A SARS-CoV-2 neutralizing antibody discovery by single cell sequencing and molecular modeling

Although vaccines have been developed, mutations of SARS-CoV-2, especially the dominant B.1.617.2 (delta) and B.1.529 (omicron) strains with more than 30 mutations on their spike protein, have caused a significant decline in prophylaxis, calling for the need for drug improvement. Antibodies are drugs preferentially used in infectious diseases and are easy to get from immunized organisms. The current study combined molecular modeling and single memory B cell sequencing to assess candidate sequences before experiments, providing a strategy for the fabrication of SARS-CoV-2 neutralizing antibodies. A total of 128 sequences were obtained after sequencing 196 memory B cells, and 42 sequences were left after merging extremely similar ones and discarding incomplete ones, followed by homology modeling of the antibody variable region. Thirteen candidate sequences were expressed, of which three were tested positive for receptor binding domain recognition but only one was confirmed as having broad neutralization against several SARS-CoV-2 variants. The current study successfully obtained a SARS-CoV-2 antibody with broad neutralizing abilities and provided a strategy for antibody development in emerging infectious diseases using single memory B cell BCR sequencing and computer assistance in antibody fabrication.Twenty-seven blood samples were collected from donors who had recovered from SARS-CoV-2 infection. Before sequencing memory B cells, all the sera were detected for antigen recognition. A: Two virus antigens, spike (S) and nucleoprotein (N), were tested in enzyme-linked immunosorbent assay (ELISA) with sera dilution ratio of 1:2000 ( P <0.001, n =27). B: Gradient dilution was performed to get a response curve and relative EC 50 for further identification of spike protein reactivity. C: The inhibition of RBD binding to its ligand ACE2 was validated for the sera in competitive ELISAs with gradient dilution and the relative IC 50 was calculated. D: The neutralization or protection was examined in pseudovirus neutralizing assay. The pseudovirus was loaded with luciferase genes, and RLU was positively related to viral survival. Samples with strong recognition, high inhibition, and neutralization were in colors while others in black. EC 50 and IC 50 were summarized in Supplementary Table 1 . For the binding and inhibition, n =27 and for the neutralization, n =10. Data are shown as mean SD. Student's t -test was used for statistical analysis. EC 50 and IC 50 were calculated using nonlinear regression, four parameters in GraphPad Prism 9.0. OD450: absorbency at 450 nm; OD665/620: fluorescence ratio at 665/620 nm; RLU: relative light unit; [Ab]: antibody concentration; EC 50 : half maximal effective concentration; IC 50 : half maximal inhibitory concentration.A: Single B cells were sorted from peripheral blood mononuclear cells (PBMCs) of sample W25 by first discarding dead cells (left panel), gating for IgG subset (middle panel), and picking out S-His positive cells (right panel). B: The radio of His positive cells in the healthy donor (right panel, 0.031%) was lower than that in sample W25 (left panel, 0.058%). C: Antibody sequences originated from separated single B cells by reverse transcription and two rounds of semi-nest PCR with specific primers. D: Forty-two sequences were left for molecular modeling analysis after PBMCs isolation (2.24 10 8 cells), B cell separation (8.2 10 6 cells), single B cells expressing IgG antibodies against S protein (198 cells) and BCR sequence libraries (128 sequences). HC: healthy control.Using molecular modeling programs, schematic plots of antibodies and antigen-antibody complex were created and displayed. A: Fragment of the variable region (Fv) model of the H52 antibody (upper left) was generated by antibody modeling cascade. Receptor binding domain (RBD) structure [ 16 ] (upper right) was used as antigen input and H52-RBD complex was created by RDOCK program (lower). B: Interaction energy was calculated for Fv-RBD and HCDR3-RBD. The interaction energy of RBD-ACE2 calculated by the program was 57.00 kcal/mol and was taken