Brisbane] (CHQ), CHU Necker - Enfants Malades [AP-HP], Universidad Fernando Pessoa Canarias [Las Palmas de Gran Canaria, Spain], Garvan Institute of medical research, Howard Hughes Medical Institute [New York] (HHMI), Howard Hughes Medical Institute (HHMI)-New York University School of Medicine, NYU System (NYU)-NYU System (NYU)-Rockefeller University [New York]-Columbia University Irving Medical Center (CUIMC), Funder(s): Agence Nationale de la Recherche Award Id(s): ANR-21-RHUS-0008, ANR-10-IAHU-01, ANR-10-LABX-62-IBEID , ANR-20-CE93-003Funder(s): Fondation pour la Recherche MédicaleAward Id(s): MEMO-COV-2-FRMFunder(s): Institut PasteurFunder(s): Agence Nationale de Recherches sur le Sida et les Hépatites ViralesFunder(s): Howard Hughes Medical InstituteFunder(s): Rockefeller UniversityFunder(s): St. Giles FoundationFunder(s): National Institutes of Health Award Id(s): R01AI088364, R01AI163029, UL1TR001866Funder(s): National Center for Advancing Translational SciencesFunder(s): Fisher Center for Alzheimer’s Research FoundationFunder(s): Meyer FoundationFunder(s): JPB FoundationFunder(s): French Foundation for Medical Research Award Id(s): EQU201903007798, EA20170638020Funder(s): European Union’s Horizon 2020 Award Id(s): 824110Funder(s): Square FoundationFunder(s): Fondation du SouffleFunder(s): French Ministry of Higher Education, Research, and Innovation Award Id(s): MESRI-COVID-19Funder(s): Institut National de la Santé et de la Recherche MédicaleFunder(s): Fondation Bettencourt SchuellerFunder(s): CSL BehringFunder(s): KU Leuven Award Id(s): C16/18/007Funder(s): http://dx.doi.org/10.13039/501100003130 Award Id(s): G0C8517N, G0B5120N, G0E8420NFunder(s): Jeffrey Modell FoundationFunder(s): National Health and Medical Research Council Award Id(s): 1176665Funder(s): Allergy and Immunology Foundation of AustraliaFunder(s): John Brown Cook FoundationFunder(s): Fondation Princesse GraceFunder(s): Comité ad-hoc de pilotage national des essais thérapeutiques et autres recherchesFunder(s): Assistance Publique—Hôpitaux de Paris Award Id(s): MEMO-COV-2Funder(s): Société Nationale de Médecine InterneFunder(s): ANRS Nord-Sud Award Id(s): ANRS-COV05Funder(s): ANR-RHU Award Id(s): ANR-21-RHUS-08 (COVIFERON)Funder(s): HORIZON-HLTH-2021-DISEASE-04 Award Id(s): 01057100Funder(s): Grandir - Fonds de solidarité pour l’enfanceFunder(s): SCOR Corporate Foundation for ScienceFunder(s): REACTing-INSERMFunder(s): University of Paris CitéFunder(s): Imagine InstituteFunder(s): VIB GC PIDFunder(s): European Research Council Award Id(s): ERC-StG MORE2ADA2, ANR-21-RHUS-0008,COVIFERON,Covid-19 and interferons: from discovery to therapy(2021), ANR-10-IAHU-0001,Imagine,Institut Hospitalo-Universitaire Imagine(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-20-CE93-0003,GENVIR,Analyse multi-omique de l'immunité anti-virale: de l'identification des circuits biologiques pertinents à la découverte de défauts monogéniques héréditaires de l'immunité chez les patients avec infections virales sévères(2020), European Project: 824110,H2020-INFRAIA-2018-1,EASI-Genomics(2019), European Project: 948959,ERC-2020-STG,MORE2ADA2(2021), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and CHU Henri Mondor

Subjects
EXPRESSION, COVID-19 Vaccines, Immunology, VECTOR, Research & Experimental Medicine, Antibodies, Viral, DISEASE, Immunology and Allergy, Humans, PSEUDOURIDINE, Autoantibodies, B-Lymphocytes, Science & Technology, SARS-CoV-2, Vaccination, COVID-19, Antibodies, Neutralizing, Medicine, Research & Experimental, Toll-Like Receptor 7, ANTIBODIES, Spike Glycoprotein, Coronavirus, Interferon Type I, [SDV.IMM]Life Sciences [q-bio]/Immunology, mRNA Vaccines, [SDV.IMM.VAC]Life Sciences [q-bio]/Immunology/Vaccinology, Life Sciences & Biomedicine
Abstract

Inborn and acquired deficits of type I interferon (IFN) immunity predispose to life-threatening COVID-19 pneumonia. We longitudinally profiled the B cell response to mRNA vaccination in SARS-CoV-2 naive patients with inherited TLR7, IRF7, or IFNAR1 deficiency, as well as young patients with autoantibodies neutralizing type I IFNs due to autoimmune polyendocrine syndrome type-1 (APS-1) and older individuals with age-associated autoantibodies to type I IFNs. The receptor-binding domain spike protein (RBD)-specific memory B cell response in all patients was quantitatively and qualitatively similar to healthy donors. Sustained germinal center responses led to accumulation of somatic hypermutations in immunoglobulin heavy chain genes. The amplitude and duration of, and viral neutralization by, RBD-specific IgG serological response were also largely unaffected by TLR7, IRF7, or IFNAR1 deficiencies up to 7 mo after vaccination in all patients. These results suggest that induction of type I IFN is not required for efficient generation of a humoral response against SARS-CoV-2 by mRNA vaccines. ispartof: JOURNAL OF EXPERIMENTAL MEDICINE vol:220 issue:1 ispartof: location:United States status: published

Published
2023

10. Tunneling nanotubes provide a novel route for SARS-CoV-2 spreading between permissive cells and to non-permissive neuronal cells

Author

Anna Pepe, Stefano Pietropaoli, Matthijn Vos, Giovanna Barba-Spaeth, Chiara Zurzolo, Trafic membranaire et Pathogénèse - Membrane Traffic and Pathogenesis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Virologie Structurale - Structural Virology, Catalent Phama Solution, Plateforme technologique Nanoimagerie / Nanoimaging Technological Platform, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), The NanoImaging Core was created with the help of a grant from the French Government’s Investissements d’Avenir658program (EQUIPEX CACSICE - Centre d’analyse de systèmes complexes dans les environnements complexes, ANR-11-EQPX-0008). We would like to thank Jean-Marie Winter (NanoImaging Core at Institut Pasteur). This work was supported by the « URGENCE COVID-19 » fundraising campaign of Institut Pasteur (Paris), ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Institut Pasteur [Paris]-Université Paris Cité (UPCité)

Subjects
chemistry.chemical_classification, Chemistry, viruses, fungi, Endocytosis, Entry into host, Exocytosis, In vitro, Cell biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Permissive, Glycoprotein, Intracellular, Actin
Abstract

SARS-CoV-2 entry into host cells is mediated by the binding of its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor, highly expressed in several organs, but very low in the brain. The mechanism through which SARS-CoV-2 infects neurons is not understood. Tunneling nanotubes (TNTs), actin-based intercellular conduits that connect distant cells, allow the transfer of cargos, including viruses. Here, we explored the neuroinvasive potential of SARS-CoV-2 and whether TNTs are involved in its spreading between cellsin vitro. We report that neuronal cells, not permissive to SARS-CoV-2 through an exocytosis/endocytosis dependent pathway, can be infected when co-cultured with permissive infected epithelial cells. SARS-CoV-2 induces TNTs formation between permissive cells and exploits this route to spread to uninfected permissive cells in co-culture. Correlative Cryo-electron tomography reveals that SARS-CoV-2 is associated with the plasma membrane of TNTs formed between permissive cells and virus-like vesicular structures are inside TNTs established both between permissive cells and between permissive and non-permissive cells. Our data highlight a potential novel mechanism of SARS-CoV-2 spreading which could serve as route to invade non-permissive cells and potentiate infection in permissive cells.

