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A combination of potently neutralizing monoclonal antibodies isolated from an Indian convalescent donor protects against the SARS-CoV-2 Delta variant [1]

['Nitin Hingankar', 'Iavi Hiv Vaccine Translational Research Laboratory', 'Iavi-Thsti Partnership Program', 'Translational Health Science', 'Technology Institute', 'Ncr Biotech Science Cluster', 'Faridabad', 'Suprit Deshpande', 'Payel Das', 'Zaigham Abbas Rizvi']

Date: 2022-07

Ethics statement

Ethics statement for use of human samples. The participants included in this study were members of DBT COVID-19 consortium cohort, organized by interdisciplinary research institutes and hospitals in the National Capital Region of India. It was coordinated by the Translational Health Science and Technology Institute. The main clinical sites were ESIC Medical College Hospital, Faridabad, and Loknayak Hospital, New Delhi. The study protocol was approved by the Institute Ethics Committees (IEC) of all participating institutions (IEC of ESIC Medical College Hospital, Faridabad, IEC of Loknayak Hospital, New Delhi and IEC of Translational Health Science & Technology Institute, Faridabad). Plasma and peripheral blood mononuclear cells (PBMCs) were prepared from blood samples obtained from eleven individuals between 6–8 weeks post recovery from SARS-CoV-2 infection who were infected in April 2020. The formal written consent was obtained from the participants.

Animals and ethics statement. Prior to the conduct of experiments to assess protective efficacy of the novel mAbs in small animals, approvals on the protocols involving dosing and animal challenge were obtained from the institutional animal ethics committee of the Translational Health Science & Technology Institute, Faridabad (approval # IAEC/THSTI/159), institutional biosafety committee of the Translational Health Science & Technology Institute, Faridabad (approval # 324/2021) and DBT Review Committee on Genetic Manipulation (RCGM; DBT RCGM approval #: IBKP UAC: TRARDSAB0214). 6–8 weeks old K18-hACE2 transgenic mice used to test the antibody efficacy were housed and maintained at the designated small animal facility (SAF) and subsequently transferred to the Animal biosafety level-3 (ABSL-3) institutional facility for infusion with mAbs and SARS-CoV2 challenge study. The animals were maintained under 12-hour light and dark cycle and fed with standard pellet diet and water ad libitum.

FACS sorting of antigen specific memory B cells. Antigen-specific single memory B cell sorting was performed in a FACS sorter (BD FACS Melody) essentially following the methods as described earlier [20]. Briefly, cryo-preserved PBMCs were first thawed at 37°C in a water bath and washed with an RPMI medium containing 10% fetal bovine sera (FBS) following incubation with fluorescently-labeled antibodies (BD Biosciences) against cell surface markers for CD3 (PE-Cy7); CD8 (PE-Cy7); CD14(PE-Cy7); CD16 (PE-Cy7); CD19 (BV421); CD20 (BV421); IgD (PerCP-Cy5.5); IgG (APC-H7) in addition to labelled RBD that was described elsewhere [20]as an antigen in FACS buffer containing PBS (pH7.4), 1% FBS, and 1.0 mM EDTA on ice. Live/Dead Fixable Aqua Blue Cell Stain (Thermo Fisher Inc.) was used to stain the cells for another 10 minutes on ice as per the manufacturer’s instructions. The avi-tagged SARS-CoV-2 RBD antigen was first labeled with biotin (Avidity, BirA500) was subsequently coupled to streptavidin-PE and streptavidin-APC (BD Biosciences) by incubating the mixture at 4°C for 1 hour at 4:1 molar ratio. The stained cells were subsequently washed with FACS buffer to remove unbound antibodies and probe and then filtered through 70-μm cell mesh (BD Biosciences) before processed in the FACS sorter. Single antigen RBD+CD3-CD8-CD14-CD16-CD19+CD20+IgD-IgG+ cells were sorted and collected into individual wells of a 96-well plate pre-filled with 20 ul of lysis buffer containing reverse transcriptase buffer (Thermo Fisher), IGEPAL (Sigma), DTT and RNAseOUT (Thermo Fisher). Plates containing sorted cells were sealed, snap-frozen on dry ice and stored at -80°C until used further.

