| Salmonella paratyphi A(SPA) is the pathogen of paratyphoid fever A, which is mainly transmitted through the fecal-oral pathway with an infectious dose of 103 ~ 109 CFU. Individuals are susceptible to SPA, which often infects children and young adults. In recent years, the incidence of enteric fever caused by SPA is rising in many parts of the world, and becomes a major public health concern. In China, the incidence of paratyphoid fever A is also higher with several outbreaks in certain areas.The polyvalent inactivated typhoid vaccine was tested to have important effects on the prevention of paratyphoid fever, however, it was stopped to use due to its severe side effects. The development of new vaccines against paratyphoid fever A is urgently needed.There are two categories of vaccines, component vaccines and live attenuated vaccines. The component vaccines were usually prepared from major protective antigens of pathogens, including the proteins and carbohydrate antigens, such as Salmonella typhi Vi polysaccharide vaccine, DPT vaccine etc. Live attenuated vaccines are generated from pathogens through genetic modification to reduce the toxicity and keep the immunogenicity. Such as Salmonella Ty21 a live attenuated vaccine, Measles vaccine and live attenuated hepatitis A vaccine. The ideal vaccines are the best balance of safety and immunogenicity. Two types of vaccines could induce strong immune responses, so as to eliminate the invading pathogens in a time-dependent manner. Live attenuated vaccine can also induce local immunity, which may effectively prevent pathogens from proliferation and colonization. Paratyphoid fever A is usually classified as "travel related disease", which can form the carrier state after infection, increase the risk of disease transmission.Therefore, the construction of a live attenuated vaccine of paratyphoid A is of importance to control the epidemic of paratyphoid fever A.In this dissertation, a live attenuated vaccine for paratyphoid A was constructed by double knockout of the ync D and aro C genes of Salmonella paratyphi A strain CMCC50093. In our previous study, ync D gene was determined to be an in vivo induced(IVI) gene of Salmonella typhi. The deletion of ync D will significantly reduce the virulence of Salmonella typhi. ync D gene is highly conserved in the Salmonella strains and may be a good candidate for the construction of live attenuated Salmonella vaccines.The aro C gene functionally regulates the synthesis of Salmonella aromatic amino acids, the deletion of aro C will affect the synthesis of corresponding amino acids, resulting in the growth arrested and the virulence reduced. The aro C gene is a common target gene used in construction of attenuated vaccine strain. After obtained the △ync D/△aro C double knockout SPA strain, the attenuated effect, the genetic stability, as well as the safety and immunogenicity of the vaccine candidate strain were evaluated.The main results are as follows.1.Identification and characterization of the wild-type SPASalmonella paratyphi A strain CMCC50093 was purchased from The National Center for Medical Culture Collections(Beijing, China) and used as the wild-type strain in this study. In order to further understand the biological characters of the wild-type strain, we first extracted its genomic DNA by CTAB method, the 16 S r RNA gene was amplified and sequenced, then characterized by whole genome sequencing. The biochemical characters and the drug resistance spectrum were also determined.The results showed that CMCC50093 is a typical SPA strain and suitable for the construction of a live attenuated vaccine strain.2.Construction of △ync D/△aro C double knockout strain.A double knockout strain was constructed by the homologous recombination method. A suicide vector p YG4 was used, which contains a π protein dependent ori R6 K derived from R6 K plasmid that restricts its replication only in the host capable expression of π protein, such as E. coli S17-1/ x pir.The positive selection marker of p YG4 plasmid is the kanamycin resistence cassette, and the reverse selection marker is a sac B gene encoding the sucrose fructan transferase from Bacillus subtilis. In this study, we used the p YG4 plasmid consecutively to knockout the genes of Salmonella paratyphi A.In the case of ync D gene knockout, two fragments of approximately 750 bp that locate ambilateral the ync D gene in SPA genomic DNA were amplified by PCR using two pair of primers. The purified PCR products were mixed as template, an over-lap