Published
2022

11. Analysis of mRNA vaccination-elicited RBD-specific memory B cells reveals strong but incomplete immune escape of the SARS-CoV-2 Omicron variant

Author

Aurélien Sokal, Matteo Broketa, Giovanna Barba-Spaeth, Annalisa Meola, Ignacio Fernández, Slim Fourati, Imane Azzaoui, Andrea de La Selle, Alexis Vandenberghe, Anais Roeser, Magali Bouvier-Alias, Etienne Crickx, Laetitia Languille, Marc Michel, Bertrand Godeau, Sébastien Gallien, Giovanna Melica, Yann Nguyen, Virginie Zarrouk, Florence Canoui-Poitrine, France Noizat-Pirenne, Jérôme Megret, Jean-Michel Pawlotsky, Simon Fillatreau, Etienne Simon-Lorière, Jean-Claude Weill, Claude-Agnès Reynaud, Félix A. Rey, Pierre Bruhns, Pascal Chappert, Matthieu Mahévas, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Virologie Structurale - Structural Virology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Hôpital Henri Mondor, Immunologie et Neurogénétique Expérimentales et Moléculaires (INEM), Université d'Orléans (UO)-Centre National de la Recherche Scientifique (CNRS), Hôpital Beaujon [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Structure Fédérative de Recherche Necker (SFR Necker - UMS 3633 / US24), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Institut Pasteur [Paris]-Université Paris Cité (UPCité), This work was initiated by a grant from the Agence Nationale de la Recherche and the Fondation pour la Recherche Médicale (ANR and MEMO-COV-2-FRM) and funded by the Fondation Princesse Grace, an ERC Advanced Investigator Grant (B-response), and a grant from the French Ministry of Health (Soutien Exceptionnel à la Recherche Clinique 2022, CAPNET [Comité ad-hoc de pilotage national des essais thérapeutiques et autres recherches]). Assistance Publique – Hôpitaux de Paris (AP-HP, Département de la Recherche Clinique et du Développement) was the promotor and the sponsor of MEMO-COV-2. Work in the Unit of Structural Virology was funded by Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur. A.S. was supported by a Poste d’accueil from INSERM, I.F. by a fellowship from the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS), M.B. by a CIFRE fellowship from the Association Nationale de la Recherche et de la Technologie (ANRT), and A.D.L.S by a SNFMI fellowship. P.B. acknowledges funding from the French National Research Agency grant ANR-14-CE16-0011 project DROPmAbs, the Institut Carnot Pasteur Microbes et Santé (ANR 11 CARN 0017-01), the Institut Pasteur, and the Institut National de la Santé et de la Recherche Médicale (INSERM)., We thank Garnett Kelsoe for providing the human cell culture system, together with invaluable advice, A. Boucharlat and the Chemogenomic and Biological Screening Core Facility headed by F. Agou, as well as P. England and the Molecular Biophysics Core Facility at the Institut Pasteur, Paris, France for their support during the course of this work, Sébastien Storck, Lucie Da Silva, and Sandra Weller for their advice and support, the physicians, Constance Guillaud, Raphael Lepeule, Frédéric Schlemmer, Elena Fois, Henri Guillet, Nicolas De Prost, and Pascal Lim, whose patients were included in this study, and Florence Guivel-Benhassine and Olivier Schwartz from the Institut Pasteur for providing authentic SARS-CoV-2 B.1.1.529 virus., ANR-20-COVI-0072,MEMO-COV-2,Lymphocytes B et T CD4 mémoires spécifiques du virus chez les patients guéris du Covid-19(2020), ANR-14-CE16-0011,DROP-mAbs,Analyse en profondeur de répertoires d'anticorps par microfluidique en gouttelettes couplé à du séquençage haut-débit pour le diagnostic et la découverte d'anticorps thérapeutiques(2014), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), IMRB - CEPIA/'Clinical Epidemiology And Ageing : Geriatrics, Primary Care and Public Health' [Créteil] (U955 Inserm - UPEC), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), and Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité)

Subjects
B cells, MBC, variants, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Omicron, SARS-CoV-2, VOC, Immunology, Vaccination, COVID-19, Antibodies, Viral, Antibodies, Neutralizing, Infectious Diseases, mRNA vaccine, germinal center, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Spike Glycoprotein, Coronavirus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, memory B cells, Immunology and Allergy, Humans, affinity, RNA, Messenger, [SDV.IMM.VAC]Life Sciences [q-bio]/Immunology/Vaccinology
Abstract

International audience; The SARS-CoV-2 Omicron variant can escape neutralization by vaccine-elicited and convalescent antibodies. Memory B cells (MBCs) represent another layer of protection against SARS-CoV-2, as they persist after infection and vaccination and improve their affinity. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant remains unclear. We assessed the affinity and neutralization potency against the Omicron variant of several hundred naturally expressed MBC-derived monoclonal IgG antibodies from vaccinated COVID-19-recovered and -naive individuals. Compared with other variants of concern, Omicron evaded recognition by a larger proportion of MBC-derived antibodies, with only 30% retaining high affinity against the Omicron RBD, and the reduction in neutralization potency was even more pronounced. Nonetheless, neutralizing MBC clones could be found in all the analyzed individuals. Therefore, despite the strong immune escape potential of the Omicron variant, these results suggest that the MBC repertoire generated by mRNA vaccines still provides some protection against the Omicron variant in vaccinated individuals.

Published
2022

12. Immune escape of SARS-CoV-2 Omicron variant from mRNA vaccination-elicited RBD-specific memory B cells

Author

Aurélien Sokal, Matteo Broketa, Annalisa Meola, Giovanna Barba-Spaeth, Ignacio Fernández, Slim Fourati, Imane Azzaoui, Andrea de La Selle, Alexis Vandenberghe, Anais Roeser, Magali Bouvier-Alias, Etienne Crickx, Laetitia Languille, Marc Michel, Bertrand Godeau, Sébastien Gallien, Giovanna Melica, Yann Nguyen, Virginie Zarrouk, Florence Canoui-Poitrine, France Noizat-Pirenne, Jérôme Megret, Jean-Michel Pawlotsky, Simon Fillatreau, Etienne Simon-Lorière, Jean-Claude Weill, Claude-Agnès Reynaud, Félix A. Rey, Pierre Bruhns, Pascal Chappert, and Matthieu Mahévas

Abstract

SummaryMemory B cells (MBCs) represent a second layer of immune protection against SARS-CoV-2. Whether MBCs elicited by mRNA vaccines can recognize the Omicron variant is of major concern. We used bio-layer interferometry to assess the affinity against the receptor-binding-domain (RBD) of Omicron spike of 313 naturally expressed monoclonal IgG that were previously tested for affinity and neutralization against VOC prior to Omicron. We report here that Omicron evades recognition from a larger fraction of these antibodies than any of the previous VOCs. Additionally, whereas 30% of these antibodies retained high affinity against Omicron-RBD, our analysis suggest that Omicron specifically evades antibodies displaying potent neutralizing activity against the D614G and Beta variant viruses. Further studies are warranted to understand the consequences of a lower memory B cell potency on the overall protection associated with current vaccines.

Published
2021

13. mRNA vaccination of naive and COVID-19-recovered individuals elicits potent memory B cells that recognize SARS-CoV-2 variants

Author

Aurélien Sokal, Giovanna Barba-Spaeth, Ignacio Fernández, Matteo Broketa, Imane Azzaoui, Andréa de La Selle, Alexis Vandenberghe, Slim Fourati, Anais Roeser, Annalisa Meola, Magali Bouvier-Alias, Etienne Crickx, Laetitia Languille, Marc Michel, Bertrand Godeau, Sébastien Gallien, Giovanna Melica, Yann Nguyen, Virginie Zarrouk, Florence Canoui-Poitrine, France Pirenne, Jérôme Mégret, Jean-Michel Pawlotsky, Simon Fillatreau, Pierre Bruhns, Felix A. Rey, Jean-Claude Weill, Claude-Agnès Reynaud, Pascal Chappert, Matthieu Mahévas, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Service de médecine interne [Mondor], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Henri Mondor-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Virologie Structurale - Structural Virology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Anticorps en thérapie et pathologie - Antibodies in Therapy and Pathology, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Collège Doctoral, Sorbonne Université (SU), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Service de bactériologie, virologie, hygiène [Mondor], Hôpital Henri Mondor, Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Hôpital Beaujon [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Clinical Epidemiology and Ageing : Geriatrie Soins Primaires et Santé Publique (CEpiA), Etablissement Français du Sang, Structure Fédérative de Recherche Necker (SFR Necker - UMS 3633 / US24), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Inovarion, This work was initiated by a grant from the Agence Nationale de la Recherche and the Fondation pour la Recherche Médicale (ANR, MEMO-COV-2 -FRM) and funded by the Fondation Princesse Grace and by an ERC Advanced Investigator Grant (B-response). Assistance Publique – Hôpitaux de Paris (AP-HP, Département de la Recherche Clinique et du Développement) was the promotor and the sponsor of MEMO-COV-2. Work in the Unit of Structural Virology was funded by the Institut Pasteur Urgence COVID-19 Fundraising Campaign. A.S. was supported by a Poste d’Accueil from the Institut National de la Santé et de la Recherche Médicale (INSERM), I.F. by a fellowship from the Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ANRS), M.B. by a CIFRE fellowship from the Association Nationale de la Recherche et de la Technologie (ANRT), and A.d.L.S by a SNFMI fellowship. P.B. acknowledges funding from ANR (ANR-14-CE16-0011–DROPmAbs), the Institut Carnot Pasteur Microbes et Santé (ANR 11 CARN 0017-01), the Institut Pasteur, and INSERM., We thank Garnett Kelsoe for providing the human cell culture system and invaluable advice. We thank A. Boucharlat and the Chemogenomic and Biological screening core facility headed by F. Agou as well as P. England and the Molecular Biophysics core facility at the Institut Pasteur, Paris, France for support during the course of this work. We also thank Sébastien Storck, Lucie Da Silva, and Sandra Weller for advices and support and the physicians, Constance Guillaud, Raphael Lepeule, Frédéric Schlemmer, Elena Fois, Henri Guillet, Nicolas De Prost, and Pascal Lim, whose patients were included in this study., ANR-20-COVI-0072,MEMO-COV-2,Lymphocytes B et T CD4 mémoires spécifiques du virus chez les patients guéris du Covid-19(2020), ANR-14-CE16-0011,DROP-mAbs,Analyse en profondeur de répertoires d'anticorps par microfluidique en gouttelettes couplé à du séquençage haut-débit pour le diagnostic et la découverte d'anticorps thérapeutiques(2014), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Collège doctoral [Sorbonne universités], Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), IMRB - CEPIA/'Clinical Epidemiology And Ageing : Geriatrics, Primary Care and Public Health' [Créteil] (U955 Inserm - UPEC), and Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)

