Basic Research On A Novel Biologically Inactivated Vaccine For Brucella And Its Differential Diagnosis

Brucellosis is a zoonotic disease caused by Brucella spp.,of which spread and distribute worldwide.B.melitensis,B.abortus and B.suis are highly pathogenic to both humans and animals with clinical manifestations of abortion,stillbirth,foetal weakness,endometritis,and mastitis in animals.Brucella can spread rapidly through the herd and cause serious economic losses.Vaccination is the optimal way to control Brucellosis in animals and humans,but the current live attenuated Brucellosis vaccines（e.g.,S19,Rev.1,S2,M5 and RB51）have shortcomings,including residual virulence,risk of infection in humans and pregnant animals,and inability to differentiate infection from vaccinated animals.The bacterial ghost（BG）has an empty shell and retain an intact bacterial structure.The bacterial ghosts were prepared by using temperature-controlled expression of the phage E gene,which could induce well immune responses.However,absolute inactivation is the bottleneck in the current ghost preparation,thus introducing the secondary killing is necessary to ensure absolute inactivation.In this study,the phage lysis gene E was cloned into a temperature-controlled plasmid and the Brucella abortus A19BG strain was constructed after electroshock transformation,which had a typical bacterial morphology and was genetically stable for 20 consecutive passages.A combination of phage lysis and 0.1%H₂O₂could reach100%of inactivation efficiency.Absolute inactivated Brucella BG were mixed with ISA206 VG adjuvant and were evaluated in a guinea pig model,bacterial ghost A19BG had a typical morphology and showed genetical stable,which showed no adverse reactions in guinea pigs after vaccination with A19BG.The levels of antibodies,interferon IFN-γ,interleukin IL-4 and CD4+T cells in guinea pigs were like those with A19.Immunization of guinea pigs and cattle with A19BG was effective and protective like the results of A19 after challenged with Brucella abortus M28,with 83.3%and60%in guinea pigs and cattle,respectively,.A mass spectrometry-based,label-free,relative quantitative proteomics approach were used to investigate the global proteomic changes between A19BG and A19.Proteomic microarrays identified 2,014 proteins,1,116 of which were differentially expressed compared to A19,with shared immunoproteins such as OMP（Bcsp31,Omp25,Omp10,Omp19,Omp28 and Omp2a）.The antibodies against HSP（Dna K,Gro S and Gro L）and Sod C were no lower than A19,these proteins may underlie the immunoprotective effects of A19BG.Antibody profiling identified more than 30specific immunogenic proteins,among which BMEII0032 and BMEI0892 proteins showed a significant difference between A19 and A19BG（p<0.05）,which could be used as differential diagnostic antigens for A19BG candidates.In summary,this study developed a complete inactivation method and prepared a reliable safety and efficacy ghost vaccine.We revealed the mechanism of protective efficacy of the A19BG,and screened two differential diagnostic markers.The results provided an alternative technical tool for the prevention and control of Brucellosis.