Evaluation And Mechanism Studies On Immune Efficacy Of Genetic Engineering Vaccine Against Foot-and-mouth Disease

Foot and mouth disease(FMD) caused by foot and mouth disease virus(FMDV) is an infectious disease affecting cloven-hoofed animals.This disease poses a serious threat for animal health and an economic devastation might result in the massive animal death and the economic sanction from other countries on account of the FMD.In countries where disease eradication has not been achieved,vaccination plays a crucial role in its control. Although inactivated virus vaccines effectively prevent FMD,their use is accompanied by dangerous residual potency problems,including incomplete viral inactivation or virus escape from vaccine-producing facilities.As a result,genetic engineering vaccination offers an attractive alternative to overcome the disadvantages of inactivated virus vaccine。The FMD is caused by foot-and-mouth disease virus(FMDV),which is a member of the picornavirus family.The virus particle contains 60 copies of each of four structural proteins that designated VP1,2,3 and 4,respectively.P1 protein which constructing the viral empty capsid contained almost all antigenic epitope,and the antigen site 1,the RGD sequence,within the VP1 G-H loop is the main neutralizing epitope.In recent studyies, genetic engineering vaccines utilizing antigen site 1 as antigen could elite high titer of antibody,but could not completely protect animals after FMDV assaulting.The results of these studies suggest the importance of other antigen sites in eliciting anti-FMDV immune response.Thus,it is better to utilize P1 as an antigen.The maturation cleavage,P1 is cleaved into VP0,VP3 and VP4,are performed by 3C protein.So in this study,we construct vaccines utilizing codon-optimizated P1 and 3C gene as antigen and evaluate their immunization effect in BALB/c mice.In this study,we synthesized the fragments of P12A3C gene.The whole-long P12A3C gene was amplified by over-lapping PCR and cloned into the transfer vector pAdTrack-CMV.The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E.coli strain BJ5183.Taking the advantage of the high efficient homologous recombinant machinery presented in bacteria,the recombinant adenoviral backbone plasmid was generated in BJ5183,and then was transfected into 293 cells. Recombinant adenovirus was propagated in 293 cells with high titers.We have also constructed the control recombinant adenovirus AdEasy.The transcription of P12A3C and expression of P12A3C protein were detected by PCR,RT-PCR and ELISA.Furthermore, we also constructed DNA vaccine pcP12A3C by cloning the P12A3C gene into plasmid pcDNA3.1(+).Six-to eight weeks old female BALB/c mice were inoculated with newly-constructed genetic engineering vaccine.Further,we evaluated their ability to induce specific humoral immune response by iELISA,liquid block ELISA and neutralization test,and their ablity to induce specific cellular immune response by detection cytocine secretion,lymphocyte proliferation assay,and specific CTL activity detection.In results,we have constructed the recombinant adenoviral vector and rencombinant plasmid DNA encoding P12A3C gene of FMDV,which both can express P12A3C antigen when in vitro infected into cells.Balb/c mice immunized with both pcP12A3C and AdP12A3C induce persistent anti-FMDV humoral and cellular immune response.The neutralizing antibody induced by recombinant adenovirus AdP12A3C was above 1:32,which suggest the antibody can effectively neutralized FMDV.However,DNA vaccine pcP12A3C only induce low level of neutralizing antibody.Both DNA vaccine and recombinant adenovirus vaccine elicited Th1 type cellular immune response and induce innoculated mice producing FMDV specific CTL activity to kill target cells.In conclusion,the recombinant adenovirus expressing P12A3C can effectively stimulate specific humoral and celluler immune response in mice and is potentially to be used as a candidate vaccine for both prevention of FMDV infection.The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science.Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve.Residual genomic DNA of engineering bacteria has been identified as a potential risk factor,so whose level must be controlled under the regulatory standards.We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines.The assay utilizes PCR amplified and digoxigenin-labeled E.coli 16S rRNA gene as probe.The sensitivity of the dot-blot hybridization assay with E.coli 16S rRNA gene probe was evaluated in comparison with single copy uidR gene probe.The optimized dot-blot hybridization assay had both low background and a suitable sensitivity,detecting 10 pg of residual E.coli DNA.The method is suitable in the routine use of measuring the levels of residual E.coli DNA in the pharmaceutical-grade DNA vaccine.