Multisystem inflammatory syndrome in children (MIS-C) is a severe, post-infectious sequela of SARS-CoV-2 infection, yet the pathophysiological mechanism connecting the infection to the broad inflammatory syndrome remains unknown. Here we leveraged a large set of MIS-C patient samples (n=199) to identify a distinct set of host proteins that are differentially targeted by patient autoantibodies relative to matched controls. We identified an autoreactive epitope within SNX8, a protein expressed primarily in immune cells that regulates an antiviral pathway associated with MIS-C pathogenesis. In parallel, we also probed the SARS-CoV-2 proteome-wide MIS-C patient antibody response and found it to be differentially reactive to a distinct domain of the SARS-CoV-2 nucleocapsid (N) protein relative to controls. This viral N region and the mapped SNX8 epitope bear remarkable biochemical similarity.  Furthermore, we find that many children with anti-SNX8 autoantibodies also have T cells cross-reactive to both SNX8 and this distinct region of the SARS-CoV-2 N protein. Together, these findings suggest that MIS-C patients develop a characteristic immune response against the SARS-CoV-2 N protein that is associated with cross-reactivity to the self-protein SNX8, demonstrating a mechanistic link from the infection to the inflammatory syndrome with implications for better understanding a range of post-infectious autoinflammatory diseases.

Patients

Patients were recruited through the prospectively enrolling multicenter Overcoming COVID-19 and Taking on COVID-19 Together study in the United States. The study was approved by the central Boston Children’s Hospital Institutional Review Board (IRB) and reviewed by IRBs of participating sites with CDC IRB reliance. A total of 292 patients were enrolled into 1 of the following independent cohorts between June 1, 2020 and September 9, 2021: 223 patients hospitalized with MIS-C (199 in the primary discovery cohort, 24 in a separate subsequent validation cohort), 29 patients hospitalized for COVID-19 in either an intensive care or step-down unit (referred to as severe acute COVID-19 in this study), and 45 outpatients (referred to as “at-risk controls” in this study) post-SARS-CoV-2 infections associated with mild or no symptoms. The demographic and clinical data are summarized in Table I, Extended Data Table 1, and Extended Data Table 2. The 2020 US Centers for Disease Control and Prevention case definition was used to define MIS-C(1). All patients with MIS-C had positive SARS-CoV-2 serology results and/or positive SARS-CoV-2 test results by reverse transcriptase quantitative PCR. All patients with severe COVID-19 or outpatient SARS-CoV-2 infections had a positive antigen test or nucleic acid amplification test for SARS-CoV-2. For outpatients, samples were collected from 36 to 190 days after the positive test (median, 70 days after the positive test; interquartile range, 56-81 days). For use as controls in the SARS-CoV-2 specific PhIP-Seq, plasma from 48 healthy, pre-COVID-19 controls was obtained as deidentified samples from the New York Blood Center. These samples were part of retention tubes collected at the time of blood donations from volunteer donors who provided informed consent for their samples to be used for research.

Human proteome PhIP-Seq

Human Proteome PhIP-Seq was performed following our previously published vacuum-based PhIP-Seq protocol (2) (https://www.protocols.io/view/scaled-high-throughput-vacuum-phip-protocol-ewov1459kvr2/v1).

Our human peptidome library consists of a custom-designed phage library of 731,724 unique T7 bacteriophage each presenting a different 49 amino-acid peptide on its surface. Collectively these peptides tile the entire human proteome including all known isoforms (as of 2016) with 25 amino-acid overlaps. 1 milliliter of phage library was incubated with 1 microliter of human serum overnight at 4C and immunoprecipitated with 25 microliters of 1:1 mixed protein A and protein G magnetic beads (Thermo Fisher, Waltham, MA, #10008D and #10009D). These beads were then washed, and the remaining phage-antibody complexes were eluted in 1 milliliter of E.Coli (BLT5403, EMD Millipore, Burlington, MA) at 0.5-0.7 OD and amplified by growing in 37C incubator. This new phage library was then re-incubated with the same individual’s serum and the previously described protocol was repeated. DNA was then extracted from the final phage library, barcoded, and PCR-amplified, and Illumina adaptors were added. Next-generation sequencing was then performed using an Illumina sequencer (Illumina, San Diego, CA) to a read depth of approximately 1 million per sample.

