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1. 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 Bilounga-Ndongo, Félix A Rey, Antoine Gessain, Marija Backovic, and Florence Buseyne
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
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
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2. 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, Félix A Rey, Laura S Handal, Summer M Diaz, Irene A Zagol-Ikapitte, Minh H Tran, W Hayes McDonald, Jens Meiler, Joseph X Reidy, Andrew Trivette, Alexander Bukreyev, Jay W Hooper, and James E Crowe
Subjects: hantavirus, bunyavirus, Sin Nombre virus, Andes virus, Hantaan virus, antibody, Medicine, Science, Biology (General), QH301-705.5
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 infections in humans are relatively rare, mortality rates range 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 human 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 to authentic hantaviruses. 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: 2023
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3. Human herpesvirus 8 molecular mimicry of ephrin ligands facilitates cell entry and triggers EphA2 signaling.

Author: Taylor P Light, Delphine Brun, Pablo Guardado-Calvo, Riccardo Pederzoli, Ahmed Haouz, Frank Neipel, Félix A Rey, Kalina Hristova, and Marija Backovic
Subjects: Biology (General), QH301-705.5
Abstract: Human herpesvirus 8 (HHV-8) is an oncogenic virus that enters cells by fusion of the viral and endosomal cellular membranes in a process mediated by viral surface glycoproteins. One of the cellular receptors hijacked by HHV-8 to gain access to cells is the EphA2 tyrosine kinase receptor, and the mechanistic basis of EphA2-mediated viral entry remains unclear. Using X-ray structure analysis, targeted mutagenesis, and binding studies, we here show that the HHV-8 envelope glycoprotein complex H and L (gH/gL) binds with subnanomolar affinity to EphA2 via molecular mimicry of the receptor's cellular ligands, ephrins (Eph family receptor interacting proteins), revealing a pivotal role for the conserved gH residue E52 and the amino-terminal peptide of gL. Using FSI-FRET and cell contraction assays, we further demonstrate that the gH/gL complex also functionally mimics ephrin ligand by inducing EphA2 receptor association via its dimerization interface, thus triggering receptor signaling for cytoskeleton remodeling. These results now provide novel insight into the entry mechanism of HHV-8, opening avenues for the search of therapeutic agents that could interfere with HHV-8-related diseases.
Published: 2021
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4. Multimeric single-domain antibody complexes protect against bunyavirus infections

Author: Paul J Wichgers Schreur, Sandra van de Water, Michiel Harmsen, Erick Bermúdez-Méndez, Dubravka Drabek, Frank Grosveld, Kerstin Wernike, Martin Beer, Andrea Aebischer, Olalekan Daramola, Sara Rodriguez Conde, Karen Brennan, Dorota Kozub, Maiken Søndergaard Kristiansen, Kieran K Mistry, Ziyan Deng, Jan Hellert, Pablo Guardado-Calvo, Félix A Rey, Lucien van Keulen, and Jeroen Kortekaas
Subjects: bunyavirus, therapy, single-domain antibody, vhh, rift valley fever virus, schmallenberg virus, Medicine, Science, Biology (General), QH301-705.5
Abstract: The World Health Organization has included three bunyaviruses posing an increasing threat to human health on the Blueprint list of viruses likely to cause major epidemics and for which no, or insufficient countermeasures exist. Here, we describe a broadly applicable strategy, based on llama-derived single-domain antibodies (VHHs), for the development of bunyavirus biotherapeutics. The method was validated using the zoonotic Rift Valley fever virus (RVFV) and Schmallenberg virus (SBV), an emerging pathogen of ruminants, as model pathogens. VHH building blocks were assembled into highly potent neutralizing complexes using bacterial superglue technology. The multimeric complexes were shown to reduce and prevent virus-induced morbidity and mortality in mice upon prophylactic administration. Bispecific molecules engineered to present two different VHHs fused to an Fc domain were further shown to be effective upon therapeutic administration. The presented VHH-based technology holds great promise for the development of bunyavirus antiviral therapies.
Published: 2020
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5. Molecular organization and dynamics of the fusion protein Gc at the hantavirus surface

Author: Eduardo A Bignon, Amelina Albornoz, Pablo Guardado-Calvo, Félix A Rey, and Nicole D Tischler
Subjects: envelope proteins, inter-spike interface, membrane fusion, viral breathing, Bunyavirales order, Medicine, Science, Biology (General), QH301-705.5
Abstract: The hantavirus envelope glycoproteins Gn and Gc mediate virion assembly and cell entry, with Gc driving fusion of viral and endosomal membranes. Although the X-ray structures and overall arrangement of Gn and Gc on the hantavirus spikes are known, their detailed interactions are not. Here we show that the lateral contacts between spikes are mediated by the same 2-fold contacts observed in Gc crystals at neutral pH, allowing the engineering of disulfide bonds to cross-link spikes. Disrupting the observed dimer interface affects particle assembly and overall spike stability. We further show that the spikes display a temperature-dependent dynamic behavior at neutral pH, alternating between ‘open’ and ‘closed’ forms. We show that the open form exposes the Gc fusion loops but is off-pathway for productive Gc-induced membrane fusion and cell entry. These data also provide crucial new insights for the design of optimized Gn/Gc immunogens to elicit protective immune responses.
Published: 2019
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6. A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

Author: Danilo Dubrau, M Alejandra Tortorici, Félix A Rey, and Norbert Tautz
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.
Published: 2017
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7. Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc.

Author: Pablo Guardado-Calvo, Eduardo A Bignon, Eva Stettner, Scott Allen Jeffers, Jimena Pérez-Vargas, Gerard Pehau-Arnaudet, M Alejandra Tortorici, Jean-Luc Jestin, Patrick England, Nicole D Tischler, and Félix A Rey
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.
Published: 2016
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8. The disulfide bonds in glycoprotein E2 of hepatitis C virus reveal the tertiary organization of the molecule.

Author: Thomas Krey, Jacques d'Alayer, Carlos M Kikuti, Aure Saulnier, Laurence Damier-Piolle, Isabelle Petitpas, Daniel X Johansson, Rajiv G Tawar, Bruno Baron, Bruno Robert, Patrick England, Mats A A Persson, Annette Martin, and Félix A Rey
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% beta-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.
Published: 2010
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9. Dermal-type macrophages expressing CD209/DC-SIGN show inherent resistance to dengue virus growth.

Author: Wing-Hong Kwan, Erika Navarro-Sanchez, Hélène Dumortier, Marion Decossas, Hortense Vachon, Flavia Barreto dos Santos, Hervé W Fridman, Félix A Rey, Eva Harris, Philippe Despres, and Christopher G Mueller
Subjects: Arctic medicine. Tropical medicine, RC955-962, Public aspects of medicine, RA1-1270
Abstract: BACKGROUND: An important question in dengue pathogenesis is the identity of immune cells involved in the control of dengue virus infection at the site of the mosquito bite. There is evidence that infection of immature myeloid dendritic cells plays a crucial role in dengue pathogenesis and that the interaction of the viral envelope E glycoprotein with CD209/DC-SIGN is a key element for their productive infection. Dermal macrophages express CD209, yet little is known about their role in dengue virus infection. METHODS AND FINDINGS: Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein. Because dermal macrophages stain for IL-10 in situ, we generated dermal-type macrophages from monocytes in the presence of IL-10 to study their infection by dengue virus. The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells. In addition, no IFN-alpha was produced in response to the virus. The inability of dengue virus to grow in the macrophages was attributable to accumulation of internalized virus particles into poorly-acidified phagosomes. CONCLUSIONS: Aborting infection by viral sequestration in early phagosomes would present a novel means to curb infection of enveloped virus and may constitute a prime defense system to prevent dengue virus spread shortly after the bite of the infected mosquito.
Published: 2008
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10. Characterization of a structural intermediate of flavivirus membrane fusion.

Author: Karin Stiasny, Christian Kössl, Jean Lepault, Félix A Rey, and Franz X Heinz
Subjects: Immunologic diseases. Allergy, RC581-607, Biology (General), QH301-705.5
Abstract: Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E) mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion.
Published: 2007
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11. Genome microevolution of chikungunya viruses causing the Indian Ocean outbreak.

Author: Isabelle Schuffenecker, Isabelle Iteman, Alain Michault, Séverine Murri, Lionel Frangeul, Marie-Christine Vaney, Rachel Lavenir, Nathalie Pardigon, Jean-Marc Reynes, François Pettinelli, Leon Biscornet, Laure Diancourt, Stéphanie Michel, Stéphane Duquerroy, Ghislaine Guigon, Marie-Pascale Frenkiel, Anne-Claire Bréhin, Nadège Cubito, Philippe Desprès, Frank Kunst, Félix A Rey, Hervé Zeller, and Sylvain Brisse
Subjects: Medicine
Abstract: BackgroundA chikungunya virus outbreak of unprecedented magnitude is currently ongoing in Indian Ocean territories. In Réunion Island, this alphavirus has already infected about one-third of the human population. The main clinical symptom of the disease is a painful and invalidating poly-arthralgia. Besides the arthralgic form, 123 patients with a confirmed chikungunya infection have developed severe clinical signs, i.e., neurological signs or fulminant hepatitis.Methods and findingsWe report the nearly complete genome sequence of six selected viral isolates (isolated from five sera and one cerebrospinal fluid), along with partial sequences of glycoprotein E1 from a total of 127 patients from Réunion, Seychelles, Mauritius, Madagascar, and Mayotte islands. Our results indicate that the outbreak was initiated by a strain related to East-African isolates, from which viral variants have evolved following a traceable microevolution history. Unique molecular features of the outbreak isolates were identified. Notably, in the region coding for the non-structural proteins, ten amino acid changes were found, four of which were located in alphavirus-conserved positions of nsP2 (which contains helicase, protease, and RNA triphosphatase activities) and of the polymerase nsP4. The sole isolate obtained from the cerebrospinal fluid showed unique changes in nsP1 (T301I), nsP2 (Y642N), and nsP3 (E460 deletion), not obtained from isolates from sera. In the structural proteins region, two noteworthy changes (A226V and D284E) were observed in the membrane fusion glycoprotein E1. Homology 3D modelling allowed mapping of these two changes to regions that are important for membrane fusion and virion assembly. Change E1-A226V was absent in the initial strains but was observed in >90% of subsequent viral sequences from Réunion, denoting evolutionary success possibly due to adaptation to the mosquito vector.ConclusionsThe unique molecular features of the analyzed Indian Ocean isolates of chikungunya virus demonstrate their high evolutionary potential and suggest possible clues for understanding the atypical magnitude and virulence of this outbreak.
Published: 2006
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12. Variants with the N501Y mutation extend SARS-CoV-2 host range to mice, with contact transmission

Author: Sylvie Behillil, Dominique Rousset, Flora Donati, Laurine Levillayer, Grégory Jouvion, Jean Jaubert, Xavier Montagutelli, Vincent Enouf, Mélanie Albert, Sylvie van der Werf, Fabiana Gámbaro, Eduard Baquero Salazar, Etienne Simon-Loriere, Félix A. Rey, Laurine Conquet, Matthieu Prot, Génétique de la souris - Mouse Genetics, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Génétique fonctionnelle des maladies infectieuses - Functional Genetics of Infectious Diseases, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), 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), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Centre National de Référence des virus respiratoires (dont la grippe et le SARS-CoV2) [Paris] (CNR - laboratoire associé), Institut Pasteur de la Guyane, Réseau International des Instituts Pasteur (RIIP), This work was supported by the « URGENCE COVID-19 » fundraising campaign of Institut Pasteur), the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases (Grant No. ANR-10-LABX-62-IBEID), the Agence Nationale de la Recherche (Grant No. ANR-20-COVI-0028-01) and the RECOVER project funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 101003589. ESL acknowledges funding from the INCEPTION programme (Investissements d’Avenir grant ANR-16-CONV-0005)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-20-COVI-0028,HuMoCID,Développement de modèles murins de COVID-19(2020), ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016), and European Project: 101003589, H2020-SC1-PHE-CORONAVIRUS-2020,RECOVER(2020)
Subjects: Infectivity, Genetics, Mutation rate, variants, reservoir, mice, Rodent, biology, Sars-CoV-2, Host (biology), [SDV]Life Sciences [q-bio], transmission, RNA, host range, Virus, biology.animal, Adaptation, Receptor
Abstract: Receptor recognition is a major determinant of viral host range, as well as infectivity and pathogenesis. Emergences have been associated with serendipitous events of adaptation upon encounters with a novel host, and the high mutation rate of RNA viruses has been proposed to explain their frequent host shifts 1. SARS-CoV-2 extensive circulation in humans has been associated with the emergence of variants, including variants of concern (VOCs) with diverse mutations in the spike and increased transmissibility or immune escape 2. Here we show that unlike the initial virus, VOCs are able to infect common laboratory mice, replicating to high titers in the lungs. This host range expansion is explained in part by the acquisition of changes at key positions of the receptor binding domain that enable binding to the mouse angiotensin-converting enzyme 2 (ACE2) cellular receptor, although differences between viral lineages suggest that other factors are involved in the capacity of SARS-CoV-2 VOCs to infect mice. This abrogation of the species barrier raises the possibility of wild rodent secondary reservoirs and provides new experimental models to study disease pathophysiology and countermeasures.
Published: 2023

