Investigation Of MRNA Vaccine And Protein-based Subunit Vaccine Of SARS-COV-2

Coronavirus Disease 2019（COVID-19）caused by severe acute respiratory syndrome coronavirus 2（SARS-CoV-2）has led to a global pandemic with serious negative impacts on global public health and economic development.Vaccine research against SARS-CoV-2 has been carried out extensively.However,the emergence of SARS-CoV-2 variants weaken the function of the existing diagnostic,therapeutic drugs and vaccines,so there is still a long way to go in developing vaccines for COVID-19.The Spike（S）protein of SARS-CoV-2 is the most important structural protein.SARS-CoV-2 infects people by binding to the receptor angiotensin-converting enzyme 2（ACE2）on the surface of the host cell through the receptor binding domain（RBD）on the S protein.Therefore,S protein and its RBD region are key targets for SARS-CoV-2 vaccines.In the classification of SARS-CoV-2 vaccine,protein-based subunit vaccine has become the most widely studied and applied type because of its easy preparation,high safety,high stability,easy production and transportation.At the same time,mRNA vaccine,as a new type of vaccine,is also widely used due to its advantages such as short research and development period,rapid adaptation to constantly emerged variants and long immune response period.Therefore,based on the strategies of protein-based subunit vaccine and mRNA vaccine,this study intends to take SARS-CoV-2 S protein and its RBD region as targets to develop vaccines which could neutralize original type and multiple variants of SARS-CoV-2.1.mRNA vaccine based on SARS-CoV-2 RBDIn this study,mRNA vaccines encoding SARS-CoV-2 S1 protein or RBD domain were constructed separately and named S1-mRNA-LNP and RBD-mRNA-LNP respectively.The expression of mRNA vaccine encoded protein was detected by Western blot,and the results showed that mRNA vaccine encoded protein had good expression and stability.S1-mRNA-LNP and RBD-mRNA-LNP were used according to Intradermal injection（I.D.）or Intramuscular injection（I.M.）pathway to immunize mice with doses of 10μg or 30μg and the sera from immunized mice were collected at 10,40,and 70 days after the second immunization.The results were as follows:1)ELISA showed that S1-mRNA-LNP and RBD-mRNA-LNP vaccines of different immune doses and different injection pathways could all induce RBD specific antibody response which had no significant decrease at 70 days after the second immunization,and the level of specific antibody in RBD-mRNA-LNP group（Antibody titer:10³～10⁵）was significantly higher than that in S1-mRNA-LNP group（Antibody titer:10²～10⁴）.2)The results of neutralization experiment indicated that RBD-mRNA-LNP vaccine with different immune doses and immune pathways could all induce neutralizing antibodies against SARS-CoV-2 original strain pseudovirus（Antibody titer:10²～10⁴）and live virus（Antibody titer:10²）which had no significant decrease at 70days after the second immunization,and the titer is significantly higher than that of S1-mRNA-LNP.3)RBD-mRNA-LNP could also induce neutralizing antibodies against SARS-CoV-2 Alpha,Beta and Gamma variants pseudoviruses,as well as Delta and Omicron variants pseudoviruses and live viruses（Antibody titer:10²～10⁴）.4)Flow cytometry was used to detect dose-dependent inhibition of sera of mice and the results showed that compared with S1-mRNA-LNP,antibodies induced by RBD-mRNA-LNP could effectively block the binding of SARS-CoV-2 RBD to ACE2 receptors which could resist the invasion of SARS-CoV-2.5)Flow cytometry showed that Tfh cells,GC B cells in mice lymphoid tissue and plasma cells in mice spleen tissue were recruited in vivo,which were more significant in RBD-mRNA-LNP immunized mice.6)Stimulation of spleen cells by overlapping peptide libraries showed that RBD-mRNA-LNP could also effectively induce RBD specific T cell response.7)Challenge assay showed that RBD-mRNA-LNP vaccine was effectively against the fatal assault of SARS2-N501Y_MA30strain.In conclusion,this part of research suggests that RBD is a key target for designing effective SARS-CoV-2 vaccines,and RBD-based mRNA vaccines have great potential in alleviating the COVID-19 pandemic.2.mRNA vaccine based on SARS-CoV-2 Omicron BA1 SBased on the previous results,an mRNA vaccine encoding SARS-CoV-2 Omicron BA1-S protein（BA1-S-mRNA-LNP）was designed in this study.Immunizing mice with BA1-S-mRNA-LNP alone or in combination or sequentially with the RBD-mRNA-LNP,and the neutralizing antibodies against VOCs in sera of immunized mice were evaluated.Firstly,the expression of BA1-S-mRNA-LNP and RBD-mRNA-LNP vaccine encoded protein were detected by flow cytometry.The results showed that the specific proteins were highly expressed in 293T cells.Then the mice were immunized with the following five regiments:1)three doses of RBD-mRNA-LNP alone,2)three doses of BA1-S-mRNA-LNP alone,3)one dose of BA1-S-mRNA-LNP followed by two doses of RBD-mRNA-LNP,4)one dose of RBD-mRNA-LNP followed by two doses of BA1-S-mRNA-LNP,5)three doses of a 1:1combination of BA1-S-mRNA-LNP and RBD-mRNA-LNP,or 6)three doses of