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1. Qualitative monitoring of SARS-CoV-2 mRNA vaccination in humans using droplet microfluidics

Author

Matteo Broketa, Aurélien Sokal, Michael Mor, Pablo Canales-Herrerias, Angga Perima, Annalisa Meola, Ignacio Fernández, Bruno Iannascoli, Guilhem Chenon, Alexis Vandenberghe, Laetitia Languille, Marc Michel, Bertrand Godeau, Sébastien Gallien, Giovanna Melica, Marija Backovic, Felix A. Rey, Jean Baudry, Natalia T. Freund, Matthieu Mahévas, and Pierre Bruhns

Subjects
Immunology, Vaccines, Medicine
Abstract

SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here, we qualitatively investigated the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19–recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual variability in affinity for RBD, with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented more than 65% of the plasmablast response at all time points. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.

Published
2023
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2. Fusogenicity and neutralization sensitivity of the SARS-CoV-2 Delta sublineage AY.4.2

Author

Nell Saunders, Delphine Planas, William H. Bolland, Christophe Rodriguez, Slim Fourati, Julian Buchrieser, Cyril Planchais, Matthieu Prot, Isabelle Staropoli, Florence Guivel-Benhassine, Françoise Porrot, David Veyer, Hélène Péré, Nicolas Robillard, Madelina Saliba, Artem Baidaliuk, Aymeric Seve, Laurent Hocqueloux, Thierry Prazuck, Felix A. Rey, Hugo Mouquet, Etienne Simon-Lorière, Timothée Bruel, Jean-Michel Pawlotsky, and Olivier Schwartz

Subjects
SARS-CoV-2, Delta, AY.4.2, Vaccines, Fusogenicity, Neutralization, Medicine, Medicine (General), R5-920
Abstract

Summary: Background: SARS-CoV-2 lineages are continuously evolving. As of December 2021, the AY.4.2 Delta sub-lineage represented 20 % of sequenced strains in the UK and had been detected in dozens of countries. It has since then been supplanted by Omicron. The AY.4.2 spike displays three additional mutations (T95I, Y145H and A222V) in the N-terminal domain when compared to the original Delta variant (B.1.617.2) and remains poorly characterized. Methods: We compared the Delta and the AY.4.2 spikes, by assessing their binding to antibodies and ACE2 and their fusogenicity. We studied the sensitivity of an authentic AY.4.2 viral isolate to neutralizing antibodies. Findings: The AY.4.2 spike exhibited similar binding to all the antibodies and sera tested, and similar fusogenicity and binding to ACE2 than the ancestral Delta spike. The AY.4.2 virus was slightly less sensitive than Delta to neutralization by a panel of monoclonal antibodies; noticeably, the anti-RBD Imdevimab showed incomplete neutralization. Sensitivity of AY.4.2 to sera from vaccinated individuals was reduced by 1.3 to 3-fold, when compared to Delta. Interpretation: Our results suggest that mutations in the NTD remotely impair the efficacy of anti-RBD antibodies. The spread of AY.4.2 was not due to major changes in spike fusogenicity or ACE2 binding, but more likely to a partially reduced neutralization sensitivity. Funding: The work was funded by Institut Pasteur, Fondation pour la Recherche Médicale, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, ANRS, the Vaccine Research Institute, Labex IBEID, ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR.

Published
2022
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3. In Vitro Viral Evolution Identifies a Critical Residue in the Alphaherpesvirus Fusion Glycoprotein B Ectodomain That Controls gH/gL-Independent Entry

Author

Melina Vallbracht, Henriette Lötzsch, Barbara G. Klupp, Walter Fuchs, Benjamin Vollmer, Kay Grünewald, Marija Backovic, Felix A. Rey, and Thomas C. Mettenleiter

Subjects
Microbiology, QR1-502
Abstract

The class III fusion protein glycoprotein B (gB) drives membrane fusion during entry and spread of herpesviruses. To mediate fusion, gB requires activation by the conserved gH/gL complex by a poorly defined mechanism.

Published
2021
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4. SARS‐CoV‐2 Omicron emergence urges for reinforced One‐Health surveillance

Author

Xavier Montagutelli, Sylvie van der Werf, Felix A Rey, and Etienne Simon‐Loriere

Subjects
Medicine (General), R5-920, Genetics, QH426-470
Abstract

Graphical Abstract SARS‐CoV‐2 Omicron harbors substitutions in the receptor binding domain of the spike which strongly suggest its capacity to infect rodents. Wild animal reservoirs could favor the emergence of new variants with risks of spillback to humans and should be closely monitored.

Published
2022
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5. Generation of plasma cells and CD27−IgD− B cells during hantavirus infection is associated with distinct pathological findings

Author

Priscilla F Kerkman, Andy Dernstedt, Lalitha Tadala, Eva Mittler, Mirjam Dannborg, Christopher Sundling, Kimia T Maleki, Johanna Tauriainen, Anne Tuiskunen‐Bäck, Julia Wigren Byström, Pauline Ocaya, Therese Thunberg, Rohit K Jangra, Gleyder Román‐Sosa, Pablo Guardado‐Calvo, Felix A Rey, Jonas Klingström, Kartik Chandran, Andrea Puhar, Clas Ahlm, and Mattias NE Forsell

Subjects
antibodies, atypical B cells, B cells, haemorrhagic fever with renal syndrome, hantavirus, plasmablasts, Immunologic diseases. Allergy, RC581-607
Abstract

Abstract Objective Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction. Methods We performed a comprehensive characterisation of longitudinal antiviral B‐cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells. Results We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27−IgD− B cells and CD27−/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection. Conclusion Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus‐infected individuals have significantly elevated levels of extracellular ATP in circulation.

Published
2021
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6. Immunization with GP1 but Not Core-like Particles Displaying Isolated Receptor-Binding Epitopes Elicits Virus-Neutralizing Antibodies against Junín Virus

Author

Gleyder Roman-Sosa, Anne Leske, Xenia Ficht, Tung Huy Dau, Julia Holzerland, Thomas Hoenen, Martin Beer, Robert Kammerer, Reinhold Schirmbeck, Felix A. Rey, Sandra M. Cordo, and Allison Groseth

Subjects
arenavirus, Junín virus, immune response, neutralizing antibodies, glycoprotein, GP1, Medicine
Abstract

New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses.