Subjects
Immunology, Antibody Affinity, Immunization, Secondary, Somatic hypermutation, Antibodies, Viral, Mass Vaccination, Article, plasma cells, RBD, Affinity maturation, 03 medical and health sciences, 0302 clinical medicine, Memory B Cells, Animals, Humans, Immunology and Allergy, Potency, B-cell memory, RNA, Messenger, Neutralizing antibody, Memory B cell, BNT162 Vaccine, Cells, Cultured, 030304 developmental biology, affinity maturation, 0303 health sciences, BNT162b2 vaccine, biology, SARS-CoV-2, Precursor Cells, B-Lymphoid, Germinal center, COVID-19, neutralizing antibody, Convalescence, Antibodies, Neutralizing, 3. Good health, somatic hypermutation, Vaccination, Infectious Diseases, germinal center, Spike Glycoprotein, Coronavirus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, biology.protein, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Antibody, [SDV.IMM.VAC]Life Sciences [q-bio]/Immunology/Vaccinology, Immunologic Memory, 030217 neurology & neurosurgery
Abstract

In addition to serum immunoglobulins, memory B cell (MBC) generation against SARS-CoV-2 represents another layer of immune protection, but the quality of MBC responses in naive and COVID-19-recovered individuals after vaccination remains ill-defined. We studied longitudinal cohorts of naive individuals and disease-recovered patients for up to 2 months after SARS-CoV-2 mRNA vaccination. We assessed the quality of the memory response by analysis of VDJ repertoires, affinity and neutralization against variants of concern (VOCs), using unbiased cultures of 2452 MBCs. Upon boosting, the MBC pool of recovered patients selectively expanded, further matured and harbored potent neutralizers against VOCs. Although naïve individuals had weaker neutralizing serum responses, half of their RBD-specific MBCs displayed high affinity towards multiple VOCs, including delta (B.1.617.2), and one-third retained neutralizing potency against beta (B.1.351). Our data suggest that an additional challenge in naive vaccinees could recall such affinity-matured MBCs and allow them to respond efficiently to VOCs., Graphical Abstract, To better understand B cell responses to SARS-CoV-2 mRNA vaccination, Sokal et al. analyzed memory B cells from COVID-19-recovered and -naive individuals. In recovered patients, vaccination amplifies a broad repertoire of matured MBCs and generates variant-neutralizing plasma cells. In naïve individuals, vaccination induces an MBC pool containing potent neutralizing clones against all current variants of concern, including beta and delta.

Published
2021

14. Memory B cells control SARS-CoV-2 variants upon mRNA vaccination of naive and COVID-19 recovered individuals

Author

Noizat-Pirenne F, Annalisa Meola, Claude-Agnès Reynaud, Magali Bouvier-Alias, Laetitia Languille, M. Broketa, Aurélien Sokal, Simon Fillatreau, Jérôme Mégret, Matthieu Mahévas, Anaïs Roeser, Jean-Claude Weill, Alexis Vandenberghe, Bertrand Godeau, Marc Michel, Giovanna Melica, Pascal Chappert, Slim Fourati, Zarrouk, Félix A. Rey, Etienne Crickx, Giovanna Barba-Spaeth, S. Gallien, Imane Azzaoui, Yann Nguyen, Florence Canoui-Poitrine, La Selle Ad, Bruhns P, Jean-Michel Pawlotsky, and Ignacio Fernandez

Subjects
Vaccination, Messenger RNA, Coronavirus disease 2019 (COVID-19), Repertoire, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Immunology, Potency, Biology, Memory B cell, Neutralization
Abstract

SummaryHow a previous SARS-CoV-2 infection may amplify and model the memory B cell (MBC) response elicited by mRNA vaccines was addressed by a comparative longitudinal study of two cohorts, naive individuals and disease-recovered patients, up to 2 months after vaccination. The quality of the memory response was assessed by analysis of the VDJ repertoire, affinity and neutralization against variants of concerns (VOC), using unbiased cultures of 2452 MBCs. Upon boost, the MBC pool of recovered patients selectively expanded, further matured and harbored potent neutralizers against VOC. Maturation of the MBC response in naive individuals was much less pronounced. Nevertheless, and as opposed to their weaker neutralizing serum response, half of their RBD-specific MBCs displayed high affinity towards multiple VOC and one-third retained neutralizing potency against B.1.351. Thus, repeated vaccine challenges could reduce these differences by recall of affinity-matured MBCs and allow naive vaccinees to cope efficiently with VOC.

Published
2021

15. Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

Author

Pierre Isnard, Giovanna Barba-Spaeth, Gérard Pierron, Christian Weber, Etienne Simon-Loriere, Sylvie Souquere, Christophe Zimmer, Jean-Baptiste Gibier, Stefano Pietropaoli, Marie-Anne Rameix-Welti, Marion Rabant, Florian Mueller, Elena Rensen, Imagerie et Modélisation - Imaging and Modeling, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Virologie Structurale - Structural Virology, Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Anatomie et Cytologie Pathologiques, CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Necker-Enfants Malades, Service d'anatomopathologie, Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Institut Pasteur [Paris], Université Paris-Saclay, Hôpital Ambroise Paré [AP-HP], Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiologie et physiopathologie des rétrovirus endogènes et infectieux (RETRO-ENDO), Institut Gustave Roussy (IGR)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), ESL acknowledges funding from the French Government’s Investissement d’Avenir program, ‘INCEPTION’ (ANR-16-CONV-0005). GBS acknowledges funding by the Institut Pasteur Coronavirus task force (don AXA COVID-19 project COVID-SPREAD). This work was funded by Institut Pasteur., and ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016)

Subjects
0303 health sciences, medicine.diagnostic_test, biology, Coronavirus disease 2019 (COVID-19), Oligonucleotide, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), viruses, RNA, RNA virus, biology.organism_classification, Virology, 3. Good health, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), medicine, In patient, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, 030217 neurology & neurosurgery, 030304 developmental biology, Fluorescence in situ hybridization
Abstract

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize viral RNA directly in infected cells are critical to analyze its replication cycle, screen for therapeutic molecules or study infections in human tissue. Here, we report the design, validation and initial application of fluorescencein situhybridization (FISH) probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy (EM). We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening and diagnostics.

Published
2021

16. Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

Author

Mickaël M. Ménager, Marine Luka, Marc Michel, Sophie Hue, Slim Fourati, Aurélien Sokal, Simon Fillatreau, Claude-Agnès Reynaud, Giovanna Barba-Spaeth, Annalisa Meola, Laetitia Languille, Jean-Michel Pawlotsky, Matthieu Mahévas, Jérôme Mégret, Félix A. Rey, Etienne Crickx, Asma Beldi-Ferchiou, Magali Bouvier-Alias, Jean-Claude Weill, Imane Azzaoui, Alexis Vandenberghe, Pascal Chappert, Samia Baloul, Anaïs Roeser, Ignacio Fernandez, Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Service de médecine interne [Mondor], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Henri Mondor-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Inovarion, Virologie Structurale - Structural Virology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Service de bactériologie, virologie, hygiène [Mondor], Molecular virology and immunology – Physiopathology and therapeutic of chronic viral hepatitis (Team 18) (Inserm U955), Institut Mondor de Recherche Biomédicale (IMRB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10, IMRB - 'Transfusion et Maladies du Globule Rouge' [Créteil] (U955 Inserm - UPEC), Hôpital Henri Mondor, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-IFR10, Vaccine Research Institute (VRI), Physiopathologie et immunothérapies dans l’infection VIH [Créteil] (Inserm U955 Équipe 16), Clinical Epidemiology and Ageing : Geriatrie Soins Primaires et Santé Publique (CEpiA), Inflammatory Responses and Transcriptomic Networks in diseases (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Structure Fédérative de Recherche Necker (SFR Necker - UMS 3633 / US24), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), This work was supported by a grant from the Agence Nationale de la Recherche and the Fondation pour la Recherche Médicale (ANR, MEMO-COV-2), by the Fondation Princesse Grace, and, in part by an ERC Advanced Investigator Grant (B-response). Assistance Publique – Hôpitaux de Paris (AP-HP, Département de la Recherche Clinique et du Développement) was the promotor and the sponsor of MEMO-COV-2. A.S. was supported by a Poste d’Accueil from Inserm, A.R. by an Année Recherche from AP-HP and by a SNFMI fellowship., ANR-20-COVI-0072,MEMO-COV-2,Lymphocytes B et T CD4 mémoires spécifiques du virus chez les patients guéris du Covid-19(2020), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR10-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Vaccine Research Institute [Créteil, France] (VRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
Adult, Somatic cell, Cell, Somatic hypermutation, Context (language use), Biology, Lymphocyte Activation, spike protein, Severity of Illness Index, Article, General Biochemistry, Genetics and Molecular Biology, plasma cells, RBD, 03 medical and health sciences, 0302 clinical medicine, Single-cell analysis, medicine, Humans, neutralizing antibodies, Memory B cell, B cell, 030304 developmental biology, B-Lymphocytes, 0303 health sciences, Germinal center, COVID-19, OC43, Middle Aged, Flow Cytometry, 3. Good health, somatic hypermutation, medicine.anatomical_structure, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, germinal center, Spike Glycoprotein, Coronavirus, Immunology, extrafollicular response, Single-Cell Analysis, HKU1, Immunologic Memory, 030217 neurology & neurosurgery
Abstract

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection., Graphical abstract, The B cell response against SARS-CoV-2 shows a temporal shift from an extrafollicular reaction that includes memory B cells against seasonal coronaviruses toward a germinal center-dependent memory response encoding (spike-specific) neutralizing antibodies.

