Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C

Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with acute COVID-19 (n=70) or MIS-C (n=141) across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between COVID-19 and MIS-C, with increased multi-organ involvement in MIS-C encompassing diverse cell types including endothelial and neuronal cells, and an enrichment of pyroptosis related genes. Whole blood RNA profiling reveals upregulation of similar pro-inflammatory pathways in COVID-19 and MIS-C, but also MIS-C specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole blood RNA in paired samples yields different yet complementary signatures for each disease state. Our work provides a systems-level view of immune responses and tissue damage in COVID-19 and MIS-C and informs the future development of new disease biomarkers. Graphical Loy et al. use cell-free RNA, whole blood RNA, and cell-free DNA sequencing to characterize distinct host response and cellular injury profiles in pediatric patients with MIS-C and/or COVID-19. This study highlights the complementary information from cell-free and whole blood RNA analyses, with broad implications for future liquid biopsy applications.

Serologic Responses to COVID-19 Vaccination in Children with History of Multisystem Inflammatory Syndrome (MIS-C)

Understanding the serological responses to COVID-19 vaccination in children with history of MIS-C could inform vaccination recommendations. We prospectively enrolled seven children hospitalized with MIS-C and measured SARS-CoV-2 binding IgG antibodies to spike protein variants longitudinally pre- and post-Pfizer-BioNTech BNT162b2 primary series COVID-19 vaccination. We found that SARS-CoV-2 variant cross-reactive IgG antibodies variably waned following acute MIS-C, but were significantly boosted with vaccination and maintained for up to 3 months. We then compared post-vaccination binding, pseudovirus neutralizing, and functional antibody-dependent cell-mediated cytotoxicity (ADCC) titers to the reference strain (Wuhan-hu-1) and Omicron variant (B.1.1.529) among previously healthy children (n=16) and children with history of MIS-C (n=7) or COVID-19 (n=8). Despite the breadth of binding antibodies elicited by vaccination in all three groups, pseudovirus neutralizing and ADCC titers were significantly reduced to the Omicron variant.

Serologic responses to COVID-19 vaccination in children with history of multisystem inflammatory syndrome (MIS-C).

Understanding the serological responses to COVID-19 vaccination in children with history of MIS-C could inform vaccination recommendations. We prospectively enrolled seven children hospitalized with MIS-C and measured SARS-CoV-2 binding IgG antibodies to spike protein variants longitudinally pre- and post-Pfizer-BioNTech BNT162b2 primary series COVID-19 vaccination. We found that SARS-CoV-2 variant cross-reactive IgG antibodies variably waned following acute MIS-C, but were significantly boosted with vaccination and maintained for up to 3 months. We then compared post-vaccination binding, pseudovirus neutralizing, and functional antibody-dependent cell-mediated cytotoxicity (ADCC) titers to the reference strain (Wuhan-hu-1) and Omicron variant (B.1.1.529) among previously healthy children (n = 16) and children with history of MIS-C (n = 7) or COVID-19 (n = 8). Despite the breadth of binding antibodies elicited by vaccination in all three groups, pseudovirus neutralizing and ADCC titers were significantly reduced to the Omicron variant.

Multiplatform analyses reveal distinct drivers of systemic pathogenesis in adult versus pediatric severe acute COVID-19.

The pathogenesis of multi-organ dysfunction associated with severe acute SARS-CoV-2 infection remains poorly understood. Endothelial damage and microvascular thrombosis have been identified as drivers of COVID-19 severity, yet the mechanisms underlying these processes remain elusive. Here we show alterations in fluid shear stress-responsive pathways in critically ill COVID-19 adults as compared to non-COVID critically ill adults using a multiomics approach. Mechanistic in-vitro studies, using microvasculature-on-chip devices, reveal that plasma from critically ill COVID-19 adults induces fibrinogen-dependent red blood cell aggregation that mechanically damages the microvascular glycocalyx. This mechanism appears unique to COVID-19, as plasma from non-COVID sepsis patients demonstrates greater red blood cell membrane stiffness but induces less significant alterations in overall blood rheology. Multiomics analyses in pediatric patients with acute COVID-19 or the post-infectious multi-inflammatory syndrome in children (MIS-C) demonstrate little overlap in plasma cytokine and metabolite changes compared to adult COVID-19 patients. Instead, pediatric acute COVID-19 and MIS-C patients show alterations strongly associated with cytokine upregulation. These findings link high fibrinogen and red blood cell aggregation with endotheliopathy in adult COVID-19 patients and highlight differences in the key mediators of pathogenesis between adult and pediatric populations.

