COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children.

BACKGROUND: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy, yet limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children. METHODS: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age (N=24) undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples) or in a convenience sample of children under 5 years of age presenting to pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation. RESULTS: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process. CONCLUSIONS: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

Bacterial Sphingolipids Exacerbate Colitis by Inhibiting ILC3-derived IL-22 Production.

BACKGROUND & AIMS: Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear. METHODS: We used dextran sodium sulfate (DSS)-induced colitis in mice monocolonized with Bacteroides fragilis strains expressing or lacking sphingolipids to assess the influence of bacterial sphingolipids on intestinal inflammation using transcriptional, protein, and cellular analyses. Colonic explant and organoid were used to study the function of bacterial sphingolipids. Host mucosal immune cells and cytokines were profiled and characterized using flow cytometry, enzyme-linked immunosorbent assay, and Western blot, and cytokine function in vivo was investigated by monoclonal antibody injection. RESULTS: B fragilis sphingolipids exacerbated intestinal inflammation. Mice monocolonized with B fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin (IL)-22 by ILC3. Mice colonized with B fragilis lacking sphingolipids following DSS treatment showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production. Protection against DSS colitis associated with B fragilis lacking sphingolipids was reversed on IL22 blockade. Furthermore, bacterial sphingolipids restricted epithelial IL18 production following DSS treatment and interfered with IL22 production by a subset of ILC3 cells expressing both IL18R and major histocompatibility complex class II. CONCLUSIONS: B fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL18 expression and concomitantly suppressing the production of IL22 by ILC3 cells.

COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children.

Background: Understanding antibody responses to SARS-CoV-2 vaccination is crucial for refining COVID-19 immunization strategies. Generation of mucosal immune responses, including mucosal IgA, could be of potential benefit to vaccine efficacy, yet limited evidence exists regarding the production of mucosal antibodies following the administration of current mRNA vaccines to young children. Methods: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples, N=116) or on convenience samples of children under 5 years of age presenting to a pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation. Results: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process. Conclusions: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

An enhanced IL17 and muted type I interferon nasal epithelial cell state characterizes severe COVID-19 with fungal coinfection.

Recent case reports and epidemiological data suggest that fungal infections represent an underappreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing data set characterizing the upper respiratory microenvironment during COVID-19 and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our previous study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals. Using matched single-cell transcriptomic profiles of these individuals' respiratory mucosa, we identify epithelial immune signatures suggestive of IL17 stimulation and anti-fungal immunity. Further, we observe a significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggest that IL17 stimulation-in part driven by Candida colonization-and blunted interferon signaling represent a common feature of severe COVID-19 infection. IMPORTANCE: In this paper, we present an analysis suggesting that symptomatic and asymptomatic fungal coinfections can impact patient disease progression during COVID-19 hospitalization. By looking into the presence of other pathogens and their effect on the host immune response during COVID-19 hospitalizations, we aim to offer insight into an underestimated scenario, furthering our current knowledge of determinants of severity that could be considered for future diagnostic and intervention strategies.

Humoral profiles of toddlers and young children following SARS-CoV-2 mRNA vaccination.

Although young children generally experience mild symptoms following infection with SARS-CoV-2, severe acute and long-term complications can occur. SARS-CoV-2 mRNA vaccines elicit robust immunoglobulin profiles in children ages 5 years and older, and in adults, corresponding with substantial protection against hospitalizations and severe disease. Whether similar immune responses and humoral protection can be observed in vaccinated infants and young children, who have a developing and vulnerable immune system, remains poorly understood. To study the impact of mRNA vaccination on the humoral immunity of infant, we use a system serology approach to comprehensively profile antibody responses in a cohort of children ages 6 months to 5 years who were vaccinated with the mRNA-1273 COVID-19 vaccine (25 µg). Responses are compared with vaccinated adults (100 µg), in addition to naturally infected toddlers and young children. Despite their lower vaccine dose, vaccinated toddlers elicit a functional antibody response as strong as adults, with higher antibody-dependent phagocytosis compared to adults, without report of side effects. Moreover, mRNA vaccination is associated with a higher IgG3-dependent humoral profile against SARS-CoV-2 compared to natural infection, supporting that mRNA vaccination is effective at eliciting a robust antibody response in toddlers and young children.

Enhanced production of eicosanoids in plasma and activation of DNA damage pathways in PBMCs are correlated with the severity of ancestral COVID-19 infection.

Background: Many questions remain unanswered regarding the implication of lipid metabolites in severe SARS-CoV-2 infections. By re-analyzed sequencing data from the nasopharynx of a previously published cohort, we found that alox genes, involved in eicosanoid synthesis, were up-regulated in high WHO score patients, especially in goblet cells. Herein, we aimed to further understand the roles played by eicosanoids during severe SARS-CoV-2 infection. Methods and findings: We performed a total fatty acid panel on plasma and bulk RNA-seq analysis on peripheral blood mononuclear cells (PBMCs) collected from 10 infected and 10 uninfected patients. Univariate comparison of lipid metabolites revealed that lipid metabolites were increased in SARS-CoV-2 patients including the lipid mediators Arachidonic Acid (AA) and Eicosapentaenoic Acid (EPA). AA, EPA and the fatty acids Docosahexaenoic acid (DHA) and Docosapentaenoic acid (DPA), were positively correlated to WHO disease severity score. Transcriptomic analysis demonstrated that COVID-19 patients can be segregated based on WHO scores. Ontology, KEGG and Reactome analysis identified pathways enriched for genes related to innate immunity, interactions between lymphoid and nonlymphoid cells, interleukin signaling and, cell cycling pathways. Conclusions: Our study offers an association between nasopharynx mucosa eicosanoid genes expression, specific serum inflammatory lipids and, subsequent DNA damage pathways activation in PBMCs to severity of COVID-19 infection.

