The Research Of Novel Coronavirus MRNA Nano-vaccines

At present,COVID-19(Coronavirus Disease 2019)caused by SARS-Co V-2(Severe Acute Respiratory Syndrome Coronavirus-2)is spreading all over the world,bringing a huge crisis to global public health security.As of April 09,2021,more than149 million innocent people have been infected with SARS-Co V-2,and the death toll was more than 3.1 million.Therefore,there is an urgent need for effective interventions,particularly vaccines.mRNA vaccine has the advantages of fast production,strong immunogenicity and good safety.Currently,six mRNA vaccines against COVID-19had entered clinical trials or received emergency government authorization for use.However,how to deliver mRNA to the body and achieve a safe and efficient immune response is still an urgent problem to be solved in the development of mRNA vaccine.The vaccine would show different efficacy and tolerability through different immune pathways and delivery vectors.However,the current marketing of coronavirus mRNA vaccines were delivered only depend on intramuscular injection and Lipid Nanoparticles(LNP).It remains to be seen whether better immune effects can be achieved by optimizing drug delivery routes(such as intradermal and subcutaneous injection)and developing other delivery vectors(such as Liposome(LP)and novel LNP).Based on this,RBD protein of SARS-Co V-2 was used as the model antigen to construct RBD mRNA,meanwhile,LP and novel LNP were used as vectors to construct the coronavirus mRNA vaccine in this study.The immune-inducing effect and antiviral ability of the constructed COVID-mRNA vaccine were evaluated by in vivo and in vitro experiments.The COVID-mRNA vaccine constructed in this project has not been reported at home and abroad,which is expected to provide new ideas for the research and development of COVID-19 vaccine.In this study,mRNA encoding RBD weas prepared by In Vitro transcription technology(IVT).First of all,the mRNA helper sequence optimized in the early stage of the research group in the influenza vaccine research was inserted into the sequence of the RBD coding gene.In order to enable the RBD to be secreted extracelluar to induce immune response,this experiment inserted the classic t PA as a secretion signal sequence to C-terminus of the RBD.Then,it was inserted into the p UC57 vector by the whole gene synthesis technology to obtain the recombinant plasmid p UC57-RBD.The recombinant plasmid was amplified and recovered,and the concentration and purity were checked.Next,RBD-mRNA(t PA-RBD mRNA)was prepared by IVT technology,and“cap”and“tail”were added.During the preparation process,each synthesis step was purified to ensure the purity of mRNA.Furthermore,in this project,the non-classical secretion signal peptide(derived from IL1β)was selected to replace the classical secretion signal peptide,and innovatively designed RBD-mRNA(IL1β-RBD mRNA)mediated by non-classical antigen secretion-promoting signal peptide.Furthermore,using the p M plasmid containing poly A(polyadenosine)tail sequence as a template and IL1β-RBD mRNA was obtained via IVT technology.IL1β-RBD mRNA didn’t required additional"tailing"operation to get the poly A tail.By monitoring the concentration and purity of mRNA during the preparation process,the t PA-RBD mRNA and IL1β-RBD mRNA constructed in this project had high purity and yield,which could meet the requirements of subsequent relevant experiments.mRNA is easily degraded in the body,therefore,in order to protect RBD-mRNA to ensure the efficiency of antigen encoding,two cationic materials,DOTAP and G0-C14 were selected in this project according to the research group’s previous expreience in the non-viral delivery vector of mRNA vaccine.Two kinds of carriers,LP(Liposome)and GP(G0-C14 liposome),were prepared for the delivery of t PA-RBD mRNA.First,LP and GP were prepared by solvent evaporation-film hydration method,and t PA-RBD mRNA was loaded by electrostatic incubation to prepare cationic lipid-mediated mRNA vaccines,that were LPX(Liposome-mRNA complex)and GPX(G0-C14 liposome-mRNA complex).Then,the RBD protein expression levels in vivo and in vitro and the in vivo immune-induced antibody titers of the two mRNA vaccines were evaluated.The results showed that LPX had higher RBD protein expression level,as well as Ig G and Ig M antibody titers.Therefore,LPX was selected for systematic pharmacy evaluation,in vivo immune effect research and safety evaluation.The particle size and potential of LPX were measured by a Malvern particle size analyzer.The results showed that the particle size of LPX was in the range of 160-170nm,and the potential was about 20 m V.LPX was irradiated with a laser beam,and it was showed that