amplification was performed and the resulting product was cloned into the p MD19-T plasmid.The homologous fragment was digested from the recombinant p MD19-T and inserted into the p YG4 plasmid to obtain p YG4-Δync D from transformed E. coli S17-1/λ pir with kanamycin selection. Then p YG4-Δync D was transformed into SPA strain CMCC50093, and the ync D deleted strain was screened out with positive and reverse selections enabled by p YG4 vector and characterized by PCR using the wild-type DNA as a control. In the operation process, we did not add any other DNA sequence into the ync D knockout strain, so the second target gene(aro C) deletion was subsequently performed to obtain the △ync D/△aro C double deleted strain.3.Determination of the growth curve and LD50 of the △ync D/△aro C double deleted strainThe growth curves of the wild-type SPA, △ync D knockout strain and △ync D/△aro C knockout strains were determined by LB liquid ordinary culture. The LD50 was determined using the gastric mucin induced BALB/c mice model. The results of growth curves showed that the growth of the double knock strains has changed compared to the wild-type strain, mainly in the growth rate and the maximum biomass after 24 h of culture. After peritoneally challenged with 2.5×101, 2.5×102, 2.5×103, 2.5×104, 2.5×105, 2.5×106, and 2.5×107 CFU of the selected strains respectively, the alive mice were counted and LD50 was calculated by regression analysis with SPSS software. The results showed that the virulence of double knockout strain was only about 1/42000 of that of the wild-type strain, showing a significantly decreased virulence.4.The preliminary safety evaluation of the △ync D/△aro C double knockout strain.The invasive and intracellular survival abilities of the △ync D/△aro C double knockout strain were determined using mononuclear macrophage THP-1, and the in vivo bacterial carriers were determined with mice abdominalinfected model. The results showed the clonal numbers of the double knockout strain infected group were significantly lower than that of the wild-type strain. The model mice were sacrificed 7 days post intraperitoneal injection with 2.5×103, 2.5×104, and 2.5×105 CFU of the wild-type and the double knockout strains,respectively, and the bacteria in the liver and spleen of the infected mice were counted with LB plates. The results showed that the invasive and survival abilities of the double knockout strain have been significantly reduced in comparison to that of the wild-type strain, suggesting a good safety for the △ync D/△aro C double knockout SPA strain.5.Evaluation of the immunogenicity of the△ync D/△aro C double knockout strainTo determine the effects of different inoculation pathways, mice were challenged intraperitoneally, nasally or orally with different doses of the double knockout strains. The anti-LPS antibodies of the infected mice were detected by ELISA. The results showed that, in addition to orall inoculation group,the intraperitoneal and nasal inoculation groups challenged with high concentration of bacteria(2×109 CFU) can induce better immune response. The anti-LPS antibody titers in the serum of immunized mice were significantly higher than that of the normal mice. LPS is considered to be one of the major protective antigen of Salmonella, and our results showed that the double knockout SPA strain has good immunogenicity.6.Evaluation of immune protection of the △ync D/△aro C double knockout strainThe higher titers of anti-LPS for Salmonella typhi and Salmonella paratyphi A in sera of the immunized mice proposed that there may be cross-protection to Salmonella typhi. The double knockout SPA strain immunized mice were attacked with Salmonella typhi strain Ty2. We found only low dose(2.5×101 CFU) of Ty2 challenged mice could be protected, whereas all mice challenged with 2.5×101, 2.5×102,2.5×103, 2.5×104, 2.5×105, and 2.5×106 CFU of wild-type SPA, respectively, could be protected. These results demonstrated that the △ync D/△aro C double knockout strain has good protection ability against the SPA infection, however, its cross protection ability against Salmonella typhi is limited. The levels of IL-6, IL-12,and TNF-a, in seraof the immunized mice challenged with wild-type SPA tested by ELISA were significantly lower with shortened regression time than those of the control group, suggesting that the double knockout SPA strain can enhance the immune clearance of the bacteria, reduce the inflammation in the host, thus has a good protective efficacy. |
PDF Full Text Request