Construction Of Pneumococcal CbpA DNA Vaccine And The Immunity Effects Of Co-Immunization With Protein Vaccine

Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. It's still a common pathogen of community acquired pneumonia, bacteremia, otitis media and meningitis. The 23 valent capsular polysacchride vaccine is useful in adults but fails to protect those at highest risk of disease at the youngest and oldest ages, and although the current 7-valent conjugate vaccines are effective against invasive disease caused by the vaccine-type strains, yet streptococcus pneumoniae can be divided into more than 90 serotypes according to the immunochemistry of their capsular polysaccharide, so vaccine coverage is limited, the cost of production is ver high and this conjugate vaccines are not applicable in the developing countries. Futhermore, replacement by invasive nonvaccine-serotypes in vaccinated individuals is a grim problem confronting us. Recently, DNA vaccine becomes appreciated because they are able to induce cellular and humoral response to the encoded antigens.CbpA is one of the most important virulence factors playing some crtical roles in the pathogenesis of pneumococcal infection. CbpA has been shown to interact with the human polymeric immunoglobulin receptor, thereby facilitating invasion of mucosa. The molecule of CbpA appears to act as a bridging element between the choline of teichoic acidor lipoteichoic acid and human cell glycoconjugates by utilizing the repeat region binding to choline and the N-terminal region binding to cells, respectively. CbpA is a surface protein that is necessary for pneumococcal meningitis.Objective:We aimed to construct a DNA vaccine encoding S.pn cbpA, which was applied to the BALB/C mice by two different strategies. The BALB/C mice were immunized with naked recombinant plasmid via the intramuscular route, specific antibodies were detected in sera of mice by ELISA; the BALB/C mice were immunized with naked recombinant plasmid combined with protein vaccine via the intramuscular route, specific antibodies were detected in sera of mice by ELISA;Method: CbpA gene was amplified from TIGR4 line by PCR, then was inserted into the eukaryotic expression vector pcDNA3.1(+) and construct recombinant expression vector pcDNA3.1/CbpA. Western blot analysis indicated the expression of the cbpA gene in transfected COS-7 cells after 48h. Then the BALB/C mice were immunized with pcDNA3.1/CbpA, CbpA protein vaccine, pcDNA3.1/CbpA plus murine interleukjn-2, pcDNA3.1/CbpA combined with CbpA protein vaccine respectively, and boosted with the same concentration on day 14, and on day 28, specific antibodies were detected in sera of mice by ELISA; After challenging with TIGR4, The mice were then monitored for death over 21 days,then count the suivival rate.Results: The results of digestion with restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene CbpA had been cloned into the eukaryotic expression vector pcDNA3.1(+). The length of the CbpA gene was 1461bp. Compared to the sequence of GeneBank, no mutation was found. Western blot analysis showed that the CbpA gene was expressed in COS-7 cells successfully. The result of mice experiment showed that specific antibodies were detected in all groups except pcDNA3.1(+) group by ELISA. The highest specific antibodies of the various groups were observed with the combination of 50ug pcDNA3.1/CbpA and 50ug protein vaccine. Active immunization protection assays, In this experiment, the survival rate for mice that were immunized with pcDNA3.1/CbpA combined with rCbpA or pcDNA3.1/CbpA combined with murine IL-2 were significantly longer than that for mice that received plasmids of pcDNA3.1(+) alone .The highest survival rate of the various groups of mice was observed with the combination of 50ug pcDNA3.1/CbpA and 50ug rCbpA.Conclusion: The CbpA gene was cloned successfully into the plasmid pcDNA3.1(+) and expressed successfully in COS-7 cells. Sequencing and sequencing analysis were done and no mutation was found. The recombinant plasmid pcDNA3.1/CbpA was proved immunogenic and it can induce protective immune response. Combination of murine IL-2 and recombinant plasmid pcDNA3.1/CbpA could enhance the antibody level and the survival rate of mice. The combination with DNA vaccine and protein vaccine could induce the higher specific antibodies and enhance protective efficacy compared with only DNA vaccine.