Materials and methods
1. Isolation of 12-2A12
The mice were immunized with CVS-N2C G gene (Protein Access Number: ADJ29911.1) plasmid mixed with adjuvant at 1:1. The immune dose was 60 μg/mouse and immunized three times, with an interval of 21 days. The serum titer was measured by flow cytometry on the 7th day after immunization.
CHO-K1 cells overexpressing G protein were prepared by transiently transforming the CVS-N2C G gene DNA plasmid into CHO-K1 cells.
The mouse spleen was fused with SP2/0 cell lines and the fused cells were cultured in 37 ℃, 8% CO2 environment. On the 12th day, positive clones binding to G protein were obtained by flow cytometry. The Murine monoclonal antibody m12-2A12 was finally obtained through multiple rounds of detection, subculture and small-scale amplification.
RNA from hybridoma cells was extracted and reverse transcribed into cDNA. The target fragment was obtained by PCR. Connect the target fragment to the human vector to obtain the antibody light chain plasmid and the antibody heavy chain plasmid (the light and heavy chain V region is the mouse antibody sequence, the constant region is the human antibody sequence, and the antibody subtype is IgG1). The obtained plasmid was transiently transferred into CHO-S cells. After 4-6 days of culture, human mouse chimeric monoclonal antibody 12-2A12 was obtained from cell supernatant.
2.Cell culture
The HEK 293T cell lines (CRL-3216) were obtained from the American Type Culture Collection. It was cultured in Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT) with 100 U/mL of penicillin-streptomycin solution (GIBCO, Grand Island, NY), 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (GIBCO), and 10% fetal bovine serum (PAN-Biotech, Aidenbach, Germany) at 37 ℃, 5% CO2 environment.
3. Construction of RABV plasmids
All the sequences of RABV G protein in the Supplementary table1 were downloaded from GeneBank. Optimized and synthesized by General Biology Company (Anhui, China) according to the mammalian expression system, and finally cloned into pcDNA3.1 (+) vector. All plasmids were introduced into Trans5α Chemically-Competent Cells (TransGen Biotech, Beijing, China) for preservation and amplification, and extracted using the Plasmid Plus Midi Kit (Qiagen, Dusseldorf, Germany).
4. Preparation, and titration of RABV pseudoviruses
In the VSV pseudovirus system, first, the HEK 293T cells in good growth condition and full growth were subcultured into the T25 cell culture flask, and cultured at 37 ℃ and 5% CO2 for about 24 hours, and transfected when the cell confluence was about 80% ~ 90%. According the Lipofectamine 3000 (Invitrogen, Carlsbad, CA) instructions, RABV plasmids transfection into 293T cells. After 24 h incubation, the transfected cells were infected with G *ΔG-vesicular stomatitis virus (Kerafast, Boston, MA), which was diluted to 7.0×104 TCID50/mL by Dulbecco’s modified Eagle’s medium (HyClone). After 2h, the 293T cells were washed with PBS (GIBCO) three times and then 5 mL new complete culture medium was added. After 24h, the pseudoviruses in the culture supernatant were harvested, filtered using a 0.45 µm pore-size membrane (Millipore, Boston, MA) and stored at −80°C.
When the pseudovirus is titrated, a 5-fold initial dilution was carried out of 96-well culture plates, followed by serial 5-fold dilutions, with the last column only containing complete culture medium. Add 250 μL PBS to the 36 wells around the 96-well culture plates to reduce the experimental error caused by edge effect. Place 96-well culture plates in an incubator with 5% CO2 and 37 ℃ for 24 hours. After 24h, discarding 100 μL/well culture medium supernatant (100μL cell supernatant remaining in the well), adding 100 μL fluorescence detection reagent, placed in dark room temperature for 2 minutes. The luminescence values (PerkinElmer, Waltham, MA) were measured, and the TCID50 was calculated as described previously. The RLU value is 3 times higher than the cell control as the cut-off value. The value higher than the cut-off value is positive, and the value lower than the cut-off value is negative. The titer of pseudovirus is calculated by Reed-Munch method and expressed by TCID50.