Published
2021

17. Maturation and persistence of the anti-SARS-CoV-2 memory B cell response

Author

Aurélien Sokal, Pascal Chappert, Anais Roeser, Giovanna Barba-Spaeth, Slim Fourati, Imane Azzaoui, Alexis Vandenberghe, Ignacio Fernandez, Magali Bouvier-Alias, Etienne Crickx, Asma Beldi Ferchiou, Sophie Hue, Laetitia Languille, Samia Baloul, France Noizat-Pirenne, Marine Luka, Jérôme Megret, Mickaël Ménager, Jean-Michel Pawlotsky, Simon Fillatreau, Felix A Rey, Jean-Claude Weill, Claude-Agnès Reynaud, and Matthieu Mahévas

Subjects
medicine.anatomical_structure, Somatic cell, Repertoire, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Cell, Immunology, medicine, Germinal center, Biology, Memory B cell, Gene, B cell
Abstract

Memory B cells play a fundamental role in host defenses against viruses, but to date, their role have been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 Spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including preexisting cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late remarkably stable memory B-cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.

Published
2020

18. Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH

Author

Elena Rensen, Stefano Pietropaoli, Florian Mueller, Christian Weber, Sylvie Souquere, Sina Sommer, Pierre Isnard, Marion Rabant, Jean-Baptiste Gibier, Fabiola Terzi, Etienne Simon-Loriere, Marie-Anne Rameix-Welti, Gérard Pierron, Giovanna Barba-Spaeth, Christophe Zimmer, Imagerie et Modélisation - Imaging and Modeling, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), National Cystic Fibrosis Reference Center [CHU Necker] (CNR - INSERM U1151), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), CHU Lille, Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Infection et inflammation (2I), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Hôpital Ambroise Paré [AP-HP], Institut Gustave Roussy (IGR), Virologie Structurale - Structural Virology, E Simon-Loriere acknowledges funding from the French Government’s Investissement d’Avenir program, 'INCEPTION’ (ANR-16-CONV-0005). G Barba-Spaeth acknowledges funding by the Institut Pasteur Coronavirus task force (don AXA COVID-19 project COVID-SPREAD). C Zimmer acknowledges funding by Fondation pour la Recherche Médicale (Equipe FRM, DEQ 20150331762), Institut Carnot Pasteur MS, and Institut Pasteur., We would like to thank Edouard Bertrand, who originally developed smiFISH and Hans Johansson for insightful discussions. We also thank Guillaume Dumenil (Ultrastructural Bioimaging UTechS of Institut Pasteur) for having suggested the application of CoronaFISH to EM and for follow-up discussions. We thank Nathalie Jolly and Nathalie Clément (Center for Translational Science, Institut Pasteur) and Cloé Giquel (Legal department, Institut Pasteur) for their help in obtaining authorization to use patient samples., ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016), Barba Spaeth, Giovanna, Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs - - INCEPTION2016 - ANR-16-CONV-0005 - CONV - VALID, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université de Paris (UP), Département Croissance et Signalisation [Paris], Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Pasteur [Paris], and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)

Subjects
[SDV]Life Sciences [q-bio], viruses, Health, Toxicology and Mutagenesis, Plant Science, Virus Replication, Antiviral Agents, Sensitivity and Specificity, Biochemistry, Genetics and Molecular Biology (miscellaneous), 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, Chlorocebus aethiops, Animals, Humans, Vero Cells, In Situ Hybridization, In Situ Hybridization, Fluorescence, Virus Release, Research Articles, 030304 developmental biology, 0303 health sciences, Alanine, Ecology, SARS-CoV-2, COVID-19, Reproducibility of Results, respiratory system, Adenosine Monophosphate, respiratory tract diseases, COVID-19 Drug Treatment, 3. Good health, [SDV] Life Sciences [q-bio], Microscopy, Electron, RNA, Viral, Caco-2 Cells, 030217 neurology & neurosurgery, Research Article
Abstract

We design an RNA-FISH method to target the plus and minus strands of SARS-CoV-2 RNA, and apply it to image the virus in cell lines, lung tissue, and nasal swabs, by fluorescence and electron microscopy., The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

Published
2022

19. La vaccination par ARNm des sujets naïfs ou convalescents de la COVID-19 permet la génération de lymphocytes B mémoires capables de reconnaître et de neutraliser les variants du SARS-CoV-2

Author

Pierre Bruhns, Marc Michel, Alexis Vandenberghe, Yann Nguyen, Ignacio Fernandez, Giovanna Barba-Spaeth, M. Broketa, Félix A. Rey, Anaïs Roeser, Aurélien Sokal, Slim Fourati, Bertrand Godeau, C.A. Reynaud, Virginie Zarrouk, A. De La Selle, Matthieu Mahevas, Pascal Chappert, Imane Azzaoui, J.C. Weill, and Etienne Crickx

Subjects
2019-20 coronavirus outbreak, Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Gastroenterology, Internal Medicine, Co087, Biology, Molecular biology
Abstract

Introduction La mémoire immunitaire est un mécanisme qui protège les individus contre la réinfection. Cette stratégie de défense de l’organisme, qui est à la base du succès des vaccins, comprend la production d’anticorps protecteurs dans le sang ainsi que la formation de cellules à mémoire, capables de se réactiver rapidement en cellules productrices d’anticorps lors d’une nouvelle infection. Les vaccins à ARNm, codant pour la protéine Spike du SARS-CoV-2, ont été rapidement déployés dans le monde entier, avec une grande efficacité clinique. Déterminer les caractéristiques de la réponse lymphocytaire B mémoire générée par ces vaccins est d’une importance majeure, notamment dans le contexte de circulation de variants du SARS-CoV-2, porteurs de mutations dans la protéine Spike. Nous avons étudié la dynamique, l’évolution clonale, et l’affinité des cellules B à mémoire chez des patients vaccinés par le vaccin à ARNm, ainsi que leur capacité à reconnaître et à neutraliser les variants du SARS-CoV-2 dans deux cohortes longitudinales de patients, l’une infectée lors de la première vague (convalescents-vaccinés), et l’autre n’ayant pas été infectée (naïfs-vaccinés). Patients et méthodes Les patients infectés lors de la première vague de la pandémie ont été inclus dans l’étude MEMO-CoV2. Une partie de ces patients avec une forme sévère hospitalisée, ou une forme modérée ambulatoire ont reçu une dose de vaccin à ANR messager (BNT162b2) un an après l’infection. De façon parallèle, une cohorte de soignants, naïfs de toute infection et avec une sérologie négative, ont été vaccinés avec deux doses de vaccin à ARNm. Ces deux cohortes ont été suivies et analysées (Sérologie, Cytométrie en flux des cellules B) longitudinalement jusqu’à 2 mois après le boost vaccinal (première injection pour les convalescents, deuxième injection pour les naïfs). Les cellules B mémoires spécifiques du domaine RBD de la protéine Spike ont été isolées, triées et cultivées en cellule unique. Pour chaque cellule anti-RBD mémoire, nous avons séquencé la chaîne lourde de l’immunoglobuline et nous avons déterminé l’affinité par Biolayer-interferometry des anticorps produits contre des variants préoccupants(α, β, γ et δ). Nous avons aussi déterminé pour certains clones leur capacité à neutraliser le virus D614G (dominant lors de la première vague) et β in vitro. Résultats La vaccination induisait une réponse sérologique IgG anti-RBD robuste chez tous les patients analysés (n = 47). L’activité neutralisante du sérum contre le virus D614G était excellente pour tous les patients. Néanmoins, la neutralisation sérique des variants β et δ était très nettement meilleure chez les patients déjà infectés, suggérant que les plasmocytes mobilisés lors du boost vaccinal proviennent de cellules mémoires matures. L’analyse en cytométrie en flux, a mis en évidence une expansion du pool mémoires chez les patients convalescents à un niveau supérieur à celui des naïfs. L’analyse de plus de 2400 séquences de la chaîne lourde de l’immunoglobuline provenant de cellules B mémoires anti-RBD cultivés en cellule unique, a révélé que la réponse vaccinale anti-RBD mobilise des cellules peu mutées, donc nouvellement générées, chez les individus naïfs. À l’inverse les cellules mémoires mobilisées après le boost chez patients convalescents arboraient de nombreuses mutations somatiques, témoignant de la mobilisation de mémoires préexistantes. L’analyse du répertoire des cellules B mémoires montrait que sa diversité était conservée après la vaccination malgré son expansion. Nous avons ensuite analysé l’affinité de 382 anticorps monoclonaux issus cellules B mémoires mobilisées par le boost vaccinal, contre le RBD de différent variants (α, β, γ, δ, κ). L’affinité des anticorps contre la RBD sauvage était plus forte chez les convalescents que chez les naïfs et corrélait avec le nombre de mutations somatiques dans la chaine lourde de l’immunoglobuline, reflétant le processus de maturation d’affinité. Des clones de très haute affinité contre tous les RBD variants étaient détectés chez tous les individus testés, y compris chez les naïfs, ainsi que des clones neutralisant le variant β, qui a la plus grande capacité à échapper à la réponse immune. Conclusion Chez les patients convalescents, la vaccination amplifie un large répertoire de cellules B mémoires matures et génère des plasmocytes neutralisant les variants. Chez les individus naïfs, la vaccination induit un pool de mémoire contenant des clones neutralisants puissants contre tous les variants préoccupants actuels, dont bêta et delta. Nos résultats suggèrent qu’une troisième dose chez les sujets naïfs permettrait de différentier en plasmocytes les lymphocytes B mémoires de grande qualité générés par le schéma vaccinal initial et ainsi d’augmenter l’activité neutralisante des sérums contre les variants du SARS-CoV-2.

