A single birth dose of Hepatitis B vaccine induces polyfunctional CD4+ T helper cells

Abstract

A single birth-dose of Hepatitis B vaccine (HepB) can protect newborns from acquiring Hepatitis B infection through vertical transmission, though several follow-up doses are required to induce long-lived protection. In addition to stimulating antibodies, a birth-dose of HepB might also induce polyfunctional CD4⁺ T-cells, which may contribute to initial protection. We investigated whether vaccination with HepB in the first week of life induced detectable antigen-specific CD4⁺ T-cells after only a single dose and following completion of the entire HepB vaccine schedule (3 doses). Using HBsAg- stimulated peripheral blood mononuclear cells from 344 infants, we detected increased populations of antigen-specific polyfunctional CD154⁺IL-2⁺TNFα⁺ CD4⁺ T-cells following a single birth-dose of HepB in a proportion of infants. Frequencies of polyfunctional T-cells increased following the completion of the HepB schedule but increases in the proportion of responders as compared to following only one dose was marginal. Polyfunctional T-cells correlated positively with serum antibody titres following the birth dose (day30) and completion of the 3-dose primary HepB vaccine series (day 128). These data indicate that a single birth dose of HepB provides immune priming for both antigen-specific B- and T cells.

Keywords: BCG; CD154; CD4 + T helper cell; Hepatitis B vaccine; T-cell activation; antibody titres; neonate.

Figure 1

Gating strategy for detection of activated CD4⁺ T-cells. Example FACS plots from SEB stimulated infant PBMSs at D128, showing the gating strategy used to measure the expression of activation markers IL-2, TNFα and CD154 (CD40L). To assess frequencies of cells expressing all three markers, Boolean gating was applied.

Figure 2

HepB- and BCG-specific CD4⁺ T-cells in infant and adult samples. PBMCs from infants at D30 and adult donors (pooled data from 3 donors) were stimulated with HBsAg, BCG or SEB for 18hrs. Expression of activation markers CD154, IL-2 and TNFα, following stimulation was compared to background (unstimulated samples). Cell frequencies were analysed based on expression of all three markers (A) or total expression of one marker (B–D). Kruskal-Wallis with Dunn’s post test, *p < 0,05; **p < 0,01; *** p < 0,005; **** p < 0,001; ns, not significant.

Figure 3

Antigen-specific CD4⁺ T-cells following completion of HepB vaccine schedule. PBMCs from infants at D30 and D128 were stimulated and stained as in Figure 2 . Background activation was removed by subtracting values from unstimulated matched samples. Cell frequencies of CD4⁺ polyfunctional T-cells (A) or frequencies based on total expression of IL-2 (B), TNFα (C) or CD154 (D) were compared. Responders to HBsAg (E) and BCG (F) stimulation were calculated as having cell frequencies above 2 x of the matched unstimulated sample. (A–D) Zero and negative values were assigned a value of 0.001 to enable Log transformation. Data are expressed as Log10 of CD4⁺ T-cell frequencies. Kruskal-Wallis with Dunn’s post-test, **p < 0,01; ***p < 0,005; ns, not significant.

Figure 4

Correlation between HepB specific CD4⁺ T-cells and antibody titres. Infants were vaccinated with HepB in the first week of life and received additional doses at 6, 10 and 14 weeks. IgG1 antibodies were measured at D30 and D128 and PBMCs from the same blood draws were stimulated with HBsAg for 18hrs and stained as in Figures 2, 3 . Cell frequencies of CD4⁺ polyfunctional T-cells (A, E) or frequencies based on total expression of IL-2 (B, F), TNFα (C, G) or CD154 (D, H) were correlated with their matched antibody titres on D30 (A–D) or D128 (E–H). Background CD4⁺ T-cell activation was removed by subtracting values from unstimulated matched samples. Only infants with undetectable HepB-specific antibody titres at birth and available matched Ab – T-cell sample (n = 179) were included. Spearman correlation was performed on Log10 of CD4⁺ T-cell frequencies and Log10 of antibody titres. P < 0,05 was considered significant.