Humoral profiles of toddlers and young children following SARS-CoV-2 mRNA vaccination.

Although young children generally experience mild symptoms following infection with SARS-CoV-2, severe acute and long-term complications can occur. SARS-CoV-2 mRNA vaccines elicit robust immunoglobulin profiles in children ages 5 years and older, and in adults, corresponding with substantial protection against hospitalizations and severe disease. Whether similar immune responses and humoral protection can be observed in vaccinated infants and young children, who have a developing and vulnerable immune system, remains poorly understood. To study the impact of mRNA vaccination on the humoral immunity of infant, we used a system serology approach to comprehensively profile antibody responses in a cohort of children ages 6 months to 5 years who were vaccinated with the mRNA-1273 COVID-19 vaccine (25 µg). Responses were compared with vaccinated adults (100 µg), in addition to naturally infected toddlers and young children. Despite their lower vaccine dose, vaccinated toddlers elicited a stronger functional antibody response than adults, including against variant of concerns (VOCs), without report of side effects. Moreover, mRNA vaccination was associated with a higher IgG3-dependent humoral profile against SARS-CoV-2 compared to natural infection, supporting that mRNA vaccination is effective at eliciting a robust antibody response in toddlers and young children.

The IL-10 receptor inhibits cell extrinsic signals necessary for STAT1-dependent macrophage accumulation during colitis.

The loss of IL-10R function leads to severe early onset colitis and, in murine models, is associated with the accumulation of immature inflammatory colonic macrophages. We have shown that IL-10R-deficient colonic macrophages exhibit increased STAT1-dependent gene expression, suggesting that IL-10R-mediated inhibition of STAT1 signaling in newly recruited colonic macrophages might interfere with the development of an inflammatory phenotype. Indeed, STAT1-/- mice exhibit defects in colonic macrophage accumulation after Helicobacter hepaticus infection and IL-10R blockade, and this was phenocopied in mice lacking IFNγR, an inducer of STAT1 activation. Radiation chimeras demonstrated that reduced accumulation of STAT1-deficient macrophages was based on a cell-intrinsic defect. Unexpectedly, mixed radiation chimeras generated with both wild-type and IL-10R-deficient bone marrow indicated that rather than directly interfering with STAT1 function, IL-10R inhibits the generation of cell extrinsic signals that promote the accumulation of immature macrophages. These results define the essential mechanisms controlling the inflammatory macrophage accumulation in inflammatory bowel diseases.

Severe COVID-19 is associated with fungal colonization of the nasopharynx and potent induction of IL-17 responses in the nasal epithelium.

Recent case reports and epidemiological data suggest fungal infections represent an under-appreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing (scRNA-seq) dataset characterizing the upper respiratory microenvironment during COVID-19, and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals, including confirmatory diagnostic testing demonstrating elevated serum (1, 3)-ß-D-glucan and/or confirmed fungal culture of the predicted pathogen. Using matched single-cell transcriptomic profiles of these individuals' respiratory mucosa, we identify epithelial immune signatures suggestive of IL-17 stimulation and anti-fungal immunity. Further, we observe significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggests that IL-17 stimulation - in part driven by Candida colonization - and blunted type I/III interferon signaling represents a common feature of severe COVID-19 infection.

Monocytes transition to macrophages within the inflamed vasculature via monocyte CCR2 and endothelial TNFR2.

Monocytes undergo phenotypic and functional changes in response to inflammatory cues, but the molecular signals that drive different monocyte states remain largely undefined. We show that monocytes acquire macrophage markers upon glomerulonephritis and may be derived from CCR2+CX3CR1+ double-positive monocytes, which are preferentially recruited, dwell within glomerular capillaries, and acquire proinflammatory characteristics in the nephritic kidney. Mechanistically, the transition to immature macrophages begins within the vasculature and relies on CCR2 in circulating cells and TNFR2 in parenchymal cells, findings that are recapitulated in vitro with monocytes cocultured with TNF-TNFR2-activated endothelial cells generating CCR2 ligands. Single-cell RNA sequencing of cocultures defines a CCR2-dependent monocyte differentiation path associated with the acquisition of immune effector functions and generation of CCR2 ligands. Immature macrophages are detected in the urine of lupus nephritis patients, and their frequency correlates with clinical disease. In conclusion, CCR2-dependent functional specialization of monocytes into macrophages begins within the TNF-TNFR2-activated vasculature and may establish a CCR2-based autocrine, feed-forward loop that amplifies renal inflammation.