5. In vitro RABV pseudovirus-based neutralization
Adjust the concentration of monoclonal antibody to 1000 ng/mL, add 96-well culture plates, and dilute 5 times in series, a total of 5 gradients. The same as the pseudovirus titer test, 265 μL PBS was added to 36 wells around the 96-well cell culture plates to reduce the experimental error caused by edge effect. RABV pseudovirus was diluted to appropriate titer with complete medium and add 50 μL/well into the 96-well culture plates. The 96-well culture plates were incubated 1 h at 37°C in an incubator with 5% CO2. After 1h, 100 µL of cells was added to each well. Following co-incubation for 24 h at 37°C with 5% CO2, the luminescence values were measured using a luminometer (PerkinElmer, Waltham, MA), and the reduction values were calculated by comparison with the control wells after 24 h. The IC50 of Abs were calculated with
the Reed–Muench method.
6. Affinity determination
The affinity of antibody 12-2A12 binding G protein was evaluated by SPR method. Preparation 10 × HBS-EP+ buffer solution, adjust pH value to 7.4. At the same time, the antibody was diluted to 1 μg/mL, using 1 × HBS-EP+, and Rabies-G-His was diluted to 1 μg/mL. Series S Sensor Chip Protein A chip was used to capture 50RU antibody by Biocore T200, and affinity analysis was performed with different concentrations of antibody (200, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0nM as control). The affinity data, including Ka, Kd and KD, were obtained after the kinetic fitting analysis of the experimental data using the evaluation software.
7. RABV G protein expression and purification
RABV-G-ΔFuLp used for crystallization were expressed by Bac-to-Bac baculovirus expression system (Invitrogen)[36]. The expression and purification steps of RABV-G-ΔFuLp are briefly described as follows, Sf9 cells were used to transfection and amplificated virus, and Hi5 cells were used to produce proteins. The cell culture supernatants were collected 48 hours after infection and passed through 0.22μm filter membrane. Then a 5 mL HisTrap HP column (GE Healthcare) were used for primary purification for the processed supernatants. Then further purified on a Source 15Q column (GE Healthcare) and a Superdex 200 Increase 10/300 GL column (GE Healthcare). Finally, RABV-G-ΔFuLp were exchanged into a buffer consisting of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl for crystallization.
8.12-2A12 Fabs preparation
12-2A12 Fabs were produced by papain digestion and further purified on a Protein A column (GE Healthcare) and a Superdex 200 10/300 GL column (GE Healthcare). Finally, 12-2A12 Fabs were exchanged into a buffer consisting of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl for crystallization.
9. Analytical gel filtration chromatography
Purified RABV-G-ΔFuLp was mixed with 12-2A12 Fabs at a molar ratio of 1:2 in a buffer consisting of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and then incubated overnight on ice. Finally, using a Superdex 200 Increase 10/300 GL (GE Healthcare) column for analysis, and identification by SDS-PAGE gel. Fractions containing 12-2A12 Fab/RABV-G-ΔFuLp complex were concentrated to 8 mg/mL for further crystallization trials.
10. Crystallization, data collection, and structure determination
Crystallization trials were established using the sitting drop vapor diffusion method with 1 μL of protein mixed with 1 μL of reservoir solution that was then equilibrated against 100 μL of reservoir solution at 18 °C. Diffractable crystals for the RBG-12-2A12 complex were obtained in the No.1-23 condition of Morpheus MD 1-46 kit (containing 30 mM Sodium fluoride, 30 mM Sodium bromide, 30 mM Sodium iodide, 50 mM Tris (base) and 50 mM BICINE (pH 8.5), 40% v/v Glycerol, and 20% w/v PEG 4000) with a protein concentration of 8 mg/mL.
Diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) BL19U (wavelength, 0.97919Å). For data collection, the crystals were cryoprotected by briefly soaking in reservoir solution supplemented with 20% (v/v) glycerol before flash-cooling in liquid nitrogen. The dataset was processed using HKL2000 software[37]. The complex structures of RBG-12-2A12 were determined according to the molecular replacement method using Phaser[38] with previously structures[22]. The atomic models were completed using Coot[39] and refined with phenix.refine in Phenix[40], and the stereochemical qualities of the final models were assessed using MolProbity[41]. The data collection, processing, and refinement statistics are summarized in Supplementary Table 2. All structural figures were generated using Pymol software (http://www.pymol.org).
11. Ethics
All animal procedures were performed in accordance with guidelines for the ethical review of laboratory animal welfare of (Number AB-101-18H0001), Biocytogen Pharmaceuticals (Beijing) Co., Ltd and accept the guidance and supervision and inspection of the laboratory animal welfare ethics review committee.