Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

Cells and viruses.293T and Vero.E6 cells were obtained from the American Type Culture Collection (ATCC) and were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Clontech), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (antibiotics mixture). THP-1 cells were obtained from the ATCC and maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS (Clontech). Sf9 insect cells (ATCC CRL-1711) were propagated in TNM-FH medium (Gemini Bio-Products) supplemented with 10% FBS and antibiotics mixture. High Five cells (BTI-TN-5B1-4 subclone; Vienna Institute of Biotechnology) were grown in serum-free SFX medium (HyClone) supplemented with antibiotics mixture (33).

VSV-EBOV was grown in Vero.E6 cells, and titers were determined by plaquing virus on Vero.E6 cell monolayers and staining with polyclonal anti-VSV mouse serum. VSV-EBOV and VSV-EBOVΔmuc are both replication-competent pseudotyped viruses derived from the VSV-EBOV-XN2 rescue system and featuring enhanced green fluorescent protein (eGFP) open reading frames (ORFs) in the first cloning site directly preceding NP (34, 35).

A recombinant Newcastle disease virus (rNDV-EBOV-GP) expressing the ectodomain of the EBOV glycoprotein (strain wt/GIN/2014/Makona-Gueckedou-C07; GenBank accession number KJ660347 ) was prepared as previously described (36). Briefly, a synthetic, codon-optimized sequence encoding the ectodomain of the EBOV-GP was fused in frame to the sequence coding the transmembrane and cytoplasmic tail of the NDV F protein and cloned as an additional transcript unit in a plasmid containing the full-length cDNA of the NDV vaccine strain LaSota. The recombinant virus was rescued by transfection following a previously described protocol (37) and amplified in specific-pathogen-free (SPF) embryonated chicken eggs (Charles River Laboratories).

All experiments with infectious EBOV were carried out in the biosafety level 4 laboratory of the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany, according to institutional biosafety regulations. All personnel performing infections wore positive-pressure biosafety suits.

Production of ebolavirus VLPs.To produce ebolavirus VLPs, 293T cells in multiple 175-cm² flasks were transfected with EBOV GP in a pCAGGS vector and VP40 in a pCAGGS vector using a ratio of 2:3. After incubating at 37°C for 48 h, the cell supernatant was harvested and clarified via low-speed centrifugation (3,000 × g for 30 min at 4°C). VLPs were pelleted through a 30% sucrose cushion (30% sucrose in NTE buffer [100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA], pH 7.4) by ultracentrifugation (Beckman L7-65 ultracentrifuge with an SW-28 rotor at 25,000 rpm for 2 h). Once all of the supernatant was aspirated, the VLP pellets were resuspended in phosphate-buffered saline (PBS) (pH 7.4). Presence of the ebolavirus GP in the final VLP preparation was confirmed via Western blotting by probing with murine positive-control MAb (supplied by the Basler Laboratory).

Recombinant proteins.Coding sequences of glycoprotein ectodomains (ending at D632 in EBOV, upstream of the ADAM17/TACE cleavage site at D637) from EBOV, an isolate of EBOVG, BDBV, SUDV, TAFV, RESTV, and MARV were amplified via PCR (primer sequences are available upon request) and inserted into a modified pFastBacDual vector in frame with a carboxy-terminal trimerization domain and a hexahistidine tag using an In-Fusion cloning kit (Clontech). The constructs obtained were Sanger sequenced and transformed into DH10Bac competent cells (ThermoFisher Scientific) to produce recombinant bacmids, which were then transfected into Sf9 cells as described previously (38, 39). Recombinant baculoviruses were propagated in Sf9 cells. Proteins were expressed in High Five cells and purified from culture supernatants following a detailed published protocol (38).