Published
2022
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7. SARS‐CoV‐2‐related bat virus behavior in human‐relevant models sheds light on the origin of COVID‐19

Author

Sarah Temmam, Xavier Montagutelli, Cécile Herate, Flora Donati, Béatrice Regnault, Mikael Attia, Eduard Baquero Salazar, Delphine Chretien, Laurine Conquet, Grégory Jouvion, Juliana Pipoli Da Fonseca, Thomas Cokelaer, Faustine Amara, Francis Relouzat, Thibaut Naninck, Julien Lemaitre, Nathalie Derreudre‐Bosquet, Quentin Pascal, Massimiliano Bonomi, Thomas Bigot, Sandie Munier, Felix A Rey, Roger Le Grand, Sylvie van der Werf, Marc Eloit, Découverte de pathogènes – Pathogen discovery, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Centre Collaborateur de l'OIE de Détection et identification chez l’homme des pathogènes animaux émergents et développement d’outils pour leur diagnostic / Collaborating Center for the Detection and identification in humans of emerging animal pathogens and development of tools for their diagnoses (CCOIE-OIECC), Institut Pasteur [Paris] (IP)-Organisation Mondiale de la Santé Animale / World Organisation Animal Health [Paris] (OIE)-Université Paris Cité (UPCité), Génétique de la souris - Mouse Genetics, Immunologie des maladies virales, auto-immunes, hématologiques et bactériennes (IMVA-HB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre National de Référence des virus des infections respiratoires (dont la grippe) - National Reference Center Virus Influenzae [Paris] (CNR - laboratoire coordonnateur), Virologie Structurale - Structural Virology, École nationale vétérinaire - Alfort (ENVA), Dynamic Microbiology - EA 7380 (DYNAMIC), École nationale vétérinaire - Alfort (ENVA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Université Paris-Est (UPE)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Biomics (plateforme technologique), Bioinformatique structurale - Structural Bioinformatics, Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Next-generation sequencing was performed with the help of Biomics Platform, C2RT, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09), IBISA, and the Illumina COVID-19 Projects' offer. The work was funded by an Institut Pasteur 'Covid Taskforce' and in part by the H2020 project 101003589 (RECOVER) and Labex IBEID (ANR-10-LABX62-IBEID) grants. The Infectious Disease Models and Innovative Therapies (IDMIT) research infrastructure is supported by the 'Programme Investissements d'Avenir', managed by the ANR under reference ANR-11-INBS-0008. The Fondation Bettencourt Schueller and the Region Ile-de-France contributed to the implementation of IDMIT's facilities and imaging technologies. The NHP model of SARS-CoV-2 infection have been developed thanks to the support from REACTing, the Fondation pour la Recherche Medicale (FRM, AM-CoV-Path)., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-11-INBS-0008,IDMIT,Infrastructure nationale pour la modélisation des maladies infectieuses humaines(2011), and European Project: 101003589, H2020-SC1-PHE-CORONAVIRUS-2020,RECOVER(2020)

Subjects
animal model, pathogenesis, [SDE]Environmental Sciences, Genetics, adaptive mutations, serology, bat coronavirus, Molecular Biology, Biochemistry
Abstract

International audience; Bat sarbecovirus BANAL-236 is highly related to SARS-CoV-2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL-236 replicates efficiently and pauci-symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS-CoV-2. BANAL-236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre-spillover event that did not occur upon replication of a SARS-CoV-2-like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS-CoV-2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats.

Published
2023

8. The crystal structure of a simian Foamy Virus receptor binding domain provides clues about entry into host cells

Author

Ignacio Fernández, Lasse Toftdal Dynesen, Youna Coquin, Riccardo Pederzoli, Delphine Brun, Ahmed Haouz, Antoine Gessain, Felix A. Rey, Florence Buseyne, Marija Backovic, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Epidémiologie et Physiopathologie des Virus Oncogènes / Oncogenic Virus Epidemiology and Pathophysiology (EPVO (UMR_3569 / U-Pasteur_3)), Cristallographie (Plateforme) - Crystallography (Platform), This work was funded by the ‘Agence Nationale de la Recherche’ (ANR-10-LABX62-IBEID, Intra-Labex Grant, M.B.), the ‘Programme de recherche transversal from Institut Pasteur’ (PTR2020-353 ZOOFOAMENV, F.B.), and recurrent funding from Institut Pasteur (F.A.R, A.G.), We thank the staff from the Utechs Cytometry & Biomarkers and Crystallography platform at the Institut Pasteur, the synchrotron source SOLEIL (Saint-Aubin, France) for granting access to the facility, and to the staff of Proxima 1 and Proxima 2A beamlines for their kindness and assistance during X-ray data collections. We are grateful to Jan Hellert, Pablo Guardado-Calvo and Philippe Afonso for the discussions and advice, with special thanks to Max Baker for reading the manuscript and English language corrections., and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], [SDV]Life Sciences [q-bio], General Physics and Astronomy, General Chemistry, General Biochemistry, Genetics and Molecular Biology
Abstract

The surface envelope glycoprotein (Env) of all retroviruses mediates virus binding to cells and fusion of the viral and cellular membranes. A structure-function relationship for the HIV Env that belongs to the Orthoretrovirus subfamily has been well established. Structural information is however largely missing for the Env of Foamy viruses (FVs), the second retroviral subfamily. FV Envs lack sequence similarity with their HIV counterpart. We present the X-ray structure of the receptor binding domain (RBD) of a simian FV Env at 2.6 Å resolution, revealing two subdomains and an unprecedented fold. We have generated a model for the organization of the RBDs within the trimeric Env which indicates that the upper subdomain is important for stabilization of the full-length Env, and have demonstrated that residues K342, R343, R359 and R369 in the lower subdomain play key roles in the interaction of the RBD and viral particles with heparan sulfate.

