The Research On Protective Effect And Immune Mechanisms Of A Novel Live Attenuated Streptococcus Pneumoniae Vaccine With A CaPi Mineral Shell

ObjectiveStreptococcus pneumoniae(S.pn) is one of the major causes of pneumonia, otitis media, sepsis and meningitis. The danger of Streptococcus pneumoniae in children and elderly is particularly prominent. Vaccination is an effective strategy to prevent and control of Streptococcus pneumoniae invasion. At present, 23-valent pneumococcal polysaccharide vaccine(PPV23) and protein-conjugate vaccines(PCVs) including PCV7, PCV9, PCV10, and PCV13 have been widely used to prevent pneumococcal diseases. However, these vaccinations have many disadvantages, such as weak immunogenicity, limited serotype coverage, serotype replacement and high cost. Therefore, to develop new and more ideal pneumococcal vaccine to prevent the infection has become an important issue.SPY1 that we previously reported, is a live attenuated vaccine of Streptococcus pneumoniae. The strain has no capsule and its virulence has been significantly reduced, its higher security and well protection effect even better than the current listing vaccines, so it has the potential of further development. But, the vaccine has poor thermal stability and weak immunogenicity, so it needs to be refrigerated preservation, and cannot play good protective effect without adjuvant. It has been reported that some researchers have used the biomimetic mineralization technology make the live yeast cells and viruses surface with a layer of an eggshell-like exterior. Further more, their thermal stability and immunogenicity were improved. Therefore, this study aims to in situ mineralization of SPY1, and then enhanced its thermal stability and immunogenicity, further to preliminary evaluate its immune protection effect, and laid a foundation to develop the pneumococcal live attenuated vaccine.MethodsThe gene of lyt A-ins was amplified by PCR with specific primers from S.pn D39 and then cloned into the expression vector p EVP3, the recombinant p EVP3-lyt A-ins was transformed into SPY1 to construct SPY1-△lyt A mutant strain; then the SPY1-△lyt A mutant strain was cultured in the calcium-rich environment, and then titrated with different concentrations of phosphate, coated itself with a layer of Ca Pi mineralization shell; scanning electron microscopy and direct fluorescent reaction was used to detecte the Ca Pi shell; the thermal stability of mineralized and non-mineralized strains was assessed by calculated the survival rate of these strains while placed them at 37℃ in different times; C57BL/6 mice were intranasally immunized with the mineralized strain SPY1-△lyt A-Ca Pi, calculated the colonization of Streptococcus pneumoniae 19 F between the mineralized vaccine and the control to preliminary evaluate the protective effect, and then preliminary evaluation of the protection mechanisms of this mineralized vaccine by detecting antigen specific Ig G and cytokine levels.ResultsPCR results and the growth characteristics of the SPY1-△lyt A mutant strain showed that the mutant strain was successfully constructed; scanning electron microscopy and direct fluorescent reaction showed the surface of the mutant strain was mineralized with Ca Pi; and the survival rate showed that the mineralized strains had well thermal stability than non-mineralized strains; after mucosal immuned C57BL/6 mice, compared with the un-mineralized strains, the mineralization vaccine can stimulated higher levels of IL-17 and IL-10, prompted that the mineralized vaccines can cause Th17 and Treg immune responses, and the mineralized strains can reduce the the colonization of Streptococcus pneumoniae 19 F in the upper respiratory tract of mice, preliminary revealed its good immune protection effect.ConclusionWe successfully construct the Streptococcus pneumoniae live attenuated SPY1-△lyt A(no autolyzed activity) mineralized strains, not only improves its stability and thermal stability, making it possible to out of the cold chain transport; at the same time make it independent on adjuvants to improve its immunogenicity. This research will provide theoretical and experimental basis for further development of highly efficient and stable Streptococcus pneumoniae vaccines.