Influence of promoter, gene copy number, and preexisting immunity on humoral and cellular responses to a vectored antigen delivered by a Salmonella enterica vaccine

Abstract

Attenuated Salmonella strains are currently in production as vaccines for protection of animals against salmonellosis. Such commercial strains offer the potential to deliver heterologous antigen to protect animals against other diseases. One vaccine strain, attenuated Salmonella enterica serovar Typhimurium (STM-1), was tested for the ability to deliver ovalbumin and to induce immune responses in mice. Two vaccine trials were performed testing the influence of promoter choice, the location of the encoding DNA (plasmid or chromosome), and the effect of preexisting homologous or heterologous immunity. The results demonstrated that humoral and T-cell responses were induced from either of two promoters, from either the plasmid or the chromosome, and that preexposure to the empty homologous vector, STM-1, or the heterologous vector, S. enterica serovar Enteritidis, had no detrimental effect on subsequent antigen-specific responses. In the case of homologous preexposure, responses were generally greater, and this was correlated with an increased uptake of Salmonella by macrophages in vitro after opsonization with immune sera.

FIG. 1.

Immune responses observed in trial 1. Groups of mice were vaccinated with the indicated constructs. Note that in groups 5 to 12 (from left), as in the control groups 1 to 4, the delivery vehicle was STM-1. This has been omitted for brevity. (A) Humoral responses elicited after vaccination with ovalbumin constructs. **, P < 0.001 compared to the respective control group. (B) Enumeration of IL-4-secreting splenocytes from mice vaccinated with ovalbumin constructs. *, P < 0.05, and **, P < 0.001 compared to the respective control group. (C) Enumeration of IFN-γ-secreting splenocytes from mice vaccinated with ovalbumin constructs. *, P < 0.05, and **, P < 0.001 compared to the respective control group. The error bars indicate standard deviation. SFC, spot-forming cells.

FIG. 2.

Humoral immune responses observed in trial 2. Groups of mice were vaccinated once or twice according to the schedule outlined in Table 4. (A) Evaluation of humoral immune responses against the carrier antigen (chicken ovalbumin) at week 6 in groups 1 to 4. Significant differences in titer were observed between groups 2 to 4 and group 1 (**, P < 0.001), and a significant difference was observed between the group that was preexposed to the vector strain (group 3) and the unexposed group (group 2) (X, P < 0.05). S.E, S. enterica serovar Enteritidis. (B) Evaluation of humoral immune responses at week 15 in groups (Gp) 1 to 8. Significant differences in titer were observed between groups 2 to 4 and group 1 and between groups 6 to 8 and group 5 (**, P < 0.001). A significant difference was also observed between a group that was preexposed to the vector strain (group 7) and the unexposed group (group 6) (P < 0.05). The error bars indicate standard deviation.

FIG. 3.

Enumeration of cytokine-secreting splenocytes in trial 2. (A) IL-4. Compared with the control groups (1 and 5), mice vaccinated with ovalbumin constructs yielded significantly higher numbers (groups 2 to 4 and 6 to 8) (**, P < 0.001). A significant difference was also observed between groups that were preexposed to the homologous vector strain (groups 3 and 7) and unexposed groups (groups 2 and 6) (P < 0.001) and between one group that was preexposed to the related strain (group 8) and group 6 (P < 0.05). S.E, S. enterica serovar Enteritidis. (B) IFN-γ. Compared with the control groups (1 and 5), mice vaccinated with ovalbumin constructs yielded significantly higher numbers (**, P < 0.001). A significant difference was also observed between groups preexposed to the vector and a related strain, respectively (groups 3 and 4 and 7 and 8), and groups 2 and 6, the unexposed groups (P < 0.05). The error bars indicate standard deviation. SFC, spot-forming cells.

FIG. 4.

Analysis of differential uptake of STM-1 by J774 cells after opsonization with naïve serum, STM-1-specific serum, and S. enterica serovar Enteritidis (S.E)-specific serum. The number of enumerated cells was normalized to 100% for the maximal value. Compared to cells from control groups (STM-1 opsonized with naïve serum), test groups (STM-1 opsonized with STM-1-specific serum and STM-1 opsonized with S. enterica serovar Enteritidis-specific serum) showed significantly greater uptake (P < 0.001). Also, significantly greater uptake of STM-1 was observed in cells opsonized with STM-1-specific serum than in cells opsonized with S. enterica serovar Enteritidis-specific serum (P < 0.05). The error bars indicate standard deviation.