Optimization Of Dengue Virus MRNA Vaccine Preparation Method And Evaluation Of Immune Effect Of Combined Adjuvant

The development of vaccines is the most effective strategy to prevent the global spread of pathogens and suppress acute and chronic infections.mRNA vaccines are widely used in the prevention and treatment of diseases such as influenza virus and malignancies for their high safety,efficacy and rapid development potential.Compared to conventional vaccines,mRNA vaccines can stimulate both humoral and cellular immunity for adaptive immunity,producing a more balanced immune effect,and are expected to replace conventional vaccines as a tool for the treatment and prevention of certain diseases.The cost of in vitro mRNA preparation methods varies with the purity and stability of the mRNA obtained,so improving the quality of in vitro transcribed mRNA while controlling costs is a key focus of research and development.However,in the current development of mRNA vaccines,bare mRNA is highly susceptible to degradation by RNA enzymes after in vitro synthesis.Lipid nanoparticles(LNPs)are currently the most widely used mRNA vaccine vectors with high nucleic acid encapsulation rate,cell transfection efficiency,enhanced tissue penetration and lower cytotoxicity and immunogenicity for effective delivery of mRNA vaccines.Vaccine adjuvants have developed rapidly in recent years,including those already used in marketed vaccines,including aluminium salt adjuvants,MF 59,Virosomes,AS04,AS03,ISATM 51,AS01 and Cp G 1018,all of which induce a more rapid and sustained immune response;there are also a growing number of adjuvants in development to address drug treatment For example,the lipophilic bisphosphonate THZ93 has shown a preventive effect as an inhibitor of farnesyl diphosphate synthase(FPPS)in a model of pathogenic influenza.Adjuvants have been studied in combination with LNP vectors designed to enhance the therapeutic efficacy of mRNA vaccines.Dengue fever(DF)is a mosquito-borne viral disease that is estimated to cause between 50 million and 100 million infections each year.Dengue virus(DENV)is made up of four different serotypes: DENV-1,DENV-2,DENV-3 and DENV-4;infection with any one serotype provokes lifelong immunity against that serotype,but the antibodies produced by the patient not only have no effect against another serotype of DENV infection,but can The antibodies produced by the patient not only have no effect against the other serotype of DENV infection,but also lead to the development of more severe dengue fever,such as Dengue hemorrhagic fever(DHF)and Dengue shock syndrome(DSS).There is currently no safe and effective dengue vaccine available against the four serotypes,so this paper proposes to optimise the preparation of an mRNA vaccine and develop a vaccine against the four DENV serotypes in combination with adjuvants.The studies are as follows:(1)In this paper,the best mRNA preparation method was screened,and the mRNA expression obtained by this method was higher and more stable.Fluorescent expression was observed in Vero cells transfected with mRNA-e GFP for 24 h,48h and 72 h.The synthesized mRNA-Fluc was loaded into mice via LNPs vector,and stronger fluorescent expression was observed in mice after 6h.(2)The dengue mRNA-EDIII vaccine synthesized by this method can successfully translate and express the target protein intracellularly,and the results of immunization of mice by LNP delivery vector showed that the dengue mRNA-EDIII vaccine synthesized by this method can stimulate high humoral immunity against the dengue pathogen.(3)In this paper,the effects of two adjuvants,Cp G and TH-Z93,on the mRNA vaccine were screened.It was found that the additional addition of Cp G adjuvant enhanced the Th1 bias of the mRNA vaccine;and the serum protected 3-day-old suckling mice from lethal infection with four serotypes of DENV after immunisation.Research results:(1)The best mRNA preparation method C was screened in this paper.The mRNA expression obtained using method C was higher and more stable.The highest quality was obtained by transcribing mRNA-e GFP in vitro using method C.Fluorescent continuous expression was observed in Vero cells when the mRNA was transfected for24 h,48h and 72 h.The synthetic mRNA-Fluc was loaded into mice via LNPs vector and stronger fluorescent expression was observed in mice after 6h.(2)The mRNA-EDIII synthesized in this paper could successfully translate and express the target protein in cells;the synthetic LNPs/mRNAs were used in immunization experiments designed in a mouse model,which showed that both LNP(EDIII)-Blank and LNP(EDIII)+LNP(Cp G)groups produced higher serum antibodies against the four serotypes of DENV at 35 days after immunization The LNP(EDIII)-Blank and LNP(EDIII)+LNP(Cp G)groups both produced higher serum antibodies against the four serotypes of DENV;the LNP(EDIII)+LNP(Cp G)group caused a Th1 bias in immunization;the sera from the LNP(EDIII)-Blank and LNP(EDIII)+LNP(Cp G)groups protected 3-day-old dairy mice from lethal infection with the four serotypes of DENV,indicating that the vaccine provided better protection against all four serotypes of DENV infection The vaccine has been shown to provide good protection against all four serotypes of dengue virus.Study conclusions:(1)In this study,the best mRNA transcription method for laboratory mRNA vaccine studies was selected by comparing three mRNA transcription methods,and the method was successfully used to prepare EDIII tandem mRNA vaccines against four serotypes of dengue and to produce highly effective humoral immune antibodies against the four serotypes of dengue pathogenic proteins.(2)Cp G adjuvant combined with LNP vector further enhances the Th1 bias of the mRNA vaccine and the immune serum provides complete protection against dengue virus infection in mice.Research significance:In this study,a better mRNA transcription method was selected based on existing mRNA transcription techniques in vitro.The method was also used to construct a delivery system of lipid nanoparticles combined with adjuvant against dengue tetravalent serotype.The immunization strategy was successful in stimulating the production of antibodies against dengue virus tetravalent serotype in mice and enhanced the Th1 bias of the mRNA vaccine,which is of great reference value for vaccine development.