The Study About Pan-SARS-CoV-2 Vaccine Based On The Recombinant RBD’epitopes Of VOC

Objective:The widespread epidemic of the severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)and its Variants of Concern(VOC)poses a serious threat to global health and economic development.Given that the first generation of Covid-19 vaccines cannot block secondary infection with VOC strains,this study intends to construct a Pan-SARS-Co V-2vaccine that can block SARS-Co V-2 and its VOC infection.Methods:First,The high-quality intact amino acid sequences of VOC S protein were downloaded from the NCBI database,we compared and analyzed the differences between those sequences and functional partition was clarified through multilpe sequence alignment software,and the amino acid sequence of RBD region was selected.The bioinformation analysis softwares were used to predict the B cell linear epitope of VOC RBD,then combined those epitopes with the amino acid concentration site region that was proved to produce neutralizing antibodies to pick out the dominant peptides.We selected the minimal cathepsin S cleavage site(LMRK or LRMK)to link those peptides to become a artificial protein named BLEP-1 and BLEP-2respectively,the physicochemical properties,secondary tertiary structure prediction of proteins were carried out.The sequences of BLEP-1 and BLEP-2 were synthesized and inserted into the prokaryotic expression vector p ET-21 a,and then plasmids p ET-21a-BLEP-1(LMRK fragment ligation)and p ET-21a-BLEP-2(LRMK fragment ligation)were obtained.The experimental condition was optimized to increase protein expression by investigating the optimal induction temperature,IPTG induction concentration,and protein expression form et all.Mix the Cathepsin S with BLEP-1 and BLEP-2 respectively,to observe whether the two proteins could be hydrolyzed by cathepsin S.The BLEP-1 and BLEP-2 proteins were multi-point injected intradermally to immunize mice at three different doses,high(40 μg),medium(20 μg)and low(10 μg),blood was taken10 days after each immunization,and frozen at-80 °C.The immunogenicity of the two proteins was evaluated and compared by ELISA,and the titer was determined at the same time.In order to determine whether it can produce cellular immunity against Wuhan-Hu-1 and Omicron,we used Wuhan-Hu-1 S protein and Omicron S1 protein to stimulate spleen cells of mice that were immuned,and then measured IFN-γ,IL-2,IL-4 cytokines expression levels.Finally,we optimized the experimental condition of the cell-cell fusion model mediated by SARS-Co V-2 S protein which was successfully constructed in the early stage,drew a time-fusion curve,selected the most suitable observation time and then to investigate whether the antibodies induced by the two proteins have inhibitory activity on cell-cell fusion model.Results:The amino acid concentration site region of the neutralizing antibody in RBD was aa402-421;aa444-460;aa470-477;aa480-505,combined with the biographical information analysis steps such as B-cell linear epitope results and antigenicity prediction,the dominant peptides containing each mutation site were screened as aa404-aa414,aa413-aa424,aa419-aa430,aa435-aa445,aa441-aa451,aa450-aa461,aa458-aa467,aa469-aa479,aa482-aa493,aa494-aa456.The physicochemical properties and secondary structure prediction results of BLEP-1 and BLEP-2 proteins were similar,and the three-dimensional structure predicted that BLEP-2 was more dominant peptide than BLEP-1 exposed to the protein surface.BL-21 was used as the expression strain,BLEP-1 was induced at 0.5 mmol/L IPTG concentration at 37°C for 12 h,and BLEP-2 was induced at 28°C 0.5 mmol/L IPTG concentration for 12 h,and both proteins were inclusion body.A large number of high-purity target proteins were successfully obtained by denaturation,on-column renaturation,and exploration of washing and elution conditions.Cathepsin S could hydrolyze BLEP-2 protein,but had no obvious effect on BLEP-1 protein.The ELISA results showed that the 20 μg dosage of BLEP-1 and BLEP-2 protein had the best immune effect.After third immunization of BLEP-1,the specific binding Ig G antibofy titer of Wuhan-Hu-1 S,Wuhan-Hu-1 S1 and Ocmicron S1 protein could reach 1:3,200,1:12,800 and 1:800,while the BLEP-2 could reach 1:6,400,1:12,800 and 1:102,400,indicate that BLEP-1 and BLEP-2 can effetively induce humoral response.Cytokine results showed that BLEP-1 and BLEP-2 could induce a Th1 polarized cellular immune response,and more often induced Th2 polarized cellular immune response.The result of cell-cell fusion inhibition test showed that 20 μg dosage of BLEP-2 had the best inhibitory activity,and it could still have a 50% inhibitory effect when diluted at 1:110.Conclusions:The humoral response of BLEP-1 is relatively limited while BLEP-2 protein have great immunogenicity,can induce the production of high-titer of anti-Wuhan-Hu-1 and Ocmicron-specific Ig G antibodies,and effectively inhibit SARS-Co V-2 S protein-mediated cell fusion.It can induce high levels of Th1/Th2 basis cellular immune response,which indicate that it has the potential to become a novel vaccine against SARS-Co V-2 and VOC.