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. Neutralization of zoonotic retroviruses by human antibodies: Genotype-specific epitopes within the receptor-binding domain from simian foamy virus

Author

Lasse Toftdal Dynesen, Ignacio Fernandez, Youna Coquin, Manon Delaplace, Thomas Montange, Richard Njouom, Chanceline Bilonga-Ndongo, Felix A. Rey, Antoine Gessain, Marija Backovic, Florence Buseyne, Epidémiologie et Physiopathologie des Virus Oncogènes / Oncogenic Virus Epidemiology and Pathophysiology (EPVO (UMR_3569 / U-Pasteur_3)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Virologie Structurale - Structural Virology, Centre Pasteur du Cameroun, Réseau International des Instituts Pasteur (RIIP), Ministère de la Santé Publique [Yaoundé, Cameroun], This work was supported by the Agence Nationale de la Recherche (ANR-10-LABX62-IBEID, Intra-Labex Grant (MB)), and the Programme de Recherche Transversal from the Institut Pasteur (PTR2020-353 ZOOFOAMENV, FB). SFV protein production was supported by the European Virus Archive-GLOBAL project, which has received funding from the EU Horizon 2020 Research and Innovation Programme (grant agreement number 871029). LTD was supported by the Pasteur-Paris-University (PPU) International Doctoral Program and the Fondation pour la Recherche Médicale, including additional supportive funding from the Danish Pasteur Society, Augustinus Fonden, Knud-Højgaards Fond, and Viet-Jacobsen Fonden., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and European Project: 871029,H2020,H2020-INFRAIA-2019-1,EVA-GLOBAL(2020)

Subjects
[SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, Virology, Immunology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Genetics, Parasitology, Molecular Biology, Microbiology
Abstract

Infection with viruses of animal origin pose a significant threat to human populations. Simian foamy viruses (SFVs) are frequently transmitted to humans, in which they establish a life-long infection, with the persistence of replication-competent virus. However, zoonotic SFVs do not induce severe disease nor are they transmitted between humans. Thus, SFVs represent a model of zoonotic retroviruses that lead to a chronic infection successfully controlled by the human immune system. We previously showed that infected humans develop potent neutralizing antibodies (nAbs). Within the viral envelope (Env), the surface protein (SU) carries a variable region that defines two genotypes, overlaps with the receptor binding domain (RBD), and is the exclusive target of nAbs. However, its antigenic determinants are not understood. Here, we characterized nAbs present in plasma samples from SFV-infected individuals living in Central Africa. Neutralization assays were carried out in the presence of recombinant SU that compete with SU at the surface of viral vector particles. We defined the regions targeted by the nAbs using mutant SU proteins modified at the glycosylation sites, RBD functional subregions, and genotype-specific sequences that present properties of B-cell epitopes. We observed that nAbs target conformational epitopes. We identified three major epitopic regions: the loops at the apex of the RBD, which likely mediate interactions between Env protomers to form Env trimers, a loop located in the vicinity of the heparan binding site, and a region proximal to the highly conserved glycosylation site N8. We provide information on how nAbs specific for each of the two viral genotypes target different epitopes. Two common immune escape mechanisms, sequence variation and glycan shielding, were not observed. We propose a model according to which the neutralization mechanisms rely on the nAbs to block the Env conformational change and/or interfere with binding to susceptible cells. As the SFV RBD is structurally different from known retroviral RBDs, our data provide fundamental knowledge on the structural basis for the inhibition of viruses by nAbs. Trial registration: The study was registered at www.clinicaltrials.gov: https://clinicaltrials.gov/ct2/show/NCT03225794/.

Published
2023

11. Novel Toscana Virus Reverse Genetics System Establishes NSs as an Antagonist of Type I Interferon Responses

Author

Franziska Woelfl, Psylvia Léger, Nadia Oreshkova, Felix Pahmeier, Stefan Windhaber, Jana Koch, Megan Stanifer, Gleyder Roman Sosa, Zina M. Uckeley, Felix A. Rey, Steeve Boulant, Jeroen Kortekaas, Paul J. Wichgers Schreur, and Pierre-Yves Lozach

Subjects
Arbovirus, Bunyavirales, interferon, neglected diseases, Phenuiviridae, Phlebovirus, Microbiology, QR1-502
Abstract

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.

Published
2020
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12. Evolutionary diversification of the HAP2 membrane insertion motifs to drive gamete fusion across eukaryotes.

Author

Juliette Fedry, Jennifer Forcina, Pierre Legrand, Gérard Péhau-Arnaudet, Ahmed Haouz, Mark Johnson, Felix A Rey, and Thomas Krey

Subjects
Biology (General), QH301-705.5
Abstract

HAPLESS2 (HAP2) is a broadly conserved, gamete-expressed transmembrane protein that was shown recently to be structurally homologous to viral class II fusion proteins, which initiate fusion with host cells via insertion of fusion loops into the host membrane. However, the functional conformation of the HAP2 fusion loops has remained unknown, as the reported X-ray structure of Chlamydomonas reinhardtii HAP2 lacked this critical region. Here, we report a structure-guided alignment that reveals diversification of the proposed HAP2 fusion loops. Representative crystal structures show that in flowering plants, HAP2 has a single prominent fusion loop projecting an amphipathic helix at its apex, while in trypanosomes, three small nonpolar loops of HAP2 are poised to interact with the target membrane. A detailed structure-function analysis of the Arabidopsis HAP2 amphipathic fusion helix defines key residues that are essential for membrane insertion and for gamete fusion. Our study suggests that HAP2 may have evolved multiple modes of membrane insertion to accommodate the diversity of membrane environments it has encountered during eukaryotic evolution.