13. A synthetic delivery vector for mucosal vaccination

Author: Anne Billet, Justine Hadjerci, Thi Tran, Pascal Kessler, Jonathan Ulmer, Gilles Mourier, Marine Ghazarian, Anthony Gonzalez, Robert Thai, Pauline Urquia, Anne-Cécile Van Baelen, Annalisa Meola, Ignacio Fernandez, Stéphanie Deville-Foillard, Ewan MacDonald, Léa Paolini, Frédéric Schmidt, Félix A. Rey, Michael S. Kay, Eric Tartour, Denis Servent, and Ludger Johannes
Abstract: The success of mRNA-based vaccines during the Covid-19 pandemic has highlighted the value of this new platform for vaccine development against infectious disease. However, the CD8+T cell response remains modest with mRNA vaccines, and these do not induce mucosal immunity, which would be needed to prevent viral spread in the healthy population. To address this drawback, we developed a dendritic cell targeting mucosal vaccination vector, the homopentameric STxB. Here, we describe the highly efficient chemical synthesis of the protein, and itsin vitrofolding. This straightforward preparation led to a synthetic delivery tool whose biophysical and intracellular trafficking characteristics were largely indistinguishable from recombinant STxB. The chemical approach allowed for the generation of new variants with bioorthogonal handles. Selected variants were chemically coupled to several types of antigens derived from the mucosal viruses SARS-CoV-2 and type 16 human papillomavirus. Upon intranasal administration in mice, mucosal immunity, including resident memory CD8+T cells and IgA antibodies was induced against these antigens. Our study thereby identifies a novel synthetic antigen delivery tool for mucosal vaccination with an unmatched potential to respond to an urgent medical need.
Published: 2023

14. Structure, function, and evolution of the Orthobunyavirus membrane fusion glycoprotein

Author: Jan Hellert, Andrea Aebischer, Ahmed Haouz, Pablo Guardado-Calvo, Sven Reiche, Martin Beer, Félix A. Rey, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Leibniz-Institut für Virologie [Hamburg] (LIV), Friedrich-Loeffler-Institut (FLI), Cristallographie (Plateforme) - Crystallography (Platform), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This research was performed as part of the Zoonoses Anticipation and Preparedness Initiative (ZAPI project, IMI grant agreement 115760) with the assistance and financial support of IMI and the European Commission and in-kind contributions from EFPIA partners. Additional funding was provided by Institut Pasteur, CNRS, and grant ANR-10-LABX-62-IBEID (to F.A.R.). J.H. received the Pasteur-Cantarini fellowship for 24 months and was further supported by the Région Ile de France (Domaine d’intêret majeur innovative technologies for life sciences, DIM 1HEALTH)., and We thank the staff of the crystallography platform at Institut Pasteur for robot-driven crystallization screening. We also thank Fabrice Agou from the Chemogenomic and Biological Screening platform at Institut Pasteur and the staff of synchrotron beamlines PX1 at SOLEIL (St. Aubin, France) and ID23-2 at the ESRF (Grenoble, France) for help during data collection.
Subjects: emerging viruses, one health, arboviruses, La Crosse virus, [SDV]Life Sciences [q-bio], membrane fusion, Schmallenberg virus, orthobunyaviruses, General Biochemistry, Genetics and Molecular Biology, X-ray crystallography, zoonoses
Abstract: International audience; La Crosse virus, responsible for pediatric encephalitis in the United States, and Schmallenberg virus, a highly teratogenic veterinary virus in Europe, belong to the large Orthobunyavirus genus of zoonotic arthropod-borne pathogens distributed worldwide. Viruses in this under-studied genus cause CNS infections or fever with debilitating arthralgia/myalgia syndromes, with no effective treatment. The main surface antigen, glycoprotein Gc (∼1,000 residues), has a variable N-terminal half (GcS) targeted by the patients’ antibody response and a conserved C-terminal moiety (GcF) responsible for membrane fusion during cell entry. Here, we report the X-ray structure of post-fusion La Crosse and Schmallenberg virus GcF, revealing the molecular determinants for hairpin formation and trimerization required to drive membrane fusion. We further experimentally confirm the role of residues in the fusion loops and in a vestigial endoplasmic reticulum (ER) translocation sequence at the GcS-GcF junction. The resulting knowledge provides essential molecular underpinnings for future development of potential therapeutic treatments and vaccines.
Published: 2023

15. Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Author: Rebecca L Brocato, Elad Binshtein, Taylor B Engdahl, Natalia A Kuzmina, Lucia M Principe, Steven A Kwilas, Robert K Kim, Nathaniel S Chapman, Monique S Porter, Pablo Guardado-Calvo, Félix A Rey, Laura S Handal, Summer M Diaz, Irene A Zagol-Ikapitte, Minh H Tran, W Hayes McDonald, Jens Meiler, Joseph X Reidy, Andrew Trivette, Alexander Bukreyev, Jay W Hooper, James E Crowe, Vanderbilt University [Nashville], U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), University of Texas Medical Branch at Galveston, Institut Pasteur [Paris] (IP), Université Paris Cité (UPCité), and The work of TBE was supported by NIH training grant 5T32GM008320-30. The work of JWH. and other USAMRIID authors was supported by the Military Infectious Disease Research Program under project number MI210048.
Subjects: General Immunology and Microbiology, General Neuroscience, [SDV]Life Sciences [q-bio], General Medicine, General Biochemistry, Genetics and Molecular Biology
Abstract: International audience; 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 infections in humans are relatively rare, mortality rates range 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 human 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 to authentic hantaviruses. 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: 2023

16. Author response: Antigenic mapping and functional characterization of human New World hantavirus neutralizing antibodies

Author: Rebecca L Brocato, Elad Binshtein, Taylor B Engdahl, Natalia A Kuzmina, Lucia M Principe, Steven A Kwilas, Robert K Kim, Nathaniel S Chapman, Monique S Porter, Pablo Guardado-Calvo, Félix A Rey, Laura S Handal, Summer M Diaz, Irene A Zagol-Ikapitte, Minh H Tran, W Hayes McDonald, Jens Meiler, Joseph X Reidy, Andrew Trivette, Alexander Bukreyev, Jay W Hooper, and James E Crowe
Published: 2023

17. [The crystal structure of the picobirnavirus capsid reveals an organization with a unique geometry]

Author: Stéphane, Duquerroy, Bruno, Da Costa, Céline, Henry, Armelle, Vigouroux, Sonia, Libersou, Jean, Lepault, Jorge, Navaza, Bermard, Delmas, and Félix A, Rey
Published: 2022

18. Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

Author: Najiby Kassis-Chikhani, Samira Fafi-Kremer, Olivier Schwartz, Laurent Bélec, Matthieu Prot, Cyril Planchais, Timothée Bruel, Laura Courtellemont, Florence Guivel-Benhassine, Artem Baidaliuk, Isabelle Staropoli, Julien Le Guen, Etienne Simon-Loriere, Laurent Hocqueloux, Delphine Planas, David Veyer, Pierre Gantner, Maaran Michael Rajah, Aurélie Velay, Thierry Prazuck, Dhiaeddine Edriss, Félix A. Rey, Françoise Porrot, Hélène Péré, Floriane Gallais, Aymeric Sève, Nicolas Robillard, Julien Puech, Hugo Mouquet, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Génomique fonctionnelle des tumeurs solides = Functional Genomics of Solid Tumors [CRC] (FunGeST), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, Institut Pasteur [Paris] (IP), Université Paris Cité (UPCité), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), CHU Strasbourg, Immuno-Rhumatologie Moléculaire, Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre Hospitalier Régional d'Orléans (CHRO), Virologie Structurale - Structural Virology, The Opera system was co-funded by the Institut Pasteur and the Région Île de France (DIM1Health). Work in the O.S. laboratory is funded by the Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche Médicale (FRM), ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR. Work in UPBI is funded by grant ANR-10-INSB-04-01 and the Région Île-de-France program DIM1-Health. D.P. is supported by the Vaccine Research Institute. The H.M. laboratory is funded by the Institut Pasteur, the Milieu Intérieur Program (ANR-10-LABX-69-01), INSERM, REACTing, EU (RECOVER) and Fondation de France (00106077) grants. The S.F.K. laboratory is funded by Strasbourg University Hospitals (SeroCoV-HUS, PRI 7782), the Programme Hospitalier de Recherche Clinique (PHRC N 2017–HUS no. 6997), the Agence Nationale de la Recherche (ANR-18-CE17-0028), Laboratoire d’Excellence TRANSPLANTEX (ANR-11-LABX-0070_TRANSPLANTEX) and the Institut National de la Santé et de la Recherche Médicale (UMR_S 1109). The E.S.-L. laboratory is funded by the Institut Pasteur and the French Government’s Investissement d’Avenir programme, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR-10-LABX-62-IBEID)., We thank N. Casartelli for critical reading of the manuscript, P. Guardado Calvo for discussion, the individuals who participated in the study, members of the Virus and Immunity Unit for discussions and help, N. Aulner and the UTechS Photonic BioImaging (UPBI) core facility (Institut Pasteur), a member of the France BioImaging network, for image acquisition and analysis, and the DRCI, CIC, Médecine du Travail and Pôle de Biologie teams (CHU de Strasbourg) for the management of the Strasbourg cohort and serology testing., ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-18-CE17-0028,HuMABK,Les anticorps monoclonaux humains : une Nouvelle approche thérapeutique contre l'infection par le virus BK et les maladies associées(2018), ANR-11-LABX-0070,TRANSPLANTEX,Nouveaux loci d'histocompatibilité/biomarqueurs en transplantation humaine: de la découverte à l'app(2011), European Project: 101003589, H2020-SC1-PHE-CORONAVIRUS-2020,RECOVER(2020), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Vaccine Research Institute (VRI), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École pratique des hautes études (EPHE), Institut Pasteur [Paris], Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPC)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPC), and Université Paris Cité (UPC)
Subjects: Male, Delta, COVID-19 Vaccines, medicine.drug_class, India, Alpha (ethology), Biology, Antibodies, Monoclonal, Humanized, Antibodies, Viral, Monoclonal antibody, Neutralization, Epitope, Epitopes, Immune system, Neutralization Tests, medicine, Humans, Immune Evasion, Multidisciplinary, COVID-19, Convalescence, Middle Aged, Antibodies, Neutralizing, Virology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Spike Glycoprotein, Coronavirus, Monoclonal, biology.protein, France, Antibody
Abstract: The SARS-CoV-2 B.1.617 lineage was identified in October 2020 in India1–5. Since then, it has become dominant in some regions of India and in the UK, and has spread to many other countries6. The lineage includes three main subtypes (B1.617.1, B.1.617.2 and B.1.617.3), which contain diverse mutations in the N-terminal domain (NTD) and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein that may increase the immune evasion potential of these variants. B.1.617.2—also termed the Delta variant—is believed to spread faster than other variants. Here we isolated an infectious strain of the Delta variant from an individual with COVID-19 who had returned to France from India. We examined the sensitivity of this strain to monoclonal antibodies and to antibodies present in sera from individuals who had recovered from COVID-19 (hereafter referred to as convalescent individuals) or who had received a COVID-19 vaccine, and then compared this strain with other strains of SARS-CoV-2. The Delta variant was resistant to neutralization by some anti-NTD and anti-RBD monoclonal antibodies, including bamlanivimab, and these antibodies showed impaired binding to the spike protein. Sera collected from convalescent individuals up to 12 months after the onset of symptoms were fourfold less potent against the Delta variant relative to the Alpha variant (B.1.1.7). Sera from individuals who had received one dose of the Pfizer or the AstraZeneca vaccine had a barely discernible inhibitory effect on the Delta variant. Administration of two doses of the vaccine generated a neutralizing response in 95% of individuals, with titres three- to fivefold lower against the Delta variant than against the Alpha variant. Thus, the spread of the Delta variant is associated with an escape from antibodies that target non-RBD and RBD epitopes of the spike protein. The SARS-CoV-2 Delta variant partially evades neutralization by several monoclonal antibodies and by sera from individuals who have had COVID-19, but two doses of anti-COVID-19 vaccines still generate a strong neutralizing response.
Published: 2021

