Preliminary Study On SARS-CoV-2 DNA Vaccine Based On RBD Epitopes Linked By Cathepsin S Cleavage Site

Objective:The global pandemic of severe acute respiratory syndrome coronavirus-2（SARS-Co V-2）poses a serious threat to human health.The receptor binding domain（RBD）of SARS-Co V-2 S protein shows promising prospects for vaccine design,and some vaccines have been clinically evaluated.Moreover,DNA vaccines show great advantages in designing,manufacture and transport.In this study,we predicted the epitope of the RBD region,proposed a novel design strategy for SARS-Co V-2 DNA vaccine based on RBD’epitopes linked by cathepsin S cleavage site which could be targeted and presented efficiently by APC.Method:1.By Bio Edit,IEBD,Vaxi Jen v2.0 and other bioinformation analysis tools and software,the dominant epitopes of SARS-Co V-2 Wuhan-Hu-1 strain was selected.Two artificial protein sequences（LREP1 and LREP2）were designed by using two minimal cathepsin S cleavage sites（LMRK and LRMK）.The physicochemical properties,functional domains and tertiary structures of the two proteins were analyzed and predicted.2.Candidate DNA vaccines pc DNA3.1-LREP1 and pc DNA3.1-LREP2 were constructed using the eukaryotic expression vector pc DNA3.1 and transfected into293T cells.The expressions of the two proteins were verified by Western blot and indirect immunofluorescence.3.Mice were immunized with high（100μg/mouse）and low（10μg/mouse）doses of two kinds of DNA vaccines.Blood samples were collected 10 days after each immunization,and serum Ig G levels reacting with SARS-Co V-2 S protein were determined.4.Mouse spleen cells were extracted and the expressions of IFN-γ,IL-4 and IL-2were detected 24h after stimulating with S protein,so as to understand whether DNA vaccine can cause T and B cell reactions.5.The eukaryotic expression plasmid p AAV-GFP-SARS-2 S,which expressed both SARS-Co V-2 S protein and green fluorescent protein,was transfected into 293T cells（293T/Wuhan-Hu-1-S/GFP）to simulate the fusion between virus and target cell Huh-7.The cell-cell fusion model was further used to detect the immune sera’s inhibitory activity of different groups,and to evaluate the immune protection on SARS-Co V-2.Results:1.IEBD and Vaxi Jen v2.0 software were used to predict and obtain 10 B cell linear epitope peptides in the RBD region of SARS-Co V-2 S protein.The composition of LREP1α-helix and random coil were 48.23%and 30.52%respectively,and the composition of LREP2α-helix and random coil were 25.07%and 33.51%respectively.The values of LREP1 and LREP2 were 59.426%and 70.320%respectively.LREP1 and LREP2 accounted for 97.2%and 97.5%of dihedral Angle drops in the optimum area and suitable area in Ramachandran MAP,indicating a reasonable tertiary structure.2.The gene coding sequences of LREP1 and LREP2 were synthesized and linked into pc DNA3.1 through Bam HⅠand XhoⅠdigestion site.After colony PCR and double digestion identification,they were sent to the company for sequencing.The sequencing results were consistent with expectations.After transfection into 293T cell,Western blot showed the expected protein expression（42k D）in the supernatant of the medium.Indirect immunofluorescence results showed that both proteins could be expressed in the cell.3.Three times of immunization with candidate DNA vaccine could induce the production of SARS-Co V-2 S specific Ig G antibody（1:200 times serum dilution）,and the antibody level of both candidates shows that the high-dose group（100μg/mouse）was higher than the low-dose group（10μg/mouse）.4.Combined with the Prime-Boost strategy,the induction ability of immune response was significantly enhanced after the initial immunization of two doses of DNA vaccine and the enhancement of immunity by S protein.SARS-Co V-2 S specific Ig G levels were detected by ELISA with 1:200 serum dilution.P/N=2.38 in the LREP1 immunized groups and 12.67 in the LREP2 immunized groups.In the positive group（P group）,the specific Ig G antibody level P/N=5.39.The level of specific Ig G enhanced by LREP2 initial S protein immunized was 2.35 times that of positive control group P（three-dose S protein immunized group）（P<0.01）.5.Cytokine detection results showed that LREP1 induced IL-2:25.42±1.19pg/m L,IL-4:33.64±2.11pg/m L,IFN-γ:224.59±9.13pg/m L;LERP2-induced IL-2:136.39±16.78pg/m L,IL-4:38.09±5.17pg/m L,IFN-γ:1195.57±8.46pg/m L.Compared with LREP1,LREP2 had higher expression levels of IFN-γ（5.32 fold,P<0.001）,IL-4（1.94fold,P=0.352）,and IL-2（5.36 fold,P<0.00）.6.After immunization with two doses of heteroprime-boost strategy with initial immunized protein of DNA vaccine,mouse serum was used to perform cell-cell fusion inhibition experiment.The results showed that the immune serum of two candidate DNA vaccines had inhibitory effects,and LREP2 was the highest with IC₅₀was 92.557±0.564than LREP2 with IC₅₀was 86.351±73.021.Conclusion:1.The RBD epitope peptide of Wuhan-Hu-1 strain was predicted successfully,and"LMRK"and"LRMK"enzyme digestion sites were used to construct candidate DNA vaccine.2.Both candidate DNA vaccines can induce a Th1/Th2 mixed immune response.3.The antibody level and T/B cell reaction intensity induced by LREP2 with cathepsin cleavage site"LRMK"were better than those induced by LREP1 with"LMRK".4.The allogeneic Prime-Boost immune strategy enhanced by LREP2 primary S protein can achieve better immune effects than S protein alone.