Amplification and cloning of variable heavy and light IgG chains. cDNA Superscript III Reverse Transcription kit (Thermo Fisher) was used to prepare from sorted cells, cDNA master mix containing dNTPs, random hexamers, IgG gene-specific primers and RT enzyme was added to generate cDNA. Heavy and light-chain variable regions of IgG were amplified in independent nested PCR reactions using specific primers. First round PCR amplification was performed using HotStar Taq DNA Polymerases (Qiagen) and second round nested PCR was performed using Phusion HF DNA polymerase (Thermo Fisher Inc.). Specific restriction enzyme cutting sites (heavy chain, 5′-AgeI/3′-SalI; kappa chain, 5′-AgeI/3′-BsiWI; and lambda chain, 5′-AgeI/3′-XhoI) were introduced in the second round PCR primers in order to clone into the respective expression vectors. Amplified PCR products were verified on the agarose gel and wells with double positives (with amplification of both Heavy and Light chain variable regions from the same well) were identified and selected for subsequent cloning experiments. PCR products were digested with specific restriction enzymes, purified and cloned in-frame into expression vectors encoding the human IgG1, Ig kappa or Ig lambda constant domains using the Quick Ligase cloning system (New England BioLabs) according to the manufacturer instructions. Ligation reactions were transformed into NEB 5-alpha competent E. coli cells, plated on LB agar plates containing ampicillin and incubated overnight at 37oC in the incubator. Colonies with desired inserts were screened by colony PCR and used for preparation of plasmid DNA. Plasmid clones with correct insert were further confirmed by restriction digestion with respective (New England Biolabs, Inc.) restriction enzymes before being selected for the subsequent transfection experiment. Confirmed heavy and light chain plasmid DNA were co-transfected in 293T cells (ATCC) using Fugene transfection reagent (Promega) in 24 well plates for preparing antibody supernatant for initial screening for their expression and antigen specificity as detailed in the following section. Sanger sequencing were carried out to obtain the nucleotide and amino acid sequences of variable heavy and light IgG chains. Analysis of mAb sequences were carried out using the IMGT (www.imgt.org) V-quest webserver tool.

Capture ELISA for the detection of IgG expression. Maxisorp high protein binding 96 well ELISA plate (Nunc, Thermo Fisher Scientific.) was coated with 2μg/mL goat anti-human Fc antibody (Thermo Fisher Scientific) and incubated overnight at 4°C. Next day after washing, plates were blocked with 3% BSA in PBS (pH 7.4) for 1 hour at room temperature. After 3 times of washing with 1 X PBS containing 0.05% tween 20 (PBST), the cell supernatants which were harvested post transfection of antibody constructs in HEK 293T cells were added and incubated for 1 hour at room temperature. This was followed by addition of alkaline phosphatase-conjugated goat anti-human F(ab’)2 antibody (Thermo Fisher Scientific) Inc.) at 1:1000 dilution in 1% bovine serum albumin (BSA) incubated for an hour at room temperature. After the final wash, phosphatase substrate (Sigma-Aldrich Inc.) was added into the wells and absorption was measured at 405 nm on a 96 well microtiter plate reader.

