Construction And Protection Evaluation Of Recombinant Salmonella Typhimurium Candidate Vaccines Expressing Heterologous O-antigens

Salmonella can cause gastrointestinal diseases in a variety of animals and it contains multiple epidemic serotypes.O-antigen is a protective antigen of Salmonella,and its specific antibodies mediate protective immunity.In this study,we constructed three recombinant Salmonella Typhimurium vaccine strains expressing Salmonella Newport O-antigens,and evaluated their immunogenicity and immunoprotective efficacies in mice.1 Construction of recombinant Salmonella typhimurium strainsThe expression plasmid pCZ11 by Gibson assembly method carrying a subset of the Salmonella Newport O-antigen gene cluster?wzx-wbaR-wbaL-wbaQ-wzy-wbaW-wbaZ?was introduced into three Salmonella Typhimurium mutants:SLT19??asd?,SLT20??asd?rfbN?,SLT22(?asd?rfbN?pagL::TaraC P_BAD rfbN),respectively.Silver staining and western blotting results showed that SLT19?pCZ11?expressed both Salmonella Typhimurium and Salmonella Newport O-antigens;SLT20?pCZ11?expressed only the heterologous O-antigen;SLT22?pCZ11?expressed both types of O-antigens in the presence of arabinose,and only the heterologous O-antigen when grown without arabinose.2 The assay for biological characteristics of recombinant Salmonella typhimurium strainsBiological characteristics experimental results showed that the three strains showed significantly lower motility and sensitivity to polymyxin B than the wild strain,but still showed comparable colonization and virulence to the wild strain.These results indicated that the expression of heterologous O-antigens reduced the ability of bacteria to migrate and their sensitivity to polymyxin B,but did not alter the colonization and virulence of the bacteria.3 Evaluation of immunogenicity and cross-protectionWe deleted the virulence gene crp gene in the three recombinant strains,generating three recombinant Salmonella Typhimurium vaccine strains,SLT25?pCZ11?,SLT26?pCZ11?,and SLT27?pCZ11?.The mice were first immunized and boosted during the first week and the seventh week,and the ELISA results showed that the three vaccine-immunized groups produced high-level of serum IgG responses against homologous and heterologous LPS in the seventh week after immunization,the heterologous LPS antibody response generated by the SLT26?pCZ11?group was the most significant.Serum complement bactericidal assay demonstrated that the sera from the three vaccine-immunized groups were able to significantly kill both the Salmonella Typhimurium and Salmonella Newport.The sera of SLT26?pCZ11?group exhibited best bactericidal effects against Salmonella Newport.After the challenge with Salmonella typhimurium,the bacterial loads in the tissues of the three vaccine-immunized groups compared with BSG group were significantly reduced,and all provided 100%homologous protection.The bacterial loads in the three vaccine-immunized groups were significantly decreased compared with BSG group after challenge with Salmonella Newport,while the bacterial number of SLT26?pCZ11?and SLT27?pCZ11?groups were lower than that of the SLT25?pCZ11?group.Correspondingly,the SLT25?pCZ11?vaccine strain provided only 50%heterologous protection,while the SLT26?pCZ11?and SLT27?pCZ11?vaccine strains conferred 100%heterologous protection.The above results indicate that the SLT26?pCZ11?and SLT27?pCZ11?vaccine strains constructed in this study can be used to prevent Salmonella typhimurium and Salmonella Newport,and also indicate that O-antigen has the ability to stimulate the body to clear bacteria,indicating that heterologous expression of O-antigen is an effective strategy for the development of Salmonella bivalent vaccines.