Construction Of Dengue Virus Multivalent Nucleic Acid Vaccine And Verification Of Immune Effect

Dengue virus（DENV）is a mosquito-borne pathogen that causes about 300 million new infections each year,with a mortality rate of 5%.However,there is no specific drug available for its infection.Therefore,the development of DENV vaccines has become a hot spot in this field.Zika virus（ZIKV）and DENV belong to the Flaviviridae family.They are similar in the structure of EDⅢ protein and easily induce cross-protective immune responses.In recent years,EDⅢ（Envelope protein domain Ⅲ）in structural protein E has been identified as the main target for inducing highly neutralizing protective antibodies and has become the basis for constructing vaccines.At present,the relatively successful DENV vaccine is live attenuated vaccine,but this kind of vaccine has the risk of virulence recovery and is easy to cause antibody dependent enhancement（ADE）effect.Compared with traditional vaccines,the development of DENV multivalent nucleic acid vaccines is lagging behind.However,nucleic acid vaccines have the advantages of good immunogenicity,no need for adjuvants,and short development cycle,which has become an important direction in the field of vaccine research.Nucleic acid vaccine is an emerging vaccine technology,including DNA vaccine and mRNA vaccine;DNA vaccine has the advantages of low cost and simple delivery,while mRNA vaccine has higher safety.The optimization of mRNA vaccine delivery system and non-coding region sequence can improve mRNA translation efficiency,stability and help to stimulate higher levels of humoral immunity and cellular immunity.Therefore,the construction of multivalent nucleic acid vaccine targeting EDⅢ lays a theoretical foundation for the development of safe and effective DENV multivalent vaccine.【Objective】1.At present,DENV nucleic acid vaccine is mainly composed of four monovalent or two bivalent vaccines,which causes low humoral immunity,weak protection and unbalanced immune response.In this study,based on the nucleic acid vaccine technology system,a DNA vaccine with low cost and simple delivery method was used as the starting point for the development of DENV nucleic acid vaccine.The EDⅢ of the four serotypes of DENV structural protein E was used as the target antigen to construct a single sequence multivalent DNA vaccine that can cause balanced immune effects against the four serotypes of DENV.The DNA vaccine was delivered to muscle tissue by electrical pulse to verify the effectiveness of the tandem antigen in order to obtain a balanced immune response.2.Due to the higher safety of mRNA vaccine and the lagging research of single sequence DENV multivalent mRNA vaccine,this study further used the antigen verified by DNA vaccine as the target antigen,added the common TBT epitope of flavivirus,and intended to develop a safer and more effective DENV multivalent mRNA vaccine through lipid nanoparticle（LNP）delivery system and sequence optimization.【Methods】1.Construction of DENV multivalent DNA vaccine and validation of vaccine efficacyBased on the sequence of DENV published on NCBI,the EDⅢ region of four serotypes of DENV was screened,and the EDⅢ region of four serotypes of DENV was connected in series with G4S3 flexible peptide.The fragment was constructed on the p VAX1 expression vector as a DNA vaccine.The expression of target protein in DC2.4 cells after plasmid transfection was detected by immunocytofluorescence.In the mouse model,the level of specific antibody IgG was detected by enzyme-linked immunosorbent assay（ELISA）,and the levels of IFN-γand IL-4 were detected by enzyme-linked immunospot assay（ELISPOT）.The protective efficacy of serum after immunization was detected in the lethal dose virus attack suckling mouse model.2.Construction of DENV multivalent mRNA vaccine and validation of vaccine efficacy（1）Determine the optimal LNP lipid nanoparticle delivery systemThe eGFP mRNA was obtained by in vitro transcription and purification of mRNA.The eGFP mRNA was packaged with different N/P formulation delivery systems containing DLin-MC3-DMA and SM-102,respectively.The entrapment efficiency of mRNA-LNP was detected by Quant-i T Ribo Green RNA Assay Kit.The particle size,dispersion coefficient,ZETA potential and particle number of each mRNA-LNP were compared by DLS laser particle size analyzer to screen out the optimal delivery system.