Fig 1.
Electron beam irradiation inactivates S. Typhimurium.
Inactivation kinetics of S. Typhimurium exposed to different doses of eBeam irradiation. The D10 value (0.19 kGy) was calculated using the negative reciprocal of regression slope of the inactivation curve. The arrow represents the lethal eBeam dose used for preparing the eBeam immune modulation formulation (EBST).
Table 1.
Inability of eBeam-inactivated S. Typhimurium (EBST) to multiply in vivo in a Salmonella susceptible mice (C57BL/6J) model.
Fig 2.
Electron beam-inactivated ST (EBST) maintains cell membrane integrity.
Membrane integrity of S. Typhimurium remains unaffected by eBeam irradiation. Staining with Live/ Dead BacLight kit reveals the presence of intact cell membrane for (A) Live ST and (B) EBST indicated by the green labelled bacterial cells. Red colored staining of (C) HKST indicates disrupted cell membrane due to heat treatment.
Fig 3.
Membrane morphology of EBST is similar to live ST.
Scanning electron microscopic analysis shows presence of intact bacterial cell membrane for (A) Live ST and (B) EBST. (C) HKST appears to be morphologically uneven with shrunken cell membrane. Transmission electron micrographs of (D) live ST (E) EBST and (F) HKST reveals the presence of surface molecules on bacterial cell membrane of both live and EBST. HKST shows a smoothened surface indicative of loss of surface molecules.
Fig 4.
EBST retains immunogenic proteins that are detected by live ST immune mice serum.
EBST stored at room temperature (ER), 4°C (E4), -20°C (E20), lyophilized (ELy), Live ST (L), heat killed ST (HK), Protein ladder (M). After eBeam-inactivated, ST was stored at multiple temperature conditions such as 4°C, -20°C, and room temperature and as lyophilized preparation. Total extracted proteins (20 μg) were separated using SDS-PAGE and analyzed by western blotting. Immunodetection was carried out using 1/1000 dilution of S. Typhimurium infected mice serum as the primary antibody and 1/ 20,000 dilution of alkaline phosphatase conjugated sheep anti mouse IgG F(ab`)2 fragment as secondary antibody.
Fig 5.
EBST remains metabolically active and retains the activity for extended time periods of storage at 4°C.
Metabolic activity of S. Typhimurium post eBeam irradiation was measured using Alamar blue assay. The reduced environment present in metabolically active cells are detected by redox indicator which fluoresces. The fluorescence readings obtained for EBST, live ST and heat killed ST (HKST) was measured on a daily basis up to 9 days and are reported as line graph. Metabolic activity of EBST cells followed by heat inactivation (EBST+HK) and HKST followed by eBeam irradiation (HKST+EB) were measured as process controls.
Table 2.
eBeam-inactivated S. Typhimurium (EBST) remains metabolically active and tests positive for standard biochemical and enzymatic assays.
Fig 6.
EBST induces efficient DC maturation and triggers proinflammatory cytokine production in DC2.4 cells and BMDC.
DC2.4 cells were coincubated with EBST, HKST and live S. Typhimurium (MOI 1:10 for 24 h for surface markers and 4h for proinflammatory cytokine) as indicated to the left side of each row of histograms (Fig 6A). Each column of histogram represents expression level of surface markers MHC-II, CD40, CD80, CD86 and proinflammatory cytokine TNFα. Percentages in the gated region indicate the proportion of DC expressing high levels of various surface markers and TNFα. Expression levels of unstimulated DC (dotted red line) is compared to the antigen stimulated (thin black line) DC2.4 cells. Data are representative of three independent experiments. Bar graph summarizes the results from three independent experiments (Fig 6B). BMDCs were coincubated with EBST, HKST, live S. Typhimurium and a commercially available live attenuated ST vaccine (MOI 1:10 for 24 h for surface markers and 4h for proinflammatory cytokine) as indicated to the left side of each row of histograms (Fig 6C). Each column of histogram represents expression level of surface markers MHC-II, CD40, CD80, CD86 and proinflammatory cytokine TNFα. Percentages in the gated region indicate the proportion of CD11c+ DC expressing high levels of various surface markers and TNFα. Expression levels of unstimulated DC (dotted red line) is compared to the antigen stimulated (thin black line) DC. Data are representative of three independent experiments. Bar graph summarizes the results from three independent experiments (Fig 6D). Bars with differing letters indicate significant (p<0.05) differences among groups for each individual activation marker.
Fig 7.
EBST immune mice exhibit Salmonella specific CD4+IFNγ+ T cell responses during virulent ST challenge.
