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.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. Methods We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of coronavirus disease (COVID-19) patients. We studied plasma from 64 COVID-19 positive patients, 17 COVID-19 negative patients, and 34 pre-pandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 COVID-19 patients. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. Results SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95%CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within one day) was statistically significant. Conclusions The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of COVID-19 positive patients to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

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Sensitive assays are essential for the accurate identification of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report a multiplexed assay for the fluorescence-based detection of seroconversion in infected individuals from less than 1 µl of blood, and as early as the day of the first positive nucleic acid test after symptom onset. The assay uses dye-encoded antigen-coated beads to quantify the levels of immunoglobulin G (IgG), IgM and IgA antibodies against four SARS-CoV-2 antigens. A logistic regression model trained using samples collected during the pandemic and samples collected from healthy individuals and patients with respiratory infections before the first outbreak of coronavirus disease 2019 (COVID-19) was 99% accurate in the detection of seroconversion in a blinded validation cohort of samples collected before the pandemic and from patients with COVID-19 five or more days after a positive nasopharyngeal test by PCR with reverse transcription. The high-throughput serological profiling of patients with COVID-19 allows for the interrogation of interactions between antibody isotypes and viral proteins, and should help us to understand the heterogeneity of clinical presentations.