LPX had a significant Tyndall effect.Observing the microstructure of LPX by transmission electron microscope(TEM),LPX was spherical and multi-layered without obvious adhesion and aggregation,and the particle size was relatively uniform,which was consistent with the measurement results of the Malvern particle size analyzer.LPX was stored at 4°C and measured the particle size at different times.The results showed that LPX stored at 4°C remained stable particle size for 28 days.Gel electrophoresis test’s results showed that there were no free mRNA in LPX from Day28,which indicated the structure of LPX remained intact.Furthermore,WB(Western Blot),FCM(Flow Cytometry),laser confocal microscopy and ELISA technology were used to investigate the level of RBD protein expression in cells.The results showed that LPX could express RBD protein in HEK293T cells.At the same time,24 hours after transfection in HEK293T cells,the transfection efficiency of LPX reached 30-40%.In order to investigate the influence of different immune routes on the RBD expression level of LPX,the ELISA technique was used to determine the level of RBD protein in mice after administration of five immune routes(i.v.,i.m.,i.p.,i.h.and i.d.).It was found that different immune pathways showed different RBD protein levels,which illustrated that the immune pathway caused impact on the level of LPX antigen coding in vivo.The results laid the foundation for further investigation of the influence of different immune pathways on the final immune effect of LPX.At the same time,in vivo Imaging System was used to observe the protein expression of LPX in various tissues and organs in vivo.The results showed that LPX demonstrated spleen targeting,which was conducive to immune-inducing effect.Next,five immunization routes were used to immunize mice to investigate the titers of specific neutralizing antibodies induced by LPX in vivo.ELISA results showed that mice could produce high titers of specific neutralizing antibodies Ig G and Ig M after LPX immunized by five immunization pathways.After the second immunization(Day15),the antibody titer was mostly 400-1000,and the differences among the groups were relatively large.The i.v.group possessed the largest Ig G antibody titer,and the i.d.group had the largest Ig M antibody titer.After the third immunization(Day 30),the titers of Ig G and Ig M antibodies in the five groups were all around 1000 without huge difference.At the same time,the titer determination results of Ig G subtypes showed that Ig G2a subtypes were dominated in the i.h.group,i.d.group and i.p.group,which mainly tended to induce cellular immune responses mediated by Th1 cells(type I helper T cells).While,the titers of Ig G2a and Ig G1 were not significantly different between i.v.group and i.m.group.In addition,the specific antibodies induced by the five immune pathways all exhibited a good neutralizing effect on the SARS-Co V-2pseudovirus.In the pharmacodynamics research,the safety of LPX was initially evaluated.The results of the MTT cytotoxicity test showed that LPX had no obvious cytotoxicity at the experimental concentration;the in vitro hemolysis test results showed that LPX had no obvious hemolysis;After immunizing mice with LPX,the HE staining results showed that the mouse heart,liver,spleen,lungs and kidneys maintained normal structure and morphology,and there were no obvious side effects.These data showed that LPX had good biocompatibility and good safety.In this research,the valuable preclinical research data of the LPX vaccine conducted was collected in a comprehensive manner,which provided the feasibility for the vaccine to achieve clinical transformation,and also provided a reference for the clinical research of other vaccines.At the same time,LPX was the first attempt to construct a new mRNA vaccine.By active exploration of the construction of the vaccine system and comprehensive evaluation plan,this study had positive and significance reference for development of the COVID-19 vaccine in the future.In order to further develop a new mRNA vaccine that could induce higher antibody titers,furthermore,this study chose a new nanotechnology developed on the basis of Lipid Nanoparticles(LNP)as a carrier for in-depth research.First of all,selected the self-developed novel cationic lipid SKLB-11 to replace DOTAP as the new positive electricity center and supplemented with DOPE(Dioleoyl-Phosphatidyl Ethanolamine)and PEG(polyethylene glycol)as a functional component,and selected IL1β-RBD mRNA as the main drug,prepared LNPX(Lipid Nanoparticles-mRNA complex)vaccine using microfluidic technology.Investigating the aspects of particle size potential,appearance,microstructure and stability,LNPX