ABSTRACT
SARS-CoV-2, like many viruses, generates syncytia. Using SARS-CoV-2 and S (S) expressing recombinant vesicular stomatitis and influenza A viruses, we show that S-mediated syncytia formation provides resistance to interferons in cultured cells, human small airway-derived air-liquid interface cultures and hACE2 transgenic mice. Amino acid substitutions that modulate fusogenicity in Delta- and Omicron-derived S have parallel effects on viral interferon resistance. Syncytia formation also decreases antibody virus neutralization activity in cultured cells. These findings explain the continued selection of fusogenic variants during SARS-CoV-2 evolution in humans and, more generally, the evolution of fusogenic viruses despite the adverse effects of syncytia formation on viral replication in the absence of innate or adaptive immune pressure.
Subject(s)
Severe Acute Respiratory Syndrome , Vesicular StomatitisABSTRACT
Objective: Assess the presence, durability, and neutralization capacity of SARS-CoV-2-specific antibodies in breastfeeding infants’ stools, mother’s plasma, and human milk following maternal vaccination. Design: Thirty-seven mothers and 25 infants were enrolled between December 2020 and November 2021 for this prospective observational study. Human milk, maternal plasma, and infants' stools were collected pre-vaccination and at periods up to 6 months following COVID-19 vaccine series initiation/completion. SARS-CoV-2 antibody levels and their neutralization capacities were assessed in collected samples. Results: SARS-CoV-2-specific IgA and IgG levels were higher in infant stool post-maternal vaccination amongst milk-fed compared to pre-COVID controls. Human milk and plasma SARS-CoV-2-specific IgA and IgG concentrations decreased over 6 months post-vaccination but remained higher than pre-vaccination levels. We observed improved neutralization capacity in milk antibodies over time. Conclusions: The presence of neutralizing SARS-CoV-2-specific antibodies in infant stool following maternal vaccination offers further evidence of the lasting transfer of these antibodies through breastfeeding and their protective effect.
Subject(s)
COVID-19ABSTRACT
The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development.
ABSTRACT
SARS-CoV-2 nucleocapsid protein (N) induces strong antibody and T cell responses. Although considered to be localized in the cytosol, we readily detect N on the surface of live cells. N released by SARS-CoV-2 infected cells or N-expressing transfected cells binds to neighboring cells by electrostatic high-affinity binding to heparan sulfate and heparin, but not other sulfated glycosaminoglycans. N binds with high affinity to 11 human chemokines, including CXCL12{beta}, whose chemotaxis of leukocytes is inhibited by N from SARS-CoV-2, SARS-CoV-1, and MERS CoV. Anti-N Abs bound to the surface of N expressing cells activate Fc receptor-expressing cells. Our findings indicate that cell surface N manipulates innate immunity by sequestering chemokines and can be targeted by Fc expressing innate immune cells. This, in combination with its conserved antigenicity among human CoVs, advances its candidacy for vaccines that induce cross-reactive B and T cell immunity to SARS-CoV-2 variants and other human CoVs, including novel zoonotic strains.
Subject(s)
Severe Acute Respiratory SyndromeABSTRACT
SARS-CoV-2 nucleocapsid protein (N) induces strong antibody and T cell responses. Although considered to be localized in the cytosol, we readily detect N on the surface of live cells. N released by SARS-CoV-2 infected cells or N-expressing transfected cells binds to neighboring cells by electrostatic high-affinity binding to heparan sulfate and heparin, but not other sulfated glycosaminoglycans. N binds with high affinity to 11 human chemokines, including CXCL12β, whose chemotaxis of leukocytes is inhibited by N from SARS-CoV-2, SARS-CoV-1, and MERS CoV. Anti-N Abs bound to the surface of N expressing cells activate Fc receptor-expressing cells. Our findings indicate that cell surface N manipulates innate immunity by sequestering chemokines and can be targeted by Fc expressing innate immune cells. This, in combination with its conserved antigenicity among human CoVs, advances its candidacy for vaccines that induce cross-reactive B and T cell immunity to SARS-CoV-2 variants and other human CoVs, including novel zoonotic strains.
ABSTRACT
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.
Subject(s)
Coronavirus Infections , Adenocarcinoma, Bronchiolo-AlveolarABSTRACT
ABSTRACT Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa. From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA developed in the US, using two-antigen SARS-CoV-2 spike protein and receptor-binding domain. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative). Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43.4% (135/311) for spike protein, 22.8% (71/312) for RBD, and 33.9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73.9% (51.6-89.8), Specificity: 99.4% (97.7-99.9)]. In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.
ABSTRACT
Background: Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa.Methods: From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of a two-antigen test developed in the US, using SARS-CoV-2 spike protein and receptor-binding domain ELISA measurements. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative).Findings: Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43·4% (135/311) for spike protein, 22·8% (71/312) for RBD, and 33·9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73·9% (51·6-89·8), Specificity: 99·4% (97·7-99·9)].Interpretation: In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.Funding: This project was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, as well as the National Cancer Institute, National Institutes of Health.Declaration of Interests: The authors declare no conflicts of interest.Ethics Approval Statement: All pre-pandemic samples were collected during NIH-sponsored studies that were approved by the Malian USTTB FMPOS human research ethics committee and the NIAID/NIH institutional review board.Convalescent PCR-confirmed COVID-19 US positive control samples (n=10) were provided by the Adventist Hospital, Maryland. De-identified residual clinical samples for non-human subject research were obtained in accordance with 45 CFR 46.Convalescent samples were collected as part of a Public Health surveillance activity in collaboration with the Malian Ministry of Health COVID-19 Coordination Unit and with the approval of the USTTB FMOS-FAPH ethics committee (No2020/114/CE/FMOS/FAPH).