Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Cross-reactive immunity against the SARS-CoV-2 Omicron variant is low in pediatric patients with prior COVID-19 or MIS-C.

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.

D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes.

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of ß2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1ß cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children.

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

Protective antigenic sites identified in respiratory syncytial virus fusion protein reveals importance of p27 domain.

Respiratory syncytial virus (RSV) vaccines primarily focused on surface fusion (F) protein are under development. Therefore, to identify RSV-F protective epitopes, we evaluated 14 antigenic sites recognized following primary human RSV infection. BALB/c mice were vaccinated with F peptides, F proteins, or RSV-A2, followed by rA2-Line19F challenge. F peptides generated binding antibodies with minimal in vitro neutralization titers. However, several F peptides (including Site II) reduced lung viral loads and lung pathology scores in animals, suggesting partial protection from RSV disease. Interestingly, animals vaccinated with peptides (aa 101-121 and 110-136) spanning the F-p27 sequence, which is only present in unprocessed F0 protein, showed control of viral loads with significantly reduced pathology compared with mock-vaccinated controls. Furthermore, we observed F-p27 expression on the surface of RSV-infected cells as well as lungs from RSV-infected mice. The anti-p27 antibodies demonstrated antibody-dependent cellular cytotoxicity (ADCC) of RSV-infected A549 cells. These findings suggest that p27-mediated immune response may play a role in control of RSV disease in vivo, and F-p27 should be considered for inclusion in an effective RSV vaccine.