Resistance of Omicron subvariants BA.2.75.2, BA.4.6 and BQ.1.1 to neutralizing antibodies (preprint)

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariants BA.2.75.2 and BQ.1.1 are expected to become predominant in many countries in November 2022. They carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lost any antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals were low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increased these titers, which remained about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increased more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raises concerns about the efficacy of most currently available mAbs.

Poor sensitivity of iPSC-derived neural progenitors and glutamatergic neurons to SARS-CoV-2 (preprint)

COVID-19 is a respiratory disease affecting multiple organs including the central nervous system (CNS), with a characteristic loss of smell and taste. Although frequently reported, the neurological symptoms remain enigmatic. There is no consensus on the extent of CNS infection. Here, we derived human induced pluripotent stem cells (hiPSC) into neural progenitor cells (NPCs) and glutamatergic neurons to study their permissiveness to SARS-CoV-2 infection. Flow cytometry and western blot analysis indicated that NPCs and neurons do not express detectable levels of the SARS-CoV-2 receptor ACE2. We thus generated cells expressing ACE2 by lentiviral transduction to analyze in a controlled manner the properties of SARS-CoV-2 infection relative to ACE2 expression. Sensitivity of parental and ACE2 expressing cells was assessed with GFP- or luciferase- carrying pseudoviruses and with authentic SARS-CoV-2 Wuhan, D614G, Alpha or Delta variants. SARS-CoV-2 replication was assessed by microscopy, RT-qPCR and infectivity assays. Pseudoviruses infected only cells overexpressing ACE2. Neurons and NPCs were unable to efficiently replicate SARS-CoV-2, whereas ACE2 overexpressing neurons were highly sensitive to productive infection. Altogether, our results indicate that primary NPCs and glutamatergic neurons remain poorly permissive to SARS-CoV-2 across the SARS-CoV-2 variants inoculated, in the absence of ACE2 expression.

Potent Human Broadly SARS-CoV-2 Neutralizing IgA and IgG Antibodies Effective Against Omicron BA.1 and BA.2 (preprint)

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.

B.1.1.7 and B.1.351 SARS-CoV-2 variants display enhanced Spike-mediated fusion (preprint)

SARS-CoV-2 B.1.1.7 (variant Alpha) and B.1.351 (variant Beta) have supplanted pre-existing strains in many countries. Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when infected cells fuse with adjacent cells. The fitness, cytopathic effects and type-I interferon (IFN) sensitivity of the variants remain poorly characterized. Here, we assessed B.1.1.7 and B.1.351 spread and fusion in cell cultures. B.1.1.7 and B.1.351 replicated similarly to D614G reference strain in Vero, Caco-2, Calu-3 and primary airway cells and were similarly sensitive to IFN. The variants formed larger and more numerous syncytia. Variant Spikes, in the absence of any other viral proteins, resulted in faster fusion relative to D614G. B.1.1.7 and B.1.351 fusion was similarly inhibited by interferon induced transmembrane proteins (IFITMs). Individual mutations present in the variant Spikes modified fusogenicity, binding to ACE2 and recognition by monoclonal antibodies. Also, B.1.1.7 and B.1.351 variants remain sensitive to innate immunity components. The mutations present in the two variants globally enhance viral fusogenicity and allow for antibody evasion.

Release of infectious virus and cytokines in nasopharyngeal swabs from individuals infected with non-B.1.1.7 or B.1.1.7 SARS-CoV-2 variants. (preprint)

The mechanisms that allowed for the SARS-CoV-2 B.1.1.7 variant to rapidly outcompete pre-existing variants in many countries remain poorly characterized. Here, we analyzed viral release, anti-SARS-CoV-2 antibodies and cytokine production in a retrospective series of 427 RTqPCR+ nasopharyngeal swabs collected in COVID-19 patients harbouring either non-B.1.1.7 or B.1.17 variants. We utilized a novel rapid assay, based on S-Fuse-T reporter cells, to quantify infectious SARS-CoV-2. With both non-B.1.1.7 and B.1.1.7 variants, viral titers were highly variable, ranging from 0 to >106 infectious units, and correlated with viral RNA levels. Lateral flow antigenic rapid diagnostic tests (RDTs) were positive in 96% of the samples harbouring infectious virus. About 67 % of individuals carried detectable infectious virus within the first two days after onset of symptoms. This proportion decreased overtime, and viable virus was detected up to 14 days. Samples containing anti-SARS-CoV-2 IgG or IgA did not generally harbour infectious virus. The proportion of individuals displaying viable virus or being RDT-positive was not higher with B.1.1.7 than with non- B.1.1.7 variants. Ct values were slightly but not significantly lower with B.1.1.7. The variant was characterized by a fast decrease of infectivity overtime and a marked release of 17 cytokines (including IFN-b, IP-10, IL-10 and TRAIL). Our results highlight differences between non-B.1.1.7 and B.1.1.7 variants. B.1.1.7 is associated with modified viral decays and cytokine profiles at the nasopharyngeal mucosae during symptomatic infection.