Comparison of Mid-turbinate Nasal Swabs, Saliva, and Nasopharyngeal Swabs for SARS-CoV-2 Reverse Transcription-Polymerase Chain Reaction Testing in Pediatric Outpatients.

CONTEXT.­: Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains integral to care, particularly for supporting return to and attendance in schools. The concordance of SARS-CoV-2 detection in children, using various specimen types, has not been widely studied. OBJECTIVE.­: To compare 3 sample types for SARS-CoV-2 polymerase chain reaction (PCR) testing in children, collected and tested at a single facility. DESIGN.­: We prospectively recruited 142 symptomatic and asymptomatic children/young adults into a sample comparison study performed in a single health care system. Each child provided self-collected saliva, and a trained health care provider collected a mid-turbinate nasal swab and nasopharyngeal (NP) swab. Specimens were assayed within 24 hours of collection by using reverse transcription-polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 on a single testing platform. RESULTS.­: Concurrently collected saliva and mid-turbinate swabs had greater than 95% positive agreement with NP swabs when obtained within 10 days of symptom onset. Positive agreement of saliva and mid-turbinate samples collected from children with symptom onset >10 days prior, or without symptoms, was 82% compared to NP swab samples. Cycle threshold (Ct) values for mid-turbinate nasal samples more closely correlated with Ct values from NP samples than from saliva samples. CONCLUSIONS.­: These findings suggest that all 3 sample types from children are useful for SARS-CoV-2 diagnostic testing by RT-PCR, and that concordance is greatest when the child has had symptoms of COVID-19 within the past 10 days. This study provides scientific justification for using sample types other than the NP swab for SARS-CoV-2 testing in pediatric populations.

Serologic and Cytokine Signatures in Children With Multisystem Inflammatory Syndrome and Coronavirus Disease 2019.

BACKGROUND: The serologic and cytokine responses of children hospitalized with multisystem inflammatory syndrome (MIS-C) vs coronavirus disease 2019 (COVID-19) are poorly understood. METHODS: We performed a prospective, multicenter, cross-sectional study of hospitalized children who met the Centers for Disease Control and Prevention case definition for MIS-C (n = 118), acute COVID-19 (n = 88), or contemporaneous healthy controls (n = 24). We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) immunoglobulin G (IgG) titers and cytokine concentrations in patients and performed multivariable analysis to determine cytokine signatures associated with MIS-C. We also measured nucleocapsid IgG and convalescent RBD IgG in subsets of patients. RESULTS: Children with MIS-C had significantly higher SARS-CoV-2 RBD IgG than children with acute COVID-19 (median, 2783 vs 146; P < .001), and titers correlated with nucleocapsid IgG. For patients with MIS-C, RBD IgG titers declined in convalescence (median, 2783 vs 1135; P = .010) in contrast to patients with COVID-19 (median, 146 vs 4795; P < .001). MIS-C was characterized by transient acute proinflammatory hypercytokinemia, including elevated levels of interleukin (IL) 6, IL-10, IL-17A, and interferon gamma (IFN-γ). Elevation of at least 3 of these cytokines was associated with significantly increased prevalence of prolonged hospitalization ≥8 days (prevalence ratio, 3.29 [95% CI, 1.17-9.23]). CONCLUSIONS: MIS-C was associated with high titers of SARS-CoV-2 RBD IgG antibodies and acute hypercytokinemia with IL-6, IL-10, IL-17A, and IFN-γ.

Limited induction of SARS-CoV-2-specific T cell responses in children with multisystem inflammatory syndrome compared with COVID-19.

Why multisystem inflammatory syndrome in children (MIS-C) develops after SARS-CoV-2 infection in a subset of children is unknown. We hypothesized that aberrant virus-specific T cell responses contribute to MIS-C pathogenesis. We quantified SARS-CoV-2-reactive T cells, serologic responses against major viral proteins, and cytokine responses from plasma and peripheral blood mononuclear cells in children with convalescent COVID-19, in children with acute MIS-C, and in healthy controls. Children with MIS-C had significantly lower virus-specific CD4+ and CD8+ T cell responses to major SARS-CoV-2 antigens compared with children convalescing from COVID-19. Furthermore, T cell responses in participants with MIS-C were similar to or lower than those in healthy controls. Serologic responses against spike receptor binding domain (RBD), full-length spike, and nucleocapsid were similar among convalescent COVID-19 and MIS-C, suggesting functional B cell responses. Cytokine profiling demonstrated predominant Th1 polarization of CD4+ T cells from children with convalescent COVID-19 and MIS-C, although cytokine production was reduced in MIS-C. Our findings support a role for constrained induction of anti-SARS-CoV-2-specific T cells in the pathogenesis of MIS-C.

Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

To provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.

SARS-CoV-2 immune repertoire in MIS-C and pediatric COVID-19.

There is limited understanding of the viral antibody fingerprint following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children. Herein, SARS-CoV-2 proteome-wide immunoprofiling of children with mild/moderate or severe coronavirus disease 2019 (COVID-19) versus multisystem inflammatory syndrome in children versus hospitalized control patients revealed differential cytokine responses, IgM/IgG/IgA epitope diversity, antibody binding and avidity. Apart from spike and nucleocapsid, IgG/IgA recognized epitopes in nonstructural protein (NSP) 2, NSP3, NSP12-NSP14 and open reading frame (ORF) 3a-ORF9. Peptides representing epitopes in NSP12, ORF3a and ORF8 demonstrated SARS-CoV-2 serodiagnosis. Antibody-binding kinetics with 24 SARS-CoV-2 proteins revealed antibody parameters that distinguish children with mild/moderate versus severe COVID-19 or multisystem inflammatory syndrome in children. Antibody avidity to prefusion spike correlated with decreased illness severity and served as a clinical disease indicator. The fusion peptide and heptad repeat 2 region induced SARS-CoV-2-neutralizing antibodies in rabbits. Thus, we identified SARS-CoV-2 antibody signatures in children associated with disease severity and delineate promising serodiagnostic and virus neutralization targets. These findings might guide the design of serodiagnostic assays, prognostic algorithms, therapeutics and vaccines in this important but understudied population.

Original antigenic sin responses to Betacoronavirus spike proteins are observed in a mouse model, but are not apparent in children following SARS-CoV-2 infection.

BACKGROUND: The effects of pre-existing endemic human coronavirus (HCoV) immunity on SARS-CoV-2 serologic and clinical responses are incompletely understood. OBJECTIVES: We sought to determine the effects of prior exposure to HCoV Betacoronavirus HKU1 spike protein on serologic responses to SARS-CoV-2 spike protein after intramuscular administration in mice. We also sought to understand the baseline seroprevalence of HKU1 spike antibodies in healthy children and to measure their correlation with SARS-CoV-2 binding and neutralizing antibodies in children hospitalized with acute coronavirus disease 2019 (COVID-19) or multisystem inflammatory syndrome (MIS-C). METHODS: Groups of 5 mice were injected intramuscularly with two doses of alum-adjuvanted HKU1 spike followed by SARS-CoV-2 spike; or the reciprocal regimen of SARS-Cov-2 spike followed by HKU1 spike. Sera collected 21 days following each injection was analyzed for IgG antibodies to HKU1 spike, SARS-CoV-2 spike, and SARS-CoV-2 neutralization. Sera from children hospitalized with acute COVID-19, MIS-C or healthy controls (n = 14 per group) were analyzed for these same antibodies. RESULTS: Mice primed with SARS-CoV-2 spike and boosted with HKU1 spike developed high titers of SARS-CoV-2 binding and neutralizing antibodies; however, mice primed with HKU1 spike and boosted with SARS-CoV-2 spike were unable to mount neutralizing antibodies to SARS-CoV-2. HKU1 spike antibodies were detected in all children with acute COVID-19, MIS-C, and healthy controls. Although children with MIS-C had significantly higher HKU1 spike titers than healthy children (GMT 37239 vs. 7551, P = 0.012), these titers correlated positively with both SARS-CoV-2 binding (r = 0.7577, P<0.001) and neutralizing (r = 0.6201, P = 0.001) antibodies. CONCLUSIONS: Prior murine exposure to HKU1 spike protein completely impeded the development of neutralizing antibodies to SARS-CoV-2, consistent with original antigenic sin. In contrast, the presence of HKU1 spike IgG antibodies in children with acute COVID-19 or MIS-C was not associated with diminished neutralizing antibody responses to SARS-CoV-2.

Multidisciplinary assessment of the Abbott BinaxNOW SARS-CoV-2 point-of-care antigen test in the context of emerging viral variants and self-administration.

