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 Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India and adopted by the Institutional 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/137/2022) were taken before commencing work.

Viruses and antibodies

SARS-CoV-2 to B.6 and delta lineage viruses were isolated as described previously [22,23]. Leo Poon provided SARS-CoV-2 Omicron isolate (sub-lineage BA.1) [24]. 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 (isotype IgG), a non-neutralizing antibody was used from our previous study [25]. The pseudotyped viral stocks of all seven alpha and beta coronaviruses were prepared as described in our previous study [26]. 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, BA.1) terminology throughout our manuscript.

Expression and purification of antibody and RBD protein

A synthetic codon optimized (for mammalian cells) nucleotide sequence of RBD-His (wildtype), spike protein (wildtype), 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 [11]. 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).

Generation of hybridoma for anti-RBD (N501Y) murine monoclonal antibodies

Six to eight week old, female BALB/c mice were immunized intramuscularly with purified RBD (N501Y) protein (30 μg in 100 μL PBS per animal) along with Quil A adjuvant (InvivoGen, USA). Mice were boosted thrice with the purified protein (30, 15 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), following the manufacturer’s protocol. The well-adapted antibody secreting clones were propagated in tissue culture flasks and vials were stored in liquid nitrogen for future use. Hybridoma clones secreting anti-RBD antibodies were further screened by ELISA.

ELISA

For the screening of hybridomas and heat inactivated mice sera (dilution starting from 1:100 to 1:218, 700)/ purified mAbs (5 to 0.002 μg mL^-1), 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 1 h at room temperature, HRP conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch, USA; diluted 1 in 2500) was added to each well. In the 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 [27,28]. All the experiments were repeated at least three times in triplicates.

Purification of anti-RBD (N501Y) murine monoclonal antibodies

Serum-free medium designated for mAb production was used to propagate the hybridoma cells (0.5 × 10⁵ cells mL^-1) 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 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 PBS three times using dialysis tubing (Thermo Fisher Scientific; MWCO = 10 kDa), and concentrated with a 50 kDa cut-off Amicon Ultra-15 centrifuge unit (Millipore). A 0.2 μm syringe filter (MDI, India) was used to filter the antibody solutions before they were used in experiments. NanoDrop spectrophotometer was used to estimate the protein concentration and purified IgG mAbs were analyzed using 12% Tris-Glycine-SDS-PAGE.

Cytopathic effect-based neutralization assay

Initial screening of heat-inactivated mice serum samples and hybridoma culture supernatants was performed as described [25,29] with slight modification. Briefly, heat-inactivated serum or purified mAbs were serially diluted two or four times and mixed with 100 TCID₅₀ of SARS-CoV-2 isolates. The serum or mAb mixture was transferred to the Vero E6 monolayer seeded in a 96-well plate in triplicate and incubated for 1 h. The cell surface was washed with serum-free medium and replenished with fresh complete medium. The plate was further incubated for 72 h at 37°C in a humidified CO₂ incubator. The cells were observed for the absence of viral cytopathic effect and was used as indicative of neutralization. The neutralization titer was defined as the dilution at which no cytopathic effect was seen. All the experiments were repeated at least three times in triplicates.

Live virus focus reduction neutralization assay

Virus neutralization assay was performed as described previously by our group [30]. Inhibitory concentration was assessed by focus reduction neutralization assay using SARS-CoV-2 Delta (GenBank accession no.: MZ356904.1) and Omicron BA.1 (GISAID accession no.: EPI_ISL_8764350) variant. The virus neutralization assay was performed in Vero E6 cells. Cells were incubated for 24 h for Delta and 32 h for the Omicron variant. The virus stock was propagated in Calu-3 cells (American Type Culture Collection, USA). Control purified IgG was used as negative control. All the experiments were repeated at least three times in triplicates.