Hybridoma generation.Female BALB/c mice (6 to 8 weeks old; Jackson Laboratory) were initially vaccinated by intramuscular administration of plasmid DNA, followed immediately by the application of electrical stimulation (TriGrid delivery system; Ichor Medical Systems) (40, 41). The spacing of the TriGrid electrode array is 2.5 mm, and the electrical field is applied at an amplitude of 250 V/cm of electrode spacing for six pulses for a total duration of 40 ms applied over a 400-ms interval. DNA vaccination comprised 40 μg of pCAGGS EBOV GP. Three weeks later, a DNA boost was performed following the same procedure. Three weeks after that, the mice were given 10⁴ PFU VSV-EBOV i.p. Approximately 3 weeks after this infection, one mouse was boosted with a unilateral intraperitoneal injection of EBOV VLPs containing EBOV GP and VP40 (100 μg total protein content) adjuvanted with 10 μg poly(I·C). Three days postboost, the mouse was sacrificed, and its spleen was sterilely removed. The spleen was flushed forcefully with serum-free DMEM (with antibiotic mixture) using a 10-ml syringe with a 20-gauge needle, followed by repeated pulverization with flat-ended forceps. Splenocytes and SP2/0 myeloma cells (in exponential growth phase) were combined in a 5:1 ratio, and cell fusion was mediated via slow, dropwise addition of 1 ml of polyethylene glycol (mass, ∼4,000 Da). The splenocyte-SP2 mixture was then resuspended in 25 ml of complete DMEM (supplemented with antibiotic mixture, FBS, and HEPES) and left to incubate for 24 h at 37°C. After this incubation, the cells were spun down, resuspended in 10 ml of complete DMEM, mixed with 90 ml of proprietary semisolid Clonacell-HY Medium D (Stemcell Technologies), and dispensed onto tissue culture dishes (10 ml each) using a 10-ml syringe with a 15-gauge Luer Stub adapter (Becton Dickinson). Individual colonies were picked 10 days later and transferred into 96-well plates containing Clonacell-HY Medium E. Five days after transfer to 96-well plates, hybridoma supernatants were individually screened by immunostaining Vero.E6 cells infected with NDV-EBOV-GP at a multiplicity of infection (MOI) of 1 and transfected 293T cells expressing EBOV GP. Positive clones were isotyped using a Pierce rapid antibody isotyping kit (Life Technologies); only the MAbs isotyped to the IgG heavy-chain subclasses were selected for further expansion and purification.

Antibody purification.Hybridoma cultures were initially expanded in Clonacell-HY Medium E but gradually switched to serum-free hybridoma medium until a final volume of 300 to 500 ml was achieved. When the cells appeared no longer viable (10 days after the final expansion step), the cultures were harvested by low-speed centrifugation (30 min; 5,500 relative centrifugal force [RCF]), and the supernatants were passed through 0.22-μm-pore-size sterile filtration units (Millipore). The filtered supernatants were passed through a gravity flow column packed with protein G-Sepharose 4 Fast Flow beads (GE Healthcare). After washing with 3 column volumes (450 ml) of sterile PBS (pH 7.4), antibodies were eluted with 45 ml of 0.1 M glycine-HCl buffer (pH 2.7), and the eluate was immediately neutralized with 5 ml of 2 M Tris-HCl buffer (pH 10). The MAbs were further concentrated and buffer exchanged against PBS (pH 7.4) using Amicon Ultra centrifugal filter units (30-kDa cutoff; Millipore). The final protein concentration was determined using a NanoDrop device (Thermo Scientific) and measuring absorbance at 280 nm.

Challenge studies in Stat2^−/− mice. Stat2 ^−/− C57BL/6 mice were kindly provided by Christian Schindler and were bred at the Icahn School of Medicine at Mount Sinai. The LD₅₀ of the VSV-EBOV pseudotyped virus was determined by injecting 6- to 8-week-old Stat2^−/− mice with 1,000, 100, 10, or 1 PFU of PBS-diluted virus i.p. (left flank) and monitoring daily for survival and weight loss until 14 days postinfection (p.i.). Mice that lost 25% or more of their initial body weight were euthanized and scored dead. The LD₅₀ was calculated using the Reed and Muench method.

To study in which organs the virus would replicate, two groups of two mice each were inoculated with 250 PFU of VSV-EBOV (i.p.; left flank) and sacrificed either 48 or 96 h postinfection for organ titer analysis. At these time points, the brain, kidneys, intestine, spleen, and liver were harvested and homogenized using a BeadBlaster 24 (Benchmark) homogenizer. Viral titers were then measured by incubating the homogenate in serial 10-fold dilutions (50-μl volume) on confluent Vero cells in triplicate. Live virus stock from cell culture was used as a positive control; 72 h after serial dilutions of homogenate were added, the cells were assessed for fluorescence to determine the 50% tissue culture infective dose (TCID₅₀).

