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3.
Virologie ; 26(2):186, 2022.
Article in English | EMBASE | ID: covidwho-1912865

ABSTRACT

Bats are natural reservoirs for numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction of coronaviruses and bat cells is, however, sparse. There is thus a need to develop bat cellular models to understand cell tropism, viral replication and virus-induced cell responses. Here, we report the first molecular study of SARS-CoV-2 infection in chiropteran cells. We investigated the ability of primary cells from Rhinolophus and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis and Nyctalus noctula, to support SARS-CoV-2 replication. None of these cells were permissive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species including humans. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that the restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or absence of bACE2 binding in these cells. By contrast, multiple restriction factors to viral replication exist in the three N. noctula cells since hACE2 expression was not sufficient to permit infection. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication, which correlated with a potent interferon response. Together, our data highlight the existence of species-specific molecular barriers to viral replication in bat cells. Our newly developed chiropteran cellular models are useful tools to investigate the interplay between viruses belonging to the SARS-CoV- 2 lineage and their natural reservoir, including the identification of factors responsible for viral restriction.

4.
American Journal of Respiratory and Critical Care Medicine ; 205:2, 2022.
Article in English | English Web of Science | ID: covidwho-1880411
5.
Topics in Antiviral Medicine ; 30(1 SUPPL):122, 2022.
Article in English | EMBASE | ID: covidwho-1880385

ABSTRACT

Background: More than 10% of patients infected with SARS-CoV-2 experience a Long COVID syndrome, characterized by the persistence of a diverse array of symptoms where fatigue predominates. The role of the adaptive immune response in Long COVID remains poorly understood, with contrasting hypotheses suggesting either an insufficient antiviral response or an excessive immune response that would trigger autoimmune damage. To address this issue, we set to characterize humoral and cellular responses in Long COVID patients prior to SARS-CoV-2 vaccination. Methods: Long COVID patients (n=36) were included based on (1) an initial SARS-CoV-2 infection documented by PCR or the conjunction of two major signs of COVID-19 and (2) the persistence or resurgence of symptoms for over 3 months. They were compared to convalescent COVID patients with resolved symptoms (n=23) and uninfected control individuals (n=20). IgG and IgA antibodies specific to the SARS-CoV-2 spike were detected by a sensitive S-flow assay, which measures antibody binding to spike-expressing 293T cells. For CD4+ T cell response analyses, cytokine production was measured by intracellular staining on primary T cell lines stimulated by immunodominant peptides derived from the S, M, and N viral proteins. Results: Antibody analyses revealed either strong or very low/undetectable amounts of spike-specific IgG in sera from Long COVID patients, thus distinguishing a seropositive and a seronegative group. Seropositive Long COVID patients (n=21) showed strong CD4 responses that tended to be of higher magnitude than those of convalescents (P<0.05 for 2 immunodominant peptides). In contrast, seronegative Long COVID patients (n=15) showed low or undetectable CD4+ T cells responses, with 4/15 patients showing responses above those observed in healthy donors. CD4+ T cell responses correlated with spike-specific IgG responses in seropositive Long COVID patients (P≤0.002) but not in convalescents, pointing to differences in immune memory persistence. Conclusion: These findings highlight divergent adaptive immune responses among Long COVID patients, with a group characterized by seroconversion and particularly strong CD4+ T cell responses, and a second group characterized by low or undetectable antibody and cellular responses. Further studies are warranted to determine whether the etiology and the duration of symptoms differ in these two groups of Long COVID patients.

6.
Embase; 2022.
Preprint in English | EMBASE | ID: ppcovidwho-338083

ABSTRACT

Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in COVID-19 convalescents combining serological, cellular and monoclonal antibody explorations, revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell, demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.