Streptavidin ELISA for anti SARS- CoV-2 (RBD) antibody detection. 2μg/mL of Streptavidin (G-Biosciences) was coated onto each wells of Nunc maxisorp high protein-binding 96 well ELISA plate (Thermo Fisher Scientific Inc.) and incubated overnight at 4°C. Next day after washing, plates were blocked with 3% BSA in PBS (pH 7.4) for 1 hour at room temperature. 2 μg/mL of Biotinylated—RBD protein was subsequently added and incubated the plate for 2 hours at room temperature. After washing the plates for three times with PBST, cell supernatants at various dilutions were added to the wells and the plate was further incubated for 1 hour at room temperature. Finally, HRP (horseradish peroxidase) conjugated anti-human IgG Fc secondary antibody was added at a dilution of 1:1000 containing 1% BSA and the plate was incubated for an hour at room temperature. After the final wash, TMB substrate (Thermo Fisher Scientific Inc.)) was added and subsequently 1N H 2 SO 4 was added to stop the reaction. The absorption was measured at 450 nm.

Preparation and purification of IgG. The IgGs representing the mAbs were produced in either HEK 293T (ATCC) or Expi293 (Thermo Scientific) cells. Plasmid DNA expressing variable heavy and light IgG chains were transiently transfected into HEK293T or Expi293 cells using polyethylenimine (PEI). After 4–5 days of incubation, supernatants were harvested by centrifugation and filtered through a 0.2 μm membrane filter. Supernatants were then flowed slowly on to the Protein A (G Biosciences) beads in the column at 4°C in order to capture the secreted antibodies. Beads in the column were washed with five column volumes of 1X PBS at room temperature. Antibodies were eluted in two to three column volumes of 100 mM Glycine (pH 2.5) and immediately neutralized with 1M Tris-HCL (pH 8.0). Eluted antibodies were dialyzed using 10K MWCO SnakeSkin dialysis tubings (Thermo Fisher Scientific) against 1X PBS thrice and then concentrated in 30kDa NMWCO Amicon Ultra-15 Centrifugal Filter Units (Millipore). Antibody solutions were finally filtered through a 0.2 μm syringe filter (Thermo Fisher Scientific) before being used for further experiments. Concentration of IgG was measured by NanoDrop spectrophotometer and IgG heavy and light chain bands were visualized with 12% SDS PAGE analysis.

Quantitative RBD-ELISA. Anti-RBD IgG ELISA was performed essentially as described in Mehdi et al. [40]. For the screening of donors, plasma samples were three-fold diluted starting from 1:25 and were assessed for the presence of RBD binding IgG antibodies. To determine the mAb concentration in mice sera, a serial dilution of respective purified mAbs with known concentration was run as standard. mAb concentrations in mice sera were calculated for each sample dilution by interpolation of OD values from respective purified antibody dilutions using GraphPad Prism.

Microneutralization screening assay. Preliminary screening of heat-inactivated plasma samples obtained from convalescent donors for their neutralization potential were assessed as described in Malladi et al. [41] with slight modification. Briefly, plasma samples were serially two-fold diluted and mixed with 100 TCID 50 of SARS-CoV-2 isolate. The virus-plasma mixture was transferred to Vero E6 monolayer seeded in 96 well plates in triplicate and incubated for 1 hour. The cell monolayer was subsequently washed with serum free media following which fresh complete medium was added. The plate was further incubated for 72 hours at 37°C in a humidified CO 2 incubator. Absence of cytopathic effect (CPE) as an indicator of virus neutralization was assessed by observing the cells under a bright field microscope. The dilution at which no CPE was observed was considered as the neutralization titer.

Pseudovirus (PSV) neutralization assay. Pseudoviruses expressing complete SARS-CoV2 spike genes were prepared by transient transfection of HEK293T cells with three plasmids: SARS-CoV2 MLV-gag/pol and MLV-CMV-luciferase plasmids using Fugene 6 (Promega Inc.) as described earlier [20]. After 48-hour post transfection, cell supernatants containing pseudotyped viruses were harvested and frozen at -80°C until further use. Neutralization assay was carried out using HeLa-hACE2 cells for the infection of SARS-CoV-2 wild type and variant pseudoviruses. The purified IgGs were serially diluted and incubated with pseudoviruses in a humidified Incubator at 370 C. After 1-hour incubation HeLa-hACE2 cells were added to the 96-well plates at 10,000 cells/well density. After 48 hours of incubation the luciferase activity was measured by adding Britelite substrate (Perkin Elmer Inc.) according to manufacturer’s instruction and RLU obtained using a luminometer (Victor X2, Perkin Elmer Inc).

