Immunobiological Properties Of The Prime-boost Strategy With DNA Vaccine Delivered By Attenuated Salmonella Typhimurium And Killed Oil-emulsified Vaccine Against H9 Subtype Of Avian Influenza Virus

Avian influenza,a highly contagious disease with worldwide distribution,can cause severe economic losses to the poultry industry with its high percent of disease and death.The killed oil-emulsified vaccines of avian influenza virus (AIV) 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,potential dangerous of virus distribution and unable to induce cellular and mucosal immune responses.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.Although DNA vaccine can stimulate the local mucosal immunity,the systemic immunity and the protective T cell immunity,but compared with the conventional vaccine,the antibody level caused by DNA vaccine is still not very high. In order to study the synergism protection of immunity of the DNA vaccine with the killed oil-emulsified vaccine of H9N2 AIV , two types of DNA vaccines named SL7207(pmcDNA-HA) and SL7207(pCAGGS-HA) were construsted, which delivered by attenuated Salmonella typhimurium 7207 respectively , and then the immunobiological properties of the prime-boost strategy with the DNA vaccine and killed oil-emulsified vaccine were evaluated in chicken model.1. Construction and identification of recombinant Salmonella typhimurium harbouring DNA vaccine against H9 subtype of avian influenza virusFirstly,a pair of primers were designed and synthesized according to the previously published sequence in GenBank and used to amplify HA gene by reverse-transcription polymerase chain reaction from the genome RNA of a H9 subtype AIV strain A/chicken/Yang Zhou/N/2005(H9N2).And then the PCR product was cloned into pcr2.1-T easy vector for sequencing.The recombinant plasmid was identified by restriction endonuclease analysis and sequencing technique.HA nucleotide sequence homology was compared with those of some H9N2 subtype AIV published in GenBank.The results showed that the highest homology was 99% and was 83% compared with A/Turkey/Wisconsin/1/1966.The amino acid sequence of HA cleavage site indicated that this strain had low virulence.Subsequently,HA gene was cloned into eukaryotic expression vector pmcDNA3.1 through Kpnâ… and Xhoâ… double digestion;HA gene were cloned into prokaryotic expression vector pET- through Hindâ…¢and Xhoâ… double digestion respectively.Then the HA gene which were digested by BamHâ… and Xhoâ… from pET-HA was also cloned into eukaryotic expression vector pCAGGS which were digested by Bg1â…¡and Xhoâ… .The recombinant plasmids pmcDNA3.1-HA and pCAGGS-HA were transfected COS-7 cell respectively , and the transient expression of HA gene was identified by indirect immunofluorescent assay.Finally,the recombinant plasmids were transformed into the attenuated salmonella typhimurium SL7207 respectively , and the recombinants were screened and designated as SL7207(pmcDNA3.1-HA) and SL7207(pCAGGS-HA),which made a bedrock to evaluate their safety and immune efficacy.2. Immunobiological properties of the prime-boost strategy with DNA vaccine delivered by attenuated Salmonella typhimurium and killed oil-emulsified vaccine against H9 subtype of avian influenza virusIn order to evaluated the protective efficacy of the vaccines,one-day-old commercial ISA brown chickens were immunized with SL7207(pmcDNA3.1-HA),SL7207(pCAGGS-HA),SL7207(pmcDNA3.1-HA) plus killed oil-emulsified vaccine of AIV,SL7207(pCAGGS-HA) plus killed oil-emulsified vaccine of AIV,SL7207 (pmcDNA3.1),SL7207(pmcDNA3.1) plus killed oil-emulsified vaccine of AIV.Negative control groups [PBS control and SL7207(pmcDNA3.1) control] and positive control group (killed oil-emulsified vaccine of AIV) were included.The serum antibodies responses specific to H9 hemagglutinin of AIV were low in chickens immunized with all recombinant bacteria on day 16 or 30 post immunization,and there was no significant difference between each group (p>0.05).Two weeks (44d) after boost immunization,The level of serum antibodies was still low in recombinant bacteria immunized groups,and the negative control groups,the difference between the groups was not statistically significant (p>0.05).On the other hand,compared with the recombinant bacteria immunized groups,the negative control groups,the level of serum antibodies was increased significantly in groups of SL7207 (pmcDNA3.1-HA) plus inactivated vaccine of AIV and SL7207(pCAGGS-HA) plus inactivated vaccine of AIV (p<0.05).The highest level of serum antibodies was the group of SL7207(pCAGGS-HA) plus killed oil-emulsified vaccine of AIV.The intestinal mucosal antibodies against avian influenza HA protein can be detected in recombinant bacteria harboring DNA vaccine groups and the prime-boost groups after two weeks of the boost immunization.There was significant difference compared with those of the negative control groups and the inactivated vaccine group (p<0.05).Among all groups,the SL7207(pCAGGS-HA) group had the highest level of the intestinal mucosal antibody titers.The immune protection assay showed that each DNA vaccine delivered by recombinant bacteria can supply protection against avian influenza virus in chickens,and there was significant difference compared with the negative control.The immune protection rate of the recombinant bacteria plus inactivated vaccine of AIV group was higher than that of the SL7207 (pmcDNA3.1-HA) group,SL7207(pCAGGS-HA) group and the killed oil-emulsified vaccine of AIV group.