Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, James E. Crowe, Jr. ( james.crowe@Vanderbilt.Edu ). Materials described in this paper are available for distribution under the Uniform Biological Material Transfer Agreement, a master agreement that was developed by the NIH to simplify transfers of biological research materials.

The animal protocols for testing of mAbs in mice, guinea pigs and ferrets were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (UTMB) in compliance with the Animal Welfare Act and other applicable federal statutes and regulations relating to animals and experiments involving animals.

Five- to six-week-old female Hartley guinea pigs were obtained from the Charles River Laboratories. Animals were housed and challenged under maximum containment in ABSL-4 facility of the Galveston National Laboratory, UTMB.

Seven- to eight-week old female BALB/c mice were obtained from the Jackson Laboratory, and 7-8 week-old 129S6/SvEv-Stat1 tm1Rds mice (STAT1 KO) were obtained from Taconic Biosciences. Mice were housed in microisolator cages and provided food and water ad libitum. Challenge studies were conducted under maximum containment in an animal biosafety level 4 (ABSL-4) facility of the Galveston National Laboratory, UTMB.

The authentic EBOV-eGFP, mouse-adapted EBOV Mayinga (EBOV-MA, GenBank: AF49101), guinea pig-adapted SUDV (SUDV-GA, GenBank: KT878488 ), SUDV strain Gulu, and BDBV strain 200706291 Uganda viruses were described previously (). The chimeric infectious EBOV/BDBV-GP and EBOV/SUDV-GP viruses expressing eGFP were obtained by replacing the gene encoding EBOV GP with that of BDBV (GenBank: KU174137 ) or SUDV (GenBank: KU174142 ), respectively (), and passaged two times in Vero-E6 cell culture monolayers. Recombinant chimeric vesicular stomatitis virus in which the G protein was replaced with EBOV GP (rVSV/EBOV-GP) were provided by Heinz Feldmann (Rocky Mountain Laboratories, NIH, Hamilton, MT) ().

Vero-E6 (monkey, female origin), THP-1 (human, male origin), and Jurkat (human, male origin) cell lines were obtained from the American Type Culture Collection. Vero-E6 cells were cultured in Minimal Essential Medium (MEM) (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin at 5% CO, 37°C. THP-1 and Jurkat cells were cultured in RPMI 1640 (GIBCO) medium supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), 1% GlutaMax (GIBCO), and 1% penicillin-streptomycin (GIBCO) at 37°C in 5% CO. The HMAA 2.5 non-secreting mouse-human heteromyeloma cell line (sex information is not available) was a kind gift from L. Cavacini and was cultured as described previously (). A 293F cell line (human, female origin) stably-transfected to express SNAP-tagged EBOV GP was described recently (). ExpiCHO (hamster, female origin) and FreeStyle 293F (human, female origin) cell lines were purchased from ThermoFisher Scientific and cultured according to the manufacturer’s protocol. The Jurkat-EBOV GP cell line stably expressing EBOV GP Makona on the surface (Davis and Ahmed, personal communication) was a kind gift from Carl Davis (Emory University, Atlanta, GA). An NIH 3T3 engineered fibroblast line (mouse, male origin) constitutively expressing cell-surface human CD154 (CD40 ligand), secreted human B cell activating factor (BAFF) and human IL-21 was kindly provided by Dr. Deepta Bhattacharya (Washington University in St. Louis, MO). All cell lines were tested on a monthly basis for Mycoplasma and found to be negative in all cases.

Human PBMCs were obtained from survivors of the 2014 EVD epidemic in Nigeria or of the 2014 Boende outbreak in the Democratic Republic of the Congo (DRC) (unpublished). A male human survivor of the 2014 EVD outbreak in Nigeria was age 31 y when infected and age 32 when PBMCs were collected. A male human survivor of the 2014 Boende outbreak in the DRC was age 36 y when infected and age 37 y when PBMCs were collected. PBMCs were collected well after the illness had resolved, following informed consent. At time of blood collection, plasma samples were tested by RT-PCR and found to be negative for the presence of viral RNA. The studies were approved by the Institutional Review Boards of Vanderbilt University Medical Center, the UCLA Fielding School of Public Health and the Kinshasa School of Public Health (DRC).