Live virus focus-reduction neutralization test (FRNT). The live virus neutralization assay was carried out following protocols as described by Bewley et al. [42]. Briefly, IgGs were serially diluted and incubated with indicated SARS-CoV-2 isolates. The virus-IgG mixtures were next added to Vero E6 cells for virus adsorption for one hour. The viral inoculum was removed, and cells were overlaid with carboxymethylcellulose and incubated for 24 hours. Cells were fixed and stained with anti-spike RBD antibody (Sino Biologicals) followed by HRP-conjugated anti-rabbit antibody (Invitrogen) and incubated with TrueBlue substrate (Sera Care). Finally, plates were washed with sterile MilliQ water, air-dried, and microplaques were quantified by AID iSPOT reader (AID GmbH, Strassberg, Germany). 50% neutralization values were calculated with four-parameter logistic regression using GraphPad Prism 7·0 software.

Cell surface spike binding assay. The binding of mAbs to the SARS-CoV-2 spikes expressed on the HEK 293T cell-surface was assessed as described previously with some modifications [20]. Briefly, HEK293T cells were transfected with the three plasmids used to generate SARS-CoV-2 pseudovirus (SARS-CoV-2 MLV-gag/pol, MLV-CMV-luciferase and SARS-CoV-2 spike plasmids). After incubation for 36–48 h at 37°C, cells were trypsinized and a single cell suspension was prepared which was distributed into 96-well U bottom plates. 3-fold serial dilutions of mAbs starting at 10 μg/ml and up to 0.041 μg/mL were prepared in 50 μl/well and added to the spike expressing as well as un-transfected 293T cells for 1 hour on ice. Cells were subsequently washed twice with FACS buffer (1x PBS, 2% FBS, 1 mM EDTA) and then stained with 50 μl/well of 1:200 dilution of R-Phycoerythrin AffiniPure F(ab’)₂ Fragment Goat Anti-Human IgG, F(ab’)₂ fragment specific antibody (Jackson ImmunoResearch Inc.) for 45 min. Cells were finally stained firstly with 1 LIVE/DEAD fixable aqua dead cell stain (ThermoFisher) in the same buffer for another 15 minutes and subsequently washed twice in plates with FACS buffer. The binding of mAbs to spikes expressing on cell surface was analyzed using flow cytometry (BD Canto Analyzer). Percent (%) PE-positive cells for antigen binding were calculated and the binding data were generated. CC12.1 (SARS-CoV-2 mAb), and CAP256.VRC26.25 antibody (HIV-1 bnAb) were used as positive and negative controls respectively for this experiment.

mAb-RBD competition assay. Inhibition of SARS-CoV-2 RBD binding by mAbs to the cell surface hACE2 was assessed by flow cytometry as described previously with some modifications [20]. Briefly, purified mAbs at 100 μg/mL and biotinylated SARS-CoV-2 RBD were mixed in 100 ul of DPBS in the molar ratio of 4:1 and incubated on ice for 1 hour. Parental HeLa and HeLa-ACE2 single cell suspension were prepared by washing cells once with DPBS and then detaching by incubation with DPBS supplemented with 5 mM EDTA. The detached HeLa and HeLa-ACE2 cell suspensions were again washed once and resuspended in FACS buffer (2% FBS and 1 mM EDTA in DPBS). 0.5 million Hela-ACE2 cells were added to the test mAb/RBD mixture and then incubated at 4°C for half an hour. 0.5 million HeLa and HeLa-ACE2 cells were incubated in separate wells with RBD alone without mAbs for use as background and positive control, respectively. After washing once with FACS buffer, HeLa and HeLa-ACE2 cells were resuspended in FACS buffer containing 1 μg/ml streptavidin-PE (BD Biosciences) and incubated for another half an hour. Cells were stained with 1:1000 final dilution of LIVE/DEAD fixable aqua dead cell stain (ThermoFisher) in the same buffer for another 15 minutes. HeLa and HeLa-ACE2 cells stained with SARS-CoV-2 RBD alone were used as background and positive control separately. The PE mean fluorescence intensity (MFI) was determined from the gate of singlet and live cells and the percentage of ACE2 binding inhibition was calculated by following formula.

