Construction And Immunological Characterization Of Recombinant Attenuated Salmonella Choleraesuis Pore Kunzendorf Biotype Vaccine Expressing PEDV S1 Fragment

Porcine epidemic diarrhea(PED)is an acute and highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV).The prevalence and transmission of PED has been controlled to a certain extent by using the inactivated PED vaccine and live attenuated vaccine.But inactivated vaccine is difficult to provide mucosal immunity,live attenuated vaccine has the problem of reversion to virulence and susceptibility to maternal antibody interference.Therefore,there is an urgent need to develop new vaccines to prevent and control PED.Salmonella vector has the advantages to induce good humoral immunity,mucosal immunity,and cellular immunity,which is an ideal vaccine design strategy to express heterologous antigen for the prevention and control of PEDV.In this study,we screened dominant Salmonella as a vector to construct an attenuated strain with aroA and asd double gene deletions and expressing the S1 fragment of PEDV strains,which will lay a foundation for the development of PED oral vaccine.1.Comparative study on biological characteristics of Salmonella Choleraesuis and Kunzendorf biotype strainsOne WG strain of S.Choleraesuis,two strains of S.Choleraesuis pore Kunzendorf biotype CZ-C,HY-13 strains,and S Choleraesuis C500 vaccine strains were evaluated by the growth curve,drug sensitivity,antibody titer,colonization ability and pathogenicity assay.The results of growth curve determination showed that the growth rate of C500 vaccine strain was significantly slower than that of the other three isolates,and HY-13 had more obvious advantages.The results of drug sensitivity showed that the isolates were resistant to more antibiotics than the C500.Mice were immunized with 108 CFU of C500 and HY-13 by gavage.The antibody in immune serum was detected by ELISA,the results showed that the antibody from immunized mice in each group reached a peak on day 14 after immunization and then slightly decreased on day 21,antibody levels induced by HY-13,CZ-C,and WG of were significantly higher than that of C500 group.Furthermore,the colonization ability of HY-13 in vivo was higher than that of others,and the colonization amount in jejunum was the most,followed by cecum and liver.The LD50 results showed the strongest pathogenicity in the group of HY-13.Through the comprehensive evaluation of bacterial growth and colonization advantages,HY-13 strain was selected as a alternative Salmonella live vector.2.Construction and biological characteristics of recombinant attenuated Salmonella Choleraesuis pore Kunzendorf biotype expressing the S1 fragment of PEDVAn attenuated strain with deletion of aroA and asd genes(HY-13ΔaroAΔasd)was firstly constructed by λ-Red homologous recombination method.Secondly,based on the balanced lethal system,the prokaryotic expression vector pYA3334 carrying asd gene and PEDV truncated S1 fragment were recombined and then electroporated to HY-13ΔaroAΔasd for constructing the recombinant strain HY-13ΔaroAΔasd(pYA3334-S1T).Finally,the truncated S1 protein expression levels,growth curve,pathogenicity,genetic stability,and colonization levels of recombinant strain were determined.The PCR results showed that HY-13ΔaroAΔasd and the recombinant strain HY-13ΔaroAΔasd(pYA3334-S1T)were successfully constructed.Western blot results showed that the HY-13ΔaroAΔasd(pYA3334-S1T)successfully expressed truncated S1 protein.The growth level of the HY-13ΔaroAΔasd(pYA3334-S1T)is significantly slower than that of the wild strain HY-13,but significantly faster than C500 vaccine strain.The murine pathogenicity results showed that the LD50 of HY-13ΔaroAΔasd(pYA3334-S1T)is about 100-fold higher than that of wild strain HY-13.Furthermore,HY-13ΔaroAΔasd(pYA3334-S1T)showed a stable deletion of the aroA and asd genes and stabe expression of the truncated S1 protein after 50 passages,suggesting a strong genetic stability.Moreover,the colonization ability in vivo of HY-13ΔaroAΔasd(pYA3334-S1T)was reduced compared with the wild strain HY-13.Hhowever,it was still higher than that of the S.Choleraesuis vaccine strain C500.3.Evaluation of the immune efficacy and protection rate of recombinant strainsThe recombinant strains were intragastrically immunized to 6-week-old BALB/c mice with a dose of 108CFU.Compared with HY-13ΔaroAΔasd(pYA3334),HY-13ΔaroAΔasd(pYA3334-S1T)significantly induced both serum IgG and intestinal sIgA specific antibodies against PEDV,and the antibody reached a peak on day 14 after the second immunization.The results of PEDV micro-neutralization assay further showed that the antibodies in serum and small intestinal lavage fluid poccessed neutralization characteristics against PEDV HY-13ΔaroaAΔasd(pYA3334-S1T)significantly produced specific serum IgG and intestinal sIgA antibodies against both HY-13 and C500 strains compared with C500 vaccine group.HY-13ΔaroAΔasd(pYA3334-S1T)also significantly enhanced the transcript expression levels of IFN-γ and IL-4 in intestinal mucosa and spleen compared with PBS group.WG or HY-13 with 100-fold LDso was injected intraperitoneally on day 14 after the first immunization for evaluating the protection level against Salmonella challenge,HY-13ΔaroAΔasd(pYA3334-S1T)vaccine group and C500 vaccine group could provide 70%/55.6%and 40%/55.6%protection against S.Choleraesuis pore Kunzendorf biotype and S.Choleraesuis challenge,respectively.In summary,HY-13ΔaroAΔasd(pYA3334-S1T)can be developed as a Salmonella live vector vaccine candidate for the prevention and control of PED and Salmonellosis.