The Study Of Typhoid O-specific Polysaccharides Bio-conjugate Vaccines

Typhi S. is the pathogen causing typhoid fever, with high infectivity and could produce endotoxin, spread by the fecal oral route. Persons with typhoid fever usually have a sustained fever and may also feel weak, or have stomach pains, headache, or loss of appetite. Typhoid was common in most parts of the world, with the improvement of living and health conditions, and the wildly use of antibiotics(especially chloramphenicol), the incidence and mortality rate had a large decline in developed countries, while remains an important public health problem in some developing countries.Typhoid fever can be prevented and usually treated with antibiotics, but the abuse of antibiotics also brings multidrug-resistant strain of Typhi. There are two vaccines to prevent typhoid in the worldwide now. One is Vi-CPS vaccine gotten by intramuscular injection, the other is a live, attenuated vaccine which is taken orally.In contrast to isolated bacterial polysaccharides, conjugate vaccines induce a long-lasting T-lymphocyte dependent immunological memory and also could provoke immune responses in infants. In recent years the notion that bacteria do not perform protein glycosylation has become obsolete. Based on the similarity between protein glycosylation process and lipopolysaccharide biosynthesis pathway, and the function been found of pgl gene cluster, which made the production of conjugate vaccines in vivo became possible.In this study, waaL deletion mutants of S. typhi 50096 was constructed by using λ-Red recombination system. And also, a waaL recovery mutant was constructed by using low copy plasmid. Phenotypic identification was performed by LPS silver staining experiment. The results showed that no ladder was found in waaL deletion mutant, indicating that the mutant could not synthesize lipopolysaccharide. Then, growth of the mutants, the recovery strain and wild type strain at 37℃ were measured respectively, and some biochemical events of them were comparatively investigated. There were no-significant differences in LB medium. Moreover, we constructed a genetically modified S.Typhi strain expressing O-specific polysaccharides(OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPAN29). Then, the expression was induced, and been tested by SDS-PAGE electrophoresis and two-dimensional electrophoresis. The results showed that, the OPS-rEPAN29 was expressed in vivo, and differential expressed proteins were non-essential. Last, the conjugates(OPS-rEPAN29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than by O-specific polysaccharides, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier.In summary, this study successfully provide a new strategy for preparing polysaccharides-protein conjugate vaccines, as well as similar bio-conjugate vaccines of other Gram-negative pathogens.