19. Dengue virus NS1 protein conveys pro‐inflammatory signals by docking onto high‐density lipoproteins

Author: Souheyla Benfrid, Kyu‐Ho Park, Mariano Dellarole, James E Voss, Carole Tamietti, Gérard Pehau‐Arnaudet, Bertrand Raynal, Sébastien Brûlé, Patrick England, Xiaokang Zhang, Anastassia Mikhailova, Milena Hasan, Marie‐Noëlle Ungeheuer, Stéphane Petres, Scott B Biering, Eva Harris, Anavaj Sakuntabhai, Philippe Buchy, Veasna Duong, Philippe Dussart, Fasséli Coulibaly, François Bontems, Félix A Rey, Marie Flamand, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes, Sorbonne Paris Cité, Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris] (IP), Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Investigation Clinique et d’Accès aux Ressources Biologiques (Plate-forme) - Clinical Investigation and Access to BioResources (ICAReB), Plateforme technologique Production et purification de protéines recombinantes – Production and Purification of Recombinant Proteins Technological Platform (PPR), University of California [Berkeley] (UC Berkeley), University of California (UC), Génétique du Développement humain - Human developmental genetics, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), Monash University [Clayton], Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), This study benefited from the financial support of the Institut Pasteur ACIP-27-16 (P.D., V.D., M.F.), the Institut Pasteur Dengue Task Force (to MF), the Institut Pasteur INNOV-44-19 (M.F), the National Natural Science Foundation of China 31600606 (X.Z.), the National Key R&D Program of China 2016YFA0501100 (X.Z.), Guangdong Provincial Key Laboratory of Brain Connectome and Behavior 2017B030301017 (X.Z.), CAS Key Laboratory of Brain Connectome and Manipulation 2019DP173024 (X.Z.), the NIAID/NIH R01 AI24493 (E.H.) and R21 AI146464 (E.H.), ANR Equipex CACSICE ANR-11-EQPX-0008 (G.P.-A.)., The authors gratefully acknowledge the staff of the Kampong Cham Referral Hospital, the patients and parents who participated in the study, and the Arbovirus Team in the Virology Unit at the Institut Pasteur du Cambodge who contributed to this study. We acknowledge the participation of the ICAReB facility in setting up the recruitment of donors and the acquisition of blood samples, in particular Gloria Morizot, Bianca Liliana Perlaza, Sophie Chaouche, Linda Sangari, Céline Chapel, Philippe Esterre and Hélène Laude. We are most grateful to Christine Girard-Blanc and Evelyne Dufour for their contribution in producing and purifying the recombinant DENV NS1 protein, to Béatrice Poirier-Beaudouin and Cartini Mardi for their help in setting up the cytokine quantification assay, to Arvind Sharma for providing purified anti-E MAbs, to Mathilde Ban for preparing Fab-bound NS1-HDL complexes and to Xavier Montagutelli and Etienne Simon-Lorière for testing an in vivo protection assay. We thank M. Nilges and the Equipex CACSICE for providing the Falcon II direct detector and David Veesler for his help in acquiring the first electron microscopy images of the bovine NS1-HDL complex. Finally, we thank Sébastien Quesney, Alexandre Pachot and Karine Kaiser for their support. The synopsis figure was created with BioRender.com., and ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011)
Subjects: Arbovirus, lipoprotein particle, viruses, accessory protein, virus diseases, Dengue Virus, Viral Nonstructural Proteins, biochemical phenomena, metabolism, and nutrition, Biochemistry, virulence factor, Dengue, molecular pathogenesis, Phagocytosis, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Genetics, Humans, [SDV.IMM]Life Sciences [q-bio]/Immunology, lipids (amino acids, peptides, and proteins), Lipoproteins, HDL, Molecular Biology, hemorrhagic fever
Abstract: International audience; The dengue virus nonstructural protein 1 (NS1) is a secreted virulence factor that modulates complement, activates immune cells and alters endothelial barriers. The molecular basis of these events remains incompletely understood. Here we describe a functional high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Collapse of the soluble NS1 hexamer upon binding to the lipoprotein particle leads to the anchoring of amphipathic NS1 dimeric subunits into the HDL outer layer. The stable complex can be visualized by electron microscopy as a spherical HDL with rod-shaped NS1 dimers protruding from the surface. We further show that the assembly of NS1-HDL complexes triggers the production of pro-inflammatory cytokines in human primary macrophages while NS1 or HDL alone do not. Finally, we detect NS1 in complex with HDL and low-density lipoprotein (LDL) particles in the plasma of hospitalized dengue patients and observe NS1-apolipoprotein E-positive complexes accumulating overtime. The functional reprogramming of endogenous lipoprotein particles by NS1 as a means to exacerbate systemic inflammation during viral infection provides a new paradigm in dengue pathogenesis.
Published: 2022

20. Bat coronaviruses related to SARS-CoV-2 and infectious for human cells

Author: Sarah Temmam, Khamsing Vongphayloth, Eduard Baquero, Sandie Munier, Massimiliano Bonomi, Béatrice Regnault, Bounsavane Douangboubpha, Yasaman Karami, Delphine Chrétien, Daosavanh Sanamxay, Vilakhan Xayaphet, Phetphoumin Paphaphanh, Vincent Lacoste, Somphavanh Somlor, Khaithong Lakeomany, Nothasin Phommavanh, Philippe Pérot, Océane Dehan, Faustine Amara, Flora Donati, Thomas Bigot, Michael Nilges, Félix A. Rey, Sylvie van der Werf, Paul T. Brey, 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), Institut Pasteur du Laos, Réseau International des Instituts Pasteur (RIIP), Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Bioinformatique structurale - Structural Bioinformatics, National University of Laos, Centre National de Référence des virus des infections respiratoires (dont la grippe) - National Reference Center Virus Influenzae [Paris] (CNR - laboratoire coordonnateur), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, École nationale vétérinaire - Alfort (ENVA), NGS was performed with the help of the Biomics Platform, Center for Technological Resources and Research, Institut Pasteur, Paris, France, supported by France Génomique (ANR-10-INBS-09-09), Inclusive Blockchain Insurance using Space Assets and the Illumina COVID-19 Projects’ offer. The work was granted access to the High Performance Computing resources of the Institute for Development and Resources in Intensive Scientific Computing under the allocation 2020-101592 made by Grand Équipement National de Calcul Intensif. We thank the Ministry of Health and the Ministry of Natural Resources and Environments, Lao People’s Democratic Republic, for their authorization of the field work and the Faculty of Environmental Science for its authorization of the field research collaboration. The work was supported by an Institut Pasteur 'Covid Taskforce' and in part by the H2020 project 101003589 (RECOVER) and Labex IBEID (ANR-10-LABX62-IBEID) grants. Field and laboratory work at IPL was also supported by a UK embassy grant (grant no. INT 2021/LOV C19 02) and a Luxembourg Development special grant (grant no. LAO/030·202324)., We thank S. Mohamed Ali, N. Da Rocha, A. Brisebarre, T. Xaybounsou and S. Chonephetsarath for their help at the bench, A. Haouz and the staff of the protein crystallogenesis facility at Institut Pasteur for help with crystallization trials, P. England from the Molecular Biophysics facility at Institut Pasteur for his support and access to the BLI equipment, P. Legrand from the PX1 beamline at Synchrotron SOLEIL for data collection support, and P. Guardado-Calvo for discussion and support with BLI experiments., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), and European Project: 101003589, H2020-SC1-PHE-CORONAVIRUS-2020,RECOVER(2020)
Subjects: Asia, viruses, MESH: Spike Glycoprotein, Coronavirus, MESH: Angiotensin-Converting Enzyme 2, Chiroptera, Animals, Humans, MESH: Protein Binding, MESH: COVID-19, MESH: Animals, MESH: SARS-CoV-2, skin and connective tissue diseases, Taxonomy, Disease Reservoirs, MESH: Disease Reservoirs, Multidisciplinary, MESH: Humans, SARS-CoV-2, MESH: Asia, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], fungi, COVID-19, MESH: Chiroptera, Biodiversity, body regions, Caves, Spike Glycoprotein, Coronavirus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Caves, Angiotensin-Converting Enzyme 2, Protein Binding
Abstract: International audience; The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV-2-related viruses in Asian Rhinolophus bats1-4, including the closest virus from R. affinis, RaTG13 (refs. 5,6), and pangolins7-9. SARS-CoV-2 has a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range10-12. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 (hACE2) pathway have not yet been identified, although they would be key in understanding the origin of the epidemic. Here we show that such viruses circulate in cave bats living in the limestone karstic terrain in northern Laos, in the Indochinese peninsula. We found that the receptor-binding domains of these viruses differ from that of SARS-CoV-2 by only one or two residues at the interface with ACE2, bind more efficiently to the hACE2 protein than that of the SARS-CoV-2 strain isolated in Wuhan from early human cases, and mediate hACE2-dependent entry and replication in human cells, which is inhibited by antibodies that neutralize SARS-CoV-2. None of these bat viruses contains a furin cleavage site in the spike protein. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses that are potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.
Published: 2022

21. Evolution and activation mechanism of the flavivirus class II membrane-fusion machinery

Author: Marie-Christine Vaney, Mariano Dellarole, Stéphane Duquerroy, Iris Medits, Georgios Tsouchnikas, Alexander Rouvinski, Patrick England, Karin Stiasny, Franz X. Heinz, Félix A. Rey, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris-Saclay, Institute of Virology (Vienna), Medizinische Universität Wien = Medical University of Vienna, Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), This work was supported by the French ANR (Agence Nationale de la Recherche), grants ANR-13-ISV8-0002-01, ANR-10-LABX-62-10 IBEID, Wellcome Trust collaborative grant (UNS22082) to FAR, as well as by Institut Pasteur and CNRS. K.S and F.X.H acknowledge support from the Austrian Science Fund FWF (I1378-B13 and P27501-B21)., ANR-13-ISV8-0002,flavistem,La protéine E des flavivirus : interactions et fusion membranaire(2013), and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)
Subjects: Furin, Multidisciplinary, Viral Envelope Proteins, [SDV]Life Sciences [q-bio], Virion, General Physics and Astronomy, General Chemistry, Membrane Fusion, General Biochemistry, Genetics and Molecular Biology, Encephalitis Viruses, Tick-Borne
Abstract: We thank the staff at beamlines PX1 and PX2 at SOLEIL synchrotron (St. Aubin, France) and at PX beamlines at the ESRF (Grenoble, France); A. Haouz and the staff at the crystallographic facility at Institut Pasteur; F. Agou from the Chemogenomic and Biological Screening platform at Institut Pasteur; P. Guardado Calvo and I. Fernandez for helping for the MALS experiments.; International audience; The flavivirus envelope glycoproteins prM and E drive the assembly of icosahedral, spiky immature particles that bud across the membrane of the endoplasmic reticulum. Maturation into infectious virions in the trans-Golgi network involves an acid-pH-driven rearrangement into smooth particles made of (prM/E)2 dimers exposing a furin site for prM cleavage into "pr" and "M". Here we show that the prM "pr" moiety derives from an HSP40 cellular chaperonin. Furthermore, the X-ray structure of the tick-borne encephalitis virus (pr/E)2 dimer at acidic pH revealed the E 150-loop as a hinged-lid that opens at low pH to expose a positively-charged prbinding pocket at the E dimer interface, inducing (prM/E)2 dimer formation to generate smooth particles in the Golgi. Furin cleavage is followed by lid-closure upon deprotonation in the neutral-pH extracellular environment, expelling pr while the 150-loop takes the relay in fusion loop protection, thus revealing the elusive flavivirus mechanism of fusion activation.
Published: 2022

22. Epitope convergence of broadly HIV-1 neutralizing IgA and IgG antibody lineages in a viremic controller

Author: Valérie Lorin, Ignacio Fernández, Guillemette Masse-Ranson, Mélanie Bouvin-Pley, Luis M. Molinos-Albert, Cyril Planchais, Thierry Hieu, Gérard Péhau-Arnaudet, Dominik Hrebík, Giulia Girelli-Zubani, Oriane Fiquet, Florence Guivel-Benhassine, Rogier W. Sanders, Bruce D. Walker, Olivier Schwartz, Johannes F. Scheid, Jordan D. Dimitrov, Pavel Plevka, Martine Braibant, Michael S. Seaman, François Bontems, James P. Di Santo, Félix A. Rey, Hugo Mouquet, Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Virologie Structurale, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Immunité Innée - Innate Immunity, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagopole (CITECH), Institut Pasteur [Paris] (IP), Microscopie Ultrastructurale (Plate-forme), Masaryk University [Brno] (MUNI), Virus et Immunité - Virus and immunity (CNRS-UMR3569), University of Amsterdam [Amsterdam] (UvA), Weill Medical College of Cornell University [New York], Massachusetts General Hospital [Boston], Harvard Medical School [Boston] (HMS), Rockefeller University [New York], Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Beth Israel Deaconess Medical Center [Boston] (BIDMC), Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), The NanoImaging Core was created with the help of a grant from the French Government’s Investissements d’Avenir program (EQUIPEX CACSICE - Centre d’analyse de systèmes complexes dans les environnements complexes, ANR-11-EQPX-0008). We thank the NIH AIDS Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH) for contributing to reagents and the Agence National de Recherche sur le SIDA et les hépatites virales (ANRS) for an equipment grant support. The Biomics platform is supported by France Génomique (ANR-10-INBS-09-09) and IBISA. I. Fernández and C. Planchais were recipients of an ANRS postdoctoral fellowship. The J.P. Di Santo laboratory received support from the Vaccine Research Institute (Créteil, France). H. Mouquet received core grants from the Institut Pasteur, Institut national de la santé et de la recherche médicale, and the Milieu Intérieur Program (ANR-10-LABX-69-01). This work was supported by the European Research Council Seventh Framework Program (ERC-2013-StG 337146), and by Gilead Sciences HIV Cure Grants (#00397)., ANR-11-EQPX-0008,CACSICE,Centre d'analyse de systèmes complexes dans les environnements complexes(2011), ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), European Project: 337146,EC:FP7:ERC,ERC-2013-StG,HUMANTIVIRUSES(2014), Medical Microbiology and Infection Prevention, and AII - Infectious diseases
Subjects: Immunology, env Gene Products, Human Immunodeficiency Virus, HIV Infections, HIV Antibodies, Antibodies, Neutralizing, Immunoglobulin A, Epitopes, Mice, Polysaccharides, Immunoglobulin G, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, HIV-1, Immunology and Allergy, Animals, Humans, Elite Controllers, Broadly Neutralizing Antibodies
Abstract: International audience; Decrypting the B cell ontogeny of HIV-1 broadly neutralizing antibodies (bNAbs) is paramount for vaccine design. Here, we characterized IgA and IgG bNAbs of three distinct B cell lineages in a viremic controller, two of which comprised only IgG+ or IgA+ blood memory B cells; the third combined both IgG and IgA clonal variants. 7-269 bNAb in the IgA-only lineage displayed the highest neutralizing capacity despite limited somatic mutation, and delayed viral rebound in humanized mice. bNAbs in all three lineages targeted the N332 glycan supersite. The 2.8-Å resolution cryo-EM structure of 7-269-BG505 SOSIP.664 complex showed a similar pose as 2G12, on an epitope mainly composed of sugar residues comprising the N332 and N295 glycans. Binding and cryo-EM structural analyses showed that antibodies from the two other lineages interact mostly with glycans N332 and N386. Hence, multiple B cell lineages of IgG and IgA bNAbs focused on a unique HIV-1 site of vulnerability can codevelop in HIV-1 viremic controllers.
Published: 2022

