Immunogenicity And Immunoprotection Study On Salmonella Paratyphi A BG Vaccine

Paratyphoid fever is a serious invasive and intracellular infection disease, which caused by S. paratyphi A and transmitting by the fecal-oral route. The incidence of paratyphoid fever is increasing in China in recent years, and clinical treatment has been difficult due to the widespread occurrence of clinically resistant and multidrug-resistant strains of Salmonella paratyphi A. However, there is no licensed vaccine to prevent S. paratyphi A disease nowadays. Traditional triple inactivated vaccine can only provide short protection and may lead to some side effects. At present, vaccines of S. paratyphi A in studying were attenuated vaccine or subunit vaccine. Attenuated vaccine with high immunogenicity may face incidence of virulence recovery. Subunit vaccine with high cost of production should be used with adjuvant for its weak immunogenicity. BG is a kind of empty bacterium which loses its cytoplasm and DNA, with only outer membranes remained. BG is produced by controlled expression of cloned lysis gene E of bacteriophage PhiX174in bacterium. BG with the same epitope as live bacterium has not only adjuvant activity but also induce system and mucosal immune response. Here, we preparared S. paratyphi A BG, studied its corresponding biological characteristics, and detected its immunogenicity and immunoprotection through the Balb/c mice vaccination and challenge experiments.While amplifying PhiX174lysis E gene by PCR, we unexpectedly get a mutant of gene E(Em), which consisting of the same75-bp of5' terminus as gene E and another unrelevant21-bp nucleotides. Further, we constructed temperature-controlling expression vector pBV220-Em. Electroporation of pBV220-Em into S. paratyphi A and temperature induction was done to get S. paratyphi A BG. Meanwhile, we explored the temperature induction condition, determined the growth curve after induction and lysis efficiency. We also observed the BG Morphology through transmission electron microscopy (TEM). The mice were divided into six groups to determine the immunogenicity of S. paratyphi A BG.S paratyphi A inactivated vaccine was devised as positive control group and PBS as negative control group. The grouped mice was immunized through oral adminstration or subcutaneous injection three times over a two-week interval. The blood serum was collected respectively on the day before immunization, and the titer of specific IgG in the serum were determined by Whole-cell ELISA. Half of the mice in each group were killed and immersed in75%alcohol for disinfection ten days after the last vaccination. Spleens were aseptically excised and homogenized to get spleen lymphocytes. The amount of IFN-y or IL-4secreting lymphocytes were determined using ELISPOT. The MLD of S. paratyphi A for native mice was determined to decide the challenge dose of S. paratyphi A for immunized mice. Another half of the immunized mice in each group were intraperitoneally challenged with twice the MLDs of S. paratyphi A(1×106) ten days after the last vaccination and the survival challenged mice were killed one week later. Mice livers, spleens, and lungs were aseptically excised, weighed and homogenized. The tissue homogenates were serially diluted,100μl of which were plated on nonresistant plates and incubated at37℃overnight. The S. paratyphi A CFUs per g of homogenized tissues were determined.If the OD600of S. paratyphi A harboring plasmid pBV220-Em at initial induction time was below0.7, OD600wouldn't go up within3h after temperature induction. Otherwise, OD600would go up as S. paratyphi A harboring plasmid pBV220. TEM showed that the cytoplasmic contents of induced pBV220-Em/S'. paratyphi A would go out through menbrane trunnel at middle or teminal side of bacterium. In oral or subcutaneous injection immunization group, amounts of IL-4secreting lymphocytes were higher than that of IFN-y secreting lymphocytes. Oral immuzation with inactive S. paratyphi A provied none protection against a lethal intraperitoneal challenge, while oral immuzation with BG provied20%protection. Subcutaneous injection immunization could povide100%protection with inactive S. paratyphi A or BG. Bacteriological analysis of organs excised from survival challenged mice showed that S. paratyphi A distributed most in the liver and least in the lung. Bacteriophage identification indicated that most bacteria in the organs were S. paratyphi A. The antigen-specific IgG titer in survival challenged mice were about2-8times higher than that after the third immunization.In summary, the S. paratyphi A BG we have got in this study is high immunogenicity and its induced immune response is maily humoral immune response. Though subcutaneous injection immunization could provide100%protection, there are also some S. paratyphi A existing in the organs of survival challenged mice. It's needed further study whether the bacteria in the organs is related with the invasion mechanism of S. paratyphi A. This paper gives a preliminary evaluation on the immunogenicity and immunoprotection of S. paratyphi A BG, and lays a foundation for the development of S. paratyphi A BG vaccine.