Method Details

Mouse challenge with EBOV Groups of 7-8-week-old female BALB/c mice (n = 5 per group) housed in microisolator cages were inoculated with 1,000 PFU of the EBOV-MA by the intraperitoneal (i.p.) route. Mice were treated i.p. with 100 μg (∼5 mg/kg) of individual mAb per mouse on 1 dpi. Human mAb DENV 2D22 (specific to an unrelated target, dengue virus) served as negative control. Mice were monitored twice daily from day 0 to 14 dpi for illness, survival, and weight loss, followed by once daily monitoring from 15 dpi to the end of the study at 28 dpi. The extent of disease was scored using the following parameters: dyspnea (possible scores 0–5), recumbence (0–5), unresponsiveness (0–5), and bleeding/hemorrhage (0–5). Moribund mice were euthanized as per the IACUC-approved protocol. All mice were euthanized on day 28 after EBOV challenge.

Mouse challenge with SUDV Groups of 7-8-week-old STAT1 KO mice (n = 5 per group) were challenged i.p. with 1,000 PFU wt SUDV (Gulu). Animals were treated i.p. with 200 μg (∼10 mg/kg) of EBOV-specific or control mAb DENV 2D22 per mouse on 1 dpi and were monitored as above.

Guinea pig challenge with SUDV Wong et al., 2015 Wong G.

He S.

Wei H.

Kroeker A.

Audet J.

Leung A.

Cutts T.

Graham J.

Kobasa D.

Embury-Hyatt C.

et al. Development and characterization of a guinea pig-adapted Sudan virus. Groups of 5- to 6-week-old Hartley guinea pigs (n = 5/group) were injected i.p. with 1,000 PFU of SUDV-GA (guinea pig adapted strain Boniface) (). mAb EBOV-520 was delivered by i.p. route at indicated time points and doses. Control groups were treated with mAb DENV 2D22 or left untreated. Animals were monitored for the illness, survival, and weight loss. All animals were euthanized at 28 dpi.

Ferret challenge with BDBV Kozak et al., 2016 Kozak R.

He S.

Kroeker A.

de La Vega M.A.

Audet J.

Wong G.

Urfano C.

Antonation K.

Embury-Hyatt C.

Kobinger G.P.

Qiu X. Ferrets infected with Bundibugyo virus or Ebola virus recapitulate important aspects of human filovirus disease. Groups of 6-month-old male and female animals were challenged intramuscularly with 1,000 PFU of BDBV, as described previously (). Animals were treated by i.p. route with 18 mg of mAb EBOV-520 or the control mAb DENV 2D22 on day 3, and the same dose of the mAb on day 6 after challenge. The disease scores were assessed as follows: healthy, 1; developing clinical disease, 2; advanced disease, 3; moribund, 4. Blood was collected from surviving animals on 0, 3, 6, 9, 14, 21, and 28 dpi to assess virus titers. Ferrets were monitored for 28 days after virus inoculation and then euthanized.

Generation of human B cell hybridomas producing mAbs Yu et al., 2008 Yu X.

McGraw P.A.

House F.S.

Crowe Jr., J.E. An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies. Yu et al., 2008 Yu X.

McGraw P.A.

House F.S.

Crowe Jr., J.E. An optimized electrofusion-based protocol for generating virus-specific human monoclonal antibodies. 2 flasks (Corning). PBMCs from heparinized blood were isolated with Ficoll-Histopaque by density gradient centrifugation. The cells were cryopreserved in the vapor phase of liquid nitrogen until use. Human B cell hybridomas were generated as described previously () with some modifications. Briefly, previously cryopreserved samples were thawed and expanded on irradiated NIH 3T3 cells that had been engineered to express human IL-21, CD40L, and BAFF in medium A (STEMCELL Technologies) supplemented with CpG, a Chk2 inhibitor (Sigma), and cyclosporine A (Sigma). After 7 days, supernatants from each well of the 384-well culture plates were assessed by ELISA for reactivity against various ebolavirus proteins using enzyme-linked immunosorbent assays (ELISAs), as described below. The next day, cells from wells with supernatants reacting with antigen in an ELISA were fused with HMMA2.5 myeloma cells using an established electrofusion technique (). After the fusion reaction, hybridoma lines were cultured in ClonaCell-HY Medium E (STEMCELL Technologies) supplemented with HAT Media Supplement (Sigma) in 384-well plates for 18 days before screening of supernatants for antibody production. Hybridoma cell lines producing ebolavirus GP-reactive antibodies were cloned biologically by single-cell fluorescence-activated cell sorting. Hybridomas were expanded in Medium E until 50% confluent in 75-cmflasks (Corning).

mAb isotype and gene sequence analysis Thornburg et al., 2016 Thornburg N.J.

