Evaluation of Oropharyngeal responses to SARS-CoV-2

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

In our original study, peripheral blood, tonsil tissues, and adenoid tissues were obtained from 110 children who underwent tonsillectomy at Children's National Hospital from late 2020 to 2021 with the goal of: 1) evaluating the prevalence of prior COVID-19 and epidemiologic risk factors for infection; 2) evaluating the nature of SARS-CoV-2-specific immune responses in the oropharyngeal tissue; and 3)comparing the immune profile in the mucosal tissue and blood of COVID-19-convalescent children and children without prior COVID-19. All patients were PCR negative for SARS-CoV-2 prior to surgery. We identified 24 participants with evidence of prior COVID-19 through serologic testing and identification of B cells that recognize the RBD region of the SARS-CoV-2 spike protein by flow cytometry. Of these, only 11 of these participants had a prior positive PCR test for SARS-CoV-2 (25-303 days prior to surgery) and were aware of their prior infection. With high dimensional flow cytometry and CITE-seq, we identified SARS-CoV-2-specific memory B cells in the tissue derived from robust geminal center (GC) responses, with evidence of somatic hypermutation and class switching; we also noted overlapping clones in the tonsil and adenoid suggesting migration of cells between these two oropharyngeal tissues. We also found persistent expansion of B and T cell populations involved in the GC and anti-viral response including GC B cells, CD57+PD-1hi GC-Tfh, and CD57+PD1+ CD8 TRM cells, as well as CXCR3+ (IFN-gamma-associated) Tfh populations in COVID-19-convalescent subjects. These changes were most notable in the adenoids, with fewer changes observed in peripheral blood. By sequencing of T cell receptor genes, we found expanded CD8+ and CD4+ T cell clones in convalescent samples, some of which had CDR3 sequences that have been reported to be SARS-CoV-2 specific. Lastly, we found persistent viral RNA by ddPCR as long as 10 months after acute infection. In summary, our results indicate persistent SARS-CoV-2-specific immunity in the upper respiratory tract weeks to months after infection (Xu et al Nat. Immunol. 2023). However, whether intramuscular vaccination elicits similar tissue immunity in the oropharyngeal lymphoid tissue remained an important question. We have extended these studies now by characterizing SARS-CoV-2-specific B cell responses in tonsils, adenoids, and peripheral blood of children who had been immunized with a SARS-CoV-2 mRNA vaccine and compared these samples with those from children infected with SARS-CoV-2 or both infected and immunized. Notably, we found SARS-CoV-2-specific B cells in the tonsils and adenoids of children both post-infection and post-vaccination. These were primarily memory B cells but we also found a small portion of GC B cells, indicating that immune memory can be found and maintained in the upper respiratory tract after intramuscular vaccination as well as after infection. Nonetheless, vaccination and infection generated different quantities and qualities of responses in both the mucosal tissues and blood, with memory B cells expressing different markers. Specifically, infection generated a higher proportion of IgA+ BSM and CXCR3+CD21+ BSM, which showed distinct spatial localization, greater clonal expansion and increased propensity for plasma cell differentiation compared to their CXCR3- counterparts, accompanied by persistent activation of innate and T follicular helper cells in the tissues. Our data provide evidence for tissue-specific B cell memory after either SARS-CoV-2 vaccination or infection, but with distinct characteristics that can influence the quality, durability, and localization of immunity. Together, these studies may provide a framework for understanding responses to vaccination and infection, including novel insight for assessing vaccine-induced mucosal immunity and mucosal vaccines (Xu et al, under revision). Finally, in collaboration with Drs. Delmonte and Notarangelo, we have used these same high dimensional flow cytometry techniques to evaluate responses to vaccination for SARS-CoV-2 in adults , assessing responses in the peripheral blood of subjects who either had or did not have a history of infection with SARS-CoV-2. Our results again support distinct features of memory B cells after infection (Wachter et al JACI 2025). We have also published a methods paper outlining our high dimensional flow analyses of CD4 Tfh-like cells, including both bench techniques and bioinformatic analyses (Xu et al, JOVE 2025).