23. A nicotinic hypothesis for Covid-19 with preventive and therapeutic implications

Author: Zahir Amoura, Jean-Pierre Changeux, Félix A. Rey, and Makoto Miyara
Subjects: 0106 biological sciences, 0301 basic medicine, Sociology of scientific knowledge, medicine.medical_specialty, Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Protective factor, Scientific literature, 01 natural sciences, Nicotine, 03 medical and health sciences, 0302 clinical medicine, medicine, 030212 general & internal medicine, Intensive care medicine, 030304 developmental biology, 0303 health sciences, business.industry, Public health, 3. Good health, Nicotinic acetylcholine receptor, 030104 developmental biology, Nicotinic agonist, business, Neuroscience, 030217 neurology & neurosurgery, medicine.drug, 010606 plant biology & botany
Abstract: SARS-CoV-2 epidemics raises a considerable issue of public health at the planetary scale. There is a pressing urgency to find treatments based upon currently available scientific knowledge. Therefore, we tentatively propose a hypothesis which hopefully might ultimately help saving lives. Based on the current scientific literature and on new epidemiological data which reveal that current smoking status appears to be a protective factor against the infection by SARS-CoV-2 [1], we hypothesize that the nicotinic acetylcholine receptor (nAChR) plays a key role in the pathophysiology of Covid-19 infection and might represent a target for the prevention and control of Covid-19 infection.
Published: 2020

24. 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

25. Considerable escape of SARS-CoV-2 Omicron to antibody neutralization

Author: Delphine Planas, Nell Saunders, Piet Maes, Florence Guivel-Benhassine, Cyril Planchais, Julian Buchrieser, William-Henry Bolland, Françoise Porrot, Isabelle Staropoli, Frederic Lemoine, Hélène Péré, David Veyer, Julien Puech, Julien Rodary, Guy Baele, Simon Dellicour, Joren Raymenants, Sarah Gorissen, Caspar Geenen, Bert Vanmechelen, Tony Wawina-Bokalanga, Joan Martí-Carreras, Lize Cuypers, Aymeric Sève, Laurent Hocqueloux, Thierry Prazuck, Félix A. Rey, Etienne Simon-Loriere, Timothée Bruel, Hugo Mouquet, Emmanuel André, Olivier Schwartz, Virus et Immunité - Virus and immunity (CNRS-UMR3569), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Vaccine Research Institute [Créteil, France] (VRI), Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), École Doctorale Bio Sorbonne Paris Cité [Paris] (ED562 - BioSPC), Université Sorbonne Paris Cité (USPC)-Université Paris Cité (UPCité), Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Immunologie humorale - Humoral Immunology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Génomique fonctionnelle des tumeurs solides = Functional Genomics of Solid Tumors [CRC] (FunGeST), Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité), Université libre de Bruxelles (ULB), University Hospitals Leuven [Leuven], Centre Hospitalier Régional d'Orléans (CHRO), Virologie Structurale - Structural Virology, Génomique évolutive des virus à ARN - Evolutionary genomics of RNA viruses, The Opera system was co-funded by Institut Pasteur and the Région ile de France (DIM1Health). Work in the O.S. laboratory is funded by Institut Pasteur, Urgence COVID-19 Fundraising Campaign of Institut Pasteur, Fondation pour la Recherche Médicale (FRM), ANRS, the Vaccine Research Institute (ANR-10-LABX-77), Labex IBEID (ANR-10-LABX-62-IBEID), ANR/FRM Flash Covid PROTEO-SARS-CoV-2 and IDISCOVR. Work in the UPBI is funded by grant ANR-10-INSB-04-01 and Région Ile-de-France program DIM1-Health. D.P. is supported by the Vaccine Research Institute. The H.M. laboratory is funded by the Institut Pasteur, the Milieu Intérieur Program (ANR-10-LABX-69-01), the INSERM, REACTing, EU (RECOVER) and Fondation de France (00106077) grants. The E.S.-L. laboratory is funded by Institut Pasteur, the INCEPTION program (Investissements d’Avenir grant ANR-16-CONV-0005) and the French Government’s Investissement d’Avenir programme, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR-10-LABX-62-IBEID). G.B. acknowledges support from the Internal Funds KU Leuven under grant agreement C14/18/094, and the Research Foundation–Flanders (Fonds voor Wetenschappelijk Onderzoek–Vlaanderen, G0E1420N, G098321N). P.M. acknowledges support from a COVID-19 research grant of ‘Fonds Wetenschappelijk Onderzoek’/Research Foundation–Flanders (grant no. G0H4420N). S.D. is supported by the Fonds National de la Recherche Scientifique (FNRS, Belgium) and also acknowledges support from the Research Foundation–Flanders (Fonds voor Wetenschappelijk Onderzoek–Vlaanderen, G098321N) and from the European Union Horizon 2020 project MOOD (grant no. 874850)., ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), ANR-20-COVI-0059,PROTEO-SARS-CoV-2,Protéomique du SARS-CoV-2(2020), ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), ANR-16-CONV-0005,INCEPTION,Institut Convergences pour l'étude de l'Emergence des Pathologies au Travers des Individus et des populatiONs(2016), European Project: 874850,H2020-SC1-2019-Single-Stage-RTD,MOOD(2020), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Vaccine Research Institute (VRI), Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut Pasteur [Paris]-Université Paris Cité (UPCité), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université Paris Cité (UPCité)-École pratique des hautes études (EPHE), and European Project: 874850,MOOD
Subjects: MESH: Humans, MESH: Mutation, Multidisciplinary, MESH: ChAdOx1 nCoV-19, MESH: Neutralization Tests, MESH: Adult, MESH: Convalescence, MESH: Male, MESH: Antibodies, Monoclonal, MESH: Antibodies, Neutralizing, MESH: Belgium, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: COVID-19, MESH: Immunization, Secondary, MESH: SARS-CoV-2, MESH: Immune Evasion, MESH: BNT162 Vaccine, MESH: Phylogeny, MESH: Travel, MESH: Female, MESH: Antibodies, Viral
Abstract: The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa1-3. It has since spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of around 32 mutations in spike-located mostly in the N-terminal domain and the receptor-binding domain-that may enhance viral fitness and enable antibody evasion. Here we isolated an infectious Omicron virus in Belgium from a traveller returning from Egypt. We examined its sensitivity to nine monoclonal antibodies that have been clinically approved or are in development4, and to antibodies present in 115 serum samples from COVID-19 vaccine recipients or individuals who have recovered from COVID-19. Omicron was completely or partially resistant to neutralization by all monoclonal antibodies tested. Sera from recipients of the Pfizer or AstraZeneca vaccine, sampled five months after complete vaccination, barely inhibited Omicron. Sera from COVID-19-convalescent patients collected 6 or 12 months after symptoms displayed low or no neutralizing activity against Omicron. Administration of a booster Pfizer dose as well as vaccination of previously infected individuals generated an anti-Omicron neutralizing response, with titres 6-fold to 23-fold lower against Omicron compared with those against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and, to a large extent, vaccine-elicited antibodies. However, Omicron is neutralized by antibodies generated by a booster vaccine dose. ispartof: NATURE vol:602 issue:7898 pages:671-+ ispartof: location:England status: published
Published: 2021

26. 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

27. The epitope arrangement on flavivirus particles contributes to Mab C10’s extraordinary neutralization breadth across Zika and dengue viruses

Author: Gavin R. Screaton, Pablo Guardado-Calvo, Félix A. Rey, Xinghong Dai, Danyang Gong, Patrick England, Alexander Rouvinski, Marie-Christine Vaney, Ahmed Haouz, Z. Hong Zhou, Wanwisa Dejnirattisai, Stéphane Duquerroy, Ren Sun, Arvind Sharma, Xiaokang Zhang, Wiyada Wongwiwat, Juthathip Mongkolsapaya, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Université Paris Cité (UPCité), Institut Pasteur de Shanghai, Académie des Sciences de Chine - Chinese Academy of Sciences (IPS-CAS), Réseau International des Instituts Pasteur (RIIP), University of Oxford, University of California [Los Angeles] (UCLA), University of California (UC), Université Paris-Saclay, Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Cristallographie (Plateforme) - Crystallography (Platform), Siriraj Hospital, Mahidol University, Mahidol University [Bangkok], This project was supported by a Wellcome Trust collaborative grant (UNS22082 to G.R.S., J.M., and F.A.R.), by the National Institute for Health Research Biomedical Research Centre Funding Scheme (to G.R.S.), and by grants from the National Institutes of Health (DE028583, AI094386, and DE025567 to Z.H.Z. and R.S.)., and We thank Fabrice Agou and the staff of the Chemogenomic and Biological Screening Platform for access to the MALS equipment as well as the staff of the Crytallography core facility at the Institut Pasteur for robot-driven crystallization screens. We acknowledge the use of synchrotron beam lines PX1 and PX2 at SOLEIL (St Aubin, France), ID29 and ID23-1 at ESRF (Grenoble, France) and X06SA at SLS (Villigen, Switzerland), and we thank their staff for assistance. We also acknowledge the use of the resources at the Electron Imaging Center for Nanomachines, supported in part by 1S10RR23057, 1S10OD018111, 1U24GM116792 and NSF DBI-1338135 and DMR-1548924 at UCLA. G.R.S. is supported as a Wellcome Trust Senior Investigator (095541/A/11/Z). F.A.R. is supported by Institut Pasteur and the CNRS.
Subjects: medicine.drug_class, [SDV]Life Sciences [q-bio], MESH: Vero Cells, MESH: Zika Virus, MESH: Dengue, Biology, Dengue virus, Monoclonal antibody, medicine.disease_cause, MESH: Dengue Virus, complex mixtures, General Biochemistry, Genetics and Molecular Biology, Epitope, Neutralization, Zika virus, MESH: Antibodies, Monoclonal, MESH: Antibodies, Neutralizing, MESH: Drosophila melanogaster, MESH: Cross Reactions, MESH: Protein Conformation, MESH: Zika Virus Infection, MESH: Chlorocebus aethiops, medicine, MESH: Protein Binding, MESH: Animals, X-ray crystallography, MESH: Humans, Flaviviruses, broadly neutralizing antibodies, biology.organism_classification, Virology, MESH: Cell Line, Flavivirus, MESH: HEK293 Cells, MESH: Viral Envelope Proteins, biology.protein, vaccine design, cryo-EM, Paratope, lipids (amino acids, peptides, and proteins), Antibody, MESH: Antibodies, Viral
Abstract: International audience; The human monoclonal antibody C10 exhibits extraordinary cross-reactivity, potently neutralizing Zika virus (ZIKV) and the four serotypes of dengue virus (DENV1-DENV4). Here we describe a comparative structure-function analysis of C10 bound to the envelope (E) protein dimers of the five viruses it neutralizes. We demonstrate that the C10 Fab has high affinity for ZIKV and DENV1 but not for DENV2, DENV3, and DENV4. We further show that the C10 interaction with the latter viruses requires an E protein conformational landscape that limits binding to only one of the three independent epitopes per virion. This limited affinity is nevertheless counterbalanced by the particle's icosahedral organization, which allows two different dimers to be reached by both Fab arms of a C10 immunoglobulin. The epitopes' geometric distribution thus confers C10 its exceptional neutralization breadth. Our results highlight the importance not only of paratope/epitope complementarity but also the topological distribution for epitope-focused vaccine design.
Published: 2021

28. Coronaviruses with a SARS-CoV-2-like receptor-binding domain allowing ACE2-mediated entry into human cells isolated from bats of Indochinese peninsula