In prophylactic antibody protection studies, groups of two male and three female Stat2^−/− mice aged 6 to 8 weeks were given a 10-mg/kg dose of purified monoclonal antibody i.p. (right flank). Negative-control mice received a 10-mg/kg dose of MAb 8H9, which is specific for influenza virus H6 hemagglutinin. At 3 h posttreatment, 5 times the LD₅₀ of VSV-EBOV-EGFP (250 PFU) diluted in PBS (pH 7.4) was injected i.p. (left flank). All the mice were then monitored daily for survival and weight loss until 14 days postinfection. MAbs were tested in batches due to limited availability of the Stat2^−/− mice. The group size for the MAb-treated groups in each separate experiment was five mice per MAb, but in every experiment, negative controls (n = 3) were included. The cumulative data for the negative controls therefore represent 18 mice. The data shown are cumulative, i.e., data from all experiments combined.

For therapeutic studies, groups of two male and two female Stat2^−/− mice aged 6 to 8 weeks were given 5 times the LD₅₀ of VSV-EBOV (250 PFU) diluted in PBS (pH 7.4) injected i.p. (left flank). At 24 and 48 h p.i., a 10-mg/kg dose of purified mouse monoclonal antibody, either 2G12 or control MAb (8H9), was injected in 200 μl i.p. (right flank). All mice were then monitored daily for survival and weight loss until 14 days postinfection. All procedures were performed in accordance with the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC) guidelines.

Generation of MAb escape mutant viruses.MAb escape mutant variants of VSV-EBOV were generated via serial passaging in Vero cells in the presence of increasing amounts of MAb, with a starting concentration of 2× IC₅₀ (as calculated from the neutralization assay against VSV-EBOV). Initially, Vero cells in 12-well tissue culture plates (Sigma) were infected with VSV-EBOV at a multiplicity of infection (MOI) of 1 in the presence of 2× IC₅₀ of MAb (performed in duplicate for each MAb) in MEM. After incubating for 72 h at 37°C, 100 μl of supernatant was collected and used to directly inoculate a fresh monolayer of Vero cells in the presence of a 2-fold increase in the MAb concentration. This process was repeated for six passages. Throughout serial passaging, successful infection was confirmed by the expression of eGFP. In the cases of both MAbs, eGFP was present after the last passage of virus. Virus was additionally passaged in the presence of an irrelevant mouse MAb against the influenza virus hemagglutinin (8H9; IgG) to control for mutational variants obtained from passaging alone. Escape mutant viruses were plaque purified once serial passaging was completed to create monoclonal stocks. These monoclonal stocks were then tested in plaque reduction neutralization assays, as described previously, against their corresponding escape MAbs, and no residual neutralization activity was detected.

Plaque reduction neutralization assays.PRNAs were performed according to a protocol established by Tan et al. (42) with modifications. Briefly, MAbs at 1:5 dilutions starting from 100 μg/ml were incubated with 70 PFU of either VSV-EBOV, VSV-EBOVΔmuc, or one of two escape mutant viruses derived from VSV-EBOV for 1 h at room temperature (RT). These dilutions were then plaqued on 90 to 100% confluent Vero.E6 cell monolayers in 12-well plates. Cells were overlaid with minimum essential medium (MEM) containing 0.64% agarose (Oxoid) and the corresponding MAb dilutions. After 4 days of incubation at 37°C, the cells were fixed with 3.7% paraformaldehyde (PFA), and the plaques were stained using a polyclonal anti-EBOV mouse serum (1:1,000), an anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) (Sigma), and Trueblue reagent (KPL). The plaques were counted in each MAb dilution, and the percent inhibition for each MAb and dilution was individually calculated based on a no-antibody control and an isotype control. All assays were performed in duplicate. The data were analyzed by using Prism software (GraphPad), and the concentration at which a MAb inhibited 50% of plaques (IC₅₀) was calculated using a nonlinear 4-parameter regression.