Zhang H.

Bangaru S.

Sapparapu G.

Kose N.

Lampley R.M.

Bombardi R.G.

Yu Y.

Graham S.

Branchizio A.

et al. H7N9 influenza virus neutralizing antibodies that possess few somatic mutations. Giudicelli and Lefranc, 2011 Giudicelli V.

Lefranc M.P. IMGT/junctionanalysis: IMGT standardized analysis of the V-J and V-D-J junctions of the rearranged immunoglobulins (IG) and T cell receptors (TR). The isotype and subclass of secreted antibodies were determined using murine anti-human IgG1, IgG2, IgG3 or IgG4 mouse antibodies conjugated with alkaline phosphatase (Southern Biotech). Antibody heavy- and light-chain variable region genes were sequenced from hybridoma lines that had been cloned biologically from flow cytometry. Briefly, total RNA was extracted using the RNeasy Mini kit (QIAGEN) and reverse-transcriptase PCR (RT-PCR) amplification of the antibody gene cDNAs was performed using the PrimeScript One Step RT-PCR kit (CLONTECH) according to the manufacturer’s protocols with gene-specific primers (). The thermal cycling conditions were as follows: 50°C for 30 min, 94°C for 2 min, 40 cycles of (94°C for 30 s, 58°C for 30 s and 72°C for 1 min). PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter) and sequenced directly using an ABI3700 automated DNA sequencer. The identities of gene segments and mutations from germlines were determined by alignment using the ImMunoGeneTics database ().

mAb production and purification Hybridoma cells secreting GP-reactive mAbs were grown in serum-free medium (Hybridoma-SFM, Life Technologies). MAbs were purified from filtered culture supernatants by fast protein liquid chromatography (FPLC) on an ÄKTA instrument using HiTrap MabSelect Sure or HiTrap Protein G columns (GE Healthcare). Purified mAbs were buffer exchanged into PBS, filtered using sterile 0.45-μm pore size filter devices (Millipore), concentrated, and stored in aliquots at −80°C until use. McLean et al., 2000 McLean G.R.

Nakouzi A.

Casadevall A.

Green N.S. Human and murine immunoglobulin expression vector cassettes. For recombinant mAb production, cDNA encoding the genes of heavy and light chains were cloned into DNA plasmid expression vectors encoding IgG (IgG1, IgG3, Ig4, or IgG1-LALA) - or Fab- heavy chain () and transformed into E. coli cells. mAb proteins were produced following transiently transfection of FreeStyle 293F or ExpiCHO cells following the manufacturer’s protocol and were purified as described above.

GP expression and purification 4 for 4 days. Protein was purified using Strep-Tactin resin (QIAGEN) via an engineered strep II tag and purified further by Superdex 200 (S200) column chromatography. Purity of recombinant GP was confirmed by SDS-PAGE. The ectodomains of EBOV GP ΔTM (residues 1-636; strain Makona; GenBank: KM233070 ), BDBV GP ΔTM (residues 1-643; strain 200706291 Uganda; GenBank: NC_014373 ), SUDV GP ΔTM (residues 1-637; strain Gulu; GenBank: NC_006432 ), and MARV GP ΔTM (residues 1-648; strain Angola2005; GenBank: DQ447653 ) were expressed transiently in Expi293F cells with a C-terminal strep II tag using the pcDNA3 plasmid vector. Secreted proteins were purified using 5 mL StrepTrap HP column (GE Healthcare) following the manufacturer’s protocol, and then purified further and buffer exchanged into PBS using Supedex200 (GE Healthcare) size exclusion chromatography. Formation of EBOV GP ΔTM trimer was confirmed by negative stain EM. For some experiments, we used EBOV GP that was produced in Drosophila Schneider 2 (S2) cells. Briefly, recombinant ectodomain of EBOV GP ΔTM in modified pMTpuro vector was transfected into S2 cells followed by stable selection of transfected cells with 6 μg/mL puromycin. GP ectodomain expression was induced with 0.5 mM CuSOfor 4 days. Protein was purified using Strep-Tactin resin (QIAGEN) via an engineered strep II tag and purified further by Superdex 200 (S200) column chromatography. Purity of recombinant GP was confirmed by SDS-PAGE.