Author: Khamsing Vongphayloth, Delphine Chrétien, Yasaman Karami, Vilakhan Xayaphet, Sarah Temmam, Félix A. Rey, Max Bonomi, Somphavanh Somlor, Khaithong Lakeomany, Bounsavane Douangboubpha, Marc Eloit, Phetphoumin Paphaphanh, Nothasin Phommavanh, Michael Nilges, Flora Donati, Paul T. Brey, Daosavanh Sanamxay, Vincent Lacoste, Thomas Bigot, Sandie Munier, Béatrice Regnault, Sylvie van der Werf, Philippe Pérot, Eduard Baquero Salazar, Institut Pasteur [Paris] (IP), National University of Laos, Institut Pasteur du Laos, Réseau International des Instituts Pasteur (RIIP), The work was funded by an Institut Pasteur 'Covid Taskforce' and in part by the H2020 project 101003589 (RECOVER) grants. Field and laboratory work at IP-Laos was also funded by a UK embassy grant (Grant No. INT 2021/LOV C19 02) and Luxembourg Development special grant (Grant No. LAO/030·202324)., The authors want to thank Souand Mohamed Ali, Nicolas Da Rocha, Thonglakhone Xaybounsou, and Somsanith Chonephetsarath for their help at the bench. 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 granted access to the HPC resources of IDRIS under the allocation 2020-101592 made by GENCI. We thank the Ministry of Health and the Ministry of Natural Resources and Environments, Lao PDR, for their authorization of the field work and the Faculty of Environmental Science for its authorization of the field research collaboration., ANR-10-INBS-0009,France-Génomique,Organisation et montée en puissance d'une Infrastructure Nationale de Génomique(2010), European Project: 101003589, H2020-SC1-PHE-CORONAVIRUS-2020,RECOVER(2020), and Institut Pasteur [Paris]
Subjects: 0303 health sciences, Indochinese peninsula, 030306 microbiology, Coronaviruses, SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), viruses, [SDV]Life Sciences [q-bio], fungi, virus diseases, Biology, Virology, cave bats, 3. Good health, Domain (software engineering), body regions, 03 medical and health sciences, spike sequence, receptor-binding domain, skin and connective tissue diseases, angiotensin-converting enzyme 2 pathway, 030304 developmental biology
Abstract: The animal reservoir of SARS-CoV-2 is unknown despite reports of various SARS-CoV-2-related viruses in Asian Rhinolophus bats, including the closest virus from R. affinis, RaTG13. Several studies have suggested the involvement of pangolin coronaviruses in SARS-CoV-2 emergence. SARS-CoV-2 presents a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain (RBD) to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 pathway have not yet been identified, though they would be key in understanding the origin of the epidemics. Here we show that such viruses indeed circulate in cave bats living in the limestone karstic terrain in North Laos, within the Indochinese peninsula. We found that the RBDs of these viruses differ from that of SARS-CoV-2 by only one or two residues, bind as efficiently to the hACE2 protein as the SARS-CoV-2 Wuhan strain isolated in early human cases, and mediate hACE2-dependent entry into human cells, which is inhibited by antibodies neutralizing SARS-CoV-2. None of these bat viruses harbors a furin cleavage site in the spike. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.
Published: 2021

29. The Viral Class II Membrane Fusion Machinery: Divergent Evolution from an Ancestral Heterodimer

Author: Pablo Guardado-Calvo, Félix A. Rey, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This research was funded by ANR, grant number ANR-18-CE11-0011, and Labex IBEID, grant number ANR-10-LABX-62-IBEID., 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: Conformational change, Bunyaviridae, Lipid Bilayers, Alphavirus, Review, bunyavirus, Genome, Viral, Microbiology, 03 medical and health sciences, Viral envelope, Virology, Animals, Humans, Lipid bilayer, class II fusion proteins, 030304 developmental biology, Glycoproteins, chemistry.chemical_classification, 0303 health sciences, structural homology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, 030306 microbiology, Chemistry, Membrane fusion protein, Virion, Lipid bilayer fusion, Virus Internalization, biology.organism_classification, Biological Evolution, QR1-502, Cell biology, Divergent evolution, Models, Structural, Infectious Diseases, Protein Multimerization, Glycoprotein, Viral Fusion Proteins
Abstract: International audience; A key step during the entry of enveloped viruses into cells is the merger of viral and cell lipid bilayers. This process is driven by a dedicated membrane fusion protein (MFP) present at the virion surface, which undergoes a membrane–fusogenic conformational change triggered by interactions with the target cell. Viral MFPs have been extensively studied structurally, and are divided into three classes depending on their three-dimensional fold. Because MFPs of the same class are found in otherwise unrelated viruses, their intra-class structural homology indicates horizontal gene exchange. We focus this review on the class II fusion machinery, which is composed of two glycoproteins that associate as heterodimers. They fold together in the ER of infected cells such that the MFP adopts a conformation primed to react to specific clues only upon contact with a target cell, avoiding premature fusion in the producer cell. We show that, despite having diverged in their 3D fold during evolution much more than the actual MFP, the class II accompanying proteins (AP) also derive from a distant common ancestor, displaying an invariant core formed by a β-ribbon and a C-terminal immunoglobulin-like domain playing different functional roles—heterotypic interactions with the MFP, and homotypic AP/AP contacts to form spikes, respectively. Our analysis shows that class II APs are easily identifiable with modern structural prediction algorithms, providing useful information in devising immunogens for vaccine design.
Published: 2021

30. Pathogenic autoantibodies to IFN-γ act through the impedance of receptor assembly and Fc-mediated response

Author: Han-Po Shih, Jing-Ya Ding, Junel Sotolongo Bellón, Yu-Fang Lo, Pei-Han Chung, He-Ting Ting, Jhan-Jie Peng, Tsai-Yi Wu, Chia-Hao Lin, Chia-Chi Lo, You-Ning Lin, Chun-Fu Yeh, Jiun-Bo Chen, Ting-Shu Wu, Yuag-Meng Liu, Chen-Yen Kuo, Shang-Yu Wang, Kun-Hua Tu, Chau Yee Ng, Wei-Te Lei, Yu-Huan Tsai, Jou-Han Chen, Ya-Ting Chuang, Jing-Yi Huang, Félix A. Rey, Hung-Kai Chen, Tse-Wen Chang, Jacob Piehler, Chih-Yu Chi, and Cheng-Lung Ku
Subjects: Interferon-gamma, Mycobacterium Infections, Immunology, Electric Impedance, Immunology and Allergy, Antibodies, Monoclonal, Humans, Autoantibodies, Receptors, Interferon
Abstract: Anti-interferon (IFN)–γ autoantibodies (AIGAs) are a pathogenic factor in late-onset immunodeficiency with disseminated mycobacterial and other opportunistic infections. AIGAs block IFN-γ function, but their effects on IFN-γ signaling are unknown. Using a single-cell capture method, we isolated 19 IFN-γ–reactive monoclonal antibodies (mAbs) from patients with AIGAs. All displayed high-affinity (KD < 10−9 M) binding to IFN-γ, but only eight neutralized IFN-γ–STAT1 signaling and HLA-DR expression. Signal blockade and binding affinity were correlated and attributed to somatic hypermutations. Cross-competition assays identified three nonoverlapping binding sites (I–III) for AIGAs on IFN-γ. We found that site I mAb neutralized IFN-γ by blocking its binding to IFN-γR1. Site II and III mAbs bound the receptor-bound IFN-γ on the cell surface, abolishing IFN-γR1–IFN-γR2 heterodimerization and preventing downstream signaling. Site III mAbs mediated antibody-dependent cellular cytotoxicity, probably through antibody–IFN-γ complexes on cells. Pathogenic AIGAs underlie mycobacterial infections by the dual blockade of IFN-γ signaling and by eliminating IFN-γ–responsive cells.
Published: 2021

31. HIV-1 hijacks the cell extracellular matrix to spread collectively and efficiently between T lymphocytes

Author: Frédéric Tangy, Faroudy Boufassa, Alexandre Dufour, Jean-Christophe Olivo-Marin, M. Mesel-Lemoine, Gianfranco Pancino, Adeline Mallet, Asier Sáez-Cirión, M.-I. Thoulouze, A. Desrames, Annie David, Olivier Lambotte, Félix A. Rey, M. Caillet, P. Bomme, Hugo Mouquet, C. Inizan, K. Bourdic, Thibault Lagache, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Sorbonne Université (SU), Dengue et Arbovirose (URE-DA), Institut Pasteur de Nouvelle-Calédonie, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pointe-à-Pitre/Abymes [Guadeloupe] -Université des Antilles (UA)-Université Paris Cité (UPCité), HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Microscopie ultrastructurale - Ultrapole (CITECH), Institut Pasteur [Paris] (IP), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Immunologie des Maladies Virales et Autoimmunes (IMVA - U1184), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de recherche en épidémiologie et santé des populations (CESP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Génomique virale et vaccination, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Régulation des Infections Rétrovirales, Réponse humorale aux pathogènes, Analyse d'images biologiques - Biological Image Analysis (BIA), Columbia University [New York], This work was funded by grants from l’AgenceNationale de Recherche sur le SIDA et les hépatites (ANRS), l’Institut Pasteur (PTR-445), as well asby institutional grants from Institut Pasteur and the Centre National de la Recherche Scientifique(CNRS). CI was supported by an Allocation de Recherche du Ministère de l'Education Nationale, de laRecherche et de la Technologie and by a bourse from the Fondation pour la Recherche Médicale. MCwas supported by a fellowship from AXA Research fund, AD by a fellowship from l’Institut Pasteur(PTR-445), and TL by a Roux Fellowship (Institut Pasteur). We gratefully acknowledge the financialsupport of the FranceBioImaging infrastructure network supported by the French National ResearchAgency (ANR-10–INSB–04, Investments for the future and ANR-10-LABX-62-IBEID) and theRégion Ile-de-France (program DIM-Malinf). The support of E. Mottez (Center for HumanImmunology, Institut Pasteur) and A.M. Rey-Cuillé (ANRS) is thankfully acknowledged., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), and ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010)
Subjects: Infectivity, biology, Cell-Extracellular Matrix, [SDV]Life Sciences [q-bio], Cell, Human immunodeficiency virus (HIV), [SDV.BC]Life Sciences [q-bio]/Cellular Biology, medicine.disease_cause, Cell biology, Extracellular matrix, medicine.anatomical_structure, In vivo, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, medicine, biology.protein, Viral spread, Antibody
Abstract: Collective transmission via structures containing several virions has recently emerged as a highly efficient mode of viral spread. Here, we demonstrate that HIV-1 spreads between T lymphocytes in the form of viral particles colonies that are concentrated and sheltered in an extracellular matrix (ECM) lattice enabling their collective transmission upon cell contacts. Intrinsically, ECM-clustered viruses infect T lymphocytes more efficiently than individual viral particles. They preserve HIV-1 transmission from antiretroviral treatment (ArT) and potent broadly neutralizing antibodies. We also show that collagen induced by HIV-1 infection controls the clustering of virions and their collective spread, thereby enhancing infectivity. CD4+ T cells from HIV-1-infected patients produce and transmit ECM-virus clusters, supporting that they could be involved in vivo. This study provides new insights into modes of HIV-1 transmission and identifies a novel fundamental role for collagen in this process. HIV-1 spread via ECM-virus clusters may have important implications for viral dissemination and persistence, including during therapy.
Published: 2021

32. Generation of plasma cells and CD27 - IgD - B cells during hantavirus infection is associated with distinct pathological findings

Author: Mirjam Dannborg, Clas Ahlm, Gleyder Roman-Sosa, Pauline Ocaya, Julia Wigren Byström, Andrea Puhar, Johanna Tauriainen, Christopher Sundling, Rohit K. Jangra, Kimia T Maleki, Félix A. Rey, Mattias Forsell, Pablo Guardado-Calvo, Therese Thunberg, Eva Mittler, Andy Dernstedt, Jonas Klingström, Anne Tuiskunen-Bäck, Kartik Chandran, Priscilla F Kerkman, Lalitha Tadala, Umeå Centre for Microbial Research (UCMR), Umeå University, Albert Einstein College of Medicine [New York], Karolinska Institutet [Stockholm], Karolinska University Hospital [Stockholm], Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the following grants: Intramural funds (AN 2.2.1.2-76-14) and Biotechnology grant (2.1.12-128714) from the Medical Faculty, Umeå University, and funds from the Swedish Society of Medicine (SLS-787091) to MF, research grants from the Västerbotten County Council (VLL-579011 and VLL-850681) to MF and CA, the Swedish Foundation for Strategic Research to CA and JK, the Knut and Alice Wallenberg Foundation (KAW 2015.0225) and Centre for Microbial Research (UCMR) and The Laboratory for Molecular Infection Medicine Sweden (MIMS) to AP, National Institutes of Health R01AI132633 to KC, the Swedish Science Council (2018-02646_3) and intramural funds from the Karolinska Institutet to JK., and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Infectious Medicine, genetic structures, animal diseases, Immunology, atypical B cells, Infektionsmedicin, plasmablasts, Immunoglobulin D, hantavirus, Microbiology in the medical area, 03 medical and health sciences, 0302 clinical medicine, Immune system, Mikrobiologi inom det medicinska området, Immunology and Allergy, Medicine, antibodies, Haemorrhagic fever, haemorrhagic fever with renal syndrome, Pathological, General Nursing, 030304 developmental biology, Hantavirus, 0303 health sciences, B cells, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], business.industry, food and beverages, virus diseases, RC581-607, 3. Good health, biology.protein, Immunologic diseases. Allergy, Antibody, Hantavirus Infection, business, 030215 immunology
Abstract: International audience; 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