Published
2018
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13. Conformational Flexibility in the Immunoglobulin-Like Domain of the Hepatitis C Virus Glycoprotein E2

Author

Ieva Vasiliauskaite, Ania Owsianka, Patrick England, Abdul Ghafoor Khan, Sarah Cole, Dorothea Bankwitz, Steven K. H. Foung, Thomas Pietschmann, Joseph Marcotrigiano, Felix A. Rey, Arvind H. Patel, and Thomas Krey

Subjects
CD81 binding site, hepatitis C virus, Ig-like domain, conformational flexibility, glycoprotein E2, monoclonal antibodies, Microbiology, QR1-502
Abstract

ABSTRACT The hepatitis C virus (HCV) glycoprotein E2 is the major target of neutralizing antibodies and is therefore highly relevant for vaccine design. Its structure features a central immunoglobulin (Ig)-like β-sandwich that contributes to the binding site for the cellular receptor CD81. We show that a synthetic peptide corresponding to a β-strand of this Ig-like domain forms an α-helix in complex with the anti-E2 antibody DAO5, demonstrating an inside-out flip of hydrophobic residues and a secondary structure change in the composite CD81 binding site. A detailed interaction analysis of DAO5 and cross-competing neutralizing antibodies with soluble E2 revealed that the Ig-like domain is trapped by different antibodies in at least two distinct conformations. DAO5 specifically captures retrovirus particles bearing HCV glycoproteins (HCVpp) and infectious cell culture-derived HCV particles (HCVcc). Infection of cells by DAO5-captured HCVpp can be blocked by a cross-competing neutralizing antibody, indicating that a single virus particle simultaneously displays E2 molecules in more than one conformation on its surface. Such conformational plasticity of the HCV E2 receptor binding site has important implications for immunogen design. IMPORTANCE Recent advances in the treatment of hepatitis C virus (HCV) infection with direct-acting antiviral drugs have enabled the control of this major human pathogen. However, due to their high costs and limited accessibility in combination with the lack of awareness of the mostly asymptomatic infection, there is an unchanged urgent need for an effective vaccine. The viral glycoprotein E2 contains regions that are crucial for virus entry into the host cell, and antibodies that bind to these regions can neutralize infection. One of the major targets of neutralizing antibodies is the central immunoglobulin (Ig)-like domain within E2. We show here that this Ig-like domain is conformationally flexible at the surface of infectious HCV particles and pseudoparticles. Our study provides novel insights into the interactions of HCV E2 with the humoral immune system that should aid future vaccine development.

Published
2017
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14. Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Author

Taylor B. Engdahl, Elad Binshtein, Rebecca L. Brocato, Natalia A. Kuzmina, Lucia M Principe, Steven A. Kwilas, Robert K. Kim, Nathaniel S. Chapman, Monique S. Porter, Pablo Guardado-Calvo, Felix A. Rey, Laura S. Handal, Summer M. Diaz, Irene A. Zagol-Ikapitte, Joseph X. Reidy, Andrew Trivette, Alexander Bukreyev, Jay W. Hooper, and James. E. Crowe

Abstract

Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While sporadic in North and South America, many infections occur in Europe and Asia, with mortality ranging from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity across hantavirus species. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines.

Published
2022

15. Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses

Author

Eva Mittler, Anna Z. Wec, Janne Tynell, Pablo Guardado-Calvo, Julia Wigren-Byström, Laura C. Polanco, Cecilia M. O’Brien, Megan M. Slough, Dafna M. Abelson, Alexandra Serris, Mrunal Sakharkar, Gerard Pehau-Arnaudet, Russell R. Bakken, James C. Geoghegan, Rohit K. Jangra, Markus Keller, Larry Zeitlin, Olli Vapalahti, Rainer G. Ulrich, Zachary A. Bornholdt, Clas Ahlm, Felix A. Rey, John M. Dye, Steven B. Bradfute, Tomas Strandin, Andrew S. Herbert, Mattias N. E. Forsell, Laura M. Walker, Kartik Chandran, Albert Einstein College of Medicine [New York], Adimab [Lebanon], Umeå University, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), Mapp Biopharmaceutical Inc., Institute of Novel and Emerging Infectious Diseases (INNT), Friedrich-Loeffler-Institut (FLI), Helsingin yliopisto = Helsingfors universitet = University of Helsinki, The University of New Mexico [Albuquerque], This research was supported by NIAID of the NIH under award number U19AI142777 (Centers of Excellence in Translational Research) to K.C., L.M.W., J.M.D., Z.A.B., A.S.H., S.B.B., M.N.E.F., and L.Z. M.N.E.F. was supported by a Consolidator grant from the Swedish Science Council (#2020-06235). C.A. was supported by a Cutting Edge Research Grant from Region Västerbotten (VLL-579011). T.S. was supported by the Academy of Finland (#321809). P.G.-C. was supported by a grant of the National French Research Agency (ANR-18-CE11-0011). A.S. was supported by a fellowship of the French Foundation pour la Recherche Médicale (FRM, fellowship FDM20170638040). F.A.R. and P.G.-C. were supported by Labex IBEID (ANR-10-LABX-62-IBEID). R.G.U. was supported by the Bundesministerium für Bildung und Forschung (BMBF) within the Research Network Zoonotic Infectious Diseases (01KI1721A and 01KI2004A)., ANR-18-CE11-0011,LISEFU,Base structurale de la détection des lipides dans la fusion virale(2018), and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)

Subjects
Orthohantavirus, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Hantavirus Infections, virus diseases, General Medicine, Antibodies, Viral, Antibodies, Neutralizing, Puumala virus, Article, Epitopes, Cricetinae, Hemorrhagic Fever with Renal Syndrome, Animals, Humans, Glycoproteins
Abstract

International audience; The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the “capping loop” of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