Human proteome PhIP-Seq analysis

All human peptidome analysis (except when specifically stated otherwise) was performed at the gene level, in which all reads for all peptides mapping to the same gene were summed, and 0.5 reads were added to each gene to allow the inclusion of genes with zero reads in mathematical analyses. Within each individual sample, reads were normalized by converting to the percentage of total reads. To normalize each sample against background non-specific binding, a fold-change (FC) over mock-IP was calculated by dividing the sample read percentage for each gene by the mean read percentage of the same gene for the AG bead-only controls. This FC signal was then used for side-by-side comparison between samples and cohorts. FC values were also used to calculate z-scores for each MIS-C patient relative to controls and for each control sample by using all remaining controls. These z-scores were used for the logistic regression feature weighting. In instances of peptide-level analysis, raw reads were normalized by calculating the number of reads per 100,000 reads.

SARS-CoV-2 proteome PhIP-Seq

SARS-CoV-2 Proteome PhIP-Seq was performed as previously described(3). Briefly, 38 amino acid fragments tiling all open reading frames from SARS-CoV-2, SARS-CoV-1, and 7 other CoVs were expressed on T7 bacteriophage with 19 amino acid overlaps. 1 milliliter of phage library was incubated with 1 microliter of human serum overnight at 4C and immunoprecipitated with 25 microliters of 1:1 mixed protein A and protein G magnetic beads (Thermo Fisher, Waltham, MA, #10008D and #10009D). Beads were washed 5 times on a magnetic plate using a P1000 multichannel pipette. The remaining phage-antibody complexes were eluted in 1 milliliter of E.Coli (BLT5403, EMD Millipore, Burlington, MA) at 0.5-0.7 OD and amplified by growing in a 37°C incubator. This new phage library was then re-incubated with the same individual’s serum and the previously described protocol was repeated for a total of 3 rounds of immunoprecipitations. DNA was then extracted from the final phage library, barcoded, and PCR-amplified, and Illumina adaptors were added. Next-generation sequencing was then performed using an Illumina sequencer (Illumina, San Diego, CA) to a read depth of approximately 1 million per sample.

Coronavirus proteome PhIP-Seq analysis

To account for differing read depths between samples, the total number of reads for each peptide fragment was converted to the number of reads per 100k (RPK). To calculate normalized enrichment relative to pre-COVID controls (FC > Pre-COVID), the RPK for each peptide fragment within each sample was divided by the mean RPK of each peptide fragment among all pre-COVID controls. These FC > Pre-COVID values were used for all subsequent analyses as described in the text and figures.

1. HAN Archive - 00432 (2021). https://emergency.cdc.gov/han/2020/han00432.asp.
2. S. E. Vazquez, S. A. Mann, A. Bodansky, A. F. Kung, Z. Quandt, E. M. N. Ferré, N. Landegren, D. Eriksson, P. Bastard, S.-Y. Zhang, J. Liu, A. Mitchell, I. Proekt, D. Yu, C. Mandel-Brehm, C.-Y. Wang, B. Miao, G. Sowa, K. Zorn, A. Y. Chan, V. M. Tagi, C. Shimizu, A. Tremoulet, K. Lynch, M. R. Wilson, O. Kämpe, K. Dobbs, O. M. Delmonte, R. Bacchetta, L. D. Notarangelo, J. C. Burns, J.-L. Casanova, M. S. Lionakis, T. R. Torgerson, M. S. Anderson, J. L. DeRisi, Autoantibody discovery across monogenic, acquired, and COVID-19-associated autoimmunity with scalable PhIP-seq. Elife 11 (2022).
3. C. R. Zamecnik, J. V. Rajan, K. A. Yamauchi, S. A. Mann, R. P. Loudermilk, G. M. Sowa, K. C. Zorn, B. D. Alvarenga, C. Gaebler, M. Caskey, M. Stone, P. J. Norris, W. Gu, C. Y. Chiu, D. Ng, J. R. Byrnes, X. X. Zhou, J. A. Wells, D. F. Robbiani, M. C. Nussenzweig, J. L. DeRisi, M. R. Wilson, ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens. Cell Rep Med 1, 100123 (2020).