Vero E6 cells were maintained in Minimal Essential Medium (MEM) containing 10% heat-inactivated fetal bovine serum (FBS), 1% penicillin-streptomycin solution, 1% Non-Essential Amino Acid seeded in a 96-well plate (25,000 cells per well) and incubated overnight at 37°C with 5% CO₂. Reagents were two-fold serially diluted (75 μL + 75 μL) in dilution medium (MEM with 1% FBS) from 5 to 0.00244 μg mL^-1. Predefined virus dilution (75 μL) was added and incubated for 1 h at 37°C with 5% CO₂. For the quality control step, pre-defined SARS-CoV-2 neutralizing antibodies positive serum was used. Virus dilution was determined to get approximately 60–200 foci with cell control. After neutralization incubation virus-reagent mixture was added over cell monolayer for virus adsorption and incubated for 1 h at 37°C with 5% CO₂. Overlaying medium was added after adsorption incubation and cells were fixed after 24 h. In the case of BA.1, cells were fixed after 28 h of incubation. Cells were permeabilized using 100 μL of IMF buffer and incubated at room temperature for 20 min. Cells were stained with the addition of 100 μL of anti-nucleocapsid antibody at 1:2000 dilution for 1 h, followed by 100 μL of 1:500 dilution of Alexa flour 488 conjugated donkey anti-mouse IgG secondary antibody. After incubation, cells were washed thrice with PBS and developed foci were quantified by AID iSpot Reader (AID GmbH, Germany) using AID EliSpot 8.0 iSpot software. Using AID raw file to find IC₅₀ by using GraphPad Prism (Version 9.3.1) and graphs were plotted with log(inhibitor) versus normalized response—Variable slope parameter. The purified P4A2 antibody was two-fold serially diluted starting from 5 to 0.00244 μg mL^-1. The virus neutralization assay was performed in Vero E6 cells. Cells were incubated for 24 h for Delta and 32 h for BA.1 variant. The virus stock was propagated in Calu-3 cells (American Type Culture Collection, USA). Control purified IgG was used as the negative control. All the experiments were repeated at least three times in triplicates.

Pseudovirus-based neutralization assay

Full length spike proteins of all alpha and beta coronaviruses were co-transfected in 1.25 × 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, RΔ8.2 backbone plasmid, pTMPRSS2). After 68–72 h culture supernatant was collected and stored at -70°C. To access the cross-neutralization potential, three-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 1 h at 37°C. Pseudovirus/ P4A2 combinations were added to 293T-ACE2 cells pre-seeded (24 h earlier) at 20,000 cells per well. After 48–72 h, relative luminescence unit (RLU) was measured on a luminometer. The percent reduction in neutralization was measured as ratio of RLU readout in the presence of P4A2 normalized to RLU readout in the absence of P4A2. Four-parameter logistic regression was used to calculate the half maximum inhibitory concentrations (IC₅₀) (GraphPad Prism version 8.3). II62 and CR3022, mAbs against SARS-CoV-2, were used as assay controls. All the experiments were repeated at least three times in triplicates.

Immunofluorescence microscopy

Vero E6 (25,000 cells per well) seeded in 96-well plate. Virus suspensions at the indicated MOIs were added. After one-hour incubation virus suspensions were removed and overlaying medium (1.5% CMC) was added. Cells were fixed with 7.4% formaldehyde after 24 hours incubation and left overnight. Cells were washed with PBS thrice and permeabilized with 100 μL permeabilization 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. Cells were stained with P4A2 as primary antibody (diluted 1 in 2000) at room temperature for 1 h. Secondary Alexa 488 conjugated antibody (anti-mouse Alexa 488, cat no.: A21206, Invitrogen; 1:500) added after washing thrice with buffer. Micro-plaques were estimated 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 BA.1 infected Calu-3 cells (90,000 per well) were seeded in 8-well chamber slides. Cells were incubated for 48 h at 37°C in CO₂ incubator. DMEM high glucose with 10% (v/v) FBS was removed 24 h later and cells were washed with PBS. Cells were infected at a MOI of 0.5 (DMEM high glucose supplemented with 2% FBS). Delta and BA.1 virus (100 μL per well) was added and incubated at 37°C on a rocker. The virus was removed and washed twice with PBS. Then, 300 μL of 10% complete DMEM was added and incubated further for 24 h at 37°C.

