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.