Biolayer interferometry for assessing RBD binding affinity. Streptavidin (SA) biosensors (Forte´ Bio) were used to assess the binding affinities of mAbs with SARS-CoV-2 RBD in PBST (PBS containing 0.02% Tween 20) at 30°C and 1,000 rpm. shaking on an Octet RED 98 instrument (Forte´ Bio Inc.). Sensors were first soaked in PBS for 15 minutes before being used to capture biotinylated SARS-CoV-2 RBD protein [20]. RBD was loaded to the biosensors up to a level of 1.0 nm. Biosensors were then immersed into PBS for 100 seconds and then immersed into wells containing specific concentrations of a mAb dissolved in PBST (PBS containing 0.02% Tween 20) for 500 seconds to measure association. A threefold dilution series with five different concentrations (33.3, 11.1, 3.7, 1.23, and 0.41 nM) was prepared for each mAb. Biosensors were next dipped into wells containing PBST for 500 seconds to measure dissociation. Data were reference- subtracted and aligned to each other using Octet Data Analysis software v10 (Forte´ Bio Inc.) based on a baseline measurement. Curve fitting was performed using a 1:1 binding model and data for all the five concentrations of mAbs. Kon, Koff and KD values were determined with a global fit.

Epitope binning. Epitope binning experiments were performed using Streptavidin (SA) biosensors (Forte´ Bio) and mAbs were binned into epitope specificities. All the incubation steps for binning experiments were performed in 1x PBS. 50–100 nM of biotinylated RBD protein antigens were loaded on streptavidin biosensors to achieve 0.9 to 1.3 nm of wavelength shift and then washed. Saturating concentration of mAbs (100μg/ml) was added for 10 min and competing mAbs at concentrations of 25 μg/ml were then added for 5 min in order to measure binding in the presence of saturating antibodies.

Site-directed mutagenesis. Point substitutions within RBD in SARS-CoV-2 spike gene were introduced by site-directed mutagenesis using the QuikChange II kit (Agilent Technologies Inc.) following the manufacturer’s protocol and by overlapping PCR strategy as described previously [43]. Successful incorporation of desired substitutions was confirmed by Sanger sequencing.

X-ray crystallography. THSC20.HVTR04 and THSC20.HVTR26 Fab domains were sub-cloned into an in-house cleavable Fab expression vector, expressed in Expi293 cells, and purified as above. Fab fragments were purified by gel filtration with equimolar amounts of SARS-CoV-2 RBD and concentrated to 10 mg/mL and 7 mg/mL respectively. THSC20.HVTR04 complexes were initially crystallized in 0.3 uL sitting drops with 50:50 mix of protein to crystallization reagent (1.5M ammonium sulfate, 12%(v/v) isopropanol, 0.1M imidazole/ hydrochloric acid pH 6.5) and optimized in 15 well hanging drop plates. Crystals were flash frozen with 10% glycerol or ethylene glycol as cryoprotectant. THSC20.HVTR26 complexes were similarly crystallized with 15% (w/v) PEG 20,000, 100 mM HEPES/ sodium hydroxide pH 7.0 as crystallization reagent. Data were collected at Diamond Light Source (UK) MX I04 using a wavelength of 0.9795 Å at ~100K and processed using their automated pipelines. The structure was solved in Phenix v1.19.2–4158, using search models from AlphaFold. Models were refined with hydrogens to minimize clashes in COOT v0.9.6 using 5–10% of the data as an R free cross-validation test set. All structural images were generated with the PyMOL Molecular Graphics System v2.5.0 (Schrodinger LLC). These data have been deposited with PDB accession codes 7Z0X for THSC20.HVTR26-RBD complex and 7Z0Y for THSC20.HVTR04-RBD complex.