ELISA binding assays Wells of microtiter plates were coated with purified, recombinant EBOV, BDBV, SUDV, or MARV GP ΔTM and incubated at 4°C overnight. Plates were blocked with 2% non-fat dry milk and 2% normal goat serum in DPBS containing 0.05% Tween-20 (DPBS-T) for 1 hr. For mAb screening assays, hybridoma culture supernatants were diluted in blocking buffer 1:5, added to the wells, and incubated for 1 hr at ambient temperature. The bound antibodies were detected using goat anti-human IgG conjugated with HRP (Southern Biotech) and TMB substrate (ThermoFisher). Color development was monitored, 1N hydrochloric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose-response and cross-reactivity assays, serial dilutions of plasma or purified mAbs were applied to the wells in triplicate or quadruplicate, as detailed above. EC 50 values for mAb binding were determined using Prism 7.2 software (GraphPad) after log transformation of antibody concentration using sigmoidal dose-response nonlinear regression analysis. Similarly, a non-linear regression analysis was performed on the resulting curves to calculate plasma dilution that yielded a half-maximum O.D. 450 nm value. Antibody titer in plasma was expressed as the inverse of plasma dilution.

Cell surface displayed GP mAb binding Jurkat-EBOV GP cells were washed with the incubation buffer containing DPBS, 2% of heat-inactivated FBS and 2 mM EDTA (pH 8.0) by centrifugation at 400 x g for 5 min at room temperature. For antibody staining, ∼5 × 104 cells were added per each well of V-bottom 96-well plate (Corning) in 5 μL of the incubation buffer. Serial dilutions of antibody were added to the cells in triplicate or quadruplicate for total volume of 50 μL per well, followed by 1 hr incubation at room temperature, or 4°C in some experiments. Unbound antibody was removed by washing with 200 μL of the incubation buffer as described above, and cells were incubated with phycoerythrin (PE)-labeled secondary goat anti-human antibodies (Southern Biotech) for 30 min at 4°C. In some experiments, cells were fixed with 4% PFA in DPBS before staining with secondary antibodies. Staining of cells was measured by flow cytometric analysis using an Intellicyt iQue high throughput cytometer (Intellicyt), or an LSRII flow cytometer (BD Biosciences). Data for up to 20,000 events were acquired, and data were analyzed with ForeCyt (Intellicyt) or FlowJo (Tree Star) software. Dead cells were excluded from the analysis on the basis of forward and side scatter gate for viable cell population. Binding to untransduced Jurkat cells, or binding of dengue antigen-specific mAb DENV 2D22 served as negative controls for most experiments. In some experiments, binding to cell surface displayed GP was assessed with mAbs that were directly fluorescently-labeled. Briefly, mAbs were labeled with Alexa Fluor 647 NHS ester (ThermoFisher) by following the manufacturer’s protocol. Labeled mAbs were purified further and buffer exchanged into the PBS using desalting Zeba columns (ThermoFisher) and stored at 4°C with 0.1% bovine serum albumin (Sigma) and 0.01% sodium azide. To assess binding of mAbs to Jurkat-EBOV GP CL , Jurkat-EBOV GP cells were counted and treated with 0.5 mg/mL of thermolysin (Promega) in PBS for 20 min at 37°C. Cell staining and flow cytometric analysis was performed as described above. Binding to untransfected Jurkat or uncleaved Jurkat-EBOV GP served as controls.

