Viruses and antibodies
SARS-CoV-2 to B.6, and delta lineage viruses were isolated as described previously 6,7. Leo Poon provided SARS-CoV-2 Omicron isolate (sub-lineage BA.1) 8. Vero E6 or Calu-3 cells were used to propagate SARS-CoV-2 variants. Dr. Raiees Andrabi (Scripps Research Institute, USA) generously provided the full-length spike proteins of SARS-CoV-1, SARS-CoV-2, MERS, HKU1, OC43, NL63, and 229E. CR3022 antibody was purchased commercially (Sino Biologicals). II62 IgG a non-neutralizing antibody was used from our previous study 9. The pseudotyped viral stocks of all seven alpha and beta coronaviruses were prepared as described in our previous study 10. We used B.1.1.7 (Alpha), B1.1.351 (Beta), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.1.529 (Omicron) terminology in throughout our manuscript.
Expression and purification of antibody and RBD protein
A synthetic codon optimized (for mammalian cells) nucleotide sequence of RBD–His (wt), spike protein (wt), and a point mutant of RBD-His (N501Y) protein (variant B.1.1.7) and RBD corresponding to other variants from SARS-CoV-2, were cloned in pcDNA3.1 plasmid vector. The constructs were expressed and produced in Expi 293 F mammalian expression system. Briefly, the cells were transiently transfected with the plasmids. The supernatant was collected after 5-6 days, and the soluble protein was purified using Ni-NTA affinity chromatography (Qiagen, Germany).
Animal ethics statement
All the mice were procured from the Small Animal Facility, THSTI, Faridabad. All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals as promulgated by CPCSEA, Government of India and adopted by the Institution Animal Ethics Committee of THSTI (IAEC Project/Protocol No: THSTI-IAEC-146, IAEC-160). The approval of the Institutional Biosafety Committee (approval # THS-354/2021) and Department of Biotechnology Review Committee on Genetic Manipulation (RCGM approval #: BT/IBKP/137/20220) were taken before commencing work.
Generation of hybridoma for anti-RBD (N501Y) murine monoclonal antibodies
Six to eight week old, female BALB/c mice were immunized intramuscularly (I.M.) with purified RBD (N501Y) protein (30 µg in 100 µL PBS per animal) along with Quil‑A adjuvant (InvivoGen, USA). Mice were boosted 3 times with the purified protein (30 µg, 15 µg and 7.5 µg in 100 µl PBS per animal), along with Quil‑A adjuvant. Sera samples from mice were collected three days after the first and second booster. The mouse with the highest titer of serum cross-neutralizing antibodies, was given a final booster injection 4 days before the spleen was aseptically removed. Splenocytes were utilized in the generation of hybridomas using ClonaCell-HY Hybridoma Generation Kit (STEMCELL Technologies, USA), in accordance with the provided protocol. The well adapted antibody secreting clones were propagated in tissue culture flasks and frozen vials of each clone were stored in liquid nitrogen for future use. Hybridoma clones secreting anti-RBD antibodies were further screened in ELISA assay.
For the screening of hybridoma clones/ heat inactivated mice sera (Dilution starting from 1:100 to 218,700)/ purified mAbs (5 µg/ mL-1 to 0.002 ), ELISA plates were coated with recombinant RBD (N501Y) protein (1 µg mL-1; 100 µL per well). Plates were blocked with 5% non-fat milk and hybridoma culture supernatant or purified mAbs were added in three fold serial dilutions. Following incubation for 1h at room temperature, goat anti-mouse-HRP conjugated secondary antibody (Jackson Immunoresearch, USA; dilution 1 in 2500) was added to each well. In case of titration experiments, purified mAbs (100 µL per well) were added as three fold serial dilutions starting with 5 μg mL-1. Standard protocols of blocking (5% skimmed milk in PBS) and washing of ELISA plates (4 times with PBS-0.05% Tween-20) were followed as described previously 11,12.
