Study On New Vaccine Nucleic Acid Adjuvant System Based On Lipid Nanoparticles

Vaccination is the principal means to prevent infectious diseases,and new vaccines such as recombinant protein vaccines can well solve the potential danger of pathogen resurrection from traditional vaccines,but the high purity of recombinant protein usually weakens its immunogenicity.Adjuvant components need to be added to enhance the uptake and presentation of antigen by antigen-presenting cells and prolong the action time of antigen.In this research,firstly,we studied the immunogenicity of RBD and RBD-Fc proteins to determine the target antigen,and then resolute the TLR9 agonists by comparing the adjuvant effects of bacterial DNA and Cp G ODN2395.Given the biodegradability of nucleic acid molecules,we selected typical four-component cationic lipid nanoparticles as the delivery carrier to prolong their half-life period;at the same time,to obtain a recombinant protein vaccine with weak side effects,the composition of lipid nanoparticles was optimized and a series of analytical techniques were used to characterize lipid nanoparticles,which proved that LNP has been successfully prepared and can be used for follow-up experiments.The experimental results show that compared with the four-component lipid nanoparticles used in m RNA vaccine,the highest antibody level can be produced when three-component ionizable lipid nanoparticles are used to deliver TLR9 agonists as vaccine adjuvant.The main contents and results are listed as follows:1.The antibody titer test showed that the antigen RBD-Fc could induce a higher antibody level than the RBD.2.The result of agarose gel electrophoresis showed that the weight of BCG-DNA extracted by the CTAB method was between 200bp～750bp,which was much bigger than that of Cp G ODN composed of only 22 deoxyribonucleotides.According to this,it proves that the BCG-DNA was likely to contain Cp G motifs,and had the ability of immunostimulation.The results of animal experiments showed that although the naked BCG genomic DNA and Cp G ODN had the adjuvant effect,the adjuvant effect of Cp G ODN was stronger.3.The four-component lipid nanoparticle used for delivering Cp G ODN was synthesized by a nanoparticle synthesis system.The size of the unloaded was between 60～150nm.TEM results showed that the surface of lipid nanoparticles was smooth and there was no aggregation between particles.The results of the Elisa assay showed that higher antibody levels could be detected by ionizable lipid Dlin-MC3-DMA replacement compared with permanent cationic lipid nanoparticles.4.Simplifying the composition of lipid nanoparticles,and proving corresponding LNP were successfully prepared by a series of characterizations assay.Compared with other four-component or two-component LNP,the results of antibody titer detection showed that the highest level of antibody was detected when using the three-component ionized LNP as the delivery carrier.5.Through the comprehensive evaluation of the adverse reactions of mice after immunization and the results of pathological analysis of tissue sections,it was found that when delivery of Cp G ODN with three-component ionized LNP as a vaccine adjuvant system has a potential significance for further research.