Published
2021

20. La diversité du répertoire des cellules B mémoires fait face aux variants du SRAS-CoV2 après la vaccination par ARNm chez les individus naïfs et guéris

Author

Pascal Chappert, Aurélien Sokal, Giovanna Barba-Spaeth, Ignacio Fernandez, Matteo Broketta, Imane Azzaoui, Andrea De La Selle, Alexis Vandenberghe, Slim Fourati, Annalisa Meola, Magali Bouvier-Alias, Etienne Cricks, Laetitia Languille, Marc Michel, Bertrand Godeau, Florence Canoui-Poitrine, France Pirenne, Jérôme Megret, Jean-Michel Pawlotsky, Pierre Bruhns, Felix Rey, Jean-Claude Weill, Claude-Agnès Reynaud, and Matthieu Mahévas

Subjects
Biochemistry (medical), Clinical Biochemistry, Hematology
Published
2021

21. Inhibition of polyamine biosynthesis is a broad-spectrum strategy against RNA viruses

Author

Matthew L. Albert, Marie-Pascale Frenkiel, Anavaj Sakuntabhai, Carole Tamietti, Anna Yakovleva, Marco Vignuzzi, Francis Delpeyroux, Bryan C. Mounce, Sandrine Vitry, Scott W. Werneke, Etienne Simon-Loriere, Cécile Khou, Everett Clinton Smith, Mark R. Denison, Nathalie Pardigon, Marie Flamand, Peter Jan Hooikaas, Enzo Z. Poirier, Teresa Cesaro, Gonzalo Moratorio, Matthieu Prot, Monique Lafon, Romain Volle, Giovanna Barba-Spaeth, Populations virales et Pathogenèse - Viral Populations and Pathogenesis, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Immunobiologie des Cellules Dendritiques, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Vanderbilt University Medical Center [Nashville], Vanderbilt University [Nashville], Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Génétique fonctionnelle des Maladies infectieuses - Functional Genetics of Infectious Diseases, Virologie Structurale - Structural Virology, Neuro-Immunologie Virale - Viral Neuro-immunology, Biologie des virus entériques (BVE), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris] (IP), This work was funded by the Laboratoire d’Excellence via Integrative Biology of Emerging Infectious Diseases (grant no. ANR-10-LABX-62-IBEID to B.C.M. and M.V.), Equipe FRM DEQ20150331759 from the French Fondation pour la Recherche Médicale (to M.V.), Institut Pasteur Defeat Dengue Program (to B.C.M. and M.V.), Erasmus Unipharma-Graduates Program (to T.C.), Amgen Scholars Program at Institut Pasteur (to A.Y.), Het Weerstandfonds (to P.J.H), NIH (grant no.R01AI108197 to M.R.D.), NIH (grant no. U19 AI109680 to M.R.D.), InstitutPasteur Programme Transversal de Recherche (grant no. ZPTR484 to F.D.), Fondation Total (grant no. S-CM15010-05B to F.D.), Roux Howard Cantarini postdoctoral fellowship (to R.V.), Seventh Framework Programme (grant no. 278433-PREDEMICS to N.P.), and Ministry of Defense, Direction Générale de l’Armement (to C.K.)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 278433,EC:FP7:HEALTH,FP7-HEALTH-2011-two-stage,PREDEMICS(2011), Vougny, Marie-Christine, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Preparedness, Prediction and Prevention of Emerging Zoonotic Viruses with Pandemic Potential using Multidisciplinary Approaches - PREDEMICS - - EC:FP7:HEALTH2011-11-01 - 2016-10-31 - 278433 - VALID, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), and Institut Pasteur [Paris]

Subjects
0301 basic medicine, MESH: Hemorrhagic Fever, Ebola/virology, viruses, Spermine, medicine.disease_cause, Virus Replication, Disease Outbreaks, chemistry.chemical_compound, Mice, Polyamines, Diethylnorspermine, MESH: Animals, MESH: Disease Outbreaks, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, education.field_of_study, Zika Virus Infection, MESH: Chikungunya virus/metabolism, MESH: Antiviral Agents/pharmacology, MESH: Spermine/pharmacology, Ebolavirus, MESH: Virus Replication/drug effects, 3. Good health, MESH: Chikungunya Fever/virology, MESH: Polyamines/metabolism, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Ebolavirus/drug effects, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Zika Virus Infection/virology, Chikungunya virus, Eflornithine, [SDV.IMM] Life Sciences [q-bio]/Immunology, Immunology, Population, MESH: Hemorrhagic Fever, Ebola/drug therapy, MESH: Chikungunya virus/drug effects, Biology, Microbiology, Antiviral Agents, Virus, Cell Line, 03 medical and health sciences, MESH: Acetyltransferases/metabolism, Acetyltransferases, MESH: Mice, Inbred C57BL, Virology, Vaccines and Antiviral Agents, medicine, Animals, Humans, RNA Viruses, MESH: Zika Virus/drug effects, education, MESH: Mice, MESH: Spermine/analogs & derivatives, Ebola virus, MESH: Humans, RNA, Zika Virus, Hemorrhagic Fever, Ebola, MESH: Zika Virus Infection/drug therapy, MESH: Cell Line, Mice, Inbred C57BL, MESH: Eflornithine/pharmacology, 030104 developmental biology, MESH: RNA Viruses/drug effects, chemistry, Viral replication, Insect Science, Chikungunya Fever, MESH: Chikungunya Fever/drug therapy, Polyamine, MESH: Ebolavirus/metabolism
Abstract

RNA viruses present an extraordinary threat to human health, given their sudden and unpredictable appearance, the potential for rapid spread among the human population, and their ability to evolve resistance to antiviral therapies. The recent emergence of chikungunya virus, Zika virus, and Ebola virus highlights the struggles to contain outbreaks. A significant hurdle is the availability of antivirals to treat the infected or protect at-risk populations. While several compounds show promise in vitro and in vivo , these outbreaks underscore the need to accelerate drug discovery. The replication of several viruses has been described to rely on host polyamines, small and abundant positively charged molecules found in the cell. Here, we describe the antiviral effects of two molecules that alter polyamine levels: difluoromethylornithine (DFMO; also called eflornithine), which is a suicide inhibitor of ornithine decarboxylase 1 (ODC1), and diethylnorspermine (DENSpm), an activator of spermidine/spermine N 1 -acetyltransferase (SAT1). We show that reducing polyamine levels has a negative effect on diverse RNA viruses, including several viruses involved in recent outbreaks, in vitro and in vivo . These findings highlight the importance of the polyamine biosynthetic pathway to viral replication, as well as its potential as a target in the development of further antivirals or currently available molecules, such as DFMO. IMPORTANCE RNA viruses present a significant hazard to human health, and combatting these viruses requires the exploration of new avenues for targeting viral replication. Polyamines, small positively charged molecules within the cell, have been demonstrated to facilitate infection for a few different viruses. Our study demonstrates that diverse RNA viruses rely on the polyamine pathway for replication and highlights polyamine biosynthesis as a promising drug target.