had been systematically evaluated.The results showed that the particle size of LNPX was in the range of 60-65nm,and the potential was-1-0 m V,as well as had obvious Tyndall phenomenon.Observed by transmission electron microscopy,LNPX was a solid nanoparticle structure without obvious adhesion and aggregation.The particle size distribution of LNPX was relatively uniform.Stability experiment results showed that LNPX could be stored stably for at least 28 days at 4°C.The results of in vitro transfection had shown that the transfection efficiencies were more than 60%in HEK293T cells and DC2.4cells treated LNPX for 24 hours,which were better than LPX.At the same time,LP was selected to incubate IL1β-RBD mRNA to construct a new liposome vaccine NLPX(New Liposome-mRNA complex)as a comparative preparation.NLPX and LNPX were administered via five immunization routes(i.v.,i.m.,i.p.,i.h.and i.d.)to investigate the expression level of RBD protein in vivo.The results showed that the RBD protein levels of NLPX immunization groups were lower than LNPX,and LNPX showed better antigen coding ability in vivo.In addition,LNPX provided favourable spleen targeting was demonstrated by in vivo distribution experiments.After the investigation of the immune effect in vivo,the antibody titers of each group were different after the administration of five immunization routes:the antibody titers of each group were above 2000 in the first immunization,and the i.v.group showed the highest antibody titer,reaching 16000.After the second boost of immunization,the antibody titer of each group reached the maximum,except for the i.v.group up to 510,000,the remaining four groups(i.m.,i.p.,i.d.and i.h.)were all around 10,000,of which the i.m.group was higher than the other 3 groups(i.p.,i.d.and i.h.).At the same time,compared with the in vivo antibody titer study of LPX,the highest antibody titer of LNPX reached 510,000,which was much higher than that of LPX(3000),indicating that LNPX had a stronger immune-inducing effect than LPX.Finally,the neutralizing antibodies induced by LNPX also had ideal affinity for the SARS-Co V-2 pseudovirus,and the i.v.and i.m.groups diluted 6400 times had a neutralization efficiency of up to 90%to the SARS-Co V-2 pseudovirus.Moreover,we collected the spleen of the mice and studied the grouping of spleen cells in this study.The results showed that LNPX not only induced antibodies with high neutralization activity,also promoted the secretion of cytokines,inculding IL-2(Interleukin-2)and IFN-γ(Interferon-γ)from CD4~+T cells,showing Th1-type cell-mediated cellular immune response.At the same time,the cytokines could promote the proliferation and differentiation of B cells into plasma cells,thereby produced specific antibodies and enhanced humoral immunity.In addition,LNPX could promote the production of cytotoxic CD8~+T cells,which had the potential to further enhance the immune effect.Finally,a preliminary assessment of the safety of LNPX was carried out.The MTT experiment showed that LNPX didn’t produce obvious cytotoxicity.At the same time,LNPX had no obvious hemolytic toxicity to rabbit blood.After immunizing the mice,LNPX had no obvious side effects on the important tissues and organs of the mice.It was preliminarily proved that the new mRNA vaccine LNPX constructed in this project had good safety.In summary,the RBD protein of SARS-Co V-2 virus was used as antigen in this study to design and synthesize RBD-mRNA modified by classical and non-classical signal secreting peptides:t PA-RBD mRNA and IL1β-RBD mRNA.Based on the research experience of our group in the field of mRNA vaccine,firstly,we used LP as the carrier to load t PA-RBD mRNA to obtain the new mRNA vaccine LPX.In vitro and in vivo evaluation proved that LPX could effectively express RBD antigen in vivo and induce specific antibody against SARS-Co V-2.However,compared with the currently marketed mRNA vaccines,the immune-induced effect of LPX still dissatisfactory.Further,the mRNA vaccine was optimized in this study,that is,the self-developed lipid nanoparticles(LNP)was used as the delivery carrier to load IL1β-RBD mRNA and constructed LNPX.Studies had shown that LNPX had a higher RBD protein expression level in vitro and in vivo than LPX,as well as had a stronger immune-inducing effect and anti-viral ability under different immune pathways.The experimental results of this study suggested that the constructed LPX and LNPX were expected to be used as the new mRNA vaccine for further preclinical study.This study could provide new therapeutic strategies for effective prevention and treatment of COVID-19.Meanwhile,this study brought new ideas for research and development of COVID-19 vaccine.