Cellular Immune Responses Induced By Attenuated Salmonella Harboring CD8～+T Epitopes

Attenuated Salmonella have been successfully used as vaccine vector to deliver exogenous antigen. It can drectly deliver antigen to immune system, and also stimulate specific humoral responses, cellular (CD4~+ T cell, CTL) immune responses and mucosal immune responses. Presently, the feasibility of attenuated Salmonella as vaccine vector has been identified, but the mechanism of immune responses stimulated by attenuated Salmonella or recombinant Salmonella has not yet elucidated completely, especially in cellular immune response. There are several methods for detecting specific cellular immune responses. And ELISPOT (enzyme-linked immunospot) assay has been considered as a means for detecting cytokines and become a new method in evaluating the function and specificity of T lymphocyte. Because of the strong specificity and high sensitivity, the assay has been used in analyzing and evaluating cellular immune responses specific for CD8~+ T cells already. In order to investigate the mechanism of immune responses induced by attenuated Salmonella delivering exogenous antigen, the ELISPOT assay for detecting the specific cellular immunity has been established, and analyzed the cellular immune responses induced by attenuated Salmonella typhimurium harboring CD8~+ T epitopes of Ovalbumin (OVA) and ofLymphocytic Choriomeningitis Virus nucleus protein (LCMV NP).1. Establishment of ELISPOT assay for detection of cellular immuneresponses specific for recombinant SalmonellaIn order to establish the ELISPOT assay for detection of cellular immune responses specific for recombinant Salmonella, prokaryotic expression plasmid ptG2F, carrying the oligonucleotide fragment encoding CD8~+ T cell epitopes of OVA 257-264aa (H-2~d), and LCMV NP 118-132aa (H-2~b) fused with green fluorescent protein (GFP) gene was constructed firstly and electro-transformed into the attenuated strain SL7207 of Salmonella typhimurium, the recombinant bacteria were named as SL7207(ptG2F). C57BL/6 and BALB/c mice were immunized intravenously with 1× 10~6cfu SL7207(ptG2F) in two weeks intervals. For establishment of the ELISPOT assay, splenocytes were collected from mice after second and third immunization, and then specific IFN- Y and IL-4 secreting cells were detected with OVA 257~264aa and LCMV NP 118~132aa peptides as stimulus. The results showed that the cellular immune responses specific for recombinant bacteria SL7207(ptG2F) can be detected by the ELISPOT assay. When OVA CD8~+ T cell epitope is presented by the immunized mice, the immune responses are biased toward Thl response after second immunization and then switch to the Thl and Th2 balance after third immunization. In contrast, when the LCMV NP CD8~+ T cell epitope is presented, the level of Th2 immune response is higher than that of Thl after both second and third immunization. Altogether, these results indicates that the ELISOPT assay can detect the specific cellular immune response induced by recombinant Salmonella, and it also shown that attenuated Salmonella can effectively deliver the CD8~+ T cell epitopes and induce specific cellular immune responses.2. Kinetic analysis of cellular immune responses initiated by recombinant Salmonella through oral immunizationTo analyze the dynamic change of cellular immune responses against CD8+ T cell epitope delivered by recombinant Salmonella after oral immunization. BALB/c mice were immunized orally with SL7207(ptG2F) in two weeks intervals. IFN- Y and IL-4 secreting cells were detected by ELISPOT assay in murine peyer's patches cells after second , third , forth and fifth immunization respectively. Meanwhile, antibodies against GFP were determined in serum, fecal extract and intestinal mucosa by ELISA. The results showed that recombinant bacteria SL7207(ptG2F) can induce cellular immune responses specific for LCMV NP CD8+ T epitope, and present the different types after different times point of immunization. IFN- Y and IL-4 secreting cells specific for LCMV NP CD8+ T cell epitope could be detected after second immunization, and the frequenc...