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Background: Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN)., Methods: Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories., Findings: High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays., Interpretation: The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines., Funding: CEPI., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

SARS-CoV-2 mRNA vaccines confer robust protection against COVID-19, but the emergence of variants has generated concerns regarding the protective efficacy of the currently approved vaccines, which lose neutralizing potency against some variants. Emerging data suggest that antibody functions beyond neutralization may contribute to protection from the disease, but little is known about SARS-CoV-2 antibody effector functions. Here, we profiled the binding and functional capacity of convalescent antibodies and Moderna mRNA-1273 COVID-19 vaccine-induced antibodies across SARS-CoV-2 variants of concern (VOCs). Although the neutralizing responses to VOCs decreased in both groups, the Fc-mediated responses were distinct. In convalescent individuals, although antibodies exhibited robust binding to VOCs, they showed compromised interactions with Fc-receptors. Conversely, vaccine-induced antibodies also bound robustly to VOCs but continued to interact with Fc-receptors and mediate antibody effector functions. These data point to a resilience in the mRNA-vaccine-induced humoral immune response that may continue to offer protection from SARS-CoV-2 VOCs independent of neutralization., Competing Interests: Declaration of interests G.A. is the founder of Seromyx Systems Inc. A.C. is an employee of Moderna Inc. D.D., P.M., A.S.M., and E.R.M. are employees of Space Exploration Technologies Corp. All other authors have declared no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined 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 a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on 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.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic continues to spread relentlessly, associated with a high frequency of respiratory failure and mortality. Children experience largely asymptomatic disease, with rare reports of multisystem inflammatory syndrome in children (MIS-C). Identifying immune mechanisms that result in these disparate clinical phenotypes in children could provide critical insights into coronavirus disease 2019 (COVID-19) pathogenesis. Using systems serology, in this study we observed in 25 children with acute mild COVID-19 a functional phagocyte and complement-activating IgG response to SARS-CoV-2, similar to the acute responses generated in adults with mild disease. Conversely, IgA and neutrophil responses were significantly expanded in adults with severe disease. Moreover, weeks after the resolution of SARS-CoV-2 infection, children who develop MIS-C maintained highly inflammatory monocyte-activating SARS-CoV-2 IgG antibodies, distinguishable from acute disease in children but with antibody levels similar to those in convalescent adults. Collectively, these data provide unique insights into the potential mechanisms of IgG and IgA that might underlie differential disease severity as well as unexpected complications in children infected with SARS-CoV-2.

Antibodies serve as biomarkers of infection, but if sustained can confer long-term immunity. Yet, for most clinically approved vaccines, binding antibody titers only serve as a surrogate of protection. Instead, the ability of vaccine induced antibodies to neutralize or mediate Fc-effector functions is mechanistically linked to protection. While evidence has begun to point to persisting antibody responses among SARS-CoV-2 infected individuals, cases of re-infection have begun to emerge, calling the protective nature of humoral immunity against this highly infectious pathogen into question. Using a community-based surveillance study, we aimed to define the relationship between titers and functional antibody activity to SARS-CoV-2 over time. Here we report significant heterogeneity, but limited decay, across antibody titers amongst 120 identified seroconverters, most of whom had asymptomatic infection. Notably, neutralization, Fc-function, and SARS-CoV-2 specific T cell responses were only observed in subjects that elicited RBD-specific antibody titers above a threshold. The findings point to a switch-like relationship between observed antibody titer and function, where a distinct threshold of activity-defined by the level of antibodies-is required to elicit vigorous humoral and cellular response. This response activity level may be essential for durable protection, potentially explaining why re-infections occur with SARS-CoV-2 and other common coronaviruses.