A Monocyte/Dendritic Cell Molecular Signature of SARS-CoV-2 Related Multisystem Inflammatory Syndrome in Children (MIS-C) with Severe Myocarditis (preprint)

SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-κ B activity, TNF-α signaling, associated with decreased gene expression of NF-κ B inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1α and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology.Funding: The study was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), by the “URGENCE COVID-19” fundraising campaign of Institut Pasteur, by the Atip-Avenir, Emergence ville de Paris program and fond de dotation Janssen Horizon and by government grants managed by the Agence National de la Recherche as part of the “Investment for the Future” program (Institut Hospitalo-Universitaire Imagine, grant ANR-10-IAHU-01, Recherche Hospitalo-Universitaire, grant ANR-18-RHUS-0010, Laboratoire d’Excellence ‘‘Milieu Intérieur”, grant ANR-10-LABX-69-01), the Centre de Référence Déficits Immunitaires Héréditaires (CEREDIH), the Agence National de la Recherche (ANR-flash Covid19 “AIROCovid” to FRL and “CoVarImm” to DD and JDS), and by the FASTFoundation (French Friends of Sheba Tel Hashomer Hospital). The LabTech Single-Cell@Imagine is supported by the Paris Region and the “Investissements d’avenir” program through the 2019 ATF funding – Sésame Filières PIA (Grant N°3877871).CdC is the recipient of a CIFRE-PhD (Sanofi). L.B. was a recipient of an Imagine institute PhD international program supported by the Fondation Bettencourt Schueller. L.B. was also supported by the EUR G.E.N.E. (reference #ANR-17-EURE-0013) and is part of the Université de Paris IdEx #ANR-18-IDEX-0001 funded by the French Government through its“Investments for the Future” program. S.M. was a recipient of an INSERM and Institut Imagine post-doctorat program supported by the Fondation pour la Recherche Médicale (FRMN°SPF20170938825). NS was a recipient of the Pasteur-Roux-Cantarini Fellowship. VGP obtained an Imagine international PhD fellowship program supported by the Fondation Bettencourt Schueller. BPP is the recipient of an ANRS post-doctoral fellowship.Conflict of Interest: DD, FRL, JT and MMM are listed as inventors on a patent application related to this technology (European Patent Application no. EP21305197, entitled “Methods of predicting multisystem inflammatory syndrome (MIS-C) with severe myocarditis in subjects suffering from a SARS-CoV-2 infection”).Ethical Approval: The study was approved by the Ethics Committee (Comité de Protection des Personnes Ouest IV, n° DC-2017-2987).

Distinct systemic and mucosal immune responses to SARS-CoV-2 (preprint)

Coordinated local mucosal and systemic immune responses following SARS-CoV-2 infection protect against COVID-19 pathologies or fail leading to severe clinical outcomes. To understand this process, we performed an integrated analysis of SARS-CoV-2 spike-specific antibodies, cytokines, viral load and 16S bacterial communities in paired nasopharyngeal swabs and plasma samples from a cohort of clinically distinct COVID-19 patients during acute infection. Plasma viral load was associated with systemic inflammatory cytokines that were elevated in severe COVID-19, and also with spike-specific neutralizing antibodies. In contrast, nasopharyngeal viral load correlated with SARS-CoV-2 humoral responses but inversely with interferon responses, the latter associating with protective microbial communities. Potential pathogenic microrganisms, often implicated in secondary respiratory infections, were associated with mucosal inflammation and elevated in severe COVID-19. Our results demonstrate distinct tissue compartmentalization of SARS-CoV-2 immune responses and highlight a role for the nasopharyngeal microbiome in regulating local and systemic immunity that determines COVID-19 clinical outcomes.