as a standard. Only two antibodies, M20 and H52 had HCDR3-RBD energy near 57.00 kcal/mol (M20: 56.49 kcal/mol; H52: 53.09 kcal/mol) and H52 had the lowest Fv-RBD energy ( 132.96 kcal/mol). LCDR3: light chain complementarity determining region 3; HCDR3: heavy chain complementarity determining region 3; RBD: receptor binding domain.Six clusters were generated by amino acid similarities. Other parameters, such as interaction energy and amino acid composition, were shown in the heat map. Interaction energies of different parts were listed in columns as a fragment of variable region (Fv), heavy chain (VH), light chain (VL), HCDR3, and LCDR3. Candidate antibodies were selected in each cluster with the consideration of the parameters. Those with lower HCDR3 interaction, longer CDR3, and more polar or alkaline/acidic amino acids were regarded as elite (red star) and those with contrary features were control groups (blue circle). Details are shown in Supplementary Table 4 , available online. LCDR3: light chain complementarity determining region 3; HCDR3: heavy chain complementarity determining region 3.Thirteen candidate antibodies that belonged to six clusters were expressed and purified. A: Antigen recognition was firstly tested by enzyme-linked immunosorbent assay (ELISA). Antibodies in red were "elite" ones while those in black were controls in the clustering analysis. B: The three antibodies, H52, M20, and H45, which were positive in antigen recognition, were further evaluated in competitive ELISA where ACE2 was involved. C: Affinity was measured using bio-layer interference (BLI) with gradient receptor binding domain concentrations and K on , K off , K d were calculated. D: The two antibodies, H52 and M20, which exhibited better binding and inhibition, were analyzed in properties of HCDR3 and LCDR3 ( * P <0.05, ** P <0.01, n =13). Data are shown as mean SD. For ELISA, three replicates were included. In BLI, six concentrations were employed, and K d was calculated according to association and disassociation curve in BLI software BLItz Pro-1.2.0.49. Statistic analysis of antibody properties was performed using one-way ANOVA in GraphPad Prism 9.0. OD450: absorbency at 450 nm; [Ab]: antibody concentration.To study the epitope of antibody H52, peptides of receptor binding domain (RBD) were synthesized with 20 amino acids for each (named P1 to P10) and 23 for the last one (named P11). A: enzyme-linked immunosorbent assay (ELISA) was performed with an antibody gradient concentration from 2 g/mL. The area under the curve (AUC) was calculated and P4, P6, and P11 showed higher binding ability. B: Three peptides, named P4-1 to P4-3 were synthesized by replacement of a single amino acid to alanine and ELISA was performed again. AUC showed deceased binding ability. C: The mutated amino acids within RBD were highlighted with yellow background for B.1.1.7, B.1.351, P.1, B.1.617.2 (delta), C.37, and B.1.1.529 (omicron). No mutation was found in P4, P6 or P11. D: In a stereo schematic diagram, P4 (red), P6 (blue), and P11 (black) lie in the opposite side, while mutation sites (purple) were focused on RBD-ACE2 interface. AUC was calculated in GraphPad Prism 9.0 by three replicates in ELISA and was shown in a single number. ACE2: angiotensin converting enzyme 2.The reactive spectrum of antibody H52 was validated. A: The RBD proteins of SARS-CoV-2 variants B.1.1.7, B.1.351, B.1.617.2 (delta), P.1, C.37, and B.1.529 (omicron) were used as antigens. Antibody H52 was diluted with a gradient in enzyme-linked immunosorbent assays (ELISAs). B: The inhibition of RBD-ACE2 interaction was detected in competitive ELISA. Ratio of the mutated RBD and ACE2 was 2:1. C: Pseudovirus neutralizing assays were employed to evaluate antibody protection against SARS-CoV-2 and its variants B.1.1.7, B.1351, P.1, B.1.617.2, and B.1.1.529. Data are shown as mean SD. Each experiment includes three replicates. IC 50 was calculated using nonlinear regression, four parameters in GraphPad Prism 9.0. RBD: receptor binding domain; ACE2: angiotensin converting enzyme 2; OD450: absorbency at 450 nm; RLU: relative light unit; [Ab]: antibody concentration; IC 50 : half maximal inhibitory concentration.