Negative stain EM analysis. A three-fold molar excess of Fab was incubated with SARS-2 CoV 6P Mut7 for 30 minutes at room temperature and deposited on a glow discharged carbon coated Cu grid. The complexes were stained with 2% uranyl formate (w/v) for 90 seconds. An FEI Tecnai Spirit at 120 keV paired with an FEI Eagle 4k x 4k CCD camera was used for data collection, and automated using the Leginon software [44]. Raw micrographs were stored in the Appion database [45]. Particles were picked with DogPicker [46] and data was processed in RELION 3.0 [47]. Figs were generated using UCSF Chimera [48].

Measuring antibody polyreactivity. The antibody polyreactivity was assessed as described earlier [20,49]. Solubilized CHO cell membrane protein (SMP) was coated onto 96-well half-area high-binding ELISA plates (Corning, 3690) at 5ug/mL in PBS overnight at 4°C. After washing, plates were blocked with PBS/3% BSA for 1 hour at room temperature (RT). Antibodies were diluted at 100ug/mL in 1% BSA with 5-fold serial dilution. Serially diluted samples were then added in plates and incubated for 1 hour at RT. After washing, alkaline phosphatase-conjugated goat anti-human IgG Fcy secondary antibody (Jackson ImmunoResearch, 109-055-008) was added in 1:1000 dilution and incubated for 1h at RT. After final wash, phosphatase substrate (Sigma-Aldrich, S0942-200TAB) was added into each well. The absorption was measured at 405 nm in a spectrophotometer. Den3 (Dengue-specific mAb) and Bococizumab (PCK9 antagonist) were included as benchmarking controls.

K18-hACE2 mice challenge. SARS-CoV-2 Wuhan (catalogue number: USA-WA1/2020) and B.1.617.2 delta (hCoV-19/USA/PHC658/2021 catalogue number NR-55611) were procured from BEI resources (https://www.beiresources.org/) and were expanded in Vero E6 cells to produce stocks required for the experiments. Mice randomly allotted to different groups (n = 5) viz, infection control and those received SARS-CoV-2 specific (THSC20.HVTR04 and THSC20.HVTR26) and non-specific (HIV CAP256.VRC26.25) monoclonal antibodies as IgG were housed in different cages. Antibody recipient groups were given intraperitoneal injection of IgG one day prior to challenge (day -1). Except for the unchallenged control group (n = 3), animals in all other groups were challenged with 105 PFU of SARS-CoV2 (Wuhan and Delta isolates) intranasally on Day 0, administered through a catheter 25 μl/ nare under anesthesia by using ketamine (150mg/kg) and xylazine (10mg/kg) inside ABSL3 facility [36,50–52]. Unchallenged control group received mock PBS (pH 7.4) intranasally.

Gross clinical parameters of SARS-CoV-2 infection. All the infected animals were euthanized on day 6 days post infection at the ABSL3. Changes in body weight, activity of the animals were observed on each day post challenge. Post sacrifice, lungs of the animals were excised and imaged for gross morphological changes. Right lower lobe of the lung was fixed in 10% neutral formalin solution and used for histological analysis. Rest of the lung was homogenized in 2ml Trizol solution for viral load estimation. The tissue samples in trizol were stored immediately at -80°C till further use. Blood of the animals were drawn through retro-orbital vein on day -1 and 0 and through direct heart puncture at the end-point. Serum samples were stored at -80°C till further use.

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