Currently available mRNA vaccines are extremely safe and effective to prevent severe SARS-CoV-2 infections. However, the emergence of variants of concerns (VOCs) has highlighted the importance of high population-based vaccine rates to effectively suppress viral transmission and breakthrough infections. While initially left out from vaccine efforts, children have become one of the most affected age groups and are key targets to stop community and household spread. Antibodies are central for vaccine-induced protection and emerging data points to the importance of additional Fc effector functions like opsononophagocytosis or cytotoxicity, particularly in the context of VOCs that escape neutralizing antibodies. Here, we observed delayed induction and reduced magnitude of vaccine-induced antibody titers in children 5-11 years receiving two doses of the age-recommended 10 μg dose of the Pfizer SARS-CoV-2 BNT162b2 vaccine compared to adolescents (12–15 years) or adults receiving the 30 μg dose. Conversely, children mounted equivalent or more robust neutralization and opsonophagocytic functions at peak immunogenicity, pointing to a qualitatively more robust humoral functional response in children. Moreover, broad cross-VOC responses were observed across children, with enhanced IgM and parallel IgG cross-reactivity to VOCs in children compared to adults. Collectively, these data argue that despite the lower magnitude of the BNT162b2-induced antibody response in children, vaccine-induced immunity in children target VOCs broadly and exhibit enhanced functionality that may contribute to the attenuation of disease. [ABSTRACT FROM AUTHOR]

Although children have been largely spared from coronavirus disease 2019 (COVID-19), the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) with increased transmissibility, combined with fluctuating mask mandates and school reopenings, has led to increased infections and disease among children. Thus, there is an urgent need to roll out COVID-19 vaccines to children of all ages. However, whether children respond equivalently to adults to mRNA vaccines and whether dosing will elicit optimal immunity remain unclear. Here, we aimed to deeply profile the vaccine-induced humoral immune response in 6- to 11-year-old children receiving either a pediatric (50 μg) or adult (100 μg) dose of the mRNA-1273 vaccine and to compare these responses to vaccinated adults, infected children, and children who experienced multisystem inflammatory syndrome in children (MIS-C). Children elicited an IgG-dominant vaccine-induced immune response, surpassing adults at a matched 100-μg dose but more variable immunity at a 50-μg dose. Irrespective of titer, children generated antibodies with enhanced Fc receptor binding capacity. Moreover, like adults, children generated cross-VOC humoral immunity, marked by a decline of omicron-specific receptor binding domain, but robustly preserved omicron spike protein binding. Fc receptor binding capabilities were also preserved in a dose-dependent manner. These data indicate that both the 50- and 100-μg doses of mRNA vaccination in children elicit robust cross-VOC antibody responses and that 100-μg doses in children result in highly preserved omicron-specific functional humoral immunity. A Kid's COVID Vaccine: As the COVID-19 pandemic has progressed, it has become increasingly apparent that children are not entirely spared from severe disease; to that end, vaccines were recently approved for children as young as 6 months old. Here, Bartsch et al. evaluated the antibody response elicited by either an adult (100 μg) or pediatric (50 μg) dose of the Moderna mRNA-1273 vaccine. The authors found that children responded to both doses in a manner similar to, but not identical to, adults. Antibodies isolated from vaccinated children exhibited both neutralizing and nonneutralizing functions, providing data to support real-world evidence for vaccine effectiveness in younger populations. [ABSTRACT FROM AUTHOR]

Allergic symptoms after messenger RNA (mRNA) coronavirus disease 2019 (COVID-19) vaccines occur in up to 2% of recipients. Compared to nonallergic controls (n = 18), individuals with immediate allergic reactions to mRNA COVID-19 vaccines (n = 8) mounted lower immunoglobulin G1 (IgG1) to multiple antigenic targets in severe acute respiratory syndrome coronavirus 2 spike following vaccination, with significantly lower IgG1 to full-length spike (P = .04). Individuals with immediate allergic reactions to mRNA COVID-19 vaccines bound Fcγ receptors similarly to nonallergic controls. Although there was a trend toward an overall reduction in opsonophagocytic function in individuals with immediate allergic reactions compared to nonallergic controls, allergic patients produced functional antibodies exhibiting a high ratio of opsonophagocytic function to IgG1 titer. [ABSTRACT FROM AUTHOR]

