Oral Typhoid Vaccination With Live-Attenuated Salmonella Typhi Strain Ty21a Generates Ty21a-Responsive and Heterologous Influenza Virus-Responsive CD4+ and CD8+ T Cells at the Human Intestinal Mucosa

Abstract

Background: Oral vaccination with live-attenuated Salmonella Typhi strain Ty21a is modestly efficacious, but the mechanisms of protection are currently unknown. While humoral and cellular immune responses are well described in peripheral blood, the cellular response at the intestinal mucosa has never been directly assessed.

Methods: We vaccinated healthy adults with Ty21a and assessed humoral and cellular immunity in vaccinated volunteers and controls after 18 days. Immunoglobulin levels were assessed in peripheral blood by an enzyme-linked immunosorbent assay. Cellular responses were assessed in peripheral blood and at the duodenal and colonic mucosa by flow cytometry.

Results: We demonstrate the generation of Ty21a-responsive and heterologous influenza virus-responsive CD4(+) and CD8(+) T cells at the duodenal mucosa. All duodenal responses were consistently correlated, and no responses were observed at the colonic mucosa. Peripheral anti-lipopolysaccharide immunoglobulin G and immunoglobulin A responses were significantly correlated with duodenal responses. The assessment of integrin β7 expression intensity among peripheral and duodenal T-cell subsets revealed varied capacities for mucosal homing and residence.

Conclusions: The breadth of duodenal cellular responses was not reflected peripherally. The direct evaluation of mucosal immune defense may yield functional correlates of protection and could provide insight into mechanisms that may be manipulated to enhance vaccine immunogenicity.

Keywords: Salmonella; T cells; Ty21a; cellular immunity; cytokines; heterologous immunity; humoral immunity; immunoglobulins; typhoid.

Figure 1.

Levels of serum immunoglobulin G (IgG) and immunoglobulin A (IgA) to specific antigens. The levels of IgG and IgA specific to Salmonella Typhi lipopolysaccharide (LPS; A) and influenza virus (B) in serum, expressed in arbitrary units (AU). For control (C; closed squares and circles) and vaccinated (V; open squares and circles) volunteers, paired comparisons were made between day 0 and day 18 values. Horizontal bars represent mean values (paired t tests were performed using logarithmically transformed data). Abbreviation: NS, not significant.

Figure 2.

Representative flow cytometric gating strategy. Dot plots are shown for cells isolated from the duodenal mucosa (A) and peripheral blood (B). Dead cells, B cells, and monocytes were removed by staining for viability (Vivid), CD19, and CD14 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 interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and/or interleukin 2 (IL-2) assessed in nonstimulated and in live-attenuated Salmonella Typhi strain Ty21a (Ty21a)-, influenza virus–, and staphylococcal enterotoxin B (SEB)–stimulated samples.

Figure 3.

Antigen-specific cytokine-producing populations at day 18. The frequency of CD4⁺ and CD8⁺ live-attenuated Salmonella Typhi strain Ty21a (Ty21a)-responsive and heterologous influenza virus–responsive populations expressing any combination of interferon γ, tumor necrosis factor α, and/or interleukin 2 above background. Staphylococcal enterotoxin B (SEB)–stimulated control data is also included. For control (C; closed squares, circles, and triangles) and vaccinated (V; open squares, circles, and triangles) volunteers, measurements were made at the duodenal mucosa (A), the colonic mucosa (B), and in peripheral blood (C). Horizontal bars represent mean values (comparisons were made using unpaired t tests). Abbreviation: NS, not significant.

Figure 3

Figure 4.

Combinations of antigen-specific cytokine production at day 18. The frequency of CD4⁺ and CD8⁺ live-attenuated Salmonella Typhi strain Ty21a (Ty21a)-responsive and heterologous influenza virus–responsive populations expressing 1 (+), 2 (++), or 3 (+++) cytokines (interferon γ, tumor necrosis factor α, and/or interleukin 2) above background. For control (C; closed squares, circles, and triangles) and vaccinated (V; open squares, circles, and triangles) volunteers, measurements were made at the duodenal mucosa (A and B) and in peripheral blood (C and D). Horizontal bars represent mean values (comparisons were made using unpaired t tests). Abbreviation: NS, not significant.

Figure 5.

Integrin β₇ expression intensity among T-cell populations at day 18. Geometric mean fluorescence intensity (MFI) expression of integrin β₇ among nonstimulated total CD4⁺ and total CD8⁺ T-cell populations (control volunteers, closed diamonds; vaccinated volunteers, open diamonds) and in live-attenuated Salmonella Typhi strain Ty21a (Ty21a)-stimulated (control volunteers, closed squares; vaccinated volunteers, open squares) and heterologous influenza virus–stimulated (control volunteers, closed circles; vaccinated volunteers, open circles) cytokine-producing subpopulations. Measurements were made at the duodenal mucosa (A) and in peripheral blood (B). Horizontal bars represent mean values (comparisons were made using unpaired t tests). Abbreviation: NS, not significant.