While there has been significant progress in the development of rapid COVID-19 diagnostics, as the pandemic unfolds, new challenges have emerged, including whether these technologies can reliably detect the more infectious variants of concern and be viably deployed in non-clinical settings as "self-tests". Multidisciplinary evaluation of the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW, a widely used rapid antigen test, included limit of detection, variant detection, test performance across different age-groups, and usability with self/caregiver-administration. While BinaxNOW detected the highly infectious variants, B.1.1.7 (Alpha) first identified in the UK, B.1.351 (Beta) first identified in South Africa, P.1 (Gamma) first identified in Brazil, B.1.617.2 (Delta) first identified in India and B.1.2, a non-VOC, test sensitivity decreased with decreasing viral loads. Moreover, BinaxNOW sensitivity trended lower when devices were performed by patients/caregivers themselves compared to trained clinical staff, despite universally high usability assessments following self/caregiver-administration among different age groups. Overall, these data indicate that while BinaxNOW accurately detects the new viral variants, as rapid COVID-19 tests enter the home, their already lower sensitivities compared to RT-PCR may decrease even more due to user error.

Secretory phospholipase A2 in SARS-CoV-2 infection and multisystem inflammatory syndrome in children (MIS-C).

Secretory phospholipase 2 (sPLA2) acts as a mediator between proximal and distal events of the inflammatory cascade. Its role in SARS-CoV-2 infection is unknown, but could contribute to COVID-19 inflammasome activation and cellular damage. We present the first report of plasma sPLA2 levels in adults and children with COVID-19 compared with controls. Currently asymptomatic adults with a history of recent COVID-19 infection (≥4 weeks before) identified by SARS-CoV-2 IgG antibodies had sPLA2 levels similar to those who were seronegative (9 ± 6 vs.17 ± 28 ng/mL, P = 0.26). In contrast, children hospitalized with severe COVID-19 had significantly elevated sPLA2 compared with those with mild or asymptomatic SARS-CoV-2 infection (269 ± 137 vs. 2 ± 3 ng/mL, P = 0.01). Among children hospitalized with multisystem inflammatory syndrome in children (MIS-C), all had severe disease requiring pediatric intensive care unit (PICU) admission. sPLA2 levels were significantly higher in those with acute illness <10 days versus convalescent disease ≥10 days (540 ± 510 vs. 2 ± 1, P = 0.04). Thus, sPLA2 levels correlated with COVID-19 severity and acute MIS-C in children, implicating a role in inflammasome activation and disease pathogenesis. sPLA2 may be a useful biomarker to stratify risk and guide patient management for children with acute COVID-19 and MIS-C. Therapeutic compounds targeting sPLA2 and inflammasome activation warrant consideration.

SARS-CoV-2 Infection and Racial Disparities in Children: Protective Mechanisms and Severe Complications Related to MIS-C.

A novel coronavirus has resulted in a pandemic with over 176 million confirmed cases and over 3.8 million recorded deaths. In the USA, SARS-CoV-2 infection has a significant burden on minority communities, especially Hispanic and Black communities, which are overrepresented in cases compared to their percentage in the population. SARS-CoV-2 infection can manifest differently in children and adults, with children tending to have less severe disease. A review of current literature was performed to identify the hypothesized protective immune mechanisms in children, and to describe the rare complication of multisystem inflammatory syndrome in children (MIS-C) that has been documented in children post-SARS-CoV-2 infection. Epidemiologic data and case studies have indicated that children are less susceptible to more severe clinical features of SARS-CoV-2 infection, a finding that may be due to differences in the cytokine response generated by the innate immune system, high amounts of ACE-2 which maintain homeostatic functions by preventing inflammation, and trained immunity acquired from regular vaccinations. Despite these protective mechanisms, children are still susceptible to severe complications, such as MIS-C. The racial disparities seen in MIS-C are extremely apparent, and certain populations are more affected. Most specifically, 33% of MIS-C patients are Hispanic/Latino, and 30% Black. Current studies published on MIS-C do not detail whether certain symptoms are more present in certain racial/ethnic groups. Knowledge of these disparities could assist health care professionals with devising appropriate strategies for post-acute SARS-CoV-2 infection follow-up in children as well as vaccine distribution in specific communities to help slow the spread of SARS-CoV-2 infection, and ultimately reduce the potential for complications such as MIS-C.

Altered amino acid profile in patients with SARS-CoV-2 infection.

