| Objective: Evaluate whether HSV-1 VP22 could enhance the humoral and cellular immune responses and immune protective effects of DNA vaccines against Pseudomonas aeruginosa OprF or Fla B.Methods: 1. Animal immunization :(1) DNA vaccines including pVAX1-VP22, pVAX1-OprF, pVAX1-OprF-VP22, pVAX1-FlaB and pVAX1-FlaB-VP22 were extrated.(2) The BALB/c mice were immunized intramuscularly by DNA vaccines mediated by the in vivo transfection reagent. The antiserum and the splenocyte T cells of mice were prepared after the third immunization. 2. Humoral immunity: The antiserum titers of IgG1 and IgG2 a were determined by indirect ELISA. 3. Cellular immunity:(1) The level of T cells proliferation activity was tested by CCK-8 analysis.(2)The level of IFN-γ and IL-4 was examined by ELISPOT. 4. Protective immunity:(1) BALB/c mice immunized by pVAX1-FlaB-VP22, pVAX1-FlaB or pVAX1-VP22 DNA vaccines were infected by Pseudomonas aeruginosa intraperitoneally, and a week later, the lungs were gathered.(2) The number of bacterials were counted to estimate the level of lung tissue infection.(3) Pathological changes of lungs were observed in HE staining.Results: 1. Humoral immunity: The indirect ELISA showed that all serum samples from mice injected with either pVAX1-OprF-VP22, pVAX1-Opr F, pVAX1-FlaB-VP22 or pVAX1-FlaB DNA vaccines were capable of inducing antigens specific IgG1 and IgG2 a antibodies, while serum samples from the PBS and pVAX1-VP22 groups could not. The pVAX1-OprF-VP22 group produced a significantly higher ritio of IgG2a/IgG1 than the pVAX1-OprF group(P<0.05). The ritio of IgG2a/IgG1 in the pVAX1-FlaB-VP22 was significantly higher than the pVAX1-FlaB group(P<0.05), indicating Th1 proliferation. 2. Cellular immunity:(1) The result of CCK-8 test showed that the SI of T cells proliferation reactivity in the pVAX1-OprF-VP22 group was significantly higher than the pVAX1-OprF group(P<0.05), the p VAX1-OprF group was significantly higher than the pVAX1-VP22 group or control(PBS) group(P<0.05), while there was no significant difference between the pVAX1-VP22 group and the control(PBS) group(P>0.05). The pVAX1-FlaB-VP22 group was significantly higher than the pVAX1-FlaB group(P<0.05), the pVAX1-Fla B group was significantly higher than the pVAX1-VP22 group or control(PBS) group(P<0.05), while there was no significant difference between the pVAX1-VP22 group and the control(PBS) group(P>0.05).(2) The ELISPOT showed mice inoculated with the pVAX1-OprF-VP22 group induced significantly higher titers of cytokine IFN-γ than mice with the pVAX1-OprF group(P<0.05), the pVAX1-OprF group was significantly higher than the pVAX1-VP22 group or control(PBS) group(P<0.05), while there was no significant difference between the pVAX1-VP22 group and the control(PBS) group(P>0.05). The pVAX1-FlaB-VP22 group was significantly higher than the pVAX1-FlaB group(P<0.05), the pVAX1-FlaB group was significantly higher than the pVAX1-VP22 group or control(PBS) group(P<0.05), while there was no significant difference between the pVAX1-VP22 group and the control(PBS) group(P>0.05). Secretions of IL-4 cytokine by T cells were extremely low in all DNA groups. 3. Protective immunity:(1) The number of bacterials from lung tissue homogenate showed p VAX1-FlaB-VP22 group was significantly lower than the pVAX1-FlaB group(P<0.05), the pVAX1-FlaB group was significantly lower than the pVAX1-VP22 group or control(PBS) group(P<0.05), while there was no significant difference between the pVAX1-VP22 group and the control(PBS) group(P>0.05).(2) HE pathological sections showed the lungs of both pVAX1-VP22 group and control(PBS) group have serious infection, alveolar structures disappeared, stromal hyperplasia, and a large quantities of necrotic tissues can be seen in the brounchus. The lungs of pVAX1-FlaB group alveolar structures relatively intact, a large number of inflammatory cells can be seen in the brounchus and interstitial, such as plasmocites and neutrophiles. Some of erythrocytes can be seen in the lungs of pVAX1-FlaB-VP22 group, indicating a slight symptom of bleeding.Conclusion:1. VP22 can significantly enhance the single expressing antigens OprF and FlaB to induce specific humoral and cellular immune responses in mice, and be more inclined to Th1 like cellular immune response. 2. The pulmonary infection model of Pseudomonas aeruginosa can be successfully established by intraperitoneal injection, the fusion DNA vaccine encoding VP22 and FlaB eliting better prevention against Pseudomonas aeruginosa to some extent. |
PDF Full Text Request