Development of T cell immunity to norovirus and rotavirus in children under five years of age

Abstract

Most of the research effort to understand protective immunity against norovirus (NoV) has focused on humoral immunity, whereas immunity against another major pediatric enteric virus, rotavirus (RV), has been studied more thoroughly. The aim of this study was to investigate development of cell-mediated immunity to NoV in early childhood. Immune responses to NoV GI.3 and GII.4 virus-like particles and RV VP6 were determined in longitudinal blood samples of 10 healthy children from three months to four years of age. Serum IgG antibodies were measured using enzyme-linked immunosorbent assay and production of interferon-gamma by peripheral blood T cells was analyzed by enzyme-linked immunospot assay. NoV-specific T cells were detected in eight of 10 children by the age of four, with some individual variation. T cell responses to NoV GII.4 were higher than those to GI.3, but these responses were generally lower than responses to RV VP6. In contrast to NoV-specific antibodies, T cell responses were transient in nature. No correlation between cell-mediated and antibody responses was observed. NoV exposure induces vigorous T cell responses in children under five years of age, similar to RV. A role of T cells in protection from NoV infection in early childhood warrants further investigation.

Figure 1

Norovirus (NoV)- and rotavirus (RV)-specific serum immunoglobulin G (IgG) end-point titers. Sera were titrated with 2-fold serial dilutions and IgG antibodies were analyzed against NoV GI.3 (a), NoV GII.4 (b) and RV VP6 (c) in enzyme-linked immunosorbent assay. Shown are end-point titers at the age of 3, 6 and 12 months, mean end-point titers at 2–3 years and 4 years of age. The horizontal line indicates geometric mean titer. Statistical differences were determined using Fisher’s exact test, and a p value of ≤0.05 was considered statistically significant (*). Each symbol denotes an individual child (▲ = Subject 1, ♦ = 2, ○ = 3, □ = 4, ×  = 5, Δ = 6, • = 7, ◊ = 8, ■ = 9, * = 10).

Figure 2

Norovirus (NoV)- and rotavirus (RV)-specific interferon gamma (IFN-γ) production by T cells. Peripheral blood mononuclear cells (PBMC) were stimulated o/n with NoV GI.3 (a) and GII.4 (b) VLPs and RV VP6 (c) (30 µg/ml each) and cytokine production analyzed by enzyme-linked immunospot (ELISPOT) assay. Due to the shortage of cells, PBMCs collected at the age of 6 and 12 months and 2 and 3 years were pooled. Shown are mean IFN-γ spot-forming cells (SFC)/10⁶ PBMCs of two replicate wells of an individual sample. The horizontal line indicates the mean of the age grouped samples. The dashed line represents the positivity cut off (≥26 SFC/10⁶ PBMCs). Statistical differences between the groups were determined by a Mann-Whitney U test, and a p value of ≤0.05 was considered statistically significant.