Construction And Characterization Of A Novel Magnesium-inducible Lysis System Of Salmonella Delivering DNA Vaccine

Highly pathogenic avian influenza (HPAI) is a highly contagious disease of poultry caused by influenza A viruses. It caused serious economic loss and negatively influenced poultry industry and international trade. Moreover, it has become a serious threat for public health. The killed vaccine has 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. potential dangerous of virus distribution and unable to induce effitive cellular and mucosal immune responses. Thus, developing a safe and high protective vaccine is necessary.Attenuated Salmonella typhimurium can be used as vectors to directly deliver DNA vaccine into immune system from mucosal approach, then the heterologous antigen can be expressed in vivo and induces the protective humoral, cellular and local mucosal immune response. Using attenuated Salmonella typhimurium as vectors to deliver DNA vaccines is a novel promising method to control avian influenza.Attenuated Salmonella carrying DNA vaccine can not rapidly release the inclusion into the cytosol of the eukaryotic cells it invaded, which restrict the development of recombinant Salmonella vaccine vectors.A magnesium-inducible lysis Salmonella which can induce to lysis by low concentration of Mg²⁺ was constructed to improve the ability to release DNA vaccine and to induce enhanced immune responses against a heterologous antigen. Then the AIV DNA vaccine delivered by attenuated Salmonella typhimurium was constructed, and the immunobiological properties of the recombinant bacteria was evaluated in mice.1. Construction and identification of a novel magnesium-inducible lysis-delivering system of SalmonellaThe novel plasmid pPL107 was constructed by inserting the fusion gene S107 which express Lambda phage lysis genes under the control of the magnesium-inducible promoter mgtCB of Salmonella into the DNA vaccine vector pPL which do not contain antibiotic resistance gene. The lysis gene S107 can be expressed in Salmonella when incubate in culture medium without Mg²⁺. The red fluorescence report protein gene DsRed was inserted respectively into the multiple cloning site (MCS) of pPL107 and pPL. These new recombinant plasmids pPL107DsRed and pPLDsRed were harvested fromΔasd E. coli X6212 in vitro and transfected into COS-7 cells, and the strong red fluorescence was observed in transfected COS-7 cells 24 hours post transfection. These recombinant plasmids pPL107DsRed and pPLDsRed were transformed intoΔasdΔsifA S. typhimurium X4550*, and the recombinants were designated as X4550*(pPL107DsRed) and X4550*(pPLDsRed) respectively.A quick lysis was observed when the recombinant bacteria X4550*(pPL107DsRed) cells were resuspended in Mg²⁺-free culture medium. About 80% of the bacteria lysed in two hours and released their intracellular content plasmids into the supernatant. In contrast, no lysis phenomena or released plasmids can be detected when used the recombinant bacteria X4550*(pPLDsRed). X4550*(pPL107DsRed) and X4550*(pPLDsRed) had similar growth curve in LB medium when Mg²⁺ was added. X4550*(pPL107DsRed) but not X4550*(pPLDsRed), could transfer eukaryotic expression plasmid into the cytosol of RAW264.7 cells to express red fluorescence protein. These results showed that this magnesium-inducible lysis-delivering system could be a candidate system to act as a eukaryotic expression plasmid carrier.2.Construction and immunobiological properties of magnesium-inducible lysis-delivering system of Salmonella harbouring DNA vaccine against H5 subtype of avian influenza virusFirstly, the BamH I-Nhe I HA gene fragment was cloned into the MCS of the recombinant plasmid pPL107 and pPL from plasmid pVAX1-HA. The recombinant plasmids pPL107HA and pPLHA were confirmed by the indirect immunofluorescent assay (IFA) and it was shown that HA protein was expressed successfully in the eukaryotic cells COS-7. Finally, pPL107HA and pPLHA were transformed intoΔasdΔsifA S. typhimurium X4550*, and the recombinants were designated as X4550* (pPL107HA) and X4550* (pPLHA).Six-week-old BALB/c mice were immunized intravenously with X4550* (pPL107HA) and X4550* (pPLHA) at the dosage of 1X107CFU and boosted two weeks later with the same dose. The serum antibodies responses specific to H5 hemagglurinin of AIV were low in mice immunized with all recombinant bacteria on day 28 but a higher antibodies could be detected after challenge with live virus in two weeks. The MTT colorimetric assay was used to measure lymphocyte proliferation of mice splenocytes. And the lymphocyte proliferation of mice immunized with recombinant bacteria X4550* (pPL107HA) were significantly higher than that of mice immunized with X4550* (pPLHA) and control on day 28 (p<0.05). These results showed that increasing the releasing ability of attenuated Salmonella carrying DNA vaccine can enhance specific immune efficacy against HA of AIV.