Published
2022

16. Structural basis of synergistic neutralization of Crimean-Congo hemorrhagic fever virus by human antibodies

Author

Akaash K. Mishra, Jan Hellert, Natalia Freitas, Pablo Guardado-Calvo, Ahmed Haouz, J. Maximilian Fels, Daniel P. Maurer, Dafna M. Abelson, Zachary A. Bornholdt, Laura M. Walker, Kartik Chandran, François-Loïc Cosset, Jason S. McLellan, Felix A. Rey, University of Texas at Austin [Austin], Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), Albert Einstein College of Medicine [New York], Adimab LLC, Mapp Biopharmaceutical Inc., This work was supported by National Institutes of Health award U19 AI142777 to J.S.M., Z.A.B., K.C., and L.M.W., as well as by Institut Pasteur, CNRS and grant ANR-10-LABX-62-10 IBEID to F.A.R. and by the LabEx Ecofect (ANR-11-LABX-0048) of the 'Université de Lyon,' within the program 'Investissements d’Avenir' (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR), to F.-L.C. Research was funded in part by Welch Foundation grant F-0003-19620604 awarded to J.S.M. The Pasteur-Cantarini 24-month fellowship was granted to J.H., who was further supported by the Région Ile de France (Domaine d’intérêt majeur - innovative technologies for life sciences, DIM 1HEALTH)., ANR-11-LABX-0048,ECOFECT,Dynamiques eco-évolutives des maladies infectieuses(2011), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-11-IDEX-0007,Avenir L.S.E.,PROJET AVENIR LYON SAINT-ETIENNE(2011), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ribierre, Hélène, Dynamiques eco-évolutives des maladies infectieuses - - ECOFECT2011 - ANR-11-LABX-0048 - LABX - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, and PROJET AVENIR LYON SAINT-ETIENNE - - Avenir L.S.E.2011 - ANR-11-IDEX-0007 - IDEX - VALID

Subjects
Models, Molecular, Protein Folding, Multidisciplinary, Protein Conformation, [SDV]Life Sciences [q-bio], Virus Internalization, Antibodies, Viral, Crystallography, X-Ray, Antibodies, Neutralizing, Article, [SDV] Life Sciences [q-bio], Epitopes, Immunoglobulin Fab Fragments, Protein Domains, Neutralization Tests, Hemorrhagic Fever Virus, Crimean-Congo, Humans, Protein Multimerization, Viral Fusion Proteins, Protein Binding
Abstract

International audience; Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. Structural information is lacking in regard to the CCHFV membrane fusion glycoprotein Gc-the main target of the host neutralizing antibody response-as well as antibody-mediated neutralization mechanisms. We describe the structure of prefusion Gc bound to the antigen-binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show the two Fabs acting in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The structures also revealed the neutralization mechanism of previously reported antibodies against CCHFV, providing the molecular underpinnings essential for developing CCHFVspecific medical countermeasures for epidemic preparedness.

Published
2022

18. Immunization with GP1 but Not Core-like Particles Displaying Isolated Receptor-Binding Epitopes Elicits Virus-Neutralizing Antibodies against Junín Virus

Author

Gleyder Roman-Sosa, Anne Leske, Xenia Ficht, Tung Huy Dau, Julia Holzerland, Thomas Hoenen, Martin Beer, Robert Kammerer, Reinhold Schirmbeck, Felix A. Rey, Sandra M. Cordo, and Allison Groseth

Subjects
Pharmacology, Infectious Diseases, Drug Discovery, Immunology, Pharmacology (medical), arenavirus, Junín virus, immune response, neutralizing antibodies, glycoprotein, GP1
Abstract

New World arenaviruses are rodent-transmitted viruses and include a number of pathogens that are responsible for causing severe human disease. This includes Junín virus (JUNV), which is the causative agent of Argentine hemorrhagic fever. The wild nature and mobility of the rodent reservoir host makes it difficult to control the disease, and currently passive immunization with high-titer neutralizing antibody-containing plasma from convalescent patients is the only specific therapy. However, dwindling supplies of naturally available convalescent plasma, and challenges in developing similar resources for other closely related viruses, have made the development of alternative antibody-based therapeutic approaches of critical importance. In this study, we sought to induce a neutralizing antibody response in rabbits against the receptor-binding subunit of the viral glycoprotein, GP1, and the specific peptide sequences in GP1 involved in cellular receptor contacts. While these specific receptor-interacting peptides did not efficiently induce the production of neutralizing antibodies when delivered as a particulate antigen (as part of hepatitis B virus core-like particles), we showed that recombinant JUNV GP1 purified from transfected mammalian cells induced virus-neutralizing antibodies at high titers in rabbits. Further, neutralization was observed across a range of unrelated JUNV strains, a feature that is critical for effectiveness in the field. These results underscore the potential of GP1 alone to induce a potent neutralizing antibody response and highlight the importance of epitope presentation. In addition, effective virus neutralization by rabbit antibodies supports the potential applicability of this species for the future development of immunotherapeutics (e.g., based on humanized monoclonal antibodies). Such information can be applied in the design of vaccines and immunogens for both prevention and specific therapies against this and likely also other closely related pathogenic New World arenaviruses.

Published
2021

19. 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

20. Rubella virus: first calcium-requiring viral fusion protein.

Author

Mathieu Dubé, Felix A Rey, and Margaret Kielian

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

Rubella virus (RuV) infection of pregnant women can cause fetal death, miscarriage, or severe fetal malformations, and remains a significant health problem in much of the underdeveloped world. RuV is a small enveloped RNA virus that infects target cells by receptor-mediated endocytosis and low pH-dependent membrane fusion. The structure of the RuV E1 fusion protein was recently solved in its postfusion conformation. RuV E1 is a member of the class II fusion proteins and is structurally related to the alphavirus and flavivirus fusion proteins. Unlike the other known class II fusion proteins, however, RuV E1 contains two fusion loops, with a metal ion complexed between them by the polar residues N88 and D136. Here we demonstrated that RuV infection specifically requires Ca(2+) during virus entry. Other tested cations did not substitute. Ca(2+) was not required for virus binding to cell surface receptors, endocytic uptake, or formation of the low pH-dependent E1 homotrimer. However, Ca(2+) was required for low pH-triggered E1 liposome insertion, virus fusion and infection. Alanine substitution of N88 or D136 was lethal. While the mutant viruses were efficiently assembled and endocytosed by host cells, E1-membrane insertion and fusion were specifically blocked. Together our data indicate that RuV E1 is the first example of a Ca(2+)-dependent viral fusion protein and has a unique membrane interaction mechanism.

Published
2014
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21. Structural basis of HCV neutralization by human monoclonal antibodies resistant to viral neutralization escape.

