Age-related Differences in Immune Reactions to SARS-CoV-2 Spike and Nucleocapsid Antigens

Abstract

Background/aim: The manifestation and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections show a clear correlation to the age of a patient. The younger a person, the less likely the infection results in significant illness. To explore the immunological characteristics behind this phenomenon, we studied the course of SARS-CoV-2 infections in 11 households, including 8 children and 6 infants/neonates of women who got infected with SARS-CoV-2 during pregnancy.

Materials and methods: We investigated the immune responses of peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells (UCBCs), and T cells against spike and nucleocapsid antigens of SARS-COV-2 by flow cytometry and cytokine secretion assays.

Results: Upon peptide stimulation, UCBC from neonates showed a strongly reduced IFN-γ production, as well as lower levels of IL-5, IL-13, and TNF-α alongside with decreased frequencies of surface CD137/PD-1 co-expressing CD4⁺ and CD+8 T cells compared with adult PBMCs. The PBMC response of older children instead was characterized by elevated frequencies of IFN-γ+ CD4⁺ T cells, but significantly lower levels of multiple cytokines (IL-5, IL-6, IL-9, IL-10, IL-17A, and TNF-α) and a marked shift of the CD4⁺/CD8⁺ T-cell ratio towards CD8⁺ T cells in comparison to adults.

Conclusion: The increased severity of SARS-CoV-2 infections in adults could result from the strong cytokine production and lower potential to immunomodulate the excessive inflammation, while the limited IFN-γ production of responding T cells in infants/neonates and the additional higher frequencies of CD8⁺ T cells in older children may provide advantages during the course of a SARS-CoV-2 infection.

Keywords: COVID-19; SARS-CoV-2; children; immune response; inflammation; neonates; pediatric immunology; spike protein.

Figure 1. Augmented IFN-γ response of SARS-CoV-2-infected adults. A) IFN-γ single (upper panel) or IFN-γ and IL-10 double (lower panel) ELISpot scans of peripheral blood mononuclear cell (PBMC) or umbilical cord blood mononuclear cell (UCBC) cultures of SARSCoV-2-infected adults (AD), children (CH), neonates and infants (NI), respectively. Cells were activated with SARS-CoV-2 spike, nucleocapsid, or control peptides. B) Distribution of IFN-γ ELISpot responding or non-responding donors against spike and nucleocapsid peptides according to (A). C) IFN-γ ELISpot counts minus ctrl. of donors reacting to spike or nucleocapsid according to (A). D) Distribution of IFN-γ or IL-10 ELISpot responding or nonresponding donors combined against spike and nucleocapsid peptides according to (A lower panel). E) IL-10 ELISpot counts minus ctrl. of donors reacting to spike and nucleocapsid according to (A lower panel). Data points represent donors with median, interquartile, and range; *p<0.05, p-values were calculated using Fisher exact test with Freeman-Halton extension (B, D), two-way ANOVA (C), or Kruskal-Wallis test (E).

Figure 2. Flow cytometry analysis of T cells. A) Gating strategy for flow cytometric analysis of T cells. B, C) Ratios of CD137 and PD-1 doubleproducing (B) or IFN-gamma producing (C) CD4+ (left) or CD8+ (right) T cells in response to SARS-CoV-2 spike and nucleocapside peptides related to negative control. D) Relative amounts of CD4+ or CD8+ subsets of T cells in controls from adult or children peripheral blood mononuclear cells (left) and their ratios (right). Data points represent donors with median, interquartile, and range (B, C) or mean with SD (D); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; p-values were calculated using one-way ANOVA (Holm-Sidak’s multiple comparisons test) (B, C left), Kruskal-Wallis test with Dunn’s correction for multiple comparison (C right), two-way ANOVA or two-sided Mann-Whitney test (D).

Figure 3. Augmented cytokine responses of SARS-CoV-2-infected adults compared to children and neonates. A, B) Cytokine levels of IL-6 (A, left), TNF-α (A, right), IL-10 (B, left), and IL-13 (B, right) in the media of peripheral blood mononuclear cell (PBMC) or umbilical cord blood mononuclear cell (UCBC) cultures of SARS-CoV-2-infected adults, children, and neonates, respectively. Cells were activated with SARS-CoV-2 spike and nucleocapside peptides. C, D) Cytokine levels as indicated in cell culture media of PBMCs obtained from donors and activated as in A, B. Data points represent donors with median, interquartile, and range; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; p-Values were calculated using Kruskal-Wallis test with Dunn’s correction for multiple comparison (A, B) or two-sided Mann-Whitney test (C, D).