Cell surface displayed GP mAb competition-binding CL cells were pre-incubated with a saturating concentration (typically 20 μg/mL) of the first unlabeled mAb at room temperature for 30 min, followed by addition of the second fluorescently-labeled mAb (typically 5 μg/mL) and incubated for an additional 30 min. The second mAb was added after the first mAb and without washing of cells to minimize a dissociation of the first mAb from cell surface GP during a prolonged incubation. Cells were washed, fixed with PFA, and cell staining was analyzed using an Intellicyt iQue flow cytometer as detailed above. Background values were determined from binding of the second labeled mAbs to untransfected Jurkat. Results are expressed as the percent of binding in the presence of competitor mAb relative to primary mAb-only control (maximal binding) minus background. The antibodies were considered competing if the presence of first antibody reduced the signal of the second antibody to less than 30% of its maximal binding or non-competing if the signal was greater than 70%. A level of 30%–70% was considered intermediate competition. Of note, mAbs from the GP base region competitor epitope group revealed much stronger binding to Jurkat-EBOV GP CL than to Jurkat-EBOV GP cells. This finding was revealed by nearly complete cross-blocking capacity of these mAbs on Jurkat-EBOV GP CL when compared to those determined for Jurkat-EBOV GP cells ( Jurkat-EBOV GP or Jurkat-EBOVcells were pre-incubated with a saturating concentration (typically 20 μg/mL) of the first unlabeled mAb at room temperature for 30 min, followed by addition of the second fluorescently-labeled mAb (typically 5 μg/mL) and incubated for an additional 30 min. The second mAb was added after the first mAb and without washing of cells to minimize a dissociation of the first mAb from cell surface GP during a prolonged incubation. Cells were washed, fixed with PFA, and cell staining was analyzed using an Intellicyt iQue flow cytometer as detailed above. Background values were determined from binding of the second labeled mAbs to untransfected Jurkat. Results are expressed as the percent of binding in the presence of competitor mAb relative to primary mAb-only control (maximal binding) minus background. The antibodies were considered competing if the presence of first antibody reduced the signal of the second antibody to less than 30% of its maximal binding or non-competing if the signal was greater than 70%. A level of 30%–70% was considered intermediate competition. Of note, mAbs from the GP base region competitor epitope group revealed much stronger binding to Jurkat-EBOV GPthan to Jurkat-EBOV GP cells. This finding was revealed by nearly complete cross-blocking capacity of these mAbs on Jurkat-EBOV GPwhen compared to those determined for Jurkat-EBOV GP cells ( Figure 4 A, B).

Cell surface displayed GP cleavage inhibition Jurkat-EBOV GP cells were pre-incubated with serial dilutions of mAbs in PBS for 20 min at room temperature, then incubated with thermolysin for 20 min at 37°C. The reaction was stopped by addition of the incubation buffer as described above. Washed cells were incubated with 5 μg/mL of fluorescently-labeled RBS-specific mAb MR78 at 4°C for 60 min. Stained cells were washed, fixed, and analyzed by flow cytometry using Intellicyt iQue. Cells were gated for the viable population. Background staining was determined from binding of the labeled mAb MR78 to Jurkat-EBOV GP (uncleaved) cells. Results are expressed as the percent of RBS exposure in the presence of tested mAb relative to labeled MR78 mAb-only control (maximal binding to Jurkat-EBOV GP CL ) minus background.

Cell surface displayed GP CL soluble NPC1-C binding inhibition Jurkat-EBOV GP CL cells were prepared as detailed above and resuspended in the incubation buffer. Approximately 5 × 104 cells per well in V-bottom 96-well plate were incubated with serial 3-fold dilutions of mAbs in a total volume of 50 μL at ambient temperature for 30 min, followed by washing and incubation with pre-titrated concentration (typically 50 μg/mL) of soluble, FLAG epitope-tagged, recombinant NPC1- C protein (Creative BioMart). Cells were washed, incubated with PE-labeled secondary mouse anti-FLAG tag antibody (BioLegend) for 2 hr at 4°C, fixed with PFA, and then analyzed by flow cytometry using LSRII cytometer equipped with 535 nm green laser. Cells were gated for the viable population. Results are expressed as the percent of NPC1-C binding inhibition in the presence of tested mAb relative to NPC1-only control (maximal binding to Jurkat-EBOV GP CL ) minus background.