7.
Embase; 2021.
Preprint in English | EMBASE | ID: ppcovidwho-336382

ABSTRACT

Serological tests are important for understanding the physiopathology and following the evolution of the Covid-19 pandemic. Assays based on flow cytometry (FACS) of tissue culture cells expressing the spike (S) protein of SARS-CoV-2 have repeatedly proven to perform slightly better than the plate-based assays ELISA and CLIA (chemiluminescent immuno-assay), and markedly better than lateral flow immuno-assays (LFIA). Here, we describe an optimized and very simple FACS assay based on staining a mix of two Jurkat cell lines, expressing either high levels of the S protein (Jurkat-S) or a fluorescent protein (Jurkat-R expressing m-Cherry, or Jurkat-G, expressing GFP, which serve as an internal negative control). We show that the Jurkat-S&R-flow test has a much broader dynamic range than a commercial ELISA test and performs at least as well in terms of sensitivity and specificity. Also, it is more sensitive and quantitative than the hemagglutination-based test HAT, which we described recently. The Jurkat-flow test requires only a few microliters of blood;thus, it can be used to quantify various Ig isotypes in capillary blood collected from a finger prick. It can be used also to evaluate serological responses in mice, hamsters, cats and dogs. Whilst the Jurkat-flow test is ill-suited and not intended for clinical use, it offers a very attractive solution for laboratories with access to tissue culture and flow cytometry who want to monitor serological responses in humans or in animals, and how these relate to susceptibility to infection, or re-infection, by the virus, and to protection against Covid-19. Note: This manuscript has been refereed by Review Commons, and modified thanks to the comments and suggestions from two referees. Those comments, and our replies, are provided at the end of the manuscript's pdf, and can also be accessed by clicking on the box with a little green number found just above the “Abstract “tab in the medRXiv window.

8.
PubMed; 2021.
Preprint in English | PubMed | ID: ppcovidwho-333758

ABSTRACT

Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity;indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost. ONE SENTENCE SUMMARY: Drug-induced phospholipidosis is a single mechanism that may explain the in vitro efficacy of a wide-variety of therapeutics repurposed for COVID-19.

9.
Embase;
Preprint in English | EMBASE | ID: ppcovidwho-326820

ABSTRACT

The SARS-CoV-2 Omicron variant was first identified in November 2021 in Botswana and South Africa 1,2. It has in the meantime spread to many countries and is expected to rapidly become dominant worldwide. The lineage is characterized by the presence of about 32 mutations in the Spike, located mostly in the N-terminal domain (NTD) and the receptor binding domain (RBD), which may enhance viral fitness and allow antibody evasion. Here, we isolated an infectious Omicron virus in Belgium, from a traveller returning from Egypt. We examined its sensitivity to 9 monoclonal antibodies (mAbs) clinically approved or in development3, and to antibodies present in 90 sera from COVID-19 vaccine recipients or convalescent individuals. Omicron was totally or partially resistant to neutralization by all mAbs tested. Sera from Pfizer or AstraZeneca vaccine recipients, sampled 5 months after complete vaccination, barely inhibited Omicron. Sera from COVID-19 convalescent patients collected 6 or 12 months post 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 titers 5 to 31 fold lower against Omicron than against Delta. Thus, Omicron escapes most therapeutic monoclonal antibodies and to a large extent vaccine-elicited antibodies.

10.
11.
La Revue de Médecine Interne ; 42:A339-A340, 2021.
Article in French | ScienceDirect | ID: covidwho-1531776