Author

Thomas Krey, Annalisa Meola, Zhen-Yong Keck, Laurence Damier-Piolle, Steven K H Foung, and Felix A Rey

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

The high mutation rate of hepatitis C virus allows it to rapidly evade the humoral immune response. However, certain epitopes in the envelope glycoproteins cannot vary without compromising virus viability. Antibodies targeting these epitopes are resistant to viral escape from neutralization and understanding their binding-mode is important for vaccine design. Human monoclonal antibodies HC84-1 and HC84-27 target conformational epitopes overlapping the CD81 receptor-binding site, formed by segments aa434-446 and aa610-619 within the major HCV glycoprotein E2. No neutralization escape was yet observed for these antibodies. We report here the crystal structures of their Fab fragments in complex with a synthetic peptide comprising aa434-446. The structures show that the peptide adopts an α-helical conformation with the main contact residues F⁴⁴² and Y⁴⁴³ forming a hydrophobic protrusion. The peptide retained its conformation in both complexes, independently of crystal packing, indicating that it reflects a surface feature of the folded glycoprotein that is exposed similarly on the virion. The same residues of E2 are also involved in interaction with CD81, suggesting that the cellular receptor binds the same surface feature and potential escape mutants critically compromise receptor binding. In summary, our results identify a critical structural motif at the E2 surface, which is essential for virus propagation and therefore represents an ideal candidate for structure-based immunogen design for vaccine development.

Published
2013
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22. 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

23. Generation of plasma cells and CD27

Author

Priscilla F, Kerkman, Andy, Dernstedt, Lalitha, Tadala, Eva, Mittler, Mirjam, Dannborg, Christopher, Sundling, Kimia T, Maleki, Johanna, Tauriainen, Anne, Tuiskunen-Bäck, Julia, Wigren Byström, Pauline, Ocaya, Therese, Thunberg, Rohit K, Jangra, Gleyder, Román-Sosa, Pablo, Guardado-Calvo, Felix A, Rey, Jonas, Klingström, Kartik, Chandran, Andrea, Puhar, Clas, Ahlm, and Mattias Ne, Forsell

Subjects
B cells, atypical B cells, virus diseases, antibodies, Original Article, plasmablasts, Original Articles, haemorrhagic fever with renal syndrome, hantavirus
Abstract

Objective Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction. Methods We performed a comprehensive characterisation of longitudinal antiviral B‐cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells. Results We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27−IgD− B cells and CD27−/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection. Conclusion Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus‐infected individuals have significantly elevated levels of extracellular ATP in circulation., We show a comprehensive study to correlate two pathologies associated with haemorrhagic fever with renal syndrome (HFRS), thrombocytopenia and kidney dysfunction, with changes in the circulating B‐cell compartment (cells and antibodies). We show that thrombocytopenia is associated with mobilization of both activated and resting plasmablasts and plasma cells in circulation. However, the level of kidney dysfunction associated with longitudinal development of neutralizing antibodies in patients and associated with the reduction of surface CD27 expression on both plasmablasts and B cells in circulation. The low CD27 expression might be explained by elevated levels of ATP that could contribute to shedding of CD27 from B cells by a MMP‐8‐dependent mechanism.

Published
2020

24. Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.

Author

Zhen-yong Keck, Jinming Xia, Yong Wang, Wenyan Wang, Thomas Krey, Jannick Prentoe, Thomas Carlsen, Angela Ying-Jian Li, Arvind H Patel, Stanley M Lemon, Jens Bukh, Felix A Rey, and Steven K H Foung

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1-6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1-6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418-446 and aa611-616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434-446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410-425. The isolation of four HC-84 HMAbs binding to the peptide, aa434-446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus.

Published
2012
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25. Characterization of reemerging chikungunya virus.

Author

Marion Sourisseau, Clémentine Schilte, Nicoletta Casartelli, Céline Trouillet, Florence Guivel-Benhassine, Dominika Rudnicka, Nathalie Sol-Foulon, Karin Le Roux, Marie-Christine Prevost, Hafida Fsihi, Marie-Pascale Frenkiel, Fabien Blanchet, Philippe V Afonso, Pierre-Emmanuel Ceccaldi, Simona Ozden, Antoine Gessain, Isabelle Schuffenecker, Bruno Verhasselt, Alessia Zamborlini, Ali Saïb, Felix A Rey, Fernando Arenzana-Seisdedos, Philippe Desprès, Alain Michault, Matthew L Albert, and Olivier Schwartz

Subjects
Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.

Published
2007
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26. Common characteristics and unique features: A comparison of the fusion machinery of the alphaherpesviruses Pseudorabies virus and Herpes simplex virus

Author

Melina, Vallbracht, Marija, Backovic, Barbara G, Klupp, Felix A, Rey, and Thomas C, Mettenleiter

Subjects
Viral Envelope Proteins, Herpesvirus 2, Human, Cell Membrane, Virus Attachment, Herpesvirus 1, Human, Virus Internalization, Herpesvirus 1, Suid, Glycoproteins
Abstract

Membrane fusion is a fundamental biological process that allows different cellular compartments delimited by a lipid membrane to release or exchange their respective contents. Similarly, enveloped viruses such as alphaherpesviruses exploit membrane fusion to enter and infect their host cells. For infectious entry the prototypic human Herpes simplex viruses 1 and 2 (HSV-1 and -2, collectively termed HSVs) and the porcine Pseudorabies virus (PrV) utilize four different essential envelope glycoproteins (g): the bona fide fusion protein gB and the regulatory heterodimeric gH/gL complex that constitute the "core fusion machinery" conserved in all members of the Herpesviridae; and the subfamily specific receptor binding protein gD. These four components mediate attachment and fusion of the virion envelope with the host cell plasma membrane through a tightly regulated sequential activation process. Although PrV and the HSVs are closely related and employ the same set of glycoproteins for entry, they show remarkable differences in the requirements for fusion. Whereas the HSVs strictly require all four components for membrane fusion, PrV can mediate cell-cell fusion without gD. Moreover, in contrast to the HSVs, PrV provides a unique opportunity for reversion analyses of gL-negative mutants by serial cell culture passaging, due to a limited cell-cell spread capacity of gL-negative PrV not observed in the HSVs. This allows a more direct analysis of the function of gH/gL during membrane fusion. Unraveling the molecular mechanism of herpesvirus fusion has been a goal of fundamental research for years, and yet important mechanistic details remain to be uncovered. Nevertheless, the elucidation of the crystal structures of all key players involved in PrV and HSV membrane fusion, coupled with a wealth of functional data, has shed some light on this complex puzzle. In this review, we summarize and discuss the contemporary knowledge on the molecular mechanism of entry and membrane fusion utilized by the alphaherpesvirus PrV, and highlight similarities but also remarkable differences in the requirements for fusion between PrV and the HSVs.