（2）Screening the dominant non-coding region UTR and signal peptide to achieve mRNA vaccine sequence optimizationTwo eGFP mRNA transcription template sequences containing R27 or ZK untranslated region（UTR）were constructed,and the target mRNA was obtained by in vitro transcription and purification.The fluorescence expression of each group after mRNA transfection in Vero cells was observed by microscope,and the dominant UTR was screened out.Gaussia luciferase（Gluc）was used as a model protein to construct Gluc mRNA transcription template containing TPA or IgE signal peptides.The target mRNA was obtained by in vitro transcription and purification.After the transfection mRNA was detected by Secrete-Pair^TM Gaussia Luciferase Assay Kit,the expression of Gluc protein in cell supernatant and whole blood of mice at each time point after intramuscular injection was detected,and the dominant signal peptide was screened.（3）Construction and immune effect evaluation of multivalent DENV mRNA vaccineUsing the four serotype tandem antigen sequences designed by DNA vaccine,carrying the common TBT epitopes and dominant UTR and signal peptide of flavivirus,a single sequence of DENV multivalent mRNA vaccine was constructed,and the optimal LNP was used for mRNA delivery.The protein expression of mRNA in cells was detected by immunocytofluorescence.In the mouse model,the level of specific antibody IgG was detected by ELISA,and the levels of IFN-γand IL-4 were detected by ELISPOT.The protective efficacy of serum after immunization was detected in the lethal dose virus attack suckling mouse model.【Results】（1）In this study,a DNA vaccine containing four serotypes of DENV EDⅢ can be effectively expressed at the cellular level and can successfully stimulate specific humoral immunity and cellular immunity against four serotypes of Dengue virus in mice.The post-immunization serum can protect suckling mice from lethal infection of four serotypes of DENV and has a partial protective effect on lethal infection of ZIKV.（2）The stability and the number of particles of the delivery system with N/P of 3:1 in DLin-MC3-DMA formulation were better than those of the other formulations.Compared with the N/P 3:1 delivery system of DLin-MC3-DMA,the N/P6:1 formulation of SM-102performed better in various characterization data.Therefore,the N/P6:1（LNP6:1）of SM-102was finally selected as the optimal mRNA vaccine delivery system.（3）The results showed that eGFP-R27 mRNA had the highest fluorescence intensity and the best persistence after transfection,and R27 was selected as the dominant UTR.TPA-Gluc mRNA expressed the highest protein content in the supernatant of transfected cells and the whole blood of mice,so TPA was selected as the dominant signal peptide.（4）The sequence-optimized DENV mRNA vaccine can effectively express antigen proteins at the cellular level and can successfully stimulate specific humoral immunity and Th1-biased cellular immunity against TBT epitopes shared by four serotypes of DENV and flavivirus in mice,which is superior to DNA vaccine.The serum after immunization can protect suckling mice from lethal infection of four serotypes of DENV,and the protective effect of serum after mRNA vaccine immunization on lethal infection of ZIKV is better than that of DNA vaccine.【Main conclusions】（1）In this study,a DENV multivalent DNA vaccine with four serotypes of DENV EDⅢ as antigen was constructed,which could successfully stimulate humoral and cellular immune effects in mice.（2）In this study,LNP6:1 of SM-102 was selected as the optimal delivery system for mRNA vaccine.（3）In this study,R27 was the dominant UTR and TPA was the dominant signal peptide.（4）In this study,a DENV multivalent mRNA vaccine with four serotypes of DENV EDⅢ and TBT as antigens was constructed.The antigen sequence was carried with the dominant UTR and signal peptide to obtain a sequence-optimized mRNA vaccine.The vaccine can successfully stimulate mice to produce better humoral and cellular immune effects than DNA vaccine.【Research significance】In this study,based on the nucleic acid vaccine technology system,a single-sequence,tandem DENV multivalent DNA vaccine was designed.It was verified that the DNA vaccine could stimulate mice to produce specific immunity against DENV,reflecting the effectiveness of the tandem DENV EDⅢ antigen sequence,which laid a solid theoretical foundation for the study of DENV multivalent mRNA vaccine.The DENV multivalent mRNA vaccine obtained by optimizing the UTR,signal peptide and delivery system of the mRNA vaccine can effectively activate the immune response against the TBT epitope shared by DENV EDⅢ and flavivirus in mice and play an immune protective role,which provides a theoretical basis and research basis for the subsequent development of DENV multivalent vaccine and flavivirus broad-spectrum vaccine.