Purification of anti-RBD (N501Y) murine monoclonal antibodies
Serum-free media designated for monoclonal antibody production was used to propagate the hybridoma cells (0.5 × 105 cells per mL) in a T175 tissue culture flask in order to purify the antibodies. For scale up of antibody production, a WHEATON CELLine flask was used for hybridoma culture, following the manufacturer’s instructions. Protein G Agarose resin (G-Biosciences) was used to purify the anti-RBD IgG mAbs from the hybridoma culture supernatant. Five column volumes of 1X PBS were used to wash the beads in the column. Two to three column volumes of 0.1 M glycine (pH 2.5) was added to elute the antibodies, followed by neutralization with 1M Tris-HCl (pH = 8.0). The purified antibodies were dialyzed against 1X PBS three times, using dialysis tubing (Thermo Fisher Scientific, MWCO 10 kDa), and concentrated with a 50 kDa MW cut-off Amicon Ultra-15 centrifuge unit (Millipore). A 0.2 μm syringe filter (Mdi, membrane Technologies) was used to filter the antibody solutions before they could be used in experiments. NanoDrop spectrophotometer was used to estimate the protein concentration and purified IgG mAbs were visualised using 12% Tris-Glycine-SDS-PAGE analysis.
Cytopathic effect based neutralization assay
Initial screening of heat-inactivated mice serum samples and hybridoma supernatants weas performed as described 9,13, with slight modification. Briefly, heat-inactivated serum or purified mAbs were serially diluted two or four times and mixed with 100 TCID50 of SARS-CoV2 isolates. The serum or mAb mixture was transferred to the Vero E6 monolayer seeded in a 96-well plates in triplicate and incubated for 1h. The cell surface was then washed with serum-free media, and fresh complete medium was applied. The plate was further incubated for 72h at 37°C in a humidified CO2 incubator. The cells were observed for the absence of viral cytopathic effect and was used as an indicative of neutralization. The neutralisation titer was defined as the dilution at which no cytopathic impact was seen.
Live Virus Focus Reduction Neutralisation Assay:
Virus neutralisation assay was performed as described previously by our group 14. Briefly, purified P4A2 antibody was twofold serially diluted starting from 20 to 0.039 µg/ mL-1 . The virus neutralization assay was performed in Vero E6 cells. Cells were incubated for 24 hours for Delta and 32 hours for Omicron variant. The virus stock was propagated in Calu-3 cells (American Type Culture Collection). Control purified IgG was used as experimental negative control.
Pseudovirus assay based neutralization assay
Full length spike proteins of all alpha and beta coronaviruses were co-transfected in 1.25 x 105 HEK293T cells, with helper plasmid expressing firefly luciferase, an HIV-1 backbone and for SARS-CoV-1 and -2, serine protease TMPRSS2 (CMV-Luc, R∆8.2 backbone plasmid, pTMPRSS2). After 68-72h culture supernatant was collected and stored at -70°C. To access the cross- neutralization potential of P4A2, 3-fold serial dilution (starting at 10 µg mL-1) of P4A2 was performed. The serially diluted P4A2 antibody was mixed with respective pseudotyped viruses for 60 minutes at 37°C. Pseudovirus/ P4A2 combinations were added to 293T-ACE2 cells pre-seeded (24h) at 20,000 cells per well. After 48–72h, relative luminescence unit (RLU) was measured on luminometer. The percent reduction in neutralization was measured as ratio of relative luminescence units (RLU) readout in the presence of P4A2 normalized to RLU readout in the absence of bnAb. Four-parameter logistic regression was used to calculate the half maximum inhibitory concentrations (IC50) (GraphPad Prism version 8.3). II62 and CR3022 mAb against SARS-CoV-2, was used as assay control.
A 96-well plate was seeded with Vero E6 (25,000 cells per well). Virus suspensions of the indicated MOIs (100 µL per well) were added and the plate was incubated for 1 h. After 24 h incubation, cells were fixed with 7.4% formaldehyde and left overnight. Wells were washed with PBS thrice. The cells were permeabilized with 100 µL buffer (20 mM HEPES, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 0.02% sodium azide) at room temperature for 20 min. The cells were incubated with mAb P4A2 (diluted 1 in 2000) at room temperature for approximately 6- mins. Secondary antibody (anti-mouse Alexa 488, cat no.: A21206, Invitrogen; 100 µL diluted 1 in 500). Micro-plaques read using AID iSpot Analyzer (Autoimmun Diagnostika GmbH) and foci were counted using AID ELISPOT 8.0 software.