Cooperative binding to cell surface displayed GP Howell et al., 2017 Howell K.A.

Brannan J.M.

Bryan C.

McNeal A.

Davidson E.

Turner H.L.

Vu H.

Shulenin S.

He S.

Kuehne A.

et al. Cooperativity enables non-neutralizing antibodies to neutralize ebolavirus. The cell surface display assay was based on principles from previously described enhanced binding ELISA assay (). Briefly, Jurkat-EBOV GP cells were incubated with a mixture of individual unlabeled glycan cap-specific mAbs at a saturating concentration (10 μg/mL) and respective dilution of fluorescently-labeled mAbs EBOV-515 or −520. Cells were washed, and antibody binding was analyzed by flow cytometry using Intellicyt iQue.

Epitope mapping using an EBOV GP alanine-scan mutation library Davidson et al., 2015 Davidson E.

Bryan C.

Fong R.H.

Barnes T.

Pfaff J.M.

Mabila M.

Rucker J.B.

Doranz B.J. Mechanism of binding to Ebola virus glycoprotein by the ZMapp, ZMAb, and MB-003 cocktail antibodies. Davidson and Doranz, 2014 Davidson E.

Doranz B.J. A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes. Epitope mapping was carried out as described previously (). Comprehensive high-throughput alanine scanning (‘shotgun mutagenesis’) was carried out on an expression construct for EBOV GP lacking the mucin-like domain (residues 311-461) (based on the Yambuku-Mayinga variant GP sequence), mutagenizing GP residues 33-310 and 462-676 to create a library of clones, each representing an individual point mutant. Residues were changed to alanine (with alanine residues changed to serine). The resulting library, covering 492 of 493 (99.9%) of target residues, was arrayed into 384-well plates, one mutant per well, then transfected into HEK293T cells and allowed to express for 22 hr. Cells, unfixed or fixed in 4% paraformaldehyde, were incubated with primary antibody and then with an Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). After washing, cellular fluorescence was detected using the Intellicyt high throughput flow cytometer (Intellicyt). mAb reactivity against each mutant EBOV GP clone was calculated relative to wild-type EBOV GP reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type GP-transfected controls. Mutated residues within clones were identified as critical to the mAb epitope if they did not support reactivity of the test mAb but did support reactivity of other control EBOV mAbs. This counter-screen strategy facilitated the exclusion of GP mutants that were misfolded locally or that exhibited an expression defect. The detailed algorithms used to interpret shotgun mutagenesis data were described previously ().

Generation of virus neutralization escape mutants To generate escape mutants for EBOV-520 and −442 mAbs, 100 PFU of EBOV-eGFP were combined with 2-fold dilutions of the respective mAb starting at 200 μg/mL in U-bottom 96-well plates and incubated for 1 hr at 37°C. Mixtures were placed on Vero-E6 cell monolayer cultures in 96-well plates and incubated for 1 hr. Supernatants were removed, freshly-diluted mAb was added at the same concentrations in 200 μL of MEM supplemented with 2% FBS, and plates were incubated for 7 days at 37°C. Viruses that replicated in the presence of the highest concentrations of mAb, as determined by monitoring eGFP fluorescence by microscopy, were collected. 20 μL aliquots were incubated with 2-fold dilutions of mAbs starting at 200 μg/mL, and viruses were propagated in the presence of mAbs as described above. The procedure was repeated once more with mAb dilutions starting at 400 μg/mL. Viruses that replicated at the highest mAb concentrations were amplified in Vero-E6 cell culture monolayers in 24-well plates in the presence of mAbs at 200 μg/mL for 7 days. Cells were used for isolation of RNA using TRIzol reagent, and cDNA copies of viral RNA encoding GP were amplified by RT-PCR and sequenced. To determine susceptibility of the isolated escape mutants to mAbs, 100 PFU of the viruses in MEM supplemented with 2% FBS in triplicate were combined in U-bottom 96-well plates with 8 to 12 two-fold dilutions of mAb, starting at 200 μg/mL, in total volumes of 50 μL, and incubated for 1 hr at 37°C. The virus/antibody mixtures then were added in triplicate to Vero-E6 cell culture monolayers in 96-well plates, incubated for 1 hr at 37°C, washed with MEM, overlaid with 200 μL of MEM containing 2% FBS and 0.8% methylcellulose, and incubated for 48 hr at 37°C. Plates were fixed with 10% phosphate-buffered formalin (Fisher). Plaques were counted using a fluorescence microscopy. To generate EBOV-515 escape mutants, aliquots containing 100 PFU of rVSV/EBOV-GP virus were pre-incubated with serial 2-fold dilutions starting from 200 μg/mL of mAb for 1 hr at 37°C and inoculated into 96-well plate Vero-E6 cell monolayers. After 48 hr, virus samples were harvested and titrated. Virus-positive samples from the highest mAb concentration were selected for the next passage. After seven passages, a 200 PFU virus aliquot was pre-incubated with mAb EBOV-515 and inoculated into a 24-well plate Vero-E6 cell monolayer culture. After 72 hr, the infected cell monolayer was solubilized in TRIzol (Ambion, Life Technologies) and subjected to total RNA isolation, RT-PCR and sequencing of EBOV GP.