ABSTRACT

Introduction L’émergence de nouvelles souches du SARS-CoV-2, telles que le variant Delta, présentant une réplication virale augmentée et la capacité d’échapper à la réponse immune soulève des inquiétudes chez les patients immunodéprimés. Cette étude avait pour objectif d’évaluer le taux de séroconversion, de neutralisation de différents variants, et la réponse lymphocytaire T en réponse à la vaccination par le BNT162b2 chez des patients avec maladies auto-immunes en fonction des traitements reçus. Patients et méthodes Étude prospective monocentrique réalisée à l’Hôpital Cochin (Paris) incluant des patients avec maladies auto-immunes traités par immunosuppresseurs et/ou immunomodulateurs, et des professionnels de santé comme contrôles. Les cas et les contrôles étaient exclus s’ils avaient une sérologie Covid-19 positive à l’inclusion. Le critère de jugement principal était la proportion d’anticorps anti-Spike et les titres de neutralisation croisée contre les variants Alpha et Delta à 3 mois (après deux doses de vaccin). Les critères de jugements secondaires étaient la réponse lymphocytaire T spécifique, la proportion d’infections à SARS-CoV-2 symptomatiques et la tolérance du vaccin. Résultats Soixante-quatre cas et 32 contrôles avec un âge médian respectif de 56 (39,5-59,5) et 52 (37,8-66,3) ans étaient inclus. Quatre groupes de traitements étaient défini: patients traités par rituximab (n=22), methotrexate (n=16), immunosuppresseurs conventionnels hors methotrexate (n=19), patients recevant des traitements connus pour ne pas avoir d’impact sur la réponse vaccinale (n=7). L’ensemble des cas avaient une production diminuée et retardée d’IgG et d’IgA anti-spike après vaccination par le BNT162b2, ceci de façon plus prononcée dans le groupe rituximab. Alors que 2 doses de vaccin induisaient une réponse humorale neutralisante contre les variants Alpha et Delta chez 100 % des contrôles, un seul patient sous rituximab (5 %) neutralisait Alpha et aucun Delta. Les autres groupes de traitements avaient une activité neutralisante partielle contre Alpha, et significativement diminuée contre Delta. Les réponses lymphocytaires T spécifiques étaient similaires entre les contrôles et les cas, à l’exception des patients sous metrotrexate qui avaient une réponse complètement abrogée après 1 dose et considérablement diminuée après 2 doses. Après 3 mois de suivi, 2 patients traités par rituximab présentaient une infection symptomatique peu sévère à SARS-CoV-2, 4 à 7jours après la seconde dose de vaccin. Quatre patients (6,3 %) présentaient une poussée de leur maladie auto-immune conduisant à une modification thérapeutique. Conclusion Le rituximab et le methotrexate impactent de façon différente l’immunogénicité du vaccin BNT162b2, en altérant respectivement les réponses humorales et cellulaires. Le variant Delta échappe complètement à la réponse humorale chez les patients traités par rituximab. Ces résultats soulignent la nécéssité de protocoles vaccinaux particuliers et d’autres traitements préventifs de l’infection dans cette population de patients.