A monocyte/dendritic cell molecular signature of SARS-CoV2-related multisystem inflammatory syndrome in children (MIS-C) with severe myocarditis (preprint)

SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-kB activity, TNF-a signaling, associated with decreased gene expression of NF-kB inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1a and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology.

Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies (preprint)

SARS-CoV-2 B.1.1.7 and B.1.351 variants emerged respectively in United Kingdom and South Africa and spread in many countries. Here, we isolated infectious B.1.1.7 and B.1.351 strains and examined their sensitivity to anti-SARS-CoV-2 antibodies present in sera and nasal swabs, in comparison with a D614G reference virus. We established a novel rapid neutralization assay, based on reporter cells that become GFP+ after overnight infection. B.1.1.7 was neutralized by 79/83 sera from convalescent patients collected up to 9 months post symptoms, almost similar to D614G. There was a mean 6-fold reduction in titers and even loss of activity against B.1.351 in 40% of convalescent sera after 9 months. Early sera from 19 vaccinated individuals were almost as potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Nasal swabs from vaccine recipients were not neutralizing, except in individuals who were diagnosed COVID-19+ before vaccination. Thus, faster-spreading variants acquired a partial resistance to humoral immunity generated by natural infection or vaccination, mostly visible in individuals with low antibody levels.

Sex differences in the decline of neutralizing antibodies to SARS-CoV-2 (preprint)

The evolution of SARS-CoV-2 humoral response in infected individuals remains poorly characterized. Here, we performed a longitudinal study of sera from 308 RT-qPCR+ individuals with mild disease, collected at two time-points, up to 6 months post-onset of symptoms (POS). We performed two anti-S and one anti-N serology assays and quantified neutralizing antibodies (NAbs). At month 1 (M1), males, individuals > 50 years of age or with a body mass index (BMI) > 25 exhibited higher levels of antibodies. Antibody levels decreased over time. At M3-6, anti-S antibodies persisted in 99% of individuals while anti-N IgG were measurable in only 59% of individuals. The decline in anti-S and NAbs was faster in males than in females, independently of age and BMI. Our results show that some serology tests are less reliable overtime and suggest that the duration of protection after SARS-CoV-2 infection or vaccination will be different in women and men.

Asymptomatic and symptomatic SARS-CoV-2 infections elicit polyfunctional antibodies. (preprint)

A large proportion of SARS-CoV-2 infected individuals remains asymptomatic. Little is known about the extent and quality of their antiviral humoral response. Here, we analyzed antibody functions in 52 asymptomatic infected individuals, 119 mild and 21 hospitalized COVID-19 patients. We measured anti-Spike antibody levels with the S-Flow assay and mapped SARS-CoV-2 Spike- and N-targeted regions by Luminex. Neutralization, complement deposition and Antibody-Dependent Cellular Cytotoxicity (ADCC) were evaluated using replication-competent SARS-CoV-2 or reporter cell systems. We show that COVID-19 sera mediate complement deposition and kill infected cells by ADCC. Sera from asymptomatic individuals neutralize the virus, activate ADCC and trigger complement deposition. Antibody levels and activities are slightly lower in asymptomatic individuals. The different functions of the antibodies are correlated, independently of disease severity. Longitudinal samplings show that antibody functions follow similar kinetics of induction and contraction, with minor variations. Overall, asymptomatic SARS-CoV-2 infection elicits polyfunctional antibodies neutralizing the virus and targeting infected cells. - Sera from convalescent COVID-19 patients activate the complement and kill infected cells by ADCC. - Asymptomatic and symptomatic SARS-CoV-2-infected individuals harbor polyfunctional antibodies. - Antibody levels and functions are slightly lower in asymptomatic individuals - The different antiviral activities of anti-Spike antibodies are correlated regardless of disease severity. - Functions of anti-Spike antibodies have similar kinetics of induction and contraction.

