Longevity of duodenal and peripheral T-cell and humoral responses to live-attenuated Salmonella Typhi strain Ty21a

Abstract

Background: We have previously demonstrated that polyfunctional Ty21a-responsive CD4⁺ and CD8⁺ T cells are generated at the duodenal mucosa 18 days following vaccination with live-attenuated S. Typhi (Ty21a). The longevity of cellular responses has been assessed in peripheral blood, but persistence of duodenal responses is unknown.

Methods: We vaccinated eight healthy adults with Ty21a. Peripheral blood and duodenal samples were acquired after a median of 1.5 years (ranging from 1.1 to 3.7 years) following vaccination. Cellular responses were assessed in peripheral blood and at the duodenal mucosa by flow cytometry. Levels of IgG and IgA were also assessed in peripheral blood by enzyme-linked immunosorbent assay.

Results: No T-cell responses were observed at the duodenal mucosa, but CD4⁺ T-cell responses to Ty21a and FliC were observed in peripheral blood. Peripheral anti-lipopolysaccharide IgG and IgA responses were also observed. Early immunoglobulin responses were not associated with the persistence of long-term cellular immune responses.

Conclusions: Early T-cell responses which we have previously observed at the duodenal mucosa 18 days following oral vaccination with Ty21a could not be detected at a median of 1.5 years. Peripheral responses were observed at this time. Immunoglobulin responses observed shortly after vaccination were not associated with cellular immune responses at 1.5 years, suggesting that the persistence of cellular immunity is not associated with the strength of the initial humoral response to vaccination.

Keywords: Cytokines; Immunoglobulins; Salmonella; T cells; Ty21a.

Fig. 1

Levels of serum immunoglobulin (Ig)G and IgA to Salmonella Typhi lipopolysaccharide (LPS). The levels of IgG and IgA specific to Salmonella Typhi LPS in serum, expressed in arbitrary units (AU). Unpaired comparisons were made between control (C; closed squares) and vaccinated (V; open squares) volunteers at baseline (unpaired t tests were performed using logarithmically transformed data). Paired comparisons were made between baseline (T0) and day 11 (T1), day 18 (T2) and approximately 1.5 years (T3) (paired t tests were performed using logarithmically transformed data). Horizontal bars represent mean values. Abbreviation: ns, not significant; *, bootstrapped 95% confidence interval does not cross 0.

Fig. 2

Representative flow cytometric gating strategy for intracellular cytokine analysis. Dot plots are shown for cells isolated from (A) peripheral blood and (B) the duodenal mucosa. Dead cells were removed by staining for viability (LIVE/DEAD) and gating on the negative population. T cells were identified according to the expression of CD3. T cells were classified according to the expression of CD4 and CD8 and the expression of IFN-γ, TNF-α, IL-2, IL-17A, and MIP-1β assessed in non-stimulated (NS) and in Ty21a, FliC and SEB stimulated samples. Values were expressed as the percentage of the parent CD4⁺ or CD8⁺ population.

Fig. 3

Antigen-specific cytokine-producing populations at 1.5 years. The frequency of CD4⁺ and CD8⁺Salmonella Typhi strain Ty21a-responsive and FliC-responsive populations expressing any combination of IFN-γ ± TNF-α ± IL-2 ± IL-17A ± MIP-1β above background. SEB-stimulated control data are also included. For control (C; closed squares, diamonds and triangles) and vaccinated (V; open squares, diamonds and triangles) volunteers, measurements were made at the duodenal mucosa (A), and in peripheral blood (B). Values are expressed as the percentage of the parent CD4⁺ or CD8⁺ population. Horizontal bars represent mean values. Abbreviation: ns, not significant; *, bootstrapped 95% confidence interval does not cross 0.

Fig. 4

Combinations of antigen-specific cytokine production in peripheral blood. The frequency of CD4⁺ and CD8⁺ Ty21a-responsive (A and B) and FliC-responsive (C and D) populations expressing one (+), or polyfunctional populations expressing two (++), three (+++), four (++++) or five (+++++) cytokines/chemokines (IFN-γ ± TNF-α ± IL-2 ± IL1-7 ± MIP-1β) above background. For control (C; closed squares and diamonds) and vaccinated (V; open squares and diamonds) volunteers. Values are expressed as the percentage of the parent CD4⁺ or CD8⁺ population. Horizontal bars represent mean values. Abbreviation: ns, not significant; *, bootstrapped 95% confidence interval does not cross 0.

Supplementary Fig. S1

Combinations of antigen-specific cytokine production at the duodenal mucosa. The frequency of CD4+ and CD8+ Salmonella Typhi strain Ty21a-responsive (A and B) and FliC-responsive (C and D) populations expressing one (+), or polyfunctional populations expressing two (++), three (+++), four (++++) or five (+++++) cytokines/chemokines (IFN-γ ± TNF-α ± IL-2 ± IL-17A ± MIP-1β) above background. For control (C; closed squares and diamonds) and vaccinated (V; open squares and diamonds) volunteers. Values are expressed as the percentage of the parent CD4+ or CD8+ population. Horizontal bars represent mean values. Abbreviation: ns, not significant; *, bootstrapped 95% confidence interval does not cross 0.

Supplementary Fig. S2

Antigen-specific production of individual cytokines at the duodenal mucosa. The frequency of CD4+ and CD8+ Ty21a-responsive (A and B) and FliC-responsive (C and D) populations expressing IFN-γ, TNF-α, IL-2, IL-17A, or MIP-1β above background. For control (C; closed squares and diamonds) and vaccinated (V; open squares and diamonds) volunteers. Values are expressed as the percentage of the parent CD4+ or CD8+ population. Horizontal bars represent mean values.

Fig. 5

Antigen-specific production of individual cytokines in peripheral blood. The frequency of CD4⁺ and CD8⁺ Ty21a-responsive (A and B) and FliC-responsive (C and D) populations expressing IFN-γ, TNF-α, IL-2, IL-17A, or MIP-1β above background. For control (C; closed squares and diamonds) and vaccinated (V; open squares and diamonds) volunteers. Values are expressed as the percentage of the parent CD4⁺ or CD8⁺ population. Horizontal bars represent mean values.