LNPs（control）.Specific antibodies in sera were detected at 10 days after the third immunization and the results showed that,except LNPs control group,the sera of the other 5 groups contained specific Ig G antibodies targeting the original strain of SARS-CoV-2,Delta and Omicron variants（Antibody titer:10³～10⁵）,among which the titer of the third experimental group was the highest（Antibody titer:10⁴～10⁵）.The neutralization test results of pseudovirus and live virus showed that,compared with other experimental groups,the sera of mice in the third experimental group contained not only neutralizing antibodies targeting the original strain of SARS-CoV-2,Alpha,Beta,Gamma and Delta variants,neutralizing antibodies targeting SARS-CoV-2 Omicron sub-variants（BA1,BA2,BA2.12.1 and BA5）can also be detected（Antibody titer:10³～10⁴）.In conclusion,this part of the study identified an effective immunization strategy（one dose of BA1-S-mRNA-LNP followed by two doses of RBD-mRNA-LNP）which could prevent the infection of various strains of SARS-CoV-2.3.Protein-based subunit vaccine based on glycosylated RBDPrevious studies showed that RBD can be used as an effective target for broad-spectrum vaccine research,and this section focused on further improving the immune protective effect of RBD.N-linked glycan probe were introduced into the predicted dominant non-neutralizing epitope,residue 519 of RBD protein of the original SARS-CoV-2 strain,to design mutant RBD subunit vaccine containing glycosylation sites at residues 519 and 521（MU-RBD）which was expected to improve the protective effect of Wild-type RBD subunit vaccine（WT-RBD）by blocking the non-neutralizing epitope.1)ELISA results showed that MU-RBD was well bound to human ACE2 protein,RBD specific serum antibody,and confirmed SARS-CoV-2 neutralizing antibody,and the binding affinity was not different from that of WT-RBD,indicating that the glycosylation site in MU-RBD did not affect its antigenicity or binding ability to specific neutralizing antibody.2)ELISA results showed that MU-RBD and WT-RBD immunized mice could induce specific Ig G antibodies binding to SARS-CoV-2WT-RBD,Delta-RBD,Omicron BA1-RBD and BA2-RBD.3)The results of neutralization experiments of pseudovirus and live virus showed that MU-RBD could not only induce neutralizing antibodies against SARS-CoV-2 Alpha,Beta,Gamma and Epsilon variant pseudovirus（Antibody titer:10³～10⁴）,neutralizing antibodies targeting original SARS-CoV-2strain,Delta and Omicron variant pseudovirus（Antibody titer:10²～10⁴）and live virus（Antibody titer:10¹～10³）were also induced,and the antibody titer was significantly higher than that of WT-RBD induced neutralizing antibody.4)MU-RBD showed stronger protective effect than WT-RBD in the challenge assay with SARS-CoV-2 original strain and Delta variant strain.In summary,this study confirmed that RBD protein residue 519 of the original strain of SARS-CoV-2 was the non-neutralizing epitopes,and MU-RBD subunit vaccine with closed epitope showed stronger neutralizing activity and protective efficacy than WT-RBD.4.Protein-based subunit vaccines based on S protein of SARS-CoV-2 original strain or Omicron variantIn this study,S protein of the original strain of SARS-CoV-2（WT-S）and S protein of Omicron BA1 sub-variant（BA1-S）were used as targets to develop protein subunit vaccines.Vaccine immunogenicity and neutralizing activity against Omicron and other VOCs were evaluated.ELISA results showed that WT-S and BA1-S proteins can bind to human,hamster and bat ACE2 receptors and S-specific serum antibodies.Mice were immunized with Alum+MPL as adjuvants with the following regimens:1)WT-S,2)BA1-S,3)one dose of WT-S followed by two doses of BA1-S;4)WT-S/BA1-S cocktail or 5)three doses of adjuvants（control）.The sera of mice at 10,30 and 90 days after immunization were collected for immune detection.Pseudovirus neutralization assay results showed that,unlike WT-S or BA1-S alone,the third sequential immunization and the fourth cocktail immunization,especially the cocktail immunization,induced effective neutralizing antibodies targeting the original SARS-CoV-2 strain and VOCs pseudovirus（Antibody titer:10³）,and the antibody titer had no significant decrease at 90 days after the third immunization.In addition,the cocktail regimen provided the best protection against lethal challenge of Delta variant.In conclusion,this part developed an effective protein subunit vaccination strategy that can induce a neutralizing antibody response against several currently circulating Omicron sub-variants,including BA5,and other SARS-CoV-2 variants.Overall,this study constructed and verified the mRNA vaccine and protein-based subunit vaccine based on the S protein and RBD region of the original and variant strains of SARS-CoV-2,and further optimized and improved the protective effect of the vaccine against the infection of the original and variant strains through the modification of glycosylation and the changes in vaccination strategy.This study could provide experimental basis and guidance for the subsequent research and development of SARS-CoV-2 vaccine.