Published
2016

22. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus

Author

Van-Mai Cao-Lormeau, Alexander Rouvinski, Félix A. Rey, Wiyada Wongwiwat, Giovanna Barba-Spaeth, Piyada Supasa, Anavaj Sakuntabhai, Wanwisa Dejnirattisai, Prida Malasit, Gavin R. Screaton, Thaneeya Duangchinda, Juthathip Mongkolsapaya, Imperial College London, Mahidol University [Bangkok], Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Génétique fonctionnelle des Maladies infectieuses - Functional Genetics of Infectious Diseases, Institut Louis Malardé [Papeete] (ILM), Institut de Recherche pour le Développement (IRD), This work was supported by the Medical Research Council, U.K., the Wellcome Trust (G.R.S.), U.K., the National Institute for Health Research Biomedical Research Centre, Funding Scheme, European Commission Seventh Framework Programme [FP7/2007-2013] for the DENFREE project under Grant Agreement n°282 378, and the Thailand Research Fund through the Royal Golden Jubilee PhD (P.S. and J.M.)., European Project: 282378,EC:FP7:HEALTH,FP7-HEALTH-2011-single-stage,DENFREE(2012), Medical Research Council (MRC), Commission of the European Communities, Wellcome Trust, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Male, OUTBREAK, viruses, MESH: Dengue, Dengue virus, medicine.disease_cause, Antibodies, Viral, Virus Replication, MESH: Dengue Virus, Zika virus, Dengue fever, Serology, MESH: Antibodies, Monoclonal, MESH: Antibodies, Neutralizing, Dengue, MESH: Cross Reactions, Viral Envelope Proteins, MESH: Child, Veterinary virology, Immunology and Allergy, BRAIN, Child, Cells, Cultured, biology, Zika Virus Infection, Antibodies, Monoclonal, virus diseases, BRAZIL, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], 3. Good health, Flavivirus, 1107 Immunology, Child, Preschool, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Microcephaly, Female, Life Sciences & Biomedicine, Protein Binding, MESH: Cells, Cultured, Adolescent, Immunology, MESH: Immunodominant Epitopes, MESH: Zika Virus, Cross Reactions, Guillain-Barre Syndrome, Virus, Article, MESH: Microcephaly, NEUTRALIZING ANTIBODIES, 03 medical and health sciences, MESH: Zika Virus Infection, medicine, Humans, MESH: Protein Binding, Antibody-dependent enhancement, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, MESH: Adolescent, MESH: Antibody-Dependent Enhancement, MESH: Guillain-Barre Syndrome, MESH: Epitope Mapping, Science & Technology, MESH: Humans, [SDV.GEN.GPO]Life Sciences [q-bio]/Genetics/Populations and Evolution [q-bio.PE], CHIKUNGUNYA VIRUS, Immunodominant Epitopes, MESH: Virus Replication, MESH: Child, Preschool, Zika Virus, MESH: South America, Dengue Virus, South America, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, medicine.disease, Virology, Antibodies, Neutralizing, Antibody-Dependent Enhancement, MESH: Male, 030104 developmental biology, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, MESH: Viral Envelope Proteins, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], MESH: Female, Epitope Mapping, MESH: Antibodies, Viral
Abstract

International audience; Zika virus (ZIKV) was discovered in 1947 and was thought to lead to relatively mild disease. The recent explosive outbreak of ZIKV in South America has led to widespread concern, with reports of neurological sequelae ranging from Guillain Barré syndrome to microcephaly. ZIKV infection has occurred in areas previously exposed to dengue virus (DENV), a flavivirus closely related to ZIKV. Here we investigated the serological cross-reaction between the two viruses. Plasma immune to DENV showed substantial cross-reaction to ZIKV and was able to drive antibody-dependent enhancement (ADE) of ZIKV infection. Using a panel of human monoclonal antibodies (mAbs) to DENV, we showed that most antibodies that reacted to DENV envelope protein also reacted to ZIKV. Antibodies to linear epitopes, including the immunodominant fusion-loop epitope, were able to bind ZIKV but were unable to neutralize the virus and instead promoted ADE. Our data indicate that immunity to DENV might drive greater ZIKV replication and have clear implications for disease pathogenesis and future vaccine programs for ZIKV and DENV.

Published
2016

23. Structural basis of potent Zika-dengue virus antibody cross-neutralization

Author

Franz X. Heinz, Juthathip Mongkolsapaya, Ahmed Haouz, Arvind Sharma, Patrick England, Van-Mai Cao-Lormeau, Wanwisa Dejnirattisai, Félix A. Rey, Anavaj Sakuntabhai, Iris Medits, Etienne Simon-Loriere, Gavin R. Screaton, Karin Stiasny, Alexander Rouvinski, Giovanna Barba-Spaeth, Marie Christine Vaney, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Division of Immunology and Inflammation, Department of Medicine, Imperial College London-Hammersmith Hospital Campus, Medizinische Universität Wien = Medical University of Vienna, Génétique fonctionnelle des Maladies infectieuses - Functional Genetics of Infectious Diseases, Emerging Infectious Diseases, Institut Louis Malardé [Papeete] (ILM), Institut de Recherche pour le Développement (IRD)-Institut de Recherche pour le Développement (IRD), Cristallographie (Plateforme) - Crystallography (Platform), Biophysique des Macromolécules et de leurs Interactions, Siriraj Hospital, Mahidol University, Mahidol University [Bangkok], We acknowledge support from the European Commission FP7 Programme for the DENFREE project under Grant Agreement number 282 378FP7 (F.A.R., J.M., G.R.S. and A.Sa.), the 'Integrative Biology of Emerging Infectious Diseases' Labex (Laboratoire d’Excellence) grant number ANR-10-LABX-62-IBEID (French Government’s 'Investissements d’Avenir' program ) (F.A.R.), the transnational ANR/FWF grant FlaviStem/I1378 (F.A.R. and K.S.), the Medical Research Council, UK (J.M.), the National Institute for Health Research Biomedical Research Centre, Funding Scheme, UK (G.R.S.), and the NEUTRAVIR grant from Région ile-de-France (DIM-Maladies Infectieuses) (P.E., A.H. and F.A.R., ANR-13-ISV8-0002,flavistem,La protéine E des flavivirus : interactions et fusion membranaire(2013), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), European Project: 282378,EC:FP7:HEALTH,FP7-HEALTH-2011-single-stage,DENFREE(2012), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Medical Research Council (MRC), Wellcome Trust, and Commission of the European Communities

Subjects
DYNAMICS, 0301 basic medicine, Models, Molecular, viruses, [SDV]Life Sciences [q-bio], Antigen-Antibody Complex, Dengue virus, medicine.disease_cause, Crystallography, X-Ray, Epitope, Zika virus, Dengue fever, Dengue, Epitopes, 0302 clinical medicine, Viral Envelope Proteins, INFECTION, Virus maturation, IN-VIVO, FLAVIVIRUSES, Phylogeny, Multidisciplinary, Zika Virus Infection, Antibodies, Monoclonal, REACTIVE ANTIBODIES, FUSION-LOOP, 3. Good health, Multidisciplinary Sciences, Flavivirus, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Science & Technology - Other Topics, Brazil, General Science & Technology, Dengue Vaccines, Biology, Cross Reactions, MATURATION, Microbiology, 03 medical and health sciences, ENHANCEMENT, medicine, Humans, Antibody-dependent enhancement, Dengue vaccine, Science & Technology, RECEPTOR, RECOGNITION, Viral Vaccines, Zika Virus, Dengue Virus, biology.organism_classification, medicine.disease, Virology, Antibodies, Neutralizing, 030104 developmental biology, 030217 neurology & neurosurgery
Abstract

International audience; Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections.

Published
2016

24. Recognition determinants of broadly neutralizing human antibodies against dengue viruses

Author

Stéphane Duquerroy, Christine Girard-Blanc, William Shepard, Wiyada Wongwiwat, Giovanna Barba-Spaeth, Stéphane Petres, Félix A. Rey, Pablo Guardado-Calvo, Ahmed Haouz, Juthathip Mongkolsapaya, M. Erika Navarro Sanchez, Carlos M. Kikuti, Wanwisa Dejnirattisai, Philippe Dussart, Fernando Arenzana-Seisdedos, Alexander Rouvinski, Marie-Christine Vaney, Philippe Desprès, Gavin R. Screaton, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11), Imperial College London, Protéopole, Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur [Paris] (IP), Pathogénie Virale - Viral Pathogenesis, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur de la Guyane, Réseau International des Instituts Pasteur (RIIP), Mahidol University [Bangkok], his work was made possible by a Pediatrics Dengue Vaccine Initiative grant to F.A.R., allowing the set up of a production facility of recombinant DENV sE. The co-crystallization with the bnAbs was done with European Union funding (DenFree consortium) to F.A.R. and G.R.S./J.M. F.A.R. acknowledges support from Insitut Pasteur, from the French Government’s ‘Investissements d’Avenir’ program: Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant number ANR-10-LABX-62-IBEID) and the CNRS. G.S.R. and J.M. were supported by the Medical Research Council, UK, the Wellcome Trust, UK, the National Institute for Health Research Biomedical Research Centre, Funding Scheme. G.R.S. is a Wellcome Trust Senior investigator. We thank staffs at beam lines PROXIMA-1 and PROXIMA-2 at the SOLEIL synchrotron (St Aubin, France) and ID23-2 and ID29 at the European Synchrotron Radiation Facility (Grenoble, France)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), Tortosa, Pablo, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Pathogénie Virale, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Medical Research Council (MRC), Wellcome Trust, Commission of the European Communities, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]

