The Study Of Dendritic Cells Targeting Conserved AIVs Antigen Expressed By Lactobacillus Plantarum NC8

Avian influenza virus(AIV) is an important zoonotic pathogen, AIVs not only could cause serious economic losses to the poultry industry but also could represent a signi?cant threat to mammals health. That drives people to develop effective vaccines against it. The commercialized vaccines protect against AIVs by inducing antibody. Because of antigen changes(shift and drift) in the virus, vaccines targeting AIV surface proteins, such as haemagglutinin(HA), only offer limited protection against AIVs. The core nucleoprotein(NP) and matrix protein(M1), highly conserved internal proteins among the different subtypes AIVs represent attractive targets for preventive and therapeutic intervention against diverse AIVs. The NP and M1 vaccine inducing subtype cross-reactive T cells to internal AIV antigens against diverse AIVs indicates the opportunity of a universal influenza vaccine capable of inducing broadly heterologous protective immunity against the conserved viral proteins.In this study, NP and genes from AIVs mH9N2 were amplified, cloned and sequence analyzed. By molecular biology techniques, we have constructed the prokaryotic expression vector, which could simultaneously express NP gene, M1 gene, and DCpep gene. Following, recombinant Lactobacillus NC8-pSIP409-NP-M1-DCpep based on Lactobacillus plantarum(NC8) as expressed vector with exogenous genes was constructed and its expression was verified by western blotting. These results indicated that we have successfully constructed recombinant Lactobacillus NC8-pSIP409-NP-M1-DCpep expressing NP-M1-DCpep genes, which express fused protein has possessed well reaction-ogenicity with monoclonal NP antibody.In next experiment, Murine bone marrow-derived DC cells were subjected to recombinant lactobacillus NC8-pSIP409-NP-M1-DCpep treatment. Subsequently, the surface marker of activated DC and the expresssion of cytokine were measured by flow cytometry and ELISA. To examine the surface marker of DC in Peyer’s patches(PP), MLN, and small intestine, additional experiments were performed using immune mice, which were orally immunized with recombinant lactobacillus NC8-pSIP409-NP-M1-DCpep by flow cytometry. Afterward, we also detected the expressing of CD4+IFN-γ+ or CD8+IFN-γ+ in MLN and spleen, and B cells secreted lgA or the activated B cells in PP using flow cytometry. The results indicated that recombinant lactobacillus NC8-pSIP409-NP-M1-DCpep could induce B7 molecule activation from murine bone marrow-derived DC in vitro and could induce DC activation of PP and small intestine in vivo. In addition, recombinant lactobacillus NC8-pSIP409-NP-M1-DCpep not only could induce specificity of T response, but also could induce B cells activation of small intestine, promoting form germinal center in PP resulting in enhanceing screting of sIgA.We hypothesized the existence of a potential heterologous protective immunity response with NC8-pSIP409-NP-M1-DCpep. To test this hypothesis, mice were challenged with adaptability homology AIVs and heterologous H1N1 after orally immunized with NC8-pSIP409-NP-M1-DCpep. The results showed that although recombinant lactobacillus NC8-pSIP409-NP-M1-DCpep could not protect mice from 10×LD50 H1N1, it could well protect BALB/c mice from homology AIVs and immuno-protect against 0.5×LD50 H1N1 AIVs. To study the mechanism of recombinant lactobacillus against AIVs, NOD-SCID-/- mice were used. Two weeks after the last immunization of mice, splenetic cells were isolated and stained using CFSE, then were subjected to special antigen treatment for 3d. The proliferation of CD4+ and CD8+ T cells and the expressing of CD8+CD107a+ were measured by flow cytometry in spleen. The single cell was stained by different CFSE concentration from in-immunized mice spleen. CFSE staining cells were subjected to special antigen treatment for 3h. For CFSE spiking experiments, splenocytes were transferred in sterile PBS into immunized mice by intravenous injection. Then, CD8+ CTL in immunized murine spleen was established by flow cytometry after 18 h. To verify whether NOD-SCID mice were or not protected from AIVs by immunizing murine T cells subset, CD4+ T and CD8+ T cells from immunized murine splenocytes were positively isolated from the resulting splenocytes using magnetic bead separation. Then, CFSE spiking splenocytes were transferred in sterile PBS into NOD-SCID recipients by intravenous injection. Furthermore, NOD-SCID recipients were challenged homology AIVs after 12 h. The above data established recombinant lactobacillus could significantly induce specificity CD4+ T and CD8+ T proliferation, enhance expression of CD8+CD107a+, and CTL response from immunized murine splenocytes. Importantly, CD8+ T alone were transferred in sterile PBS into NOD-SCID recipients by intravenous injection, resulting in protecting NOD-SCID mice from homology AIVs. But, CD4+ T cells and CD4+ T cells and CD8+ T(ratio1:1) could not protectNOD/SCID mice from homology AIVs.In this study, recombinant lactobacillus NC8-pSIP409-NP-M1-DCpep, expressing NP gene, M1 gene, and DCpep, has been constructed. The recombinant lactobacillus could induce both specificity of T cell response and mucosal immune response, resulting in partly protecting mice from homology and heterologous AIVs. Additional experiments were performed using NOD-SCID mice. The results indicated that CD8+ T cells played a major role against...