Published
2019

27. Immune correlates of protection for dengue: State of the art and research agenda

Author

Leah C. Katzelnick, Eva Harris, Ralph Baric, Beth-Ann Coller, Josefina Coloma, James E. Crowe, Derek A.T. Cummings, Hansi Dean, Aravinda de Silva, Michael S. Diamond, Anna Durbin, Neil Ferguson, Peter B. Gilbert, Aubree Gordon, Duane J. Gubler, Bruno Guy, M. Elizabeth Halloran, Scott Halstead, Nicholas Jackson, Richard Jarman, Shee-mei Lok, Nelson L. Michael, Eng Eong Ooi, Athanasios Papadopoulos, Stanley Plotkin, Alexander R. Precioso, Robert Reiner, Felix A. Rey, Isabel Rodríguez-Barraquer, Alan Rothman, Alexander C. Schmidt, Gavin Screaton, Alessandro Sette, Cameron Simmons, Ashley L. St. John, Wellington Sun, Stephen Thomas, Joseph Torresi, John S. Tsang, Kirsten Vannice, Stephen Whitehead, Annelies Wilder-Smith, In Kyu Yoon, and Simmons, C

Subjects
0301 basic medicine, and promotion of well-being, Disease, Dengue virus, medicine.disease_cause, Antibodies, Viral, Medical and Health Sciences, Dengue fever, Dengue, Immune correlates of protection, Viral, Neutralizing, Biological Sciences, Phase III as Topic, Infectious Diseases, 3.4 Vaccines, Immune correlates, Molecular Medicine, Immune correlates of risk, Infection, Context (language use), Dengue Vaccines, Antibodies, Severe dengue, Article, Vaccine Related, 03 medical and health sciences, Rare Diseases, Clinical Trials, Phase II as Topic, Biodefense, Virology, Environmental health, medicine, Humans, Clinical Trials, Severe Dengue, Dengue vaccine, Agricultural and Veterinary Sciences, General Veterinary, General Immunology and Microbiology, business.industry, Prevention, Phase II as Topic, Public Health, Environmental and Occupational Health, Congresses as Topic, Dengue Virus, Prevention of disease and conditions, Vaccine efficacy, medicine.disease, Antibodies, Neutralizing, Vector-Borne Diseases, Natural infection, Emerging Infectious Diseases, Good Health and Well Being, 030104 developmental biology, Clinical Trials, Phase III as Topic, Immunology, Participants in the Summit on Dengue Immune Correlates of Protection, Immunization, business, Vaccine
Abstract

Dengue viruses (DENV1-4) are mosquito-borne flaviviruses estimated to cause up to ∼400 million infections and ∼100 million dengue cases each year. Factors that contribute to protection from and risk of dengue and severe dengue disease have been studied extensively but are still not fully understood. Results from Phase 3 vaccine efficacy trials have recently become available for one vaccine candidate, now licensed for use in several countries, and more Phase 2 and 3 studies of additional vaccine candidates are ongoing, making these issues all the more urgent and timely. At the "Summit on Dengue Immune Correlates of Protection", held in Annecy, France, on March 8-9, 2016, dengue experts from diverse fields came together to discuss the current understanding of the immune response to and protection from DENV infection and disease, identify key unanswered questions, discuss data on immune correlates and plans for comparison of results across assays/consortia, and propose a research agenda for investigation of dengue immune correlates, all in the context of both natural infection studies and vaccine trials.

Published
2017

28. The Therapeutic Antibody LM609 Selectively Inhibits Ligand Binding to Human α

Author

Andrew J, Borst, Zachary M, James, William N, Zagotta, Mark, Ginsberg, Felix A, Rey, Frank, DiMaio, Marija, Backovic, and David, Veesler

Subjects
Models, Molecular, Binding Sites, Bone Density Conservation Agents, Amino Acid Motifs, Genetic Vectors, Antibodies, Monoclonal, Gene Expression, Angiogenesis Inhibitors, Crystallography, X-Ray, Integrin alphaVbeta3, Ligands, Antiviral Agents, Recombinant Proteins, Article, Immunoglobulin Fab Fragments, Escherichia coli, Humans, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Antigens, Cloning, Molecular, Oligopeptides, Protein Binding
Abstract

The LM609 antibody specifically recognizes αVβ3 integrin and inhibits angiogenesis, bone resorption, and viral infections in an arginine-glycine-aspartate independent manner. LM609 entered phase II clinical trials for the treatment of several cancers and was also used for αVβ3-targeted radio-immunotherapy. To elucidate the mechanisms of recognition and inhibition of αVβ3 integrin, we solved the structure of the LM609 antigen-binding fragment by X-ray crystallography and determined its binding affinity for αVβ3. Using single-particle electron microscopy we show that LM609 binds at the interface between the β-propeller domain of the αV chain and the βI domain of the β3 chain, near the RGD-binding site, of all observed integrin conformational states. Integrating this data with fluorescence size-exclusion chromatography, we demonstrate that LM609 sterically hinders access of large ligands to the RGD binding pocket, without obstructing it. This work provides a structural framework to expedite future efforts utilizing LM609 as a diagnostic or therapeutic tool.

