A Preliminary Study Of SARS-CoV2 RBD-DC Targeting Peptide Fusion Protein As A Subunit Vaccine

The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has seriously threatened human health and social development since it outbreak.SARS-CoV-2 infects host by the binding of the Receptor binding domain(RBD)on the surface spike glycoprotein with the angiotensin converting enzyme 2(ACE2)receptor.And spike glycoproteins exist as homologous trimers on the surface of the virus.In order to effectively block the infection and transmission of SARS-CoV-2,this study mainly designed RBD and WH-RBD trimer protein vaccines against SARS-CoV-2 wild type strain and Delta variant strain,and evaluated their immunogenicity in mice.In this study,we designed a secreted RBD trimer protein which was self-assembled by the T4 trimer domain.Then we fused WH peptide,a targeted polypeptide specifically bound to dendritic cell surface specific receptor C-type lectin domain family 9A(Clec9A),onto WT-RBD trimer and Delta-RBD trimer recombinant proteins.We also expressed and purified WT-RBD trimer,Delta-RBD trimer,WH-WT-RBD trimer and WH-Delta-RBD trimer protein by insect cell expression system.BALB/c mice were immunized with the four recombinant proteins with aluminum hydroxide adjuvant for three times,and peripheral blood was taken from the mice at the second week after each immunization.In order to evalute the efficency of the antibodies,we used SARS-CoV-2 pseudoviruses as antigen which were successfully constructed in our laboratory and detected the titer of IgG by enzyme linked immunosorbent.We found that the IgG antibody of mice was higher after the second immunization,and the IgG antibody titer was significantly increased after the third immunization.Compared with the WT-RBD trimer group,IgG antibody titers in the WH-WT-RBD trimer group were 2.3 times higher after the second immunization and 1.6 times higher after the third immunization.Whereas compared with Delta-RBD trimer group,WH-Delta-RBD trimer group showed no significant difference at IgG antibody level after the second and third immunization.The results of pseudovirus neutralization experiment showed that the serum of mice immunized with the four protein vaccines had good neutralization effect on both wild type and Delta(B.1.617.2)pseudovirus,but the neutralization effect on Omicron(B.1.1.529)pseudovirus decreased significantly.Compared with the WT-RBD trimer group,the neutralization titer in WH-WT-RBD trimer group against wild type,Delta and Omicron pseudovirus were about 1.6 times higher.However,the neutralization effect of serum between Delta-RBD trimer group and WH-Delta-RBD trimer group was not significantly changed.In addition,by comparing the neutralization titers against homologous and heterologous pseudoviruses,it was found that the neutralization and protection effect of the Delta-RBD/WH-Delta-RBD trimer serum groups was better than that of the WT-RBD/WH-RBD trimer serum groups.In conclusion,this study prepared four kinds of COVID-19 subunit vaccines,WT-RBD trimer,Delta-RBD trimer,WH-WT-RBD trimer and WH-Delta-RBD trimer by insect cell expression system.It was found that WH peptide could improve the immune effect of WT-RBD trimer protein vaccine,and had little effect on the immunity effect of Delta-RBD trimer protein vaccine,so further optimization was needed.The design of WH peptide vaccine provides a new idea for the design and development of novel efficient vaccines.