In Vivo Visualization of Bacterial Colonization, Antigen Expression, and Specific T-Cell Induction following Oral Administration of Live RecombinantSalmonella entericaSerovar Typhimurium

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Abstract
Live attenuatedSalmonellastrains that express a foreign antigen are promising oral vaccine candidates. Numerous genetic modifications have been empirically tested, but their effects on immunogenicity are difficult to interpret since important in vivo properties of recombinantSalmonellastrains such as antigen expression and localization are incompletely characterized and the crucial early inductive events of an immune response to the foreign antigen are not fully understood. Here, methods were developed to directly localize and quantitate the in situ expression of an ovalbumin model antigen in recombinantSalmonella entericaserovar Typhimurium using two-color flow cytometry and confocal microscopy. In parallel, the in vivo activation, blast formation, and division of ovalbumin-specific CD4+T cells were followed using a well-characterized transgenic T-cell receptor mouse model. This combined approach revealed a biphasic induction of ovalbumin-specific T cells in the Peyer's patches that followed the local ovalbumin expression of orally administered recombinantSalmonellacells in a dose- and time-dependent manner. Interestingly, intactSalmonellacells and cognate T cells seemed to remain in separate tissue compartments throughout induction, suggesting a transport of killedSalmonellacells from the colonized subepithelial dome area to the interfollicular inductive sites. The findings of this study will help to rationally optimize recombinantSalmonellastrains as efficacious live antigen carriers for oral vaccination.