The Study Of Pneumolysin As A Component Of Pneumococcal Protein-based Vaccine And The Application Of The Bacteria-like Particles Technology

As a common pathogenic bacteria, Streptococcus pneumoniae is responsible for alot of diseases. It’s the most important community-acquired pneumonia pathogens, andalso causes sinusitis, otitis media and meningitis. In HIV infection, Streptococcuspneumoniae can also cause bacteremia and other diseases. In many developingcountries, pneumonia caused by S.pneumonia is one of the main killers of the infantand the old. While in developed countries, otitis media caused by S.pneumonia is alsoquite common in pediatrics. Pneumococcal conjugate vaccine, the currently wildly usedvaccine, bound the pneumoniae capsular polysaccharide to a carrier protein to enhancethe immunogenicity. But due to the serotype-limited and high price, the conjugatevaccine could not be promoted in developing countries.Pneumolysin, one of the virulence factors of S. pneumoniae, plays an importantrole in the invasion and colonization of S. pneumoniae. At the asme time, it has beenfound a very good conservation in different strains of S. pneumoniae. Due to thisadvantages, the Pneumolysin is a potential candidate of the pneumonia vaccine. Due tothe hemolytic toxicity of the wild type Ply, it could not be used in vaccine directly.2amino acid on the Trp-rich loop of the Ply domain4have been mutanted for a detoxedPly, which is called PlyM2. This research has constructed, expressed, and purified boththe detoxed mutant derivant PlyM2and its wild type Ply. By detection of variabletemperature fluorescence spectrum and circular dichroism spectrum, we proved that thestructure of PlyM2changed little from PlyWT, only a slightly loss of its thermalstability. By attenuated toxin safety experiment, we proved that the PlyM2has noobvious cytotoxicity. The attenuation is successful. Mice was immuned with PlyM2inAlOH adjuvant subcutaneously, the serum was tested and proved that PlyM2has a goodimmunogenicity. In a S. pneumonia serotype14intranasal challenge mice model, the PlyM2immune group achieves a similar survival rate with the pneumonia conjugatevaccine group.In order to further improve the protection, this research also attempts to use a noveladjuvant system, the BLP technology, to reformulate the protein-based subcutaneousvaccine into a mucosis vaccine. The BLP technology use a highly safe bacteria-likeparticle, which is obtained from the food class Lactococcus lactis as the backbone. Theantigen is fusioned with a peptidoglycan binding tag, the bind to the BLP. The antigen-BLP formed a particle which just like the pathogen. It has a very good immunogenicity,and is perfect for mucosis immune. In this research, we successfully constructed PlyPA,which is the PlyM2fusioned with the peptidoglycan binding tag. Then the PlyPA isbinded to BLP and we have proved it thourgh immune fluorescent confocal microscopicobservation. BLP-PlyPA immuned the mice intranasally, and the IgA of PlyM2in thebronchoalveolar lavage fluid is detected. The BLP-PlyPA intranasally immuned grouphas a much higher IgA level the the PlyM2-Al subcutaneously immuned group.In this study, we proved that the Ply is a potential candidate of the novelpneumoniae protein-based vaccine, and we have presented that the BLP technology isa reseanable adjuvant system for the application of Ply in pneumoniae protein-basedvaccine.