| Attenuated mutant strains of pathogenic bacteria, like Salmonella, Escherichia coli(E.coli), Shigella and Listeria, have been shown that they can act as live vectors to present heterologous antigens or DNA encoding them to the targeting immune sites or cells. Immunization of mice or other animals with recombinant forms of such attenuated bacteria induced effective immune responses, including cellular, humoral and mucosal immunity against heterologous antigens. But to date, it is still unclear that the mechanisms of stimulation of immune system with these organisms. A clearly knowledge of the mechanisms would be useful for the design of vaccine, especially for those intracellular pathogens induced diseases. To address these questions, the recombinant E. coli strain 13 A or 25A and recombinant Salmonella typhimurium strain SL7207 expressing CD8+ T cell epitopes of Lymphocytic Choriomeningitis Virus (LCMV) and Ovalbumin (OVA), which were fused at the terminal of green fluorescent protein (GFP) marker, or delivering DNA vaccines encoding these epitopes were constructed, and to study the efficient delivery of antigen to major histocompatibility complex class I (MHC I) pathway by bacteria vectors in order to stimulate CD8+ T cell immunity.Newcastle disease (ND), a highly contagious disease with worldwide distribution, can cause severe economic losses to the poultry industry. Some traditional vaccines have been verified that they are important for controlling the infection and epidemic of this disease, but several disadvantages are unable to be avoided, e.g. expensive cost orvirus distribution. In order to develop new ND mucosal vaccines which are safe and with high protective efficacy, firstly, the fusion protein (F) gene of a virulent NDV strain JS/5/01 (JS5) isolated from goose was cloned, sequenced and analyzed, secondly, the NDV DNA vaccine delivered by attenuated Salmonella typhimurium was constructed, and finally, the immunobiological properties of this recombinant bacteria were evaluated in mice and chickens.1. Mechanisms of in vitro delivering CD8+ T epitopes through attenuated bacteria.To verify the correct of eukaryotic expression plasmid constructs, pG2F, pDG2F or pG1B were transfected into LKb and LLd fibroblast cells with Liposome Fugene 6. GFP expression was observed in cells by FACS. The results of in vitro antigen presentation assay showed that OVA and LCMV CD8+ T cell epitopes were allowed to be presented to T hybridoma B3Z and nV1H7 by LKb and LLd cells respectively. It suggested that all plasmid constructs can express OVA and LCMV T cell epitopes fused at the N or C-terminal of GFP gene in eukaryotic expression system and allow processing and presentation of these epitopes to CD8+T cell.Following infection of LKb cells, LLd cells or bone marrow dendritic cell (BMDC) with recombinant E.coli and Salmonella strains harboring different plasmids, about 90% of cells were GFP positive in prokaryotic expression system , and 0-5% of GFP positive cells was observed in eukaryotic expression system. The results indicated that attenuated E.coli strain 13A, 25A and attenuated Salmonella strain SL7207 had better invasive capacity to LKb and LLd cells, and BMDC had strong capacity for engulfing recombinant E.coli.The results verified that recombinant bacteria can deliver the eukaryotic expression plasmids into mammalian cells.The efficiency of MHC class I presentation of OVA and LCMV CD8+ T cell epitopes from recombinant E.coli and Salmonella were analyzed using LKb and LLd cells as antigen presenting cell (APC) infected with 13A (ptG2F), 25A(ptG2F), SL7207(ptG2F) or SL7207(pDG2F). OVA CD8+ T cell epitope presented on the surface of those LKb cells infected with 13A (ptG2F), SL7207(ptG2F) and SL7207(pDG2F) were recognized by B3Z T hybridoma specific for OVA peptidep257-264 at 2h post infection, but the efficiency of MHC class I presentation of OVA CD8+ T cell epitope was lower at 48h post infection. LCMV CD8+ T cell epitope presented on the surface of those LLd cells infected with 13A(ptG2F) was recogn... |
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