Subjects
Serotype, MECHANISM, Models, Molecular, MONOCLONAL-ANTIBODY, FAB FRAGMENTS, Protein Conformation, viruses, DROSOPHILA S2 CELLS, Dengue virus, medicine.disease_cause, Antibodies, Viral, Crystallography, X-Ray, Epitope, Dengue fever, Epitopes, FUSION, Viral Envelope Proteins, Virus maturation, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0303 health sciences, Multidisciplinary, REFINEMENT, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], BORNE ENCEPHALITIS-VIRUS, ENVELOPE GLYCOPROTEIN, 3. Good health, Multidisciplinary Sciences, Flavivirus, IMMATURE FLAVIVIRUS PARTICLES, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Science & Technology - Other Topics, [SDV.IMM]Life Sciences [q-bio]/Immunology, [SDV.IMM] Life Sciences [q-bio]/Immunology, General Science & Technology, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Sequence Data, Biology, Cross Reactions, Virus, MATURATION, 03 medical and health sciences, Species Specificity, MD Multidisciplinary, medicine, Humans, Antibody-dependent enhancement, 030304 developmental biology, Science & Technology, 030306 microbiology, Dengue Virus, medicine.disease, biology.organism_classification, Virology, Antibodies, Neutralizing, Solubility, Mutation, Protein Multimerization
Abstract

International audience; Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

Published
2015

25. Interferons α and λ Inhibit Hepatitis C Virus Replication With Distinct Signal Transduction and Gene Regulation Kinetics

Author

Tobias Marcello, Charles M. Rice, Arash Grakoui, Sergei V. Kotenko, Erica S. Machlin, Margaret R. MacDonald, and Giovanna Barba-Spaeth

Subjects
Gene Expression Regulation, Viral, Carcinoma, Hepatocellular, Hepatitis C virus, Receptor expression, Hepacivirus, Biology, Virus Replication, medicine.disease_cause, Antiviral Agents, Virus, Interferon, Cell Line, Tumor, Gene expression, medicine, Humans, Interleukin 28, Oligonucleotide Array Sequence Analysis, Regulation of gene expression, Dose-Response Relationship, Drug, Hepatology, Interleukins, Liver Neoplasms, Gastroenterology, Interferon-alpha, Virology, Molecular biology, Cytokines, RNA, Viral, Interferons, Signal transduction, Signal Transduction, medicine.drug
Abstract

Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. Current therapy with pegylated interferon alpha (IFN-alpha) in combination with ribavirin is associated with adverse effects and often fails to induce a sustained response. IFN-lambdas, recently discovered IFN gene family members, exhibit antiviral and cell stimulatory activities similar to IFN-alpha. We aimed to determine whether IFN-lambda exhibits antiviral activity toward HCV and to compare the signal transduction and effector gene pathways with those of IFN-alpha.Using the HCV replicon system and cell culture infectious reporter virus, we compared IFN-alpha and IFN-lambda effects on HCV RNA replication and protein expression, as measured by quantitative reverse-transcriptase polymerase chain reaction, luciferase expression, and Western blot. Receptor expression and signaling pathways were explored using flow cytometry and Western blot. IFN-alpha- and IFN-lambda-mediated gene expression changes were compared using microarray analyses.IFN-lambda exhibited dose- and time-dependent HCV inhibition, independent of types I and II IFN receptors. The kinetics of IFN-lambda-mediated signal transducers and activators of transcription (STAT) activation and induction of potential effector genes were distinct from those of IFN-alpha. IFN-lambda induced steady increases in levels of known interferon stimulated genes (ISGs), whereas IFN-alpha ISGs peaked early and declined rapidly. IFN-lambda inhibited replication of HCV genotypes 1 and 2 and enhanced the antiviral efficacy of subsaturating levels of IFN-alpha.These results demonstrate distinct differences in IFN-lambda- and IFN-alpha-induced antiviral states. Understanding these differences may prove useful for developing new HCV treatment strategies.

Published
2006

26. Yellow fever 17D as a vaccine vector for microbial CTL epitopes

Author

Charles M. Rice, Urvashi Rai, Giovanna Barba-Spaeth, Victor Nussenzweig, Deng Tao, and Ruth S. Nussenzweig

Subjects
Immunology, Yellow fever vaccine, Antigens, Protozoan, CD8-Positive T-Lymphocytes, Recombinant virus, Epitope, Article, Epitopes, Mice, Immune system, Immunity, medicine, Immunology and Allergy, Animals, Humans, biology, Immunogenicity, Yellow Fever Vaccine, Plasmodium yoelii, biology.organism_classification, Virology, Malaria, Circumsporozoite protein, Yellow fever virus, medicine.drug, T-Lymphocytes, Cytotoxic
Abstract

The yellow fever vaccine 17D (17D) is safe, and after a single immunizing dose, elicits long-lasting, perhaps lifelong protective immunity. One of the major challenges facing delivery of human vaccines in underdeveloped countries is the need for multiple injections to achieve full efficacy. To examine 17D as a vector for microbial T cell epitopes, we inserted the H-2Kd–restricted CTL epitope of the circumsporozoite protein (CS) of Plasmodium yoelii between 17D nonstructural proteins NS2B and NS3. The recombinant virus, 17D-Py, was replication competent and stable in vitro and in vivo. A single subcutaneous injection of 105 PFU diminished the parasite burden in the liver by ∼70%. The high level of protection lasted between 4 and 8 wk after immunization, but a significant effect was documented even 24 wk afterwards. Thus, the immunogenicity of a foreign T cell epitope inserted into 17D mimics some of the remarkable properties of the human vaccine. Priming with 17D-Py followed by boosting with irradiated sporozoites conferred sterile immunity to 90% of the mice. This finding indicates that the immune response of vaccine-primed individuals living in endemic areas could be sustained and magnified by the bite of infected mosquitoes.

Published
2005

27. Functional and evolutionary insight from the crystal structure of rubella virus protein E1

Author

M. Alejandra Tortorici, Marie-Christine Vaney, Thomas Krey, Félix A. Rey, Rana Al Kurdi, Rebecca M. DuBois, Giovanna Barba-Spaeth, Virologie Structurale, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was mainly funded by grant ANR-05-MIIM-012-02-Dentry to F.A.R., who also acknowledges support from Merck-Serono from the French Government’s Investissements d’Avenir program: Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR-10-LABX-62-IBEID)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Viral membrane fusion, MESH: Hydrogen-Ion Concentration, viruses, Crystallography, X-Ray, medicine.disease_cause, Membrane Fusion, Viral Envelope Proteins, MESH: Animals, MESH: Evolution, Molecular, 0303 health sciences, Multidisciplinary, MESH: Protein Multimerization, virus diseases, Rubella virus, Hydrogen-Ion Concentration, Biological Evolution, 3. Good health, Rubella Infection, Drosophila melanogaster, Metals, Viral evolution, MESH: Models, Molecular, Viral protein, Rubella Syndrome, Congenital, MESH: Rubella Syndrome, Congenital, MESH: Biological Evolution, Biology, MESH: Membrane Fusion, Rubella, Measles, Cell Line, MESH: Drosophila melanogaster, Evolution, Molecular, 03 medical and health sciences, medicine, Animals, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Virus classification, 030304 developmental biology, X-ray crystallography, Congenital rubella syndrome, Binding Sites, MESH: Metals, 030306 microbiology, medicine.disease, MESH: Crystallography, X-Ray, Virology, MESH: Cell Line, MESH: Rubella virus, MESH: Binding Sites, MESH: Viral Envelope Proteins, Liposomes, MESH: Liposomes, Protein Multimerization
Abstract

International audience; Little is known about the three-dimensional organization of rubella virus, which causes a relatively mild measles-like disease in children but leads to serious congenital health problems when contracted in utero. Although rubella virus belongs to the same family as the mosquito-borne alphaviruses, in many respects it is more similar to other aerosol-transmitted human viruses such as the agents of measles and mumps. Although the use of the triple MMR (measles, mumps and rubella) live vaccine has limited its incidence in western countries, congenital rubella syndrome remains an important health problem in the developing world. Here we report the 1.8 Å resolution crystal structure of envelope glycoprotein E1, the main antigen and sole target of neutralizing antibodies against rubella virus. E1 is the main player during entry into target cells owing to its receptor-binding and membrane-fusion functions. The structure reveals the epitope and the neutralization mechanism of an important category of protecting antibodies against rubella infection. It also shows that rubella virus E1 is a class II fusion protein, which had hitherto only been structurally characterized for the arthropod-borne alphaviruses and flaviviruses. In addition, rubella virus E1 has an extensive membrane-fusion surface that includes a metal site, reminiscent of the T-cell immunoglobulin and mucin family of cellular proteins that bind phosphatidylserine lipids at the plasma membrane of cells undergoing apoptosis. Such features have not been seen in any fusion protein crystallized so far. Structural comparisons show that the class II fusion proteins from alphaviruses and flaviviruses, despite belonging to different virus families, are closer to each other than they are to rubella virus E1. This suggests that the constraints on arboviruses imposed by alternating cycles between vertebrates and arthropods resulted in more conservative evolution. By contrast, in the absence of this constraint, the strictly human rubella virus seems to have drifted considerably into a unique niche as sole member of the Rubivirus genus.