Individual EBST immune, AroA immune, and Sham immune mice were infected with virulent ST and at 3 days and 7 days post infection. Splenocytes were measured for production of IFNγ by antigen specific CD4+ T cells using intracellular cytokine staining (ICS). Antigens used were EBST and HKST (2 x107 cells/ well) and anti-CD3 antibody as a positive control and unstimulated splenocytes as a negative control. Percentages in the gated region of dot plots indicate the proportion of CD4+IFNγ+ of total CD4+ splenocytes (Fig 7A). Data are representative of three-five individual mice per group. The bar graph data represents mean ± SEM of 3–5 mice per group. The Y axis represents percentage of CD4+IFNγ+ of total CD4+ splenocytes (Fig 7B) on days 3 and 7. Statistics were determined for comparisons between group for each antigen at the indicated day. *, p≤0.05.
Fig 8.
EBST immune mice exhibit Salmonella specific CD4+TNF+ T cell responses during virulent ST challenge.
Individual EBST immune, AroA immune, and Sham immune mice were infected with virulent ST and at 3 days and 7 days post infection splenocytes were measured for production of TNFa by antigen specific CD4+ T cells using intracellular cytokine staining (ICS). Antigens used were EBST and HKST (2 x107 cells/ well) and anti-CD3 antibody as positive control. Unstimulated splenocytes were included as control. Percentages in the gated region of dot plots indicate the proportion of CD4+TNF+ of total CD4+ splenocytes (Fig 8A) The data represents three-five individual mice per group. The bar graph data represents the mean ± SEM of 3–5 mice per group. Y axis represents percentage of CD4+TNF+ of total CD4+ splenocytes (Fig 8B) on days 3 and 7. Statistics were determined for comparisons between group for each antigen at the indicated day. *, p≤0.05.
Fig 9.
Salmonella specific CD8+IFNγ+ T cell responses in EBST, Live ST and Sham mice during virulent ST challenge.
Individual EBST immune, AroA immune, and Sham immune mice were infected with virulent ST and at 3 days and 7 days post infection splenocytes were measured for production of IFNγ by antigen specific CD8+ T cells using intracellular cytokine staining (ICS).Antigens used were EBST and HKST (2 x107 cells/ well) and anti-CD3 antibody as positive control. Unstimulated splenocytes were included as control. Percentages in the gated region of dot plots indicate the proportion of CD8+IFNγ+ of total CD8+ splenocytes (Fig 9A). The data represents three-five individual mice per group. The bar graphs represents mean ± SEM of 3–5 mice per group. Y axis represents percentage of CD8+IFNγ+ of total CD8+ splenocytes (Fig 9B) on days 3 and 7. Statistics were determined for comparisons between group for each antigen at the indicated day. *, p≤0.05.
Fig 10.
Salmonella specific CD8+TNF+ T cell responses in EBST, Live ST and Sham mice during virulent ST challenge.
Individual EBST immune, AroA immune, and Sham immune mice were infected with virulent ST and at 3 days and 7 days post infection splenocytes were measured for production of TNFa by antigen specific CD8+ T cells using intracellular cytokine staining (ICS). Antigens used were EBST and HKST (2 x107 cells/ well) and anti-CD3 antibody as positive control. Unstimulated splenocytes were included as control. Percentages in the gated region of dot plots indicate the proportion of CD8+TNF+ of total CD8+ splenocytes (Fig 10A). The data represents three-five individual mice per group. The bar graph represents mean ± SEM of 3–5 mice per group. Y axis represents percentage of CD8+TNF+ of total CD8+ splenocytes (Fig 10B) on days 3 and 7. Statistics were determined for comparisons between group for each antigen at the indicated day. *, p≤0.05.
Fig 11.
EBST immune mice exhibit Salmonella specific multifunctional CD4+ T cells during virulent Salmonella challenge.
Simultaneous production of cytokine IFNγ and TNF by antigen specific CD4+ T cells was measured using multiparameter flow cytometry and intracellular cytokine staining (ICS) of splenocytes for individual EBST immune, AroA immune and Sham immune mice at 7 days post infection with virulent ST (Fig 11A). For comparison, naïve mice splenocytes were included. Antigens used were EBST and HKST (2 x107 cells/well) and anti-CD3 antibody as positive control. Unstimulated splenocytes were included as control. The percentages in the gated region of dot plots indicate the proportion of CD4+IFNγ+TNF+ of total CD4+ splenocytes. Data are representative of three individual mice per group. The bar graphs represent mean ± SEM of 3 mice per group. Y axis represents percentage of CD4+IFNγ+TNF+ of total CD4+ splenocytes (Fig 11B).
Fig 12.
EBST remains immunogenically stable during storage at room temperature for extended time period.
EBST formulation stored at different temperature conditions such as room temperature (RT), 4C, -20°C and after lyophilization induced stable maturation of DC2.4 cells indicated by the upregulation of (A) MHC-II, (B) CD40, (C) CD80, and (D) CD86 for up to 6 months of storage. Freshly prepared live S. Typhimurium was included as a positive control to stimulate DC2.4 cells, unstimulated DC2.4 cell provided base level expression of different surface markers on DC2.4 cells. Data represents mean values of proportion of cells positive for indicated surface marker. Bars with differing letters indicate significant (p≤0.05) differences among groups for each individual activation marker.