The Omicron variant of SARS-CoV-2 has been shown to evade neutralizing antibodies elicited by vaccination or infection. Despite the global spread of the Omicron variant, even among highly vaccinated populations, death rates have not increased concomitantly. These data suggest that immune mechanisms beyond antibody-mediated virus neutralization may protect against severe disease. In addition to neutralizing pathogens, antibodies contribute to control and clearance of infections through Fc effector mechanisms. Here, we probed the ability of vaccine-induced antibodies to drive Fc effector activity against the Omicron variant using samples from individuals receiving one of three SARS-CoV-2 vaccines. Despite a substantial loss of IgM, IgA, and IgG binding to the Omicron variant receptor binding domain (RBD) in samples from individuals receiving BNT162b2, mRNA-1273, and CoronaVac vaccines, stable binding was maintained against the full-length Omicron Spike protein. Compromised RBD binding IgG was accompanied by a loss of RBD-specific antibody Fcγ receptor (FcγR) binding in samples from individuals who received the CoronaVac vaccine, but RBD-specific FcγR2a and FcγR3a binding was preserved in recipients of mRNA vaccines. Conversely, Spike protein–specific antibodies exhibited persistent but reduced binding to FcγRs across all three vaccines, although higher binding was observed in samples from recipients of mRNA vaccines. This was associated with preservation of FcγR2a and FcγR3a binding antibodies and maintenance of Spike protein–specific antibody-dependent natural killer cell activation. Thus, despite the loss of Omicron neutralization, vaccine-induced Spike protein–specific antibodies continue to drive Fc effector functions, suggesting a capacity for extraneutralizing antibodies to contribute to disease control. Effective Responses to Omicron: The Omicron variant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is capable of evading neutralizing antibodies elicited by vaccination or prior infection. However, the role of non-neutralizing antibodies in conferring protection against disease is not clear. Here, Bartsch and colleagues showed that vaccination with BNT162b2, mRNA-1273, or CoronaVac induced antibodies that were still capable of binding to the Omicron variant Spike protein. These antibodies were also able to elicit Fc-mediated effector functions, suggesting that they may play a role in controlling disease after infection with the Omicron variant. [ABSTRACT FROM AUTHOR]

Emergent SARS-CoV-2 variants and waning humoral immunity in vaccinated individuals have resulted in increased infections and hospitalizations. Children are not spared from infection nor complications of COVID-19, and the recent recommendation for boosters in individuals ages 12 years or older calls for broader understanding of the adolescent immune profile after mRNA vaccination. We tested the durability and cross-reactivity of anti-SARS-CoV-2 serologic responses over a six-month time course in vaccinated adolescents against the SARS-CoV-2 D614G ("wild type") and Omicron antigens. Serum from 77 adolescents showed that anti-Spike antibodies wane significantly over six months. After completion of a two-vaccine series, cross-reactivity against Omicron-specific receptor-binding domain (RBD) was seen. Functional humoral activation against wild type and Omicron SARS-CoV-2 also declines over time in vaccinated adolescent children. Evidence of waning mRNA-induced vaccine immunity underscores vulnerabilities in long-term pediatric protection against SARS-CoV-2 infection, while cross-reactivity highlights the additional benefits of vaccination. Characterization of adolescent immune signatures post-vaccination will inform guidance on vaccine platforms and timelines, and ultimately optimize immunoprotection of children. [ABSTRACT FROM AUTHOR]