Low plasma arginine bioavailability has been implicated in endothelial dysfunction and immune dysregulation. The role of arginine in COVID-19 is unknown, but could contribute to cellular damage if low. Our objective was to determine arginine bioavailability in adults and children with COVID-19 vs. healthy controls. We hypothesized that arginine bioavailability would be low in patients with COVID-19 and multisystem inflammatory syndrome in children (MIS-C). We conducted a prospective observational study of three patient cohorts; arginine bioavailability was determined in asymptomatic healthy controls, adults hospitalized with COVID-19, and hospitalized children/adolescents <21 y old with COVID-19, MIS-C, or asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection identified on admission screen. Mean patient plasma amino acids were compared to controls using the Student's t test. Arginine-to-ornithine ratio, a biomarker of arginase activity, and global arginine bioavailability ratio (GABR, arginine/[ornithine+citrulline]) were assessed in all three groups. A total of 80 patients were included (28 controls, 32 adults with COVID-19, and 20 pediatric patients with COVID-19/MIS-C). Mean plasma arginine and arginine bioavailability ratios were lower among adult and pediatric patients with COVID-19/MIS-C compared to controls. There was no difference between arginine bioavailability in children with COVID-19 vs. MIS-C. Adults and children with COVID-19 and MIS-C in our cohort had low arginine bioavailability compared to healthy adult controls. This may contribute to immune dysregulation and endothelial dysfunction in COVID-19. Low arginine-to-ornithine ratio in patients with COVID-19 or MIS-C suggests an elevation of arginase activity. Further study is merited to explore the role of arginine dysregulation in COVID-19.

Quantitative SARS-CoV-2 Serology in Children With Multisystem Inflammatory Syndrome (MIS-C).

OBJECTIVES: We aimed to measure severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological responses in children hospitalized with multisystem inflammatory syndrome in children (MIS-C) compared with those with coronavirus disease 2019 (COVID-19), those with Kawasaki disease (KD), and hospitalized pediatric controls. METHODS: From March 17, 2020, to May 26, 2020, we prospectively identified hospitalized children with MIS-C (n = 10), symptomatic COVID-19 (n = 10), and KD (n = 5) and hospitalized controls (n = 4) at Children's Healthcare of Atlanta. With institutional review board approval, we obtained prospective and residual blood samples from these children and measured SARS-CoV-2 spike receptor-binding domain (RBD) immunoglobulin M and immunoglobulin G (IgG), full-length spike IgG, and nucleocapsid protein antibodies using quantitative enzyme-linked immunosorbent assays and SARS-CoV-2 neutralizing antibodies using live-virus focus-reduction neutralization assays. We statistically compared the log-transformed antibody titers among groups and performed linear regression analyses. RESULTS: All children with MIS-C had high titers of SARS-CoV-2 RBD IgG antibodies, which correlated with full-length spike IgG antibodies (R 2 = 0.956; P < .001), nucleocapsid protein antibodies (R 2 = 0.846; P < .001), and neutralizing antibodies (R 2 = 0.667; P < .001). Children with MIS-C had significantly higher SARS-CoV-2 RBD IgG antibody titers (geometric mean titer 6800; 95% confidence interval 3495-13 231) than children with COVID-19 (geometric mean titer 626; 95% confidence interval 251-1563; P < .001), children with KD (geometric mean titer 124; 95% confidence interval 91-170; P < .001), and hospitalized controls (geometric mean titer 85; P < .001). All children with MIS-C also had detectable RBD immunoglobulin M antibodies, indicating recent SARS-CoV-2 infection. RBD IgG titers correlated with the erythrocyte sedimentation rate (R 2 = 0.512; P < .046) and with hospital (R 2 = 0.548; P = .014) and ICU lengths of stay (R 2 = 0.590; P = .010). CONCLUSIONS: Quantitative SARS-CoV-2 serology may have a role in establishing the diagnosis of MIS-C, distinguishing it from similar clinical entities, and stratifying risk for adverse outcomes.

The VACCINES Act: Deciphering Vaccine Hesitancy in the Time of COVID-19.

Since the COVID-19 pandemic first hit Wuhan, China, in December 2019, scientists have been racing to develop and test novel vaccines to protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The speed of scientific discovery related to COVID-19 is unprecedented. With several vaccine candidates already being tested in clinical trials, we pose the question: what will the vaccine hesitant do in the face of this pandemic?