To check the binding of P4A2 with Delta and Omicron infected Calu 3 cells (90,000 in each well) were seeded in 8 well chamber slides. Cells were incubated for 48 h at 37°C in an incubator with supply of 5% CO2. DMEM high glucose with 10% (v/v) fetal bovine serum (FBS) was removed 24 h later and cells were washed with 1x PBS. Cells were infected at 0.5 MOI (DMEM High glucose supplemented with 2% FBS). Added Delta and Omicron virus (100 µL per well) and incubate at 37°C on a rocker. Removed the virus and washed twice with 1x PBS. Added 300 µL of 10 % complete DMEM and incubated for 24 h at 37 °C.
Biolayer interferometry binding assay
Binding assays were carried out using an Octet Red instrument (Forte'Bio) using biolayer interferometry (BLI), as previously described 9,15. Briefly, mouse Fc sensors (ForteBio Inc.) were used to capture the P4A2 mAb at 10 μg mL-1 in 1 x kinetics buffer [1x PBS, pH 7.4, 0.01% (w/v) BSA and 0.002% (v/v) Tween-20] and incubated with different concentrations of RBD. Associations and dissociations has been reported, depending on the analyte. Data was analysed using the software ForteBio Data Analysis. The starting concentration of RBD was 300 nM followed by three-fold serial dilution.
Purification, crystallization, data collection, refinement and analysis
A total of 30 mg of P4A2 mAb was digested for Fab preparation. The Fab preparation was performed by Pierce™ Fab Preparation Kit (cat. No. 44985) as per the manufacturer protocol. The purified Fab and RBD proteins were mixed at 1:1.7 ratio and incubated overnight at 4 ℃. This was then subjected to size exclusion chromatography on 16/600 Superdex 200 column (Cytiva) in a buffer containing 20mM HEPES 7.5 and 150 mM NaCl. The peak corresponding to P4A2 Fab:RBD complex was concentrated to 10 mg mL-1 and stored at -80℃ by flash freezing. The purified complex was subjected to crystallization trials using commercially available screens and trays were set up using Mosquito Crystallization Robot (TTP Labtech). The hits obtained in different screens were further expanded to produce single crystals which were tested for diffraction using a METALJET X-ray home source (Bruker Inc.). The condition that provided crystals with best diffraction quality was composed of 0.2 M magnesium formate dihydrate and 10% PEG 5KMME. These crystals were frozen with 20% glycerol as cryo-protectant. X-ray diffraction data could be collected to a maximal resolution of 3.0 Å at the automated ID30A-1 beamline in ESRF, France 16. The diffraction data was processed using IMOSFLM 17 and AIMLESS 18 programs of the CCP4 suite (Supplementary Table S1).
The structure was determined by molecular replacement using PHASER 19 and the search model was the STEC90-C11 Fab:RBD complex 20. This model was subjected to iterative model building and refinement using COOT 21 and PHENIX 22, respectively and the sequence of the STEC90-C11 Fab was slowly changed to that of P4A2. The final Rfree and Rwork are 28.2 and 23.0%, respectively. The refined structure was deposited with Protein Data Bank with the accession code 7WVL.
The structure was visualized and analysed using PYMOL (Schrödinger Corp.), and the interactions were identified using the CONTACT program of CCP4 23. Mutations were created in silico in the RBD structure using PYMOL (Schrodinger Inc.) to obtain models of P4A2 Fab bound to RBD corresponding to different SARS-CoV-2 strains. These models were subjected to energy minimization using the DESMOND module of the Schrodinger suite (Schrodinger Inc.) and then analyzed. Two models of P4A2 bound to Spike Trimer with the RBD in the up and down conformation were prepared using 7TM0 24 and 7TOU 25 and these models were also subjected to energy minimization using the DESMOND module. All the figures were prepared using PYMOL.
