Design And Evaluation Of Broadspectrum MRNA Vaccine Based On Novel Coronavirus RBD

With the evolution of novel coronavirus（SARS-CoV-2）,many variants with faster spreading speed and more fatal virulence are also emerging.This situation has reduced the immune efficacy of existing vaccines,which brings great challenges to the prevention and control of epidemic situation in the world.Thus,it is important to develop new SARS-CoV-2 vaccine with broad-spectrum protection against existing and potential variants.As a new type of nucleic acid vaccine,mRNA vaccine can activate humoral immunity and cellular immunity and has a rapid development speed.For example,two kinds of WHO-approved SARS-CoV-2 mRNA vaccine have shown good safety and protection ability.Most SARS-CoV-2 vaccines target SARS-CoV-2 spike protein receptor binding domain（RBD）,which binds to the Angiotensin-converting enzyme 2receptor on the surface of host cells.In this thesis,we explore techniques to enhance the immunogenicity and sequence coverage of mRNA vaccines through glycosylation modification and monomer cascade.Our research includes:（1）We establish the synthesis system and characterization system of mRNA in vitro.Using EGFP as the coding model,we obtain mRNA with high purity and molecular integrity by plasmid template construction,plasmid linearization,mRNA transcription in vitro and purification of mRNA synthesis products.Transfection experiments showed that the synthesized mRNA-EGFP is functional and could be efficiently expressed in cell lines.The experiments of particle size distribution and entrapment efficiency showed that the prepared mRNA liposome nanoparticles have uniform properties.（2）We explore the effect of RBD deglycosylation on the immunogenicity and broad spectrum property of mRNA vaccines.Based on the RBD glycosylation site,we first construct the deglycosylated sequence RBD_206（w）（I332-K537）by truncating the RBD_219（w）（R319-K537）sequence.Then we further construct two strands of RBD_219（w+w）-dimer and RBD_206（w+w）-dimer.In the evaluation experiments of mouse model,we show that although the recombinant-protein-form deglycosylated RBD can improve the immunogenicity,the immunogenicity and broad spectrum of RBD deglycosylated mRNA vaccines are lower than RBD glycosylated mRNA vaccines due to unexpected translation efficiency,which suggestes that the immunogenicity and expression level of target antigen should be taken into account in mRNA vaccine design.Similar to the recombinant protein vaccines,the immunogenicity and broad-spectrum of mRNA vaccines of homologous two-stranded RBD are significantly higher than that of monomer RBD mRNA vaccines.This finding provides a basis for the design of multi-strand mRNA vaccine covering different mutant RBD.（3）From the view of sequence coverage improvement,we explore the effect of multi-strand RBD on the immunogenicity and broad-spectrum of mRNA vaccines.According to the prevalence of different variants,we construct RBD219（γ+δ）-dimer,RBD219（α+γ+δ）-trimer and RBD219（α+β+γ+δ）-tetramer vaccines.The detection results of specific Ig G antibodies and pseudoviral neutralizing antibodies against different variants showed that the three multistrand RBD vaccines all induced high titers of specific antibodies.Specifically for omicron variants,the pseudoviral neutralizing antibodies of RBD219（γ+δ）-dimer,RBD219（α+γ+δ）-trimer and RBD219（α+β+γ+δ）-tetramer are significantly higher than those of RBD_219（w+w）-dimer,among which RBD219（α+γ+δ）-trimer candidate vaccine had the strongest immunogenicity and the best broad spectrum.To sum up,based on the technical system of mRNA vaccine,this study explored the effects of RBD deglycosylation and monomer series on the immunogenicity and sequence coverage of SARS-CoV-2 vaccine,which can be used as an example for the design of new broad-spectrum SARS-CoV-2 vaccine.