The Screening of Broadly Neutralizing Antibodies Targeting the SARS-CoV-2 Spike Protein by mRNA Immunization in Mice

Figure 1. A schematic representation of design, optimization, and preparation of mRNA vaccines. (a) Antigen design of England and South Africa Chimera (ESC), Delta and Omicron BA.1 spike vaccines. In the ESC antigen, mutations in the Alpha variant are marked in red, the Beta variant in blue, and shared mutations in purple. The RRAR at the furin-cleavage site was replaced with GSAS. All mutations in Delta and Omicron BA.1 spikes are marked in black. (b) Verification of expression of pseudouridine (marked as pseudo) and N1-methyl-pseudouridine (marked as N1) modified mRNAs in HEK293T cells by western blot (WB) analysis. (c) Verification of expression of Delta, Omicron BA.1, and ESC mRNAs in HEK293T cells by WB which were used for mice vaccination. (d) The distribution of the diameters of lipid nanoparticles. Every sample was detected thrice. (e) The structure of mRNA LNPs under the cryo-TEM. Scale bar = 200 nm. (f) The polydispersity indexes (PDIs) of Delta, Omicron BA.1, and ESC LNPs. NTD, N-terminal domain; RBD, receptor binding domain; CTD, C-terminal domain; FP, fusion peptide; HR, heptad repeat; CH, central helix; CD, connector domain; CT, cytoplasmic terminal.

Figure 2. Mouse immunization and antibody titers in different groups. (a) Timeline of the mouse immunization. The diagram was created on Biorender.com. (b) OD450 values of enzyme-linked immunosorbent assay (ELISA) with sera from double-vaccinated mice during serial dilution. Individual curves are shown on the left, whereas grouped curves are shown on the right. (c) ELISA titers of grouped data from double vaccinated sera. (d) OD450 values of ELISA with sera from triple-vaccinated mice in serial dilutions. Individual curves from triple-vaccinated sera are shown on the left while grouped curves are shown on the right. (e) ELISA titers of grouped data from triple vaccinated sera. (f) Comparison of ELISA titers between double- and triple-vaccinated sera in each group. (g) Paired comparison of ELISA titers of ESC triple-vaccinated mice sera collected two weeks (2w) and six months (6m) after the final vaccination. The dilution endpoint cutoff was determined using the mean OD450 and threefold SD of the negative control group at the initial dilution, which was 0.557 in (c), 0.394 in (e), and 0.248 in (g). All data are presented as mean ± standard deviation (SD). The difference in the endpoint…

Figure 3. Neutralizing assay of mice sera from different groups against variant pseudoviruses. (a,b) Common logarithm of 50% pseudovirus neutralization (log pNT50) of triple vaccinated sera of groups A, B, C, and D against wild-type (a) and Beta (b) pseudoviruses. (c) Log pNT50 of triple vaccinated sera of groups A, C, D, and double-vaccinated sera of group D (2×Omicron BA.1) against Omicron BA.1 pseudovirus. (d) Log pNT50 of triple vaccinated sera from groups A and B against Delta pseudovirus. (e) ELISA titers of the anti-spike antibody in quadruple-vaccinated sera from groups C and D. The endpoint cutoff was determined by MeanNC + 3SD, which was 0.241. (f,g) Log pNT50 of quadruple-vaccinated sera from groups C and D against Beta (f) and Omicron BA.1 (g) pseudoviruses. All data are presented as mean ± SD. The difference among different experimental groups were determined by Welch’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. n = 5.

Figure 4. Acquisition and verification of monoclonal broadly neutralizing antibody (nAb). (a) OD450 values of ELISA with a standard sample at different concentrations (red triangles) and positive hybridoma media (black triangles). (b) Inhibition rate of hybridoma supernatants against wild-type, Beta, Delta, and Omicron BA.1 pseudoviruses. (c) Neutralizing rate (%) of the 6D6 monoclonal nAb to wild-type, Beta, and Delta pseudoviruses. The IC50s of 6D6 nAb against wild-type, Beta, and Delta pseudoviruses are 0.1189, 0.1089, and 0.5239 ng/μL, respectively. (d) Inhibition rate of ascites culturing the 6D6 hybridoma against wild-type pseudovirus. (e) The bands of amplified variable region of kappa chain (6D6 IGKV) and heavy chain (6D6 IGHV) on the agarose gel. (f) OD450 values of the ELISA with the medium of 293T cells transfected with expressing vectors of the 6D6 antibody. NC, the medium of untreated 293T cells, and PC, a clinically used SARS-CoV-2 nAb. (g) Inhibition rate of the media of 293T cells transfected with 6D6 plasmid, mRNA and 6D6 hybridoma medium to wild-type, Beta and Delta pseudoviruses. The inhibition rate and neutralizing rate are determined by the relative reduction of RLU in…