SARS-CoV-2 infection in primary schools in northern France: A retrospective cohort study in an area of high transmission (preprint)

Background: The extent of SARS-CoV-2 transmission among pupils in primary schools and their families is unknown. Methods: Between 28-30 April 2020, a retrospective cohort study was conducted among pupils, their parents and relatives, and staff of primary schools exposed to SARS-CoV-2 in February and March 2020 in a city north of Paris, France. Participants completed a questionnaire that covered sociodemographic information and history of recent symptoms. A blood sample was tested for the presence of anti-SARS-CoV-2 antibodies using a flow-cytometry-based assay. Results: The infection attack rate (IAR) was 45/510 (8.8%), 3/42 (7.1%), 1/28 (3.6%), 76/641 (11.9%) and 14/119 (11.8%) among primary school pupils, teachers, non-teaching staff, parents, and relatives, respectively (P = 0.29). Prior to school closure on February 14, three SARS-CoV-2 infected pupils attended three separate schools with no secondary cases in the following 14 days among pupils, teachers and non-teaching staff of the same schools. Familial clustering of cases was documented by the high proportion of antibodies among parents and relatives of infected pupils (36/59 = 61.0% and 4/9 = 44.4%, respectively). In children, disease manifestations were mild, and 24/58 (41.4%) of infected children were asymptomatic. Interpretation: In young children, SARS-CoV-2 infection was largely mild or asymptomatic and there was no evidence of onwards transmission from children in the school setting.

Serologic responses to SARS-CoV-2 infection among hospital staff with mild disease in eastern France (preprint)

Background: The serologic response of individuals with mild forms of SARS-CoV-2 infection is poorly characterized. Methods: Hospital staff who had recovered from mild forms of PCR-confirmed SARS-CoV-2 infection were tested for anti-SARS-CoV-2 antibodies using two assays: a rapid immunodiagnostic test (99.4% specificity) and the S-Flow assay (~99% specificity).The neutralizing activity of the sera was tested with a pseudovirus-based assay. Results: Of 162 hospital staff who participated in the investigation, 160 reported SARS-CoV- 2 infection that had not required hospital admission and were included in these analyses. The median time from symptom onset to blood sample collection was 24 days (IQR: 21-28, range 13-39). The rapid immunodiagnostic test detected antibodies in 153 (95.6%) of the samples and the S-Flow assay in 159 (99.4%), failing to detect antibodies in one sample collected 18 days after symptom onset (the rapid test did not detect antibodies in that patient). Neutralizing antibodies (NAbs) were detected in 79%, 92% and 98% of samples collected 13-20, 21-27 and 28-41 days after symptom onset, respectively (P=0.02). Conclusion: Antibodies against SARS-CoV-2 were detected in virtually all hospital staff sampled from 13 days after the onset of COVID-19 symptoms. This finding supports the use of serologic testing for the diagnosis of individuals who have recovered from SARS-CoV-2 infection. The neutralizing activity of the antibodies increased overtime. Future studies will help assess the persistence of the humoral response and its associated neutralization capacity in recovered patients.

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

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 Hopital 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.

Cluster of COVID-19 in northern France: A retrospective closed cohort study (preprint)

Background: The Oise department in France has been heavily affected by COVID-19 in early 2020. Methods: Between 30 March and 4 April 2020, we conducted a retrospective closed cohort study among pupils, their parents and siblings, as well as teachers and non-teaching staff of a high-school located in Oise. Participants completed a questionnaire that covered history of fever and/or respiratory symptoms since 13 January 2020 and had blood tested for the presence of anti-SARS-CoV-2 antibodies. The infection attack rate (IAR) was defined as the proportion of participants with confirmed SARS-CoV-2 infection based on antibody detection. Blood samples from two blood donor centres collected between 23 and 27 March 2020 in the Oise department were also tested for presence of anti-SARS-CoV-2 antibodies. Findings: Of the 661 participants (median age: 37 years), 171 participants had anti-SARS-CoV-2 antibodies. The overall IAR was 25.9% (95% confidence interval (CI) = 22.6-29.4), and the infection fatality rate was 0% (one-sided 97.5% CI = 0-2.1). Nine of the ten participants hospitalised since mid-January were in the infected group, giving a hospitalisation rate of 5.3% (95% CI = 2.4-9.8). Anosmia and ageusia had high positive predictive values for SARS-CoV-2 infection (84.7% and 88.1%, respectively). Smokers had a lower IAR compared to non-smokers (7.2% versus 28.0%, P <0.001). The proportion of infected individuals who had no symptoms during the study period was 17.0% (95% CI = 11.2-23.4). The proportion of donors with anti-SARS-CoV-2 antibodies in two nearby blood banks of the Oise department was 3.0% (95% CI = 1.1-6.4). Interpretation: The relatively low IAR observed in an area where SARS-CoV-2 actively circulated weeks before confinement measures indicates that establishing herd immunity will take time, and that lifting these measures in France will be long and complex.