Figure 1.
Co-stimulatory molecule expression on DC following antigen pulsing.
a) Upregulation of MHC-II and co-stimulatory molecules on murine BMDCs. Costimulatory molecules on DCs were routinely upregulated by rPA and CpG following overnight co-culture whilst heat killed B. anthracis induced weak responses. Samples were analysed using a one-way-Anova with a Tukey post-hoc test (* p<0.05, ** p<0.01, *** p<0.001). b) Upregulation of CD40 and CD80 on human DC with rPA. Graphs represent mean±SEM proportion of DC expressing CD40 (n = 7), CD80 (n = 10) and CCR7 (n = 8). Levels of CD40 expression are also represented by mean±SEM positive intensity (PI) ratio (n = 7). One-way ANOVA (repeated measures) with ad-hoc Bonferroni corrections was applied (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). c) Confirmatory experiments representing effects of rPA/CpG combined on levels of CD40 and CD80 expression, compared to rPA alone (and basal conditions/medium only). d) Confirmatory experiments representing effects of combination of vaccine antigens (rPA and HK B. anthracis) with CpG compared to rPA/CpG combination (n = 2).
Figure 2.
Stimulation of T cell responses.
a and b) DCs were stimulated with rPA only (DC vaccine rPA only) or with PA, heat killed B. anthracis and CpG oligonucleotides (DC vaccine) and administered i.d. to A/J mice. Spleens were taken at 7 days (a) and 14 days (b) post- vaccination and restimulated ex vivo with rPA. A one-way Anova with Tukey multiple comparison post-hoc test was performed on the results (* p<0.05, ** p<0.01, *** p<0.001). Error bars represent the standard error of the mean calculated from the means of three replicates from five animals. c) Proliferation of CD4+ naive human T cells following 5-day co-culture with syngeneic human DC pulsed with medium only, B. cereus or rPA (n = 5). After 2-way ANOVA analysis with Bonferroni corrections, there were no significant differences between B. cereus and medium only (basal)-pulsed DC regarding stimulatory capacity but rPA-pulsed DC were significantly more stimulatory than basal conditions at 4% and 6% (p<0.05 in both cases) and significantly more stimulatory than B. cereus pulsed DC at 6% (p<0.05). d) CD4+ naive human T-cell proliferation following 5-day co-culture with syngeneic DC pulsed with CpG only, rPA with CpG or combination of rPA, CpG and HK B. anthracis (n = 3). After 2-way ANOVA with Bonferroni corrections, there were no significant differences between CpG and rPA/CpG-pulsed human DC regarding stimulatory capacity but rPA/CpG/HK B. anthracis-pulsed human DC were significantly more stimulatory than both other conditions at 6% (p<0.001 in both cases).
Figure 3.
Cytokine production by human T-cells.
Cytokine production by human T-cells stimulated by 6% medium (basal) or B.cereus or rPA-pulsed syngeneic DC (n = 5) (Panel A) or by 6% CpG-, rPA/CpG- or rPA/CpG/HK STI-pulsed syngeneic DC (n = 3) (Panel B) following 5-day co-culture. Paired t-test was applied, p<0.05 was considered statistically significant (*p<0.05, **p<0.01, ***p<0.001).
Figure 4.
IFNγ spot- forming cells as measured by ELISPOT.
DCs were stimulated with rPA, heat killed B. anthracis and CpG oligonucleotides and administered i.d. in combination with rPA and alum given i.m to A/J mice. Spleens were taken seven and fourteen days post vaccination and restimulated with rPA. A one-way Anova with Tukey multiple comparison post-hoc test was performed on the results (* p<0.05, ** p<0.01, *** p<0.001). Error bars represent the standard error of the mean calculated from the means of three replicates from five animals.
Figure 5.
Antibody responses 14 days after either stimulated DCs, or rPA and alum, or both, were administered.
Each point represents the mean of three replicates from five animals. A one-way Anova with Tukey multiple comparison post-hoc test was performed on the results (* p<0.05, ** p<0.01, *** p<0.001).
Figure 6.
Groups of 5 A/J mice immunised with a DC vaccine (comprising of DCs stimulated overnight with rPA, heat-killed B. anthracis and CpG), rPA in alum, or rPA in alum plus DC vaccine, or left naïve, were challenged at 14 days post-immunisation with 3×104 CFU B. anthracis STI (i.p.) and monitored for survival over the subsequent 8 days. There was a statistically significant difference in the survival curves for the negative control (alum+BMDC&CpG) group compared with the group actively immunised with rPA& alum and also receiving rPA&HK B.anthracis-pulsed DC (p<0.002) by both the Mantel-Cox and Gehan-Breslow-Wilcoxon tests. Comparison of survival curves between the negative control (alum+BMDC&CpG) group and the group receiving rPA&HK B.anthracis-pulsed DC only, was also statistically significant (p<0.01) when both tests were applied.
Figure 7.
Enumeration of viable B. anthracis in the spleens of cohorts of mice two days post challenge.
Each point represents the mean of three replicates from five animals, four from the naïve group due to an early death. A one-way Anova with Tukey multiple comparison post-hoc test was performed on the results (* p<0.05, ** p<0.01, *** p<0.001).