Published
2017

29. Macromolecular assemblies

Author

Felix A, Rey and Wesley I, Sundquist

Subjects
0303 health sciences, 03 medical and health sciences, Macromolecular Substances, Structural Biology, Multiprotein Complexes, 010402 general chemistry, 01 natural sciences, Molecular Biology, 030304 developmental biology, 0104 chemical sciences
Published
2013

30. Dengue virus: two hosts, two structures

Author

Felix A, Rey

Subjects
Dengue, Culicidae, Viral Envelope Proteins, Animals, Humans, Viral Vaccines, Dengue Virus, Protein Multimerization, Antibodies, Neutralizing, Antigens, Viral, Host Specificity, Body Temperature, Insect Vectors
Published
2013

31. Macromolecular assemblages

Author

Felix A, Rey, Wesley I, Sundquist, Virologie Structurale, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry, University of Utah, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0303 health sciences, 03 medical and health sciences, 0302 clinical medicine, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Macromolecular Substances, Structural Biology, Molecular Biology, 030217 neurology & neurosurgery, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

International audience

Published
2011

32. Transcytosis subversion by M cell-to-enterocyte spread promotes Shigella flexneri and Listeria monocytogenes intracellular bacterial dissemination

Author

Yuen-Yan Chang, Jost Enninga, Patricia Latour-Lambert, Hugo Varet, Camille Rey, Caroline Proux, Rachel Legendre, Jean-Yves Coppée, Dynamique des Interactions Hôte-Pathogène - Dynamics of Host-Pathogen Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Transcriptome et Epigénome (PF2), Institut Pasteur [Paris] (IP), CR acknowledges a grant from Danone Research. YYC is supported through a postdoctoral fellowship from the Fondation pour la Recherche Médicale (FRM). JE acknowledges grant support from Institut Pasteur (GPF 'MCellHTLV'), the European Union (ERC CoG 'EndoSubvert'), and the ANR (Grants 'StopBugEntry' and 'AutoHostPath'). The DIHP unit is member of the IBEID and MI LabExes., We thank Felix A. Rey and John Rohde for critical comments of the manuscript. We thank Philippe Sansonetti, Javier Pizarro-Cerda and Pascale Cossart for bacterial strains. We thank Marie-Agnes Dillies for advice on single cell transcriptomic statistical analysis, Valentina Libri and the Center for Translational Science (CRT) / Cytometry and Biomarkers Unit of Technology and Service (CB UTechS) at Institut Pasteur for support in conducting this study, and Jean-Yves Tinevez and the Imagopole France–BioImaging infrastructure, supported by the French National Research Agency (ANR 10-INSB-04-01, Investments for the Future), for advice and access to microscopes. Finally, we thank Gianfranco Grompone and Muriel Derrien for enabling this study., ANR-15-CE15-0017,StopBugEntry,Identification des nouvelles molécules cellulaires cibles pour combattre les infections bactériennes(2015), ANR-15-CE15-0018,AutoHostPath,Rôles alternatifs pour les récepteurs de l'autophagie dans les interactions hôte-pathogène(2015), European Project: 682809,EndoSubvert(2017), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris]

Subjects
Cell Lines, Pathology and Laboratory Medicine, Shigella flexneri, White Blood Cells, Gastrointestinal tract, Animal Cells, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Medicine and Health Sciences, Listeriosis, Pathogen motility, Biology (General), Caco-2 cells, Microfold cell, Fluorescence microscopy, Microscopy, 0303 health sciences, 030302 biochemistry & molecular biology, Light Microscopy, Acquired immune system, Bacterial Pathogens, 3. Good health, Cell biology, Transcytosis, Medical Microbiology, Biological Cultures, Pathogens, Cellular Types, Anatomy, Intracellular, Research Article, QH301-705.5, Virulence Factors, Immune Cells, Immunology, Biology, Research and Analysis Methods, Microbiology, 03 medical and health sciences, Immune system, Virology, Genetics, Humans, Antibody-Producing Cells, Microbial Pathogens, Molecular Biology, Tropism, Dysentery, Bacillary, 030304 developmental biology, Intracellular pathogens, B cells, Blood Cells, Bacteria, Intracellular parasite, Organisms, Biology and Life Sciences, Epithelial Cells, Cell Biology, RC581-607, biology.organism_classification, Listeria monocytogenes, Enterocytes, Parasitology, Shigella, Immunologic diseases. Allergy, Digestive System
Abstract

Microfold (M) cell host-pathogen interaction studies would benefit from the visual analysis of dynamic cellular and microbial interplays. We adapted a human in vitro M cell model to physiological bacterial infections, expression of fluorescent localization reporters and long-term three-dimensional time-lapse microscopy. This approach allows following key steps of M cell infection dynamics at subcellular resolution, from the apical onset to basolateral epithelial dissemination. We focused on the intracellular pathogen Shigella flexneri, classically reported to transcytose through M cells to initiate bacillary dysentery in humans, while eliciting poorly protective immune responses. Our workflow was critical to reveal that S. flexneri develops a bimodal lifestyle within M cells leading to rapid transcytosis or delayed vacuolar rupture, followed by direct actin motility-based propagation to neighboring enterocytes. Moreover, we show that Listeria monocytogenes, another intracellular pathogen sharing a tropism for M cells, disseminates in a similar manner and evades M cell transcytosis completely. We established that actin-based M cell-to-enterocyte spread is the major dissemination pathway for both pathogens and avoids their exposure to basolateral compartments in our system. Our results challenge the notion that intracellular pathogens are readily transcytosed by M cells to inductive immune compartments in vivo, providing a potential mechanism for their ability to evade adaptive immunity., Author summary Microfold (M) epithelial cells are important for the onset of infections and induction of immune responses in many mucosal diseases. We extended a human in vitro M cell model to apical infections, expression of fluorescent host and microbial reporters and real-time fluorescence microscopy. Focusing on the human intracellular pathogen S. flexneri, responsible for bacillary dysentery, this workflow allowed us to uncover that the bacterium can subvert the immunological sampling function of M cells by promoting a cytosolic lifestyle and spreading directly to neighboring enterocytes. This mechanism was shared with the etiologic agent of listeriosis, the intracellular pathogen L. monocytogenes and allowed both pathogens to avoid exposure to underlying immune compartments. These results may provide a mechanism for the ability of intracellular pathogens to evade adaptive immunity in vivo, emphasizing the importance of advanced studies of M cell host-pathogen interactions to understand early steps of mucosal invasion and their consequences on immunity.

Published
2020

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