Biolayer interferometry binding assay

Binding assays were carried out using an Octet Red instrument (ForteBio Inc.) using BLI as previously described [25,31]. Briefly, mouse Fc sensors (ForteBio Inc.) were used to capture P4A2 at 10 μg mL^-1 in 1 × kinetics buffer [PBS, pH 7.4, 0.01% (w/v) BSA and 0.002% (v/v) Tween-20] and incubated at the indicated 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. All the experiments were repeated at least three times.

Purification, crystallization, data collection, refinement and analysis

A total of 30 mg of P4A2 was digested for Fab preparation. The Fab preparation was performed by Pierce Fab Preparation Kit (Cat. no.: 44985) as per the manufacturer’s protocol. The purified Fab and RBD proteins were mixed at a molar ratio of 1:1.7 and incubated overnight at 4°C. The sample was subjected to size exclusion chromatography on 16/600 Superdex 200 column (Cytiva) in a buffer containing 20 mM HEPES (pH 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°C 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 [32]. The diffraction data was processed using IMOSFLM [33] and AIMLESS [34] programs of the CCP4 suite (S3 Table).

The structure was determined by molecular replacement using PHASER [35] and the search model was the STEC90-C11 Fab:RBD complex [36]. This model was subjected to iterative model building and refinement using COOT [37] and PHENIX [38], respectively and the sequence of the STEC90-C11 Fab was slowly changed to that of P4A2. The final R_free and R_work 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 [39]. Mutations were created in silico in the RBD structure using PYMOL to obtain models of P4A2 Fab bound to RBD corresponding to different SARS-CoV-2 strains and Omicron lineages. These models were subjected to energy minimization using the DESMOND module of the Schrodinger suite (Schrodinger Inc.) and analyzed. Two models of P4A2 bound to spike trimer with the RBD in the up and down conformation were prepared using 7TM0 [40] and 7TOU [41] 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 Institutional Animal Ethics Committee (IAEC), Institutional Biosafety Committee (IBSC) and Review Committee on Genetic Manipulation (RCGM) guidelines. In prophylactic treatment, antibody recipient groups were given intraperitoneal infusion of P4A2 one day prior to challenge (day ‘-1’), except for the control group where PBS was given (no virus challenge). In therapeutic treatment group, the mAb was administrated 12 h post-infection.

Clinical spectrum of SARS-CoV-2 infection

The mouse experiments were done in an 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 2 mL Trizol solution. The homogenates were stored immediately at -80°C 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 -80°C 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. The iScript cDNA synthesis kit (Bio-Rad, USA) was used for cDNA synthesis. Briefly, 1 μg total RNA was reverse-transcribed into cDNA. The qPCR was performed on diluted cDNAs (1:5) using the KAPA SYBR FAST qPCR Master Mix (5×) Universal Kit (KK4600) and 7500 Dx real-time PCR equipment (Applied Biosystems, USA). The results were analysed with SDS2.1 software as previously described [42]. 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 [43]. The copy number of N gene was calculated by using pre-titrated SARS-CoV-2 genomic RNA and expressed as N 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 transcripts expressed in B cell hybridoma P4A2 was RT-PCR amplified and sequenced following the protocol and primers described previously [44]. 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 Biolabs, 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 V_H (or V_L 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. 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.

Sequence analysis

The nucleotide sequence was analyzed using Sequencher (version 5.4.5; Gene Codes, USA) and MacVector, (version 17.5.4; MacVector Inc., USA) software. The V, D and J gene segment assignment was done using IMGT/V-QUEST (https://www.imgt.org/IMGT_vquest/input) [45,46] and IgBlast (https://www.ncbi.nlm.nih.gov/igblast/) [47] using default parameters.