Abstract Background We recently reported that SARS-CoV-2 infection triggered robust adaptive immune responses in the tonsils and adenoids of children. However, whether intramuscular mRNA vaccination induces immunity in these tissues is unknown. Methods We collected peripheral blood, tonsils, and adenoids from 21 children undergoing tonsillectomy/adenoidectomy in 2022 who had previously received a COVID-19 mRNA vaccination. From questionnaires and serology, we identified 10 vaccinated subjects who had not been infected; the remaining 11 were both infected and vaccinated. We characterized SARS-CoV-2-specific B cells that bound fluorescently-labelled spike proteins (S1 and RBD) in the blood and tissues of these participants using high dimensional flow cytometry, and compared them to SARS-CoV-2-specific B cells from 24 COVID-19 convalescent children recruited in 2020-2021. Results We identified SARS-CoV-2-specific B cells (S1+RBD+) in the blood, tonsils, and adenoids of nearly all infected and vaccinated subjects. Tonsils and adenoids post-infection had a higher percentage of S1+RBD+ memory B cells than post-vaccination, but differences in the percentage of S1+RBD+ cells were not noted in the peripheral blood. Furthermore, we found that only 20% of vaccinated only subjects had SARS-CoV-2-specific germinal center B cells in their tissues compared to over 70% of infected subjects and subjects with hybrid immunity. Unsupervised analyses of the high dimensional flow cytometry data revealed differences in the characteristics of SARS-CoV-2-specific B cells post-vaccination and post-infection; S1+RBD+ B cells from infected subjects had a higher portion of CXCR3+ and IgA+ memory B cells, while those from vaccinated subjects had a greater proportion of CD21lo memory B cells in the blood and tissues, implying a greater extrafollicular response post-vaccination but stronger mucosal IgA and IFN-γ-induced B cell responses post-infection. Conclusion We found tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract lymphoid tissue after mRNA vaccination, but vaccination induced B cell phenotypes distinct from that of natural infection which may affect the quality and duration of immunity in the blood and at the mucosal surface. Disclosures Pedro Milanez-Almeida, PhD, Novartis: Salary

Neutralization capacity of antibodies against Omicron after a prior SARS-CoV-2 infection in children and adolescents is not well studied. Therefore, we evaluated virus-neutralizing capacity against SARS-CoV-2 Alpha, Beta, Gamma, Delta and Omicron variants by age-stratified analyses (<5, 5-11, 12-21 years) in 177 pediatric patients hospitalized with severe acute COVID-19, acute MIS-C, and in convalescent samples of outpatients with mild COVID-19 during 2020 and early 2021. Across all patients, less than 10% show neutralizing antibody titers against Omicron. Children <5 years of age hospitalized with severe acute COVID-19 have lower neutralizing antibodies to SARS-CoV-2 variants compared with patients >5 years of age. As expected, convalescent pediatric COVID-19 and MIS-C cohorts demonstrate higher neutralization titers than hospitalized acute COVID-19 patients. Overall, children and adolescents show some loss of cross-neutralization against all variants, with the most pronounced loss against Omicron. In contrast to SARS-CoV-2 infection, children vaccinated twice demonstrated higher titers against Alpha, Beta, Gamma, Delta and Omicron. These findings can influence transmission, re-infection and the clinical disease outcome from emerging SARS-CoV-2 variants and supports the need for vaccination in children.

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.