Fluorescence-based neutralization assays.Vero.E6 cells were plated overnight at 37°C to achieve 100% confluence in sterile, flat-bottom, 96-well tissue culture plates (Sigma). Individual MAbs were serially diluted 1:2 in MEM, in duplicate, from a starting concentration of 600 μg/ml in separate 96-well, sterile, flat-bottom tissue culture plates (final volume, 75 μl/well). Extra dilutions were performed for 2G12 in a separate assay once it became clear neutralization activity did not reach 0%. VSV-EBOV was diluted to 20 times the half-maximal effective concentration (EC₅₀) in MEM and added to the MAb dilution plates (75 μl/well). The plates were briefly tapped (for mixing) and incubated at RT for 1 h. Next, medium from the Vero cells was aspirated and replaced with 100 μl of the antibody-virus mixture. The plates were incubated at 37°C for 48 h and then read by an acumen Cellista Laser Scanning Imaging Cytometer (TPP Labtech). Readouts of the eGFP mean intensity were plotted and analyzed using Prism software (GraphPad), and the IC₅₀ was defined as the concentration at which 50% of the mean intensity of eGFP was inhibited.

ELISAs.Immulon 4 HBX plates were coated overnight with recombinant purified glycoprotein from each ebolavirus species at 4°C at a concentration of 2 μg/ml (50 μl/well) in coating buffer (0.1 M Na₂CO₃-NaHCO₃, pH 9.4). After removal of the coating buffer, the plates were washed three times with PBS (pH 7.4) containing 0.1% Tween 20 (TPBS). The plates were then blocked for 1 h with blocking buffer (TPBS plus 3% dry milk powder). After washing the plates three times with TPBS, purified IgG was diluted 1:3 in the plates, starting with 100 μg/ml in blocking buffer. Positive controls (serum from the vaccinated animal postboost) and negative controls (isotype control antibody; 8H9 [anti-influenza]) were used to evaluate standardization. Control serum was diluted 1:3 in the plates, with a 1:100 starting concentration in blocking buffer. After a 2-h incubation at RT, the plates were washed three times with TPBS, and secondary horseradish peroxidase-conjugated anti-mouse IgG antibody (Rockland; 610-603-002; 1:3,000) diluted in blocking buffer was added. After a 1-h incubation, the plates were washed four times with TPBS and developed with SigmaFast o-phenylenediamine dihydrochloride (OPD) (Sigma; 100 μl per well). After 10 min of development, the reaction was stopped with 3 M HCl. The optical density at 490 nm (OD₄₉₀) was read on a plate reader, and background levels were analyzed in Microsoft Excel. The minimal binding concentration was calculated as the lowest dilution of antibody that rose above background (the average of blank wells on each plate plus 3 standard deviations). Minimal binding concentrations were imaged as a heat map using Interactive Tree of Life (iTOL) v3 (European Molecular Biology Laboratory) (43).

Phagocytosis assay.The phagocytosis assay was performed as previously described by Ackerman et al. (44). Briefly, duplicates of serial 4-fold dilutions of tested mouse IgG antibodies starting with 10 μg/ml were incubated for 2 h at 37°C with 1-μm fluorescent neutravidin beads (Invitrogen) conjugated with biotinylated EBOV-GP antigen (biotinylated using EZ-Link-NHS-PEG₄ biotin in 1:1 molar ratio; ThermoFisher Scientific) in 96-well plate format. THP-1 cells (2 × 10⁴ per well) were added and incubated for 12 h in an incubator at 37°C and 5% CO₂. At the end of the incubation time, the plates were fixed by adding 100 μl of 3.7% PFA/well, and samples were measured using the yellow 2 laser channel of an LSRIII flow cytometer (BD Biosciences). Analysis was performed using FCS Express software (De Novo Software).

ADCC reporter assay.ADCC was measured using the ADCC Reporter Bioassay kit (Promega) according to the manufacturer's instructions. Briefly, 3 × 10⁴ Vero cells per well were seeded in solid white 96-well plates (Corning Costar) and then 24 h later infected with VSV-EBOV at an MOI of 5. Serial 3-fold antibody dilutions in triplicates starting at 90 μg/ml were prepared in a separate 96-well U-bottom plate and transferred to the infected cells (1:3 of the total volume/well; therefore, the final starting concentration of antibodies was 30 μg/ml). After the addition of effector cells (part of the Reporter Bioassay kit; Promega), the plates were incubated at 37°C and 5% CO₂ for 6 h, Bio-Glo Luciferase reagent (Promega) was added, and luminescence was measured immediately using a Synergy Hybrid Reader (BioTek).

