Protective Effect Of DNA Vaccine Encoding Pseudomonas Exotoxin A And PcrV Against Acute Pulmonary P. Aeruginosa Infection

Objective:Construct novel DNA vaccine encoding Pseudomonas Exotoxin A and PcrV and identify the expression of two genes in eukaryotic cells. Vaccinate Balb/c mice with DNA plasmids, plasmids plus adjuvant CpG ODN1826and control pIRES and PBS, detect immune responses induced by vaccines. Attack vaccinated mice with PAO1pathogen and test the efficacy of protect mice from pneumonia injury. Methods:The toxA gene and pcrV gene were amplified from genome of PAO1using PCR and then inserted into multiple clone sites of plasmid pIRES. The constructions of novel plasmids were identified by gel electrophoresis and gene sequencing. Transfection assay in eukaryotic cells and western blotting were applied to confirm the feasibility of novel vaccines. Following that plasmids were injected into Balb/c mice for three times at two week interval, besides the adjuant CpG ODN1826were added for immune enhancement. Vaccine groups were divided into controls, vaccines and vaccine plus adjuvant. Sera were collected and antigen-specific IgG were tested using ELISA; meanwhile, the collected murine splenic cells were stimulated by antigens to evaluate proliferation index and cytokines secretion. The immune protective effect of novel vaccines were test through bacteria counting and histologic observation of lung tissues removed from PAO1challenged mice1week after the final vaccination.Results:Gel electrophoresis and sequencing demonstrated the target gene fragments encoding Exotoxin A and PcrV. Western blotting exhibited proteins encoded toxA and pcrV expressed by HEK293cells. In vaccinated Balb/c mice, not only significant antigen-specific IgG levels in sera but also remarkable proliferation of splenic cells as well as predominant Thl-type cytokines in splenic supernatants were detected. For pseudomonas pneumonia mice, both bacteria loading and histologic injury of lungs were eased in vaccine groups. Additionally, in plasmid plus CpG ODN1826adjuvant groups, significant elevation in efficacy of inducing immune response and providing protection from infection were observed.Conclusions:The construction of novel DNA vaccines encoding Exotoxin A and PcrV were proved to be feasible in eukaryotic expression system. In Balb/c mice, DNA vaccines were capable to induce both humoral and cellular immunity, furthermore were effective in protecting mice from P. aeruginosa infection. In addition, the CpG ODN1826was indicated to be a potent immune adjuvant.