ABSTRACT
Objective To prepare the chitosan-potD nanoparticles and to evaluate its protective efficacy against pneumococcal nasopharyngeal colonization. Methods potD gene was amplificated from pneumococcal genome and was inserted into pVAX1 expression vectors to construct pVAX1-potD recombinant plasmid which was then transfected into 293T cell using LipofectAMINE 2000 to analyze transient potD gene expression in vitro by RT-PCR and Western blot. Chitosan-potD nanoparticles were freshly prepared by coacervation methods at each time and the characterizations of the nanoparticles were then evaluated. BALB/c mice were immunized with chitosan-potD, naked potD DNA or pVAX1 for 4 times at two-week intervals. Anti-PotD IgG, IgG1 and IgG2a levels in serum and IgA levels in nasal washes, bronchoalveolar lavage fluids (BALF) and middle ear lavages(MEL) were detected by indirect enzyme-linked immunosorbent assay (ELISA). IL-17A, IL-4 and IFN-γ levels in splenocytes were determined by double sandwich ELISA. Mice were intrannsally challenged with Streptococcus pneumoniae ATCC6303, and Pneumococci were recovered from the nasopharyngeal niche at the fifth day after challenge. Results potD gene was successfully amplificated by PCR and the sequence was confimed to be consistent with that in the Genbank. The pVAX1-potD recombinant plasmid was successfully constructed and was expressed in eukaryocytes in vitro. The mean size and zeta potential of chitosan-potD nanoparticles was 430 nm and + 20.5 mv, respectively. Chitosan-potD nanoparticles were not digested by DNase Ⅰ , while naked potD DNA was completely digested. The levels of antibodies inculding IgG, IgG1, IgG2a, IgA and cytokines including IL-17A, IL-4 and IFN-γ were significantly higher in mice immunized with chitosan-potD nanoparticles than mice with naked potD or pVAX1 ( P <0.05) only. More importantly, much less Pneumococci were recovered from mice immunized with chitosan-potD nanoparticles than the other groups(P <0.05). Conclusion Chitosan-potD nanoparticles significantly enhanced the immunogenicity and protection efficacy of DNA vaccines by intranasal immunization and could be used as a potential mucosal vaccine to prevent pneumococcal infection.
ABSTRACT
Objective To study the immunogenicity of Streptococcus pneumoniae pneumolysin DNA vaccine in Rhesus macaques. Methods The deletion of the gene sequence encoding for the 11 amino acids at the carboxyl terminal of pneumolysin (PN) from its wild type gene (pn) by PCR resulted in a mu-tant pneumolysin gene (pnd). The wild type pn gene encoding PN and the mutant gene (pnd) encoding PND were cloned into pVAX1 vector respectively and then tested as Ppn and Ppnd DNA vaccines. The PN and PND proteins were purified from recombinant E. coli. Rhesus macaques were immunized by intramuscu-larly (i.m.) injection of Ppn or Ppnd DNA vaccine with electroporation (EP). Results Because of the deletion of the gene sequence encoding for the eleven amino acids at the carboxyl terminal of the PN from pn gene, the recombinant PND antigen lost its hemolytic activity while its antigenicity was remained. The spe-cific humoral immunity against pneumolysin was induced by injecting monkey with 500 μg DNA followed by EP. Boosting the Ppn or Ppnd DNA/EP primed animals with corresponding recombinant protein, PN or PND, evoked strong immune response at about 4 fold increase in the antibody titer. Conclusion Specific antibody responses were induced in the monkeys by DNA vaccination and electroporation. The immunogenic-ity of the DNA vaccines were significantly enhanced when PN or PND protein boost was applied 10 d after third DNA vaccination.
ABSTRACT
OBJECTIVE To prepare pneumolysin(Pn)by genetic engineering and thereby establish the basis for the study of vaccines against otitis media. METHODS A pair of primers including two restriction sites was designed based on the pneumolysin gene sequence reported by Walker in 1987. The pneumolysin gene was PCR-amplified from pneumococcal DNA. The resulting fragment, digested by restriction enzymes, was ligated into the vector PET-28a and then transformed into host cell E.coli JM109(DE3). RESULTS The sequence of the inserted pneumolysin gene was confirmed by DNA sequencing. CONCLUSION The pneumolysin gene was successfully cloned into the host cell.