Animal protection studies
Eight to ten week old K18-hACE2 transgenic mice were pebbled and randomly allotted to different groups (n = 5) viz., infection control and those receiving P4A2 in different cages. The animal experiments and procedures were performed in accordance with the IAEC, IBSC and RCGM guidelines. In prophylactic treatment, antibody recipient groups were given intraperitoneal (IP) infusion of P4A2 mAb one day before challenge (day ‘-1’), except for the control group where PBS was given (No virus challenge). In therapeutic treatment group, the mAb was administrated 6-12 h post infection.
Clinical spectrum of SARS-CoV-2 infection
The mouse experiments were done at the Animal Biosafety Laboratory (ABSL)-3. Change in daily body weight, activity and clinical symptoms of all the animals were monitored post infection. On day 6, all the infected animals were euthanized, the lungs were collected and imaged for gross morphological studies. The Right lower lobe of the lung was immersed in a 10% (v/v) neutral formalin solution and subjected to immunohistochemistry analysis. The viral load parameters were analysed using homogenized lung tissues in a 2 mL Trizol solution. The homogenates were stored immediately at -80ºC till further use. Blood was drawn from the animals via the retro-orbital vein on days ‘-1’ and ‘0’, and via direct heart puncture after euthanizing the animal. Serum samples were stored at -80ºC for future experiments.
Quantification of viral load in lung
RNA was isolated from homogenised lung tissues using the Trizol-chloroform technique according to the manufacturer's procedure, and quantified using Nanodrop. Using the iScript cDNA synthesis kit (Biorad, USA) was used for cDNA synthesis. Briefly, 1 mg total RNA was reverse-transcribed into cDNA. The qPCR was performed on diluted cDNAs (1:5) using the KAPA SYBR FAST qPCR Master Mix (5x) Universal Kit (KK4600) and 7500 Dx real-time PCR equipment (Applied Biosystems, USA).The results were analysed with SDS2.1 software as previously described 26. For virus load estimation, the CDC-approved SARS-CoV-2 N gene primers 5′-GACCCCAAAATCAGCGAAAT-3′ (Forward) and 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ (Reverse) were used as previously described 27. The log10 copy number of N gene was calculated by using pre-titrated SARS-CoV-2 genomic RNA and expressed as Log10 copy number/ lung mass (mg). To produce the standard curve for absolute quantification, a known copy number of viral RNA was employed as a standard.
Reverse transcriptase - polymerase chain reaction and nucleotide sequencing
The variable region of the immunoglobulin heavy and light chain genes expressed in B cell hybridoma P4A2 were PCR amplified and sequenced following the protocol and primers described previously 28. Briefly, cDNA was synthesized from 10 to 50 snap frozen hybridoma cells, using a commercially available kit (Qiagen, Germany) with isotype specific antisense primers, each at a concentration of 0.75 μM. The 20 μL reaction was performed at 42°C for 30 min. Reverse transcriptase was inactivated by incubating at 95°C for 3 min. The nested PCR amplification was performed using Q5 DNA polymerase (New England Labs, USA). The cDNA (4 μL) was used as template in a 50 μL first round PCR which comprised of external antisense primer (0.25 μM) and a cocktail of VH (or VL as the case may be) family specific external sense primers, each at a final concentration of 0.1 μM, 1 ´ Q5 DNA polymerase buffer, dNTPs (200 μM) and Q5 DNA polymerase (0.5 U) as recommended by the manufacturer. The details regarding the primers and touchdown PCR are described previously 28. Two microliter of the first round PCR product was used as template in a 50 μL second round nested PCR following the protocol described above for the first round. The second round PCR product was column purified following the manufacturer’s instructions (Invitrogen, USA) and sequenced.
The nucleotide sequence was analyzed using Sequencher (version 5.4.5; Gene Codes, USA) and MacVector (version 17.5.4; MacVector, USA) software. The V, D and J gene segment assignment was done using IMGT/V-QUEST (https://www.imgt.org/IMGT_vquest/input) 29,30 and IgBlast (https://www.ncbi.nlm.nih.gov/igblast/) 31 at default parameters.