The Study On MRNA Vaccine Against Foot And Mouth Disease Type O

Foot and mouth disease（FMD）,one of the important infectious diseases,can affect all cloven-hoofed animals.Vaccination is still the first choice for the prevention and control of FMD.Due to the large number of serotypes and easy variation of foot and mouth disease virus（FMDV）,low antigen matching between candidate vaccine strains and epidemic strains will affect the immune efficacy of vaccine and the effect of disease control.The mRNA vaccine has the advantages of fast speed,easy preparation,rapid design,synthesis and large-scale production in vitro,inducing high-efficiency protective immune response with low biosafety risk.Therefore,it is of great significance to develop its mRNA vaccine emergency reserve in order to deal with immune escape caused by FMDV mutation.The following studies were carried out in this study:1.Construction of in vitro transcription system for mRNA vaccine against FMDV type O.According to the molecular and functional characteristics of structural protein VP1 gene,the VP1 gene was amplified and ligated into p SFV1 vector to construct the recombinant plasmid p SFV1-VP1 using the inactivated Porcine FMDV type O strain as the template.The plasmid was transformed into E.coli DH5α,and the plasmid was extracted.The linearized DNA template digested with Spe I was transcribed into mVP1 in vitro.Subsequently,the mRNA was transfected into BHK-21 cells and verified its expression properly.The results suggested that BHK-21 cells transfected with mRNA after 24 h showed specific bright green cytoplasmic fluorescence using immunofluorescence assay（IFA）,while the control group had no fluorescence.Simultaneously,the target band verified by western blot appeared at 35k D in the mRNA transfected cells.These results showed that the recombinant mRNA can be effectively expressed in vitro,with evidence of the feasibility of this transcription system.2.Construction of mRNA vaccine delivery system and preparation of FMDV LNP/mVP1 vaccine.A large number of nano biomaterials have been constructed to deliver mRNA as a result of the ubiquitous RNases.Cationic lipid nanoparticles can bind mRNA by electrostatic adsorption,protecting mRNA from nuclease degradation,delivering mRNA to cells by membrane fusion.A cationic lipid nanoparticles（LNP）was constructed in this study,and its morphology,particle diameter,zeta potential,cytotoxicity and binding ability to mRNA were determined.Further,its function was evaluated by cell transfection in vitro.Cell experiment in vitro showed that LNP could protect p CMV-N-EGFP from degradation and smoothly deliver it to cells to translate antigen proteins.Subsequently,LNP and mVP1 previously obtained were self-assembled into FMDV LNP/mVP1 vaccine.The result showed FMDV LNP/mVP1 vaccine can be expressed in BHK-21 cells in vitro using immunofluorescence assay.In this study,vaccines were investigated by particle size and potential.The particle size of FMDV LNP/mVP1 vaccine was about 202.2 nm,and potential was 17.9 m V,which laid the foundation for subsequent animal experiments.3.The study on immunogenicity of FMDV LNP/mVP1 vaccine.Guinea pigs were used as immune animal model to compare guinea pigs serum antigen-specific Ig G antibody titers,virus neutralizing antibody titer,Th1 cytokine IFN-γ,and Th2cytokine IL-4 and the changes in the T cell subsets of the spleen cells in LNP,FMDV LNP/mVP1,FMDV CV and PBS group.The guinea pigs immunized were challenged at 35 dpv to evaluate the protective effect of FMDV LNP/mVP1 vaccine.The results showed that the antibody titer of FMDV LNP/mVP1 vaccine group was increased and reached the peak in the detection cycle at 35 dpv,and the expression levels of cytokines IFN-γand IL-4 were significantly increased（P<0.01）compared with LNP and PBS control group.The proportion of CD4⁺T cells and CD8⁺T cells of the spleen cells in the FMDV LNP/mVP1 group were increased compared with the LNP and PBS control group,with CD4⁺T cells increased significantly（P<0.05）.Observation of guinea pigs after challenge for 14 days,the result showed that all guinea pigs in PBS group and LNP group had obvious lesions.Among the animals immunized with FMDV LNP/mVP1 vaccine,one guinea pig（1/4）against 100 GPID₅₀（50%Guinea pig infectious dose）virus challenge had mild clinical symptoms,and the other animals were protected.The mRNA vaccine could provide 4/4 protection against 50 GPID₅₀virus challenge in vaccinated guinea pigs and 3/4 protection against 100 GPID₅₀virus challenge.Those results suggested that the FMDV LNP/mVP1 vaccine constructed in this study induced robust humoral immunity and cellular immune responses.At the same time,guinea pigs immunized with mRNA vaccine can free from FMDV challenge with an ideal protective effect.In conclusion,the developed mRNA vaccine prepared with FMDV VP1 gene as the candidate gene in this study can elicit humoral and cellular immune response with sound immunogenicity,providing a new approach for the development of emergency vaccine against FMDV,and giving experimental and theoretical basis for the design of other mRNA vaccines and the formulation of immunization strategies.