Published
2013

28. Erratum: Structural basis of potent Zika–dengue virus antibody cross-neutralization

Author

Giovanna Barba-Spaeth, Wanwisa Dejnirattisai, Alexander Rouvinski, Marie-Christine Vaney, Iris Medits, Arvind Sharma, Etienne Simon-Lorière, Anavaj Sakuntabhai, Van-Mai Cao-Lormeau, Ahmed Haouz, Patrick England, Karin Stiasny, Juthathip Mongkolsapaya, Franz X. Heinz, Gavin R. Screaton, and Félix A. Rey

Subjects
Multidisciplinary
Published
2016

29. Hepacivirus

Author

Giovanna Barba Spaeth, Catherine L. Murray, and Charles M. Rice

Published
2011

30. Immunogenicity and protective efficacy of a recombinant yellow fever vaccine against the murine malarial parasite Plasmodium yoelii

Author

Silvia Beatriz Boscardin, Giovanna Barba-Spaeth, Charles M. Rice, Cristina Stoyanov, Stephanie Deroubaix, Michel C. Nussenzweig, Ruth S. Nussenzweig, and David Franco

Subjects
Immunization, Secondary, Protozoan Proteins, Yellow fever vaccine, Mice, Transgenic, Biology, CD8-Positive T-Lymphocytes, Recombinant virus, Vaccines, Attenuated, complex mixtures, Article, Microbiology, law.invention, Interferon-gamma, Mice, law, parasitic diseases, Malaria Vaccines, medicine, Animals, Immunity, Cellular, Mice, Inbred BALB C, General Veterinary, General Immunology and Microbiology, Immunogenicity, fungi, Yellow Fever Vaccine, Public Health, Environmental and Occupational Health, Plasmodium yoelii, biology.organism_classification, Virology, Adoptive Transfer, Malaria, Vaccination, Circumsporozoite protein, Infectious Diseases, Immunization, Recombinant DNA, Molecular Medicine, Interleukin-2, Female, medicine.drug, Plasmids
Abstract

The live-attenuated yellow fever vaccine (YF17D) is one of the safest and most effective vaccines available today. Here, YF17D was genetically altered to express the circumsporozoite protein (CSP) from the murine malarial parasite Plasmodium yoelii. Reconstituted recombinant virus was viable and exhibited robust CSP expression. Immunization of naive mice resulted in extensive proliferation of adoptively transferred CSP-specific transgenic CD8(+) T-cells. A single immunization of naive mice with recombinant YF17D resulted in robust production of IFN-gamma by CD8(+) T-cells and IFN-gamma and IL-2 by CD4(+) T-cells. A prime-boost regimen consisting of recombinant virus followed by a low-dose of irradiated sporozoites conferred protection against challenge with P. yoelii. Taken together, these results show that recombinant YF17D can efficiently express CSP in culture, and prime a protective immune response in vivo.

Published
2009

31. Hepacivirus

Author

Giovanna Barba Spaeth and Charles M. Rice

Published
2006

32. Live attenuated yellow fever 17D infects human DCs and allows for presentation of endogenous and recombinant T cell epitopes

Author

Charles M. Rice, Giovanna Barba-Spaeth, Randy S. Longman, Matthew L. Albert, Laboratory of Virology and Infectious Disease, Rockefeller University [New York]-Center for the Study of Hepatitis C, Immunobiologie des Cellules Dendritiques, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by the Greenberg Medical Research Institute and the Grand Challenges in Global Health. We are grateful to the donors who participated in this study., Vougny, Marie-Christine, and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
T-Lymphocytes, Epitopes, T-Lymphocyte, Apoptosis, Epitope, Immunology and Allergy, Neutralizing antibody, Cells, Cultured, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, 0303 health sciences, Antigen Presentation, Vaccines, Synthetic, Attenuated vaccine, MESH: Dendritic Cells, Yellow Fever Vaccine, 3. Good health, medicine.anatomical_structure, MESH: Calcium, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, Yellow fever virus, medicine.drug, MESH: Cells, Cultured, [SDV.IMM] Life Sciences [q-bio]/Immunology, MESH: Vaccines, Synthetic, T cell, Immunology, Antigen presentation, Yellow fever vaccine, Biology, Vaccines, Attenuated, MESH: Epitopes, T-Lymphocyte, 03 medical and health sciences, Immunity, MESH: Vaccines, Attenuated, medicine, Humans, 030304 developmental biology, MESH: Humans, 030306 microbiology, MESH: Apoptosis, Brief Definitive Report, Dendritic Cells, Virology, MESH: Yellow fever virus, MESH: Yellow Fever Vaccine, MESH: T-Lymphocytes, MESH: Antigen Presentation, biology.protein, Calcium, CD8
Abstract

International audience; The yellow fever (YF) 17D vaccine is one of the most successful live attenuated vaccines available. A single immunization induces both long-lasting neutralizing antibody and YF-specific T cell responses. Surprisingly, the mechanism for this robust immunity has not been addressed. In light of several recent reports suggesting flavivirus interaction with dendritic cells (DCs), we investigated the mechanism of YF17D interaction with DCs and the importance of this interaction in generating T cell immunity. Our results show that YF17D can infect immature and mature human DCs. Viral entry is Ca(2+) dependent, but it is independent of DC-SIGN as well as multiple integrins expressed on the DC surface. Similar to infection of cell lines, YF infection of immature DCs is cytopathic. Although infection itself does not induce DC maturation in vitro, TNF-alpha-induced maturation protects DCs from YF-induced cytopathogenicity. Furthermore, we show that DCs infected with YF17D or YF17D carrying a recombinant epitope can process and present antigens for CD8(+) T cell stimulation. These findings offer insight into the immunologic mechanisms associated with the highly capable YF17D vaccine that may guide effective vaccine design.

Published
2005

33. Tunneling nanotubes provide a route for SARS-CoV-2 spreading

Author

Anna Pepe, Stefano Pietropaoli, Matthijn Vos, Giovanna Barba-Spaeth, and Chiara Zurzolo

Subjects
Multidisciplinary
Abstract

Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection represent a major issue in long coronavirus disease. How SARS-CoV-2 gains access to the brain and how infection leads to neurological symptoms are not clear because the principal means of viral entry by endocytosis, the angiotensin-converting enzyme 2 receptor, are barely detectable in the brain. We report that human neuronal cells, nonpermissive to infection through the endocytic pathway, can be infected when cocultured with permissive infected epithelial cells. SARS-CoV-2 induces the formation of tunneling nanotubes (TNTs) and exploits this route to spread to uninfected cells. In cellulo correlative fluorescence and cryo–electron tomography reveal that SARS-CoV-2 is associated with TNTs between permissive cells. Furthermore, multiple vesicular structures such as double-membrane vesicles, sites of viral replication, are observed inside TNTs between permissive and nonpermissive cells. Our data highlight a previously unknown mechanism of SARS-CoV-2 spreading, likely used as a route to invade nonpermissive cells and potentiate infection in permissive cells.

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34. Structure of the Yellow Fever Virus Membrane Fusion Envelope E

Author

Stéphane Duquerroy, Eric Crampon, Félix A. Rey, and Giovanna Barba-Spaeth

Subjects
chemistry.chemical_classification, biology, Biophysics, Lipid bilayer fusion, Alphavirus, biology.organism_classification, Fusion protein, Virology, Protein tertiary structure, Flavivirus, Viral envelope, Ectodomain, chemistry, Glycoprotein
Abstract

Enveloped viruses enter into cells by a membrane fusion mechanism which leads to the release of their viral genome into the cytosol of the host . Yellow fever virus (YFV), of a flavivirus of approximately 50 nm in diameter, displays on its surface 180 envelope glycoproteins E arranged in a herringbone pattern , which are responsible for both receptor recognition and membrane fusion. It causes 200,000 illnesses and 30,000 deaths every year. A vaccine is avalaible, which differs from the wild-type by 12 mutations in E out of 400 residues. We were interested in understanding changes induces by these mutationsWe produced the recombinant E protein in Drosophila Schneider 2 (S2). We determined the crystal structure of the ectodomain of the YFVE for both the wild-type Asibi and vaccinial 17D strains at 2.75A and 3.5A respectively. The overall tertiary structure of the YFV-E is typical of class II membrane fusion proteins observed for other flavivirus E and alphavirus E1 proteins. YFV-E has no N-glycosylation site as other Flavivirus E proteins, but interestingly it presents unexpected O-mannosylation. Furthermore, improvement of crystal resolution has been obtained after urea denaturation and renaturation, which is an unusual approach for improving crystal resolution.The structure of Asibi WT YFV-E and its comparison to vaccinial 17D strain as well as to other class II fusion proteins will be presented to stress on its salient characteristics.

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