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Background:  Clostridium perfringens has been engineered to express high levels of SIV antigenic protein for delivery to the mouse terminal ileum. Dendritic cells (DC) resident in Peyer’s patches (PP) in the terminal ileum are key mediators in mucosal immune response development. Thus, it is imperative that protein delivery via C. perfringens has the capacity to stimulate DC to mature and prime T cells.

Methods:  A modified pJRC200 vector with the cytotoxic regions of the cpe gene replaced by the SIV p27 gene was electroporated into C. perfringens to create bacteria producing high levels of p27. Bone marrow-derived DC (BMDC) and murine PP tissues were exposed to lysates of sporulating cultures of recombinant bacteria expressing p27, bacteria carrying a control plasmid, or media alone for 2 to 4 hours. BMDC and PP tissue sections were subjected to immunofluoresence staining using α-CD11c and α-p27 antibodies for the detection of DC and p27, respectively. The fate of internalized p27 was monitored by Western blot analysis with α-p27 serum. CD80, CD86, CD40, MHC class II, and internalized dextran were detected on/in CD11c-positive DC using flow cytometry. ELISA were performed to measure IL-12, IL-10, and TNF-α. T-cell reactivity was assessed by IFN-γ ELISpot and CD107a and b flow cytometry.

Results:  BMDC and PP DC were shown via immunofluorescence to internalize SIV p27 after exposure to lysates of sporulating C. perfringens vaccine. Degradation of p27 occurred in BMDC within 24 hours following exposure to the vaccine. Maturation of DC was evidenced by up-regulation of co-stimulatory and MHC class II molecules and reduced phagocytic activity. These DC-produced enhanced levels of proinflammatory cytokines, as compared with control DC. BMDC exposed to recombinant bacterial lysates, stimulated IFN-γ-producing p27-specific T cells. T cells also showed cytolytic activity against target cells displaying p27 epitopes.

Conclusions:  These data demonstrate that SIV p27 delivered via our recombinant C. perfringens can be taken up and processed by immature DC. Maturation of DC is stimulated in response to the vaccine. In addition, mature DC stimulate IFN-γ-producing and cytolytically active p27-specific T cells. These results show that DC can mediate the formation of an immune response against SIV p27 when exposed to the C. perfringens vaccine in vitro and suggest that DC will mediate strong responses in vivo following oral immunization.

Keywords: Clostridium perfringens; dendritic cell; immunity