An Experimental Study On The Preparation Of Pneumolysin DNA Vaccine And The Immunogenicity Of This Vaccine In Mice And Non-human Primates

Objective:Streptococcus pneumonia(S.pneumonia) is a major public health problem which causes bacterial pneumonia,bacteremia,meningitis,sinusitis and acute otitis media.The 7 or 11-valent polysaccharide and protein conjugate vaccines currently administered against S.pneumonia has been shown to be highly effective in high risk-groups,but its use in developing countries will probably not be possible due to high costs.Plasmid DNA vaccination is emerging as a safe and cost-effective method in vaccinology.DNA vaccines have shown to induce strong immune response in small and large animals which in some cases also are protective for different diseases. Pneumolysin,a sulfhydryl-activated cytolytic toxin,is produced by all types of S.pneumonia and is considered a putative virulence factor in pneumococcal infections. The Pn gene encoding for this conserved pneumolysin protein which may have potential advantages for the cross protection against various serotypes of S.pneumonia was used in present experiment for the genetic vaccination in mice and monkeys.This strategy is a promising approach for the development of a cost-effective vaccine against S.pneumonia.Methods:The Pn gene was amplified from Pneumococcal genomic DNA using conventional PCR technology.The PCR product was then cloned into the pVAX1 vector and resulted in the PN DNA vaccine.However,pneumolysin is a cytotoxic pore-forming toxin which cannot be used directly as a vaccine antigen without modification due to safety concerns.In order to get rid of this protein's hemolytic activity,the gene sequence encoding for the eleven amino acids from the carboxyl terminal of Pn was deleted.This mutated gene was also cloned into the pVAX1 vector and resulted in the PND DNA vaccine.Gene cloning technology was used for the gene expression of PN and PND in E.coli.The recombinant PN and PND proteins were then purified for testing their hemolytic activity.Western blotting was also used for testing the antigenicity of PN and PND DNA vaccines.In vivo electroporation(EP) has proven to be an efficient method for delivering genes into animal cells.In our study,mice and rhesus macaques were immunized by intramuscularly(i.m.) injection of PN or PND DNA vaccine with electroporation.PN or PND protein boost without adding adjuvant was applied ten days after third DNA vaccination.Results:The PN had an obvious hemolytic activity on rabbit erythrocytes while the PND had none.This result indicated that the absence of 11 amino acids from the carboxyl terminal of pneumolysin gene removed its hemolytic activity completely which was critical for being a safe vaccine candidate.The recombinant PN and PND were applied for Western blotting by using anti-PN and anti-PND antisera.The result indicated that the absence of 11 amino acids from the carboxyl terminal of PN did not reduced the antigenicity of PND.More important is that specific antibody response against PN was induced both in mice and monkeys by the immunization with the PND DNA vaccine.Conclusions:The PND DNA vaccine in which the gene sequence encoding for the eleven amino acids from the carboxyl terminal of Pn was deleted is safe and immunogenic both in mice and non-human primates.