33. Structure-function relations of the SARS-CoV-2 spike protein and impact of mutations in the variants of concern

Author: Félix A. Rey
Subjects: 0106 biological sciences, 0301 basic medicine, Population, Plasma protein binding, Biology, medicine.disease_cause, 01 natural sciences, Virus, 03 medical and health sciences, medicine, Humans, education, Coronavirus, Genetics, Mutation, education.field_of_study, SARS-CoV-2, Lipid bilayer fusion, COVID-19, General Medicine, Acquired immune system, 030104 developmental biology, Viral evolution, Spike Glycoprotein, Coronavirus, 010606 plant biology & botany, Protein Binding
Abstract: This review covers the main features of the severe acquired respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, its interaction with the main entry receptor, the human angiotensin converting enzyme 2 (ACE2), and the subsequent membrane fusion step. The focus is on the structural organization of these proteins and mechanistic aspects of their interactions that lead to cytoplasmic release of the viral genome. The most potently neutralizing antibodies against SARS-CoV-2 were shown to interfere with the spike/ACE2 interaction. I thus also review the location and the potential impact of mutations in the spike protein observed in the variants of concern that emerged concomitantly with acquired immunity in the population after one year of virus circulation. Understanding how these interactions affect the spike/ACE2 interactions and the subsequent spike-protein-induced membrane fusion reaction is important to stay one step ahead of the virus evolution and develop efficient countermeasures.Cette revue couvre les principales caractéristiques de la protéine de spicule du coronavirus 2 du syndrome respiratoire acquis sévère (SRAS-CoV-2), son interaction avec le principal récepteur d’entrée, l’enzyme de conversion de l’angiotensine 2 (ACE2) humaine, et l’étape subséquente de fusion membranaire. L’accent est mis sur l’organisation structurale de ces protéines et les aspects mécanistiques de leurs interactions qui conduisent à la libération cytoplasmique du génome viral. Les anticorps neutralisants les plus puissants contre le SARS-CoV-2 bloquent l’interaction spike / ACE2. Je passe donc également en revue la localisation et l’impact potentiel des mutations sur le spicule observées dans les variants préoccupants qui sont apparus en même temps que l’immunité acquise dans la population après un an de circulation du virus. Comprendre comment ces mutations affectent les interactions spicule / ACE2 et la catalyse de la réaction de fusion membranaire est important pour garder une longueur d’avance sur l’évolution du virus et développer des contre-mesures efficaces.
Published: 2021

34. Species-specific gamete recognition initiates fusion-driving trimer formation by conserved fusogen HAP2

Author: Jennifer F. Pinello, Ignacio Fernandez, Juliette Fedry, Eduard Baquero, Jun Zhang, Félix A. Rey, and William J. Snell
Subjects: Conformational change, Science, General Physics and Astronomy, Trimer, Membrane Fusion, General Biochemistry, Genetics and Molecular Biology, Article, Cell Fusion, 03 medical and health sciences, 0302 clinical medicine, Viral envelope, Species Specificity, medicine, 030304 developmental biology, Plant Proteins, 0303 health sciences, Fusion, Multidisciplinary, biology, Chemistry, Arabidopsis Proteins, Chlamydomonas, Cell adhesion, Membrane Proteins, General Chemistry, Plants, biology.organism_classification, Fusion protein, Recombinant Proteins, Cell biology, medicine.anatomical_structure, Germ Cells, Fertilization, Gamete, Carrier Proteins, 030217 neurology & neurosurgery, Function (biology)
Abstract: Recognition and fusion between gametes during fertilization is an ancient process. Protein HAP2, recognized as the primordial eukaryotic gamete fusogen, is a structural homolog of viral class II fusion proteins. The mechanisms that regulate HAP2 function, and whether virus-fusion-like conformational changes are involved, however, have not been investigated. We report here that fusion between plus and minus gametes of the green alga Chlamydomonas indeed requires an obligate conformational rearrangement of HAP2 on minus gametes from a labile, prefusion form into the stable homotrimers observed in structural studies. Activation of HAP2 to undergo its fusogenic conformational change occurs only upon species-specific adhesion between the two gamete membranes. Following a molecular mechanism akin to fusion of enveloped viruses, the membrane insertion capacity of the fusion loop is required to couple formation of trimers to gamete fusion. Thus, species-specific membrane attachment is the gateway to fusion-driving HAP2 rearrangement into stable trimers., HAP2 is essential for gamete fusion during fertilization and is conserved among eukaryotes. Here the authors show that species-specific adhesion between Chlamydomonas plus and minus gametes initiates HAP2 to undergo a fusogenic conformational change into homotrimers via a molecular mechanism akin to that of enveloped viruses.
Published: 2021

35. 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

36. Orthobunyavirus spike architecture and recognition by neutralizing antibodies

Author: Jan Hellert, Martin Beer, Ahmed Haouz, Kerstin Wernike, Félix A. Rey, Sven Reiche, Pablo Guardado-Calvo, Emiliana Brocchi, Andrea Aebischer, Virologie Structurale - Structural Virology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institute of Diagnostic Virology (IVD), Friedrich-Loeffler-Institut (FLI), Cristallographie (Plateforme) - Crystallography (Platform), Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna 'Bruno Ubertini' (IZSLER), This research was performed as part of the Zoonoses Anticipation and Preparedness Initiative (ZAPI project, IMI Grant Agreement n°115760), with the assistance and financial support of IMI and the European Commission, and in kind contributions from EFPIA partners. F.A.R. also received funding by the Région Ile de France (Domaine d’intêret majeur Innovative technologies for life sciences, DIM 1HEALTH). Additional funding was provided by Institut Pasteur, the CNRS and the GIS IBiSA (Infrastructures en biologie santé et agronomie). J.H. received the Pasteur-Cantarini fellowship for 24 months, and was later supported by the DIM 1HEALTH grant., We thank Patrick England from the Molecular Biophysics facility and Fabrice Agou from the Chemogenomic and Biological Screening platform at Institut Pasteur and the staff of synchrotron beamlines PX1 and PX2 at SOLEIL (St. Aubin, France) and ID29 and ID30B at the ESRF (Grenoble, France) for help during data collection. We thank Xiaohong Shi and Richard M. Elliott from Glasgow, UK, for the BUNV Gc gene., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, 0301 basic medicine, Orthobunyavirus, General Physics and Astronomy, MESH: Cricetinae, 02 engineering and technology, MESH: Amino Acid Sequence, Crystallography, X-Ray, medicine.disease_cause, Genome, MESH: Mice, Knockout, MESH: Antibodies, Monoclonal, MESH: Antibodies, Neutralizing, Mice, MESH: Protein Structure, Tertiary, Viral Envelope Proteins, MESH: Chlorocebus aethiops, Cricetinae, Chlorocebus aethiops, MESH: Animals, Neutralizing antibody, lcsh:Science, Mice, Knockout, Multidisciplinary, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Antibodies, Monoclonal, Schmallenberg virus, Ruminants, 021001 nanoscience & nanotechnology, 3. Good health, MESH: Ruminants, Female, Antibody, 0210 nano-technology, Viral protein, Science, MESH: Glycoproteins, MESH: Orthobunyavirus, MESH: Vero Cells, Article, General Biochemistry, Genetics and Molecular Biology, Antigenic drift, 03 medical and health sciences, MESH: Viral Structures, MESH: Mice, Inbred C57BL, medicine, Animals, Amino Acid Sequence, Vero Cells, MESH: Mice, Viral Structures, Glycoproteins, General Chemistry, biology.organism_classification, MESH: Crystallography, X-Ray, Antibodies, Neutralizing, Virology, MESH: Male, Protein Structure, Tertiary, Mice, Inbred C57BL, 030104 developmental biology, MESH: Viral Envelope Proteins, biology.protein, Vero cell, lcsh:Q, MESH: Female
Abstract: Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses., Orthobunyaviruses (OBVs) cause severe disease in humans and farm animals, but the molecular basis for infection is not fully understood. Here, the authors present crystal structures of free and antibody-bound OBV envelope glycoproteins and show that their domains enable efficient immunization in a mouse model.
Published: 2019

37. Author Correction: Bat coronaviruses related to SARS-CoV-2 and infectious for human cells

Author: Sarah Temmam, Khamsing Vongphayloth, Eduard Baquero, Sandie Munier, Massimiliano Bonomi, Béatrice Regnault, Bounsavane Douangboubpha, Yasaman Karami, Delphine Chrétien, Daosavanh Sanamxay, Vilakhan Xayaphet, Phetphoumin Paphaphanh, Vincent Lacoste, Somphavanh Somlor, Khaithong Lakeomany, Nothasin Phommavanh, Philippe Pérot, Océane Dehan, Faustine Amara, Flora Donati, Thomas Bigot, Michael Nilges, Félix A. Rey, Sylvie van der Werf, Paul T. Brey, and Marc Eloit
Subjects: Multidisciplinary
Published: 2022

38. Reduced sensitivity of infectious SARS-CoV-2 variant B.1.617.2 to monoclonal antibodies and sera from convalescent and vaccinated individuals

Author: Olivier Schwartz, Cyril Planchais, Aurélie Velay, Françoise Porrot, Thierry Prazuck, Laura Courtellemenont, Matthieu Prot, Julien Le Guen, Maaran Michael Rajah, Julien Puech, Hugo Mouquet, Floriane Gallais, Aymeric Sève, Florence Guivel-Benhassine, Laurent Hocqueloux, Samira Fafi-Kremer, Etienne Simon-Loriere, Dhiaeddine Edriss, Hélène Péré, Artem Baidaliuk, Félix A. Rey, Delphine Planas, David Veyer, Nicolas Robillard, Timothée Bruel, Najibi Kassis-Chikhani, Laurent Bélec, Isabelle Staropoli, and Pierre Gantner
Subjects: Lineage (genetic), Immune system, Coronavirus disease 2019 (COVID-19), medicine.drug_class, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), medicine, biology.protein, Biology, Antibody, Monoclonal antibody, Virology, Epitope, Neutralization
Abstract: The SARS-CoV-2 B.1.617 lineage emerged in October 2020 in India1–6. It has since then become dominant in some indian regions and further spread to many countries. The lineage includes three main subtypes (B1.617.1, B.1617.2 and B.1.617.3), which harbour diverse Spike mutations in the N-terminal domain (NTD) and the receptor binding domain (RBD) which may increase their immune evasion potential. B.1.617.2 is believed to spread faster than the other versions. Here, we isolated infectious B.1.617.2 from a traveller returning from India. We examined its sensitivity to monoclonal antibodies (mAbs) and to antibodies present in sera from COVID-19 convalescent individuals or vaccine recipients, in comparison to other viral lineages. B.1.617.2 was resistant to neutralization by some anti-NTD and anti-RBD mAbs, including Bamlanivimab, which were impaired in binding to the B.1.617.2 Spike. Sera from convalescent patients collected up to 12 months post symptoms and from Pfizer Comirnaty vaccine recipients were 3 to 6 fold less potent against B.1.617.2, relative to B.1.1.7. Sera from individuals having received one dose of AstraZeneca Vaxzevria barely inhibited B.1.617.2. Thus, B.1.617.2 spread is associated with an escape to antibodies targeting non-RBD and RBD Spike epitopes.
Published: 2021

39. The dengue virus NS1 protein conveys pro-inflammatory signals by docking onto human high-density lipoproteins

Author: X Zhang, Tamietti C, Stéphane Petres, Anastassia Mikhailova, Fasséli Coulibaly, Benfrid S, Park K, Eva Harris, Duong, Mariano Dellarole, James E. Voss, Marie Flamand, Philippe Buchy, Sakunthabaï A, Gérard Pehau-Arnaudet, Patrick England, Philippe Dussart, Félix A. Rey, Scott B. Biering, Sébastien Brûlé, Marie-Noëlle Ungeheuer, François Bontems, and Bertrand Raynal
Subjects: biology, Chemistry, viruses, virus diseases, biochemical phenomena, metabolism, and nutrition, Dengue virus, medicine.disease_cause, medicine.disease, Virulence factor, Complement system, Dengue fever, Cell biology, Pathogenesis, Immune system, medicine, biology.protein, lipids (amino acids, peptides, and proteins), Antibody, Lipoprotein
Abstract: The nonstructural NS1 protein is a virulence factor secreted by dengue virus (DENV)-infected cells. NS1 is known to alter the complement system, activate immune cells and perturb endothelial barriers. Here we show that pro-inflammatory signals are triggered by a high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Electron microscopy images of the NS1-HDL complexes show spherical HDL particles with rod-shaped NS1 protrusions on their surface. These complexes are readily detectable in the plasma of hospitalized dengue patients using anti-apolipoprotein A-I (ApoA-I) antibodies specific of the HDL moiety. The functional reprogramming of HDL particles by the NS1 protein as a means to exacerbate systemic inflammation during DENV infection provides a new paradigm linking the human lipoprotein network to dengue pathogenesis.
Published: 2021

