The Construction Of Live Recombinant Attenuated S.Typhimurium Vaccines For Major Salmonellosis Infections Prevention

Salmonella enterica serovars are important zoonotic gram-negative pathogens.These bacteria are responsible for a huge global health and economic burden through two forms of invasive illness: invasive nontyphoidal Salmonella(i NTS)diseases and typhoidal Salmonella diseases(enteric fever).i NTS diseases are mostly caused by S.Typhimurium and S.Enteritidis,while enteric fever are principally caused by S.Typhi and S.Paratyphi A.In this thesis,we aimed at contructing new types of recombinant attenuated S.Typhimurium vaccines,so that they could simutatineously express and present more than one types of homologous or heterologous protective antigens and thus provided broad-spectrum protection.The major research results are as follows.1.Reversible S.Typhimurium O-antigen synthesis and outer membrane proteins cross-immunogenicity enhancementIn the study of this chapter,we first verified that the cps G gene,which located in cps gene cluster,and the rfb K gene,which located in rfb gene cluster,were actually isozyme genes by LPS silver staining assay.S.Typhimurium could no longer synthesize full length of O-antigens after deleting cps G and rfb K.However,if the mannose was added to the media,this mutant strain could restore its ability to synthesize intact O-antigens.Therefore,the reversible synthesis of O-antigens could be achieved via mannose supplement.We latter introduced Δcps G Δrfb K to a live attenuated S.Typhimurium vaccine S738(Δcrp Δcya)to evaluate whether or not the cease synthesis of Colanic acid and O-antigens would contribute to the outer membrane proteins cross-immunity enhancement.We found that,compared with its parent strain,live attenuated S.Typhimurium vaccine S112(Δcrp Δcya Δcps G Δrfb K)could significantly induce higher levels of anti-S.Typhimurium,anti-S.Enteritidis and antiS.Choleraesuis OMP Ig G antibodies.All vaccinated mouse could survive from oral challenge of wild type virulent S.Typhimurium strain.Meanwhile,the mouse could partially survive from oral challenge of wild type virulent S.Choleraesuis and S.Enteritidis strains.The results in this study had shown that the deletion mutation of cps G and rfb K genes could enhance the corss-protection of live attenuated S.Typhimurium vaccines.2.The construction of live attenuated S.Typhimurium vaccines expressing S.Enteritidis(O9),S.Newport(O7)and S.Choleraesuis(O8)O-antigensIn the study of this chapter,the immunodominant O4 O-serotype of S.Typhimurium was converted into S.Enteritidis O9,S.Choleraesuis O7 and S.Newport O8 serotypes by genetic recombination.The construction of new types of live attenuated S.Typhimurium vaccines based on the strategies of O-serotype conversion could induce high levels of heterologous O-polysaccharide-specific Ig G antibodies.In addition,the complement deposition assay and the macrophages uptake assay all confirmed that these specific Ig G antibodies are biologically functional.Vaccinated mice could survived from a challenge of 100 times the 50% lethality dose(LD50)of wild-type S.Typhimurium.Protective efficacy against heterologous virulent Salmonella challenges was highly O-serotype related.Furthermore,broad-spectrum protection against S.Typhimurium,S.Enteritidis and S.Choleraesuis was observed by co-vaccination of O9 and O7 O-serotype-converted vaccine candidates.The results in this study highlighted the strategy of expressing heterologous Opolysaccharides via genetic engineering in developing live attenuated S.Typhimurium vaccines against the major serovars of i NTS diseases.3.The construction of live attenuated S.Typhimurium vaccines expressing S.Typhi O9 O-antigen and Vi capsularIn the study of this chapter,new types of live attenuated S.Typhimurium vaccines were constructed to synthesize Vi capsular antigen in vivo and produces the immunodominant O9 O-antigen polysaccharides instead of its native O4.The constructed live attenuated S.Typhimurium vaccines were effective in stimulating anti-Vi and anti-O9 antibodies in a mouse model,and the surface Vi capsular expression did not affect the immune responses against the O9 O-antigen polysaccharides.Moreover,the in vitro serum sensitive assay proved that the resulting anti-Vi and anti-O9 antibodies were effective at killing S.Typhi and other Salmonella spp expressing Vi or O9 O-antigen polysaccharides,and provided efficient protection against lethal challenge by S.Typhimurium and S.Enteritidis.The results in this study highlighted the strategy of targeting the Vi capsular and O9 O-antigens simultaneously when developing live attenuated S.Typhimurium vaccines to prevent the innate immune evasion during S.Typhi initial infection.4.The construction of live attenuated S.Typhimurium vaccines expressing S.Paratyphi A O2 O-antigenIn the study of this chapter,we successfully converted the O4 O-serotype of S.Typhimurium into S.Paratyphi A O2 O-serotype by genetic recombination.We found that S.Typhimurium could not effectively utilize O2 O-units to synthesis O2 O-antigen polysaccharides,and the deleterious effect on S.Typhimurium due to O2 O-units discrimination could be ameliorated by replacing the abe,wzx B1 and wba VB1 genes with prttyv A1,wzx A1 and wba VA1,respectively.Consequently,multiple live vaccine candidates were designed with the goal of ameliorating the deleterious effects and balancing the immune response towards the O2 and O4 epitopes.The results had showed that vaccine candidates could induce high levels of S.Paratyphi A and/or S.Typhimurium LPS-specific Ig G responses in murine model.Moreover,the functional properties of serum antibodies were evaluated using in vitro C3 complement deposition and opsonophagocytic assays.The results in this study highlights the potential for developing S.Typhimurium live vaccines against S.Paratyphi A through O2 O-serotype conversion or regulation.In summary,the most vital results in this thesis was breaking down the immunoprotection limitation of tranditional live attenuated vaccines,i.e.they were largely monovalent vaccine.By reconstructing and rearranging the most important outer membrane protective antigens,we have constructed a series of new live attenuated S.Typhimurium vaccines.When these vaccines were vaccinated separately or cooperatively,they could induce stronge immunoprotection not only against the main non-typhoid Salmonella desease,suh as,S.Typhimurium,S.Enteritidis,S.Choleraesuis and S.Newport,but alos against the main typhoid Salmonella desease,such as,S.Typhi and S.Paratyphi A.The limited results in this thesis will put forward the fundamental research of bacteria vaccines and leave behind some practical construction strategies for bacteria live vaccines cross-immunity developments.