12.
La Revue de Médecine Interne ; 42:A275-A276, 2021.
Article in French | ScienceDirect | ID: covidwho-1531761

ABSTRACT

Introduction La restauration immunitaire induite par les inhibiteurs de checkpoints immunitaires ont révolutionné le pronostic des cancers métastatiques [1]. La contribution de cette stratégie thérapeutique pour la prise en charge des infections reste toutefois controversée, malgré quelques études en faveur de leur utilisation, notamment dans le contexte de paralysie immunitaire au décours d’un sepsis sévère [2], [3], ou dans les situations d’épuisement immunitaire au cours des infections virales chroniques [4], [5]. Au cours de la COVID-19, des études suggèrent qu’un état d’épuisement immunitaire en lien avec une anergie et/ou une déplétion des lymphocytes T serait partiellement responsable de la virulence du SARS-CoV-2 [6], [7], [8], [9], [10]. Certains proposent donc l’utilisation des anti-PD1 comme stratégie thérapeutique tandis que d’autres suggèrent qu’au contraire, elle pourrait aggraver l’hyperinflammation [11], [12]. Patients et méthodes Nous avons suivi de façon prospective 292 patients atteints de mélanome lors de la première vague de COVID-19 (de mars à juin 2020) dont la moitié était traitée par immunothérapie (anti-PD1±anti-CTLA4). Les patients présentant des symptômes de COVID-19 étaient dépistés par PCR. Une sérologie SARS-CoV-2 était recherchée de façon systématique. Les patients présentant une infection symptomatique active (<21 jours du début des symptômes, PCR positive) ou convalescente (>21 jours du début des symptômes, PCR négative, sérologie positive) ont été inclus pour une étude approfondie de la réponse immunitaire par une analyse transcriptionnelle (Nanostring), protéomique (SIMOA, Luminex) et cellulaire (cytométrie de masse). Résultats Quinze patients atteints de COVID-19 ont été identifiés (infection active ou convalescente) avec une estimation de la séroprévalence à 8,6 % de la cohorte. Quatre patients sur 15 ont nécessité une hospitalisation (26,7 %). Les données cliniques ne retrouvaient pas d’éléments en faveur d’une forme plus sévère de COVID-19 lors d’un traitement par anti-PD1, seul un patient ayant également une leucémie lymphoïde chronique, a développé une forme sévère de la COVID-19 et est décédé de défaillance respiratoire. L’analyse de la réponse immunitaire, en comparaison avec une cohorte de patients non traités par immunothérapie, retrouvait une réponse immunitaire innée semblable dans les deux cohortes. De même, le taux d’anticorps anti-Spike (IgG et IgA), la capacité neutralisante ainsi que la longévité des anticorps (suivi du taux sur une période d’1 an) étaient similaires en présence ou non d’un traitement par immunothérapie. En revanche, l’analyse de la réponse cellulaire mettait en évidence, chez les patients traités par immunothérapie, une expansion de la population de lymphocytes T CD8+ effecteurs mémoires, une augmentation de l’activation des lymphocytes T CD4+ et CD8+, et une augmentation la production d’IFN-gamma lors d’une stimulation ex-vivo par des peptides issus du SARS-CoV-2. Conclusion Nos résultats sont en faveur d’une augmentation de la réponse cellulaire T anti-SARS-CoV-2 lors d’un traitement par anti-PD1 chez les patients suivis pour mélanome, et de l’absence d’une exacerbation de la réponse inflammatoire. Il est nécessaire de confirmer ces résultats avec un plus grand nombre de patients, dans d’autres types de cancers et dans d’autres centres.

13.
Br J Dermatol ; 185(6): 1176-1185, 2021 12.
Article in English | MEDLINE | ID: covidwho-1455515

ABSTRACT

BACKGROUND: The outbreak of chilblain-like lesions (CLL) during the COVID-19 pandemic has been reported extensively, potentially related to SARS-CoV-2 infection, yet its underlying pathophysiology is unclear. OBJECTIVES: To study skin and blood endothelial and immune system activation in CLL in comparison with healthy controls and seasonal chilblains (SC), defined as cold-induced sporadic chilblains occurring during 2015 and 2019 with exclusion of chilblain lupus. METHODS: This observational study was conducted during 9-16 April 2020 at Saint-Louis Hospital, Paris, France. All patients referred with CLL seen during this period of the COVID-19 pandemic were included in this study. We excluded patients with a history of chilblains or chilblain lupus. Fifty patients were included. RESULTS: Histological patterns were similar and transcriptomic signatures overlapped in both the CLL and SC groups, with type I interferon polarization and a cytotoxic-natural killer gene signature. CLL were characterized by higher IgA tissue deposition and more significant transcriptomic activation of complement and angiogenesis factors compared with SC. We observed in CLL a systemic immune response associated with IgA antineutrophil cytoplasmic antibodies in 73% of patients, and elevated type I interferon blood signature in comparison with healthy controls. Finally, using blood biomarkers related to endothelial dysfunction and activation, and to angiogenesis or endothelial progenitor cell mobilization, we confirmed endothelial dysfunction in CLL. CONCLUSIONS: Our findings support an activation loop in the skin in CLL associated with endothelial alteration and immune infiltration of cytotoxic and type I IFN-polarized cells leading to clinical manifestations.


Subject(s)
COVID-19 , Chilblains , Interferon Type I , COVID-19/immunology , Chilblains/virology , France , Humans , Interferon Type I/immunology , Pandemics
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