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

Author: Marija Backovic, Barbara G. Klupp, Kay Grünewald, Thomas C. Mettenleiter, Henriette Lötzsch, Benjamin Vollmer, Félix A. Rey, Melina Vallbracht, Walter Fuchs, Friedrich-Loeffler-Institut (FLI), Heinrich Pette Institute [Hamburg], Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), These studies were supported by a grant from the Deutsche Forschungsgemeinschaft (DFG grant Me 854/11-2). F.A.R. and M.B. acknowledge funding from Institut Pasteur, CNRS and Labex IBEID (grant ANR-10-IHUB-0002)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: herpesviruses, viruses, [SDV]Life Sciences [q-bio], membrane fusion, autonomous fusion, Microbiology, 03 medical and health sciences, Viral entry, Virology, glycoprotein B, bovine herpesvirus, 030304 developmental biology, 0303 health sciences, 030306 microbiology, Chemistry, Mutagenesis, Lipid bilayer fusion, gH/gL complex, pseudorabies virus, Fusion protein, Phenotype, QR1-502, Cell biology, Ectodomain, Viral evolution, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, fusion regulation, viral entry, Signal transduction, Research Article
Abstract: International audience; Herpesvirus entry and spread requires fusion of viral and host cell membranes, which is mediated by the conserved surface glycoprotein B (gB). Upon activation, gB undergoes a major conformational change and transits from a metastable prefusion to a stable postfusion conformation. Although gB is a structural homolog of low-pH-triggered class III fusogens, its fusion activity depends strictly on the presence of the conserved regulatory gH/gL complex and nonconserved receptor binding proteins, which ensure that fusion occurs at the right time and space. How gB maintains its prefusion conformation and how gB fusogenicity is controlled remain poorly understood. Here, we report the isolation and characterization of a naturally selected pseudorabies virus (PrV) gB able to mediate efficient gH/gL-independent virus-cell and cell-cell fusion. We found that the control exerted on gB by the accompanying viral proteins is mediated via its cytosolic domain (CTD). Whereas gB variants lacking the CTD are inactive, a single mutation of a conserved asparagine residue in an alpha-helical motif of the ectodomain recently shown to be at the core of the gB prefusion trimer compensated for CTD absence and uncoupled gB from regulatory viral proteins, resulting in a hyperfusion phenotype. This phenotype was transferred to gB homologs from different alphaherpesvirus genera. Overall, our data propose a model in which the central helix acts as a molecular switch for the gB pre-to-postfusion transition by conveying the structural status of the endo-to the ectodomain, thereby governing their cross talk for fusion activation, providing a new paradigm for herpesvirus fusion regulation. IMPORTANCE 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. A detailed molecular-level understanding of herpesvirus membrane fusion is of fundamental virological interest and has considerable potential for the development of new therapeutics blocking herpesvirus cell invasion and spread. Using in vitro evolution and targeted mutagenesis of three different animal alphaherpesviruses, we identified a single conserved amino acid in a regulatory helix in the center of the gB ectodomain that enables efficient gH/gL-independent entry and plays a crucial role in the pre-to-postfusion transition of gB. Our results propose that the central helix is a key regulatory element involved in the intrastructural signal transduction between the endo- and ectodomain for fusion activation. This study expands our understanding of herpesvirus membrane fusion and uncovers potential targets for therapeutic interventions.
Published: 2021

41. 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

42. The prefusion structure of herpes simplex virus glycoprotein B

Author: Thomas C. Mettenleiter, E. E. Jefferys, A. Hernandez-Duran, Barbara G. Klupp, Vojtěch Pražák, Melina Vallbracht, Marija Backovic, Félix A. Rey, Daven Vasishtan, Kay Grünewald, Maya Topf, Benjamin Vollmer, University of Oxford [Oxford], Heinrich Pette Institute [Hamburg], Birkbeck College [University of London], Friedrich-Loeffler-Institut (FLI), Virologie Structurale - Structural Virology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Universität Hamburg (UHH), We gratefully acknowledge funding from the Wellcome Trust (209250/Z/17/Z to M.T. and K.G., 107806/Z/15/Z to K.G., 203815/Z/16/A to E.E.J., and Core Award 203141/Z/16/Z), from the MRC (MR/M019292/1 to M.T. and K.G. and MR/R017875/1 to B.V. and K.G.), the DFG [a Cluster of Excellence RESIST (EXC 2155) project to K.G., Me 854/11-2 to T.C.M., and INST 152/772-1, 774-1, 775-1, 776-1 FUGG to the CSSB cryo-EM facility], the BMBF (05K18BHA to K.G.), and a Marie Curie Individual Fellowship (KGBVIFEF to B.V.). Work by M.B. and F.A.R. was supported by IP recurrent funding and F.A.R. by the ANR (ANR-10-LABX-62-IBEID)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), University of Oxford, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Protein Conformation, [SDV]Life Sciences [q-bio], Herpesvirus 1, Human, medicine.disease_cause, Virus, Conserved sequence, 03 medical and health sciences, Viral envelope, medicine, Humans, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Mutation, Fusion, Multidisciplinary, biology, 030306 microbiology, Chemistry, Herpes Simplex, Virus Internalization, biology.organism_classification, Cell biology, Herpes simplex virus, Vesicular stomatitis virus, Glycoprotein
Abstract: International audience; Cell entry of enveloped viruses requires specialized viral proteins that mediate fusion with the host membrane by substantial structural rearrangements from a metastable pre- to a stable postfusion conformation. This metastability renders the herpes simplex virus 1 (HSV-1) fusion glycoprotein B (gB) highly unstable such that it readily converts into the postfusion form, thereby precluding structural elucidation of the pharmacologically relevant prefusion conformation. By identification of conserved sequence signatures and molecular dynamics simulations, we devised a mutation that stabilized this form. Functionally locking gB allowed the structural determination of its membrane-embedded prefusion conformation at sub-nanometer resolution and enabled the unambiguous fit of all ectodomains. The resulting pseudo-atomic model reveals a notable conservation of conformational domain rearrangements during fusion between HSV-1 gB and the vesicular stomatitis virus glycoprotein G, despite their very distant phylogeny. In combination with our comparative sequence-structure analysis, these findings suggest common fusogenic domain rearrangements in all class III viral fusion proteins.
Published: 2020

43. Extensive flavivirus E trimer breathing accompanies stem zippering of the post‐fusion hairpin

Author: Félix A. Rey, Marie-Christine Vaney, Franz X. Heinz, Martial Rey, Karin Stiasny, Julia Chamot-Rooke, Alexander Rouvinski, Iris Medits, Medizinische Universität Wien = Medical University of Vienna, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), We acknowledge support from the Austrian Science Fund FWF (I1378-B13, P27501-B21), French ANR (grant ANR-13-ISV8-0002-01), Institut Pasteur and CNRS., ANR-13-ISV8-0002,flavistem,La protéine E des flavivirus : interactions et fusion membranaire(2013), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Virologie Structurale
Subjects: Zipper, [SDV]Life Sciences [q-bio], membrane fusion, fusion intermediate, Trimer, Biochemistry, Article, Encephalitis Viruses, Tick-Borne, Conserved sequence, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Genetics, flavivirus E trimer, Molecular Biology, 030304 developmental biology, 0303 health sciences, Fusion, class II fusion protein, Chemistry, Membrane fusion protein, Lipid bilayer fusion, Articles, Microbiology, Virology & Host Pathogen Interaction, Transmembrane protein, 3. Good health, dynamic behavior, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Membrane, stem zippering, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Biophysics, 030217 neurology & neurosurgery
Abstract: Flaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic trimers. The rod‐like E subunits bend into “hairpins” to bring the fusion loops next to the C‐terminal transmembrane (TM) anchors, with the TM‐proximal “stem” element zippering the E trimer to force apposition of the membranes. The structure of the complete class II trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E‐protein trimers from the tick‐borne encephalitis flavivirus with sequential stem truncations. Our thermostability and antibody‐binding data suggest that the stem “zipper” ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to trimer stability. We further identified a highly dynamic behavior of E trimers C‐terminally truncated upstream the CS, which, unlike fully stem‐zippered trimers, undergo rapid deuterium exchange at the trimer interface. These results thus identify important “breathing” intermediates in the E‐protein‐driven membrane fusion process., The flavivirus envelope protein E exhibits a dynamic “breathing” behavior not only as a dimer at the surface of mature virions, but also as a trimeric intermediate during membrane fusion in the endosome.
Published: 2020

44. A molecular determinant of West Nile virus secretion and morphology as a target for viral attenuation

Author: Justine, Basset, Julien, Burlaud-Gaillard, Maxence, Feher, Philippe, Roingeard, Félix A, Rey, Nathalie, Pardigon, Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris] (IP), Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, Plateforme IBISA de Microscopie Electronique [CHRU de Tours] (UNIV Tours), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT), Morphogénèse et antigénicité du VIH et du virus des Hépatites (MAVIVH - U1259 Inserm - CHRU Tours ), Centre Hospitalier Régional Universitaire de Tours (CHRU Tours)-Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Cellule d'Intervention Biologique d'Urgence - Laboratory for Urgent Response to Biological Threats (CIBU), Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Financial support was provided in part by Institut Carnot-Pasteur Microbes and Health, pardigon, nathalie, Institut Pasteur [Paris], Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Université de Tours, Centre Hospitalier Régional Universitaire de Tours (CHRU TOURS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Tours, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Models, Molecular, Protein Conformation, alpha-Helical, viruses, Gene Expression, Antibodies, Viral, Mice, Viral Proteins, Cell Line, Tumor, Chlorocebus aethiops, Animals, membrane protein, Amino Acid Sequence, Vero Cells, attenuation, Neurons, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Mice, Inbred BALB C, Binding Sites, Membrane Glycoproteins, Sequence Homology, Amino Acid, Flavivirus, virus diseases, Antibodies, Neutralizing, Survival Analysis, Virus-Cell Interactions, secretion, Disease Models, Animal, Mutation, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Female, Protein Conformation, beta-Strand, Sequence Alignment, West Nile virus, West Nile Fever, Protein Binding
Abstract: West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health., West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo. Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains. IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.
Published: 2020

45. SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors

Author: Lucie Le Fevre, Diane Descamps, Sylvie van der Werf, Nicolas Escriou, Delphine Planas, Olivier Schwartz, Flora Donati, Cyril Planchais, Rémy Robinot, Isabelle Staropoli, Bernadette Crescenzo, Bruno Hoen, Mireille Nowakowski, Marion Gransagne, Mélanie Albert, Jérémy Dufloo, Marc Eloit, Stéphane Petres, Camille Besombes, Ludivine Grzelak, Yazdan Yazdanpanah, Hugo Mouquet, Sarah Temmam, Arnaud Fontanet, Pierre Charneau, Marija Backovic, Vincent Enouf, Pascal Morel, Françoise Porrot, Quentin Le Hingrat, Audrey Lemaitre, Timothée Bruel, Philippe Souque, Maxime Chazal, Guillaume Mellon, Laura Tondeur, Anabelle Pourbaix, Simon Rolland, Félix A. Rey, Christèle Huon, Julian Buchrieser, Sylvie Behillil, Marie-Noëlle Ungeheuer, François Anna, Maaran Michael Rajah, Lila Bouadma, Florence Guivel, Jacques Bellalou, Marie-Aude Creach, Caroline Demeret, and Kuang-Yu Chen
Subjects: 0303 health sciences, education.field_of_study, biology, 030306 microbiology, business.industry, Population, Antibody titer, Virology, Asymptomatic, 3. Good health, Serology, 03 medical and health sciences, Antigen, biology.protein, Medicine, Seroprevalence, Antibody, Seroconversion, medicine.symptom, education, business, 030304 developmental biology
Abstract: It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their antibody response profile. Here, we performed a pilot study to assess the levels of anti-SARS-CoV-2 antibodies in samples taken from 491 pre-epidemic individuals, 51 patients from Hôpital Bichat (Paris), 209 pauci-symptomatic individuals in the French Oise region and 200 contemporary Oise blood donors. Two in-house ELISA assays, that recognize the full-length nucleoprotein (N) or trimeric Spike (S) ectodomain were implemented. We also developed two novel assays: the S-Flow assay, which is based on the recognition of S at the cell surface by flow-cytometry, and the LIPS assay that recognizes diverse antigens (including S1 or N C-terminal domain) by immunoprecipitation. Overall, the results obtained with the four assays were similar, with differences in sensitivity that can be attributed to the technique and the antigen in use. High antibody titers were associated with neutralisation activity, assessed using infectious SARS-CoV-2 or lentiviral-S pseudotypes. In hospitalized patients, seroconversion and neutralisation occurred on 5-14 days post symptom onset, confirming previous studies. Seropositivity was detected in 29% of pauci-symptomatic individuals within 15 days post-symptoms and 3 % of blood of healthy donors collected in the area of a cluster of COVID cases. Altogether, our assays allow for a broad evaluation of SARS-CoV2 seroprevalence and antibody profiling in different population subsets.
Published: 2020