Immunofluorescence.To test for antibody binding via immunofluorescence, 293T cells were plated overnight in sterile, flat-bottom, 96-well tissue culture plates (Sigma) and transfected with Zaire ebolavirus GP in the pCAGGS vector. After incubating for 24 h at 37°C, the cells were fixed with 3.7% formaldehyde for at least 1 h at 4°C. Next, the formaldehyde was discarded and the cell monolayer was blocked with 3% milk in PBS for at least 1 h. For the primary antibody step, the plates were incubated with either experimental anti-EBOV MAb (30 μg/ml), positive-control anti-EBOV MAb 8G9 (30 μg/ml), or no antibody as a negative control in 1% milk (100 μl/well) for 1 h at room temperature. The plates were washed 3 times with PBS and incubated with Alexa Fluor 488 goat anti-mouse secondary antibody in PBS, 1% milk (100 μl/well) for 1 h at room temperature in the dark. Finally, after washing 3 additional times, the cells were visualized via fluorescence microscopy.

To test for antibody binding to wild-type Zaire ebolavirus (Mayinga variant), Vero.E6 cells were grown on glass coverslips and infected at an MOI of 0.1 in a BSL4 laboratory. After incubating for 72 h at 37°C, the cells were washed with PBS and stained with tetramethyl rhodamine isocyanate (TRITC)-labeled cell mask (1:1,000) for 10 min. The cells were washed three times with PBS and fixed with 4% formaldehyde for 30 to 45 min at room temperature. Next, the formaldehyde was discarded, and 0.1% Triton X-100 in PBS plus 10% fetal calf serum (FCS) was added to permeabilize for 30 min. For the primary antibody step, the cells were incubated with either experimental anti-EBOV MAb (10 μg/ml) or positive-control anti-EBOV polyclonal mouse serum (which detects EBOV nucleoprotein) for 1 h at room temperature. The antibodies were diluted in PBS. The cells were then washed 3 times with PBS and incubated with FITC anti-mouse secondary and DAPI (4′,6-diamidino-2-phenylindole) (1:10.000) in PBS for 1 h at room temperature in the dark. Finally, after washing 3 additional times, the cells were mounted on glass slides with Prolong Antifade and visualized via confocal fluorescence microscopy.

Western blotting.Binding of MAbs to EBOV glycoprotein was analyzed on SDS-PAGE (10% polyacrylamide; Mini Protean TGX gels; Bio-Rad) under reducing conditions. Three hundred nanograms of either EBOV GP (lane 1) or influenza virus H7 hemagglutinin (HA) (lane 2; control) was added to the gel and electrophoresed at 200 V for 40 min (Fig. 2). After electrophoresis, each gel was transferred to a polyvinylidene difluoride (PVDF) membrane via an Owl HEP series semidry electroblotting system (Thermo Scientific), according to the manufacturer's instructions. The membranes were washed in 1× PBS and blocked with 10% nonfat milk-PBS for 2 h at room temperature. The membranes were then washed three times with 1× TPBS. One of eight anti-EBOV MAbs (10 μg/ml) or a control anti-polyhistidine antibody (Sigma-Aldrich; 1:1,000 in blocking buffer) was then incubated with the membrane for 1 h at RT and washed three times in TPBS. An anti-mouse IgG-HRP conjugate (1:3,000 in blocking buffer) was then incubated with the membrane for 1 h at RT. Subsequently, ECL substrate (Pierce) was added and chemiluminescence was detected on a Proteinsimple FluorChem E imager.

Visualization of escape mutants on three-dimensional (3D) structures and statistical analysis.Escape mutations were visualized based on a Protein Data Bank (PDB) file derived from a structure of the human MAb KZ52 bound to the Zaire ebolavirus GP trimer (3INU ) crystallized by Lee et al. (45) using the PyMOL Molecular Graphics System (version 1.8) from Schrödinger, LLC.

Data were evaluated for statistical significance using Prism v7 software (Graphpad). Survival curves were assessed using the log-rank (Mantel-Cox) test, with adjustments made for multiple comparisons using the Bonferroni correction.