Background: Weeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called Multisystem Inflammatory Syndrome in Children (MIS-C).Gastrointestinal symptoms are common in MIS-C patients and severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not previously been identified.Methods: Here, we analyzed biospecimens from 100 children: 19 children with MIS-C, 26 with acute COVID-19, and 55 controls. Stool was assessed for SARS-CoV-2 by RT-PCR and plasma was assessed for markers of breakdown of mucosal barrier integrity, including zonulin.Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As proof of concept, we treated a MIS-C patient with larazotide, a zonulin antagonist, and monitored impact on antigenemia and clinical response. Results:We showed that in MIS-C, prolonged presence of SARS-CoV-2 in the GI tract leads to release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The MIS-C patient treated with larazotide displayed a coinciding decrease in plasma SARS-CoV-2 Spike antigen levels, inflammatory markers, and a resultant clinical improvement above that achieved with currently available treatments. Conclusion:These mechanistic data of MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

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Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals.U sing Single Molecule Arrays( Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibodymediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in ab iosafety level 2l aboratory within two hours.W eu sed this assayt oa ssess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for as hort period of time and showthat neutralization and antibody levels increase over time.Wealso adapted the assayfor SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy,C OVID-19 infected, and vaccinated individuals.T his assayi sh ighly adaptable for clinical applications,s uch as vaccine development and epidemiological studies.

Coronavirus disease 2019 (COVID-19) manifests with high clinical variability and warrants sensitive and specific assays to analyze immune responses in infected and vaccinated individuals.U sing Single Molecule Arrays( Simoa), we developed an assay to assess antibody neutralization with high sensitivity and multiplexing capabilities based on antibodymediated blockage of the ACE2-spike interaction. The assay does not require live viruses or cells and can be performed in ab iosafety level 2l aboratory within two hours.W eu sed this assayt oa ssess neutralization and antibody levels in patients who died of COVID-19 and patients hospitalized for as hort period of time and showthat neutralization and antibody levels increase over time.Wealso adapted the assayfor SARS-CoV-2 variants and measured neutralization capacity in pre-pandemic healthy,C OVID-19 infected, and vaccinated individuals.T his assayi sh ighly adaptable for clinical applications,s uch as vaccine development and epidemiological studies.