46. Multimeric single-domain antibody complexes protect against bunyavirus infections

Author: Frank Grosveld, Sara Rodriguez Conde, Paul J. Wichgers Schreur, Dorota Kozub, Jan Hellert, Kieran K Mistry, Erick Bermúdez-Méndez, Martin Beer, Pablo Guardado-Calvo, Félix A. Rey, Karen Brennan, Ziyan Deng, Michiel M. Harmsen, Olalekan Daramola, Sandra G. P. van de Water, Jeroen Kortekaas, Dubravka Drabek, Maiken Søndergaard Kristiansen, Andrea Aebischer, Lucien van Keulen, Kerstin Wernike, Wageningen BioVeterinary Research, Wageningen University and Research [Wageningen] (WUR), Erasmus University Medical Center [Rotterdam] (Erasmus MC), Harbour Antibodies B.V, Institute of Diagnostic Virology (IVD), Friedrich-Loeffler-Institut (FLI), AstraZeneca [Cambridge, UK], Virologie Structurale - Structural Virology, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Innovative Medicines Initiative (ZAPI (grant agreement no. 115760))Ministerio de Ciencia Tecnología y Telecomunicaciones (Student Fellowship (PEM-066-2015-II))Universidad de Costa Rica (Student Fellowship (OAICE-CAB-05-56-2016)), European Project: 115760,EC:FP7:SP1-JTI,IMI-JU-11-2013,ZAPI(2015), Cell biology, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, 0301 basic medicine, Rift Valley fever virus, Laboratory of Virology, bunyavirus, MESH: Antibodies, Neutralizing, Mice, schmallenberg virus, MESH: Animals, Biology (General), Microbiology and Infectious Disease, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], General Neuroscience, Bacteriologie, Schmallenberg virus, Bacteriology, Host Pathogen Interaction & Diagnostics, General Medicine, PE&RC, Virology & Molecular Biology, 3. Good health, MESH: Camelids, New World, Medicine, Female, Antibody, Camelids, New World, Research Article, MESH: Antiviral Agents, QH301-705.5, Science, infectious disease, 030106 microbiology, virus, Bunyaviridae Infections, Antiviral Agents, MESH: Single-Domain Antibodies, General Biochemistry, Genetics and Molecular Biology, Virus, World health, Laboratorium voor Virologie, MESH: Bunyaviridae Infections, 03 medical and health sciences, Emerging pathogen, SDG 3 - Good Health and Well-being, Animals, Humans, Life Science, MESH: Mice, single-domain antibody, Host Pathogen Interaction & Diagnostics, therapy, MESH: Humans, General Immunology and Microbiology, microbiology, vhh, Bacteriology, Single-Domain Antibodies, biology.organism_classification, Antibodies, Neutralizing, Virology, Host Pathogen Interactie & Diagnostiek, MESH: Male, Virologie & Moleculaire Biologie, 030104 developmental biology, Single-domain antibody, Infectious disease (medical specialty), Bacteriologie, Host Pathogen Interactie & Diagnostiek, biology.protein, MESH: Female, rift valley fever virus
Abstract: International audience; The World Health Organization has included three bunyaviruses posing an increasing threat to human health on the Blueprint list of viruses likely to cause major epidemics and for which no, or insufficient countermeasures exist. Here, we describe a broadly applicable strategy, based on llama-derived single-domain antibodies (VHHs), for the development of bunyavirus biotherapeutics. The method was validated using the zoonotic Rift Valley fever virus (RVFV) and Schmallenberg virus (SBV), an emerging pathogen of ruminants, as model pathogens. VHH building blocks were assembled into highly potent neutralizing complexes using bacterial superglue technology. The multimeric complexes were shown to reduce and prevent virus-induced morbidity and mortality in mice upon prophylactic administration. Bispecific molecules engineered to present two different VHHs fused to an Fc domain were further shown to be effective upon therapeutic administration. The presented VHH-based technology holds great promise for the development of bunyavirus antiviral therapies.
Published: 2020

47. Author response: Multimeric single-domain antibody complexes protect against bunyavirus infections

Author: Ziyan Deng, Kerstin Wernike, Dorota Kozub, Sandra G. P. van de Water, Frank Grosveld, Pablo Guardado-Calvo, Jeroen Kortekaas, Sara Rodriguez Conde, Andrea Aebischer, Paul J. Wichgers Schreur, Jan Hellert, Kieran K Mistry, Olalekan Daramola, Martin Beer, Félix A. Rey, Karen Brennan, Erick Bermúdez-Méndez, Dubravka Drabek, Lucien van Keulen, M.M. Harmsen, and Maiken Søndergaard Kristiansen
Subjects: Single-domain antibody, Chemistry, Virology
Published: 2020

48. Structure of the prefusion-locking broadly neutralizing antibody RVC20 bound to the rabies virus glycoprotein

Author: Olivier Schwartz, Amalio Telenti, Guilherme D. Melo, Florence Larrous, Julian Buchrieser, Davide Corti, Leah Soriaga, Félix A. Rey, Ahmed Haouz, Jan Hellert, Patrick England, Hervé Bourhy, Andrea Minola, Virologie Structurale - Structural Virology, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Virus et Immunité - Virus and immunity (CNRS-UMR3569), Lyssavirus, épidémiologie et neuropathologie - Lyssavirus Epidemiology and Neuropathology, Institut Pasteur [Paris] (IP), Humabs BioMed SA, Vir Biotechnology Inc [San Francisco], Biophysique Moléculaire (plateforme) - Molecular Biophysics (platform), Cristallographie (Plateforme) - Crystallography (Platform), This work was funded by Institut Pasteur. F.A.R. received additional funding from the CNRS, the Labex IBEID (ANR-10-IHUB-0002), the GIS IBiSA (Infrastructures en biologie santé et agronomie), grant ANR-13-ISV8-0002-01 and the Région Ile de France in order to support the work of J.H. (Domaine d’intérêt majeur innovative technologies for life sciences, DIM 1HEALTH). O.S. received funding from ANRS, Sidaction, the Vaccine Research Institute (ANR-10-LABX-77), the Labex IBEID (ANR-10-IHUB-0002), the 'TIMTAMDEN' ANR-14-CE14-0029, the 'CHIKV-Viro-Immuno' ANR-14-CE14-0015-01, L’Oréal Sponsorship, the CNRS and the Gilead HIV cure program. H.B., F.L. and G.D.M. received funding from the INFECT-ERA 2016 project ANR 16-IFEC-0006-01 ToRRENT.Part of this work was performed at the UtechS Photonic BioImaging (PBI) platform, member of France Life Imaging network (grant ANR-11-INBS-0006)., ANR-13-ISV8-0002,flavistem,La protéine E des flavivirus : interactions et fusion membranaire(2013), ANR-10-LABX-0077,VRI,Initiative for the creation of a Vaccine Research Institute(2010), ANR-14-CE14-0029,TIMTAMDEN,Rôle des récepteurs TIM et TAM dans l'infection des cellules cibles par le virus de la dengue(2014), ANR-14-CE14-0015,CHIKV-Viro-Immuno,Multiplication et Relation avec l'hôte du virus Chikungunya(2014), ANR-16-IFEC-0006,ToRRENT,Towards a combined post-exposure prophylaxis and successful treatment of rabies in humans(2016), ANR-11-INBS-0006,FLI,France Life Imaging(2011), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Virus et Immunité - Virus and immunity, Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]
Subjects: Viral membrane fusion, 0301 basic medicine, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Antibody Affinity, General Physics and Astronomy, MESH: Amino Acid Sequence, Antibodies, Viral, Crystallography, X-Ray, medicine.disease_cause, MESH: Antibodies, Neutralizing, Epitopes, MESH: Antibody Affinity, MESH: Animals, lcsh:Science, MESH: Mutagenesis, chemistry.chemical_classification, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 3. Good health, MESH: Rabies virus, Perfusion, Antibody, Encephalitis, MESH: Perfusion, Protein Binding, MESH: Epitopes, medicine.drug_class, Science, 030106 microbiology, MESH: Glycoproteins, Biology, Monoclonal antibody, Article, Antibodies, General Biochemistry, Genetics and Molecular Biology, Cell Line, Viral Proteins, 03 medical and health sciences, medicine, Animals, Humans, MESH: Protein Binding, Amino Acid Sequence, Post-exposure prophylaxis, X-ray crystallography, Glycoproteins, MESH: Humans, Rabies virus, Lipid bilayer fusion, General Chemistry, medicine.disease, MESH: Crystallography, X-Ray, Antibodies, Neutralizing, Virology, MESH: Viral Proteins, MESH: Cell Line, 030104 developmental biology, chemistry, Viral infection, Mutagenesis, biology.protein, Rabies, lcsh:Q, Glycoprotein, MESH: Antibodies, Viral
Abstract: Rabies virus (RABV) causes fatal encephalitis in more than 59,000 people yearly. Upon the bite of an infected animal, the development of clinical disease can be prevented with post-exposure prophylaxis (PEP), which includes the administration of Rabies immunoglobulin (RIG). However, the high cost and limited availability of serum-derived RIG severely hamper its wide use in resource-limited countries. A safe low-cost alternative is provided by using broadly neutralizing monoclonal antibodies (bnAbs). Here we report the X-ray structure of one of the most potent and most broadly reactive human bnAbs, RVC20, in complex with its target domain III of the RABV glycoprotein (G). The structure reveals that the RVC20 binding determinants reside in a highly conserved surface of G, rationalizing its broad reactivity. We further show that RVC20 blocks the acid-induced conformational change required for membrane fusion. Our results may guide the future development of direct antiviral small molecules for Rabies treatment., Rabies virus (RABV) infection of unvaccinated individuals can be treated with post exposure prophylaxis. Here, Hellert et al. analyze the structure of a broadly neutralizing human antibody in complex with its target domain in the RABV glycoprotein and demonstrate that it blocks the acid-induced conformational change required for membrane fusion.
Published: 2020

49. High-Resolution Description of the Hantavirus Surface Glycoprotein Lattice and Its Membrane Fusion Control Mechanism

Author: Rohit K. Jangra, Alexandra Serris, Robert Stass, Jean-Claude Manuguerra, Félix A. Rey, Nicolas A. Muena, Juha T. Huiskonen, Sai Li, Kartik Chandran, Nicole D. Tischler, Eduardo A Bignon, and Pablo Guardado-Calvo
Subjects: chemistry.chemical_classification, 050208 finance, Immunogen, viruses, 05 social sciences, Andes virus, Lipid bilayer fusion, Endocytosis, Virus, chemistry, Viral entry, 0502 economics and business, Biophysics, 050207 economics, Glycoprotein, Hantavirus
Abstract: Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. The hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimeric protomers associated into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the Gn head / Gc heterodimer, and of the homotetrameric Gn base. Docking them into an 11.4 A resolution cryo- electron tomography map of the hantavirus surface accounted for the complete extracellular portion of the viral glycoprotein shell and provided unprecedented detail of the surface organization of a pleomorphic virus. These results, which further revealed an in-built mechanism controlling Gc membrane-insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses
Published: 2020
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50. Novel toscana virus reverse genetics system establishes NSS as an antagonist of type I interferon responses

Author: Megan L. Stanifer, Paul J. Wichgers Schreur, Felix Pahmeier, Pierre-Yves Lozach, Zina M. Uckeley, Stefan Windhaber, Gleyder Roman Sosa, Psylvia Léger, Steeve Boulant, Félix A. Rey, Franziska Woelfl, Nadia Oreshkova, Jeroen Kortekaas, Jana Koch, Infections Virales et Pathologie Comparée - UMR 754 (IVPC), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), CellNetworks (Heidelberg) German Research Foundation (DFG)LO-2338/1-1LO-2338/3-1INRAE starter funds IDEX-Impulsion 2020 (University of Lyon) FINOVI (Fondation pour l'Universite de Lyon) Institut Pasteur, CNRS Labex IBEID ANR-10-IHUB-0002German Research Foundation (DFG)BO 4340/1-1SFB1129240245660SFB 1129Brigitte-Schlieben Lange Program from Baden Wurttemberg, Germany German Research Foundation (DFG)European CommissionSTA 1536/2-1ANR-18-CE92-0006-02 'PHLEBO', and ANR-13-ISV8-0002,flavistem,La protéine E des flavivirus : interactions et fusion membranaire(2013)
Subjects: 0301 basic medicine, Phlebovirus, Insecta, lcsh:QR1-502, Phenuiviridae, Viral Nonstructural Proteins, Antibodies, Viral, Kidney, medicine.disease_cause, lcsh:Microbiology, Bunyavirales, Sand fly fever, Interferon, Cricetinae, Chlorocebus aethiops, 2.1 Biological and endogenous factors, 2.2 Factors relating to the physical environment, Viral, Aetiology, Arbovirus, Toscana virus, Genome, biology, interferon, DNA-Directed RNA Polymerases, sand fly fever, PE&RC, 3. Good health, Virology & Molecular Biology, Infectious Diseases, Neglected diseases, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Infection, Biotechnology, medicine.drug, Viral protein, 030106 microbiology, Genome, Viral, Microbiology, Antibodies, Article, Virus, Cell Line, Vaccine Related, reverse genetics, Viral Proteins, 03 medical and health sciences, Virology, neglected diseases, Genetics, medicine, Animals, Humans, Vero Cells, Messenger RNA, RNA, Sandfly fever Naples virus, Interferon-beta, biology.organism_classification, Reverse genetics, Virologie & Moleculaire Biologie, Good Health and Well Being, 030104 developmental biology, A549 Cells, Mutation, Immunization
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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