Background: SARS-CoV-2 infection is associated with enhanced disease severity in pregnant women. Despite the potential of COVID-19 vaccines to reduce severe disease, vaccine uptake remained relatively low among pregnant women. Just as coordinated messaging from the Centers for Disease Control and Prevention and leading obstetrics organizations began to increase vaccine confidence in this vulnerable group, the evolution of SARS-CoV-2 variants of concerns, including the Omicron variant, raised new concerns about vaccine efficacy because of their ability to escape vaccine-induced neutralizing antibodies. Early data point to a milder disease course following infection with the Omicron variant in vaccinated individuals. Thus, these data suggest that alternate vaccine-induced immunity beyond neutralization may continue to attenuate Omicron variant-induced disease, such as Fc-mediated antibody activity.Objective: This study aimed to test whether vaccine-induced antibodies raised during pregnancy continue to bind to and leverage Fc receptors to protect against variants of concern including the Omicron variant.Study Design: The receptor binding domain or whole spike-specific antibody isotype binding titers and Fc gamma receptor binding directed toward variants of concern, including the Omicron variant, were analyzed in pregnant women after receiving the full dose regimen of either the Pfizer/BioNTech BNT62b2 (n=10) or Moderna mRNA-1273 (n=10) vaccination using a multiplexing Luminex assay.Results: Reduced isotype recognition of the Omicron receptor binding domain was observed following administration of either vaccine with relatively preserved, albeit reduced, recognition of the whole Omicron spike by immunoglobulin M and G antibodies. Despite the near complete loss of Fc receptor binding to the Omicron receptor binding domain, Fc receptor binding to the Omicron spike was more variable but largely preserved.Conclusion: Reduced binding titers to the Omicron receptor binding domain aligns with the observed loss of neutralizing activity. Despite the loss of neutralization, preserved, albeit reduced, Omicron spike recognition and Fc receptor binding potentially continue to attenuate disease severity in pregnant women. [ABSTRACT FROM AUTHOR]

Background: Cases of adolescents and young adults developing myocarditis after vaccination with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–targeted mRNA vaccines have been reported globally, but the underlying immunoprofiles of these individuals have not been described in detail. Methods: From January 2021 through February 2022, we prospectively collected blood from 16 patients who were hospitalized at Massachusetts General for Children or Boston Children's Hospital for myocarditis, presenting with chest pain with elevated cardiac troponin T after SARS-CoV-2 vaccination. We performed extensive antibody profiling, including tests for SARS-CoV-2–specific humoral responses and assessment for autoantibodies or antibodies against the human-relevant virome, SARS-CoV-2–specific T-cell analysis, and cytokine and SARS-CoV-2 antigen profiling. Results were compared with those from 45 healthy, asymptomatic, age-matched vaccinated control subjects. Results: Extensive antibody profiling and T-cell responses in the individuals who developed postvaccine myocarditis were essentially indistinguishable from those of vaccinated control subjects, despite a modest increase in cytokine production. A notable finding was that markedly elevated levels of full-length spike protein (33.9±22.4 pg/mL), unbound by antibodies, were detected in the plasma of individuals with postvaccine myocarditis, whereas no free spike was detected in asymptomatic vaccinated control subjects (unpaired t test; P <0.0001). Conclusions: Immunoprofiling of vaccinated adolescents and young adults revealed that the mRNA vaccine–induced immune responses did not differ between individuals who developed myocarditis and individuals who did not. However, free spike antigen was detected in the blood of adolescents and young adults who developed post-mRNA vaccine myocarditis, advancing insight into its potential underlying cause. [ABSTRACT FROM AUTHOR]

Humoral responses in coronavirus disease 2019 (COVID-19) are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined post mortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers and a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ T FH cell differentiation together with an increase in T-bet+ T H1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific "disease-related" B cell populations. These data identify defective Bcl-6+ T FH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections, and suggest that achieving herd immunity through natural infection may be difficult. • Germinal centers are lost in lymph nodes and spleens in acute COVID-19 • Bcl-6+ GC B cells and Bcl-6+ T follicular helper cells are markedly diminished • Abundant T H1 cells and aberrant TNF-α production are seen in COVID-19 lymph nodes • SARS-CoV-2-specific activated B cells accumulate in the blood of patients Shiv Pillai and colleagues show that in acute COVID-19, there is a striking loss of germinal centers in lymph nodes and spleens and depletion of Bcl-6+ B cells but preservation of AID+ B cells. A specific block in germinal center type Bcl-6+ T follicular helper cell differentiation may explain the loss of germinal centers and the accumulation of non-germinal-center-derived activated B cells. These data suggest an underlying basis for the lower quality and lack of durability of humoral immune responses observed during natural infection with SARS-CoV-2 and have significant implications for expectations of herd immunity. [ABSTRACT FROM AUTHOR]