Construction of a Whole-Cell Pneumococcal Vaccine Candidate

Background: Pneumococcal infections are a major public health problem, and the current pneumococcal vaccines do not cover all pathogenic strains. The pneumococcal whole cell vaccine which is based on non-capsular antigens common to all strains induces serotype-independent immunity. In the present study, we developed a new candidate for a whole cell pneumococcal vaccine in which two important virulence factors, the capsule and pneumolysin, were deleted. Methods: We developed two homologous recombinants to knockout the Wzy gene for disruption of capsule production and knockout the PLY gene. For homologous recombination of Wzy with the blasticidin (Blas) antibiotic-resistant gene, a cassette composed of fragments upstream and downstream of Wzy and Blas gene was constructed. The second cassette designed to knockout the PLY gene was composed of a fragment upstream of PLY, Kanamycin (Kan) resistant gene and a fragment downstream of the PLY. Electro transformation was used for gene replacement. Results: The upstream and downstream regions of the Wzy and Blas genes were successfully ligated and a 1277 bp fragment was constructed for removal of the Wzy gene. The PLY homologous recombinant was constructed as a 1981bp fragment using the flanking regions of the PLY and Kan genes for removal of the PLY gene. The Wzy gene was replaced by the blasticidin resistant gene and capsule production was disrupted. The PLY gene was replaced by the kanamycin-resistant gene. Conclusion: The mutant strain will overcome the limitations of available polysaccharide vaccines.

Construction of a Whole-Cell Pneumococcal Vaccine Candidate Keywords:

Streptococcus pneumoniae , Vaccine