Neutralization assays Ilinykh et al., 2016 Ilinykh P.A.

Shen X.

Flyak A.I.

Kuzmina N.

Ksiazek T.G.

Crowe Jr., J.E.

Bukreyev A. Chimeric filoviruses for identification and characterization of monoclonal antibodies. Towner et al., 2005 Towner J.S.

Paragas J.

Dover J.E.

Gupta M.

Goldsmith C.S.

Huggins J.W.

Nichol S.T. Generation of eGFP expressing recombinant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening. 2 pH 8.0) and divided into 2 aliquots: one aliquot was treated with 0.5 mg/mL of thermolysin (Promega), another one – with equal volume of thermolysin digestion buffer (mock-treated virus) for 40 min at 37°C. The reactions were stopped by addition of EDTA up to the final concentration 10 mM. Virus samples were re-pelleted through a 25% sucrose cushion and were washed by ultracentrifugation in buffer containing 10 mM Tris (pH 8.0) and 0.1 M NaCl for 1 hr at 175,000 x g at 4°C. Virus pellets were resuspended in the same buffer, incubated with serial mAb dilutions for 1 hr at 37°C, or mock-incubated, and titrated by applying to Vero-E6 cell culture monolayers in triplicate. Antibody neutralization assays were performed in a high-throughput or plaque reduction format using the recombinant EBOV-eGFP, rVSV/EBOV-GP, or chimeric EBOV viruses in which GP was replaced with its counterpart from BDBV or SUDV as described previously (). For the assays with thermolysin-cleaved virus, rVSV/EBOV-GP virus was propagated in Vero-E6 cells. At 48 hr after infection, virus suspension was harvested and clarified from cell debris by centrifugation for 10 min at 10,000 x g. Next, the supernatant was ultracentrifuged through a 25% sucrose cushion for 2 hr at 175,000 x g at 4°C. Pelleted virus was resuspended in thermolysin digestion buffer (50 mM Tris, 0.5 mM CaClpH 8.0) and divided into 2 aliquots: one aliquot was treated with 0.5 mg/mL of thermolysin (Promega), another one – with equal volume of thermolysin digestion buffer (mock-treated virus) for 40 min at 37°C. The reactions were stopped by addition of EDTA up to the final concentration 10 mM. Virus samples were re-pelleted through a 25% sucrose cushion and were washed by ultracentrifugation in buffer containing 10 mM Tris (pH 8.0) and 0.1 M NaCl for 1 hr at 175,000 x g at 4°C. Virus pellets were resuspended in the same buffer, incubated with serial mAb dilutions for 1 hr at 37°C, or mock-incubated, and titrated by applying to Vero-E6 cell culture monolayers in triplicate.

Antibody-mediated cellular phagocytosis by human monocytes (ADCP) Recombinant EBOV GP ΔTM (IBT Bioservices) was biotinylated and coupled to Alexa Fluor 488 Neutravidin beads (Life Technologies). Antibodies were diluted to 5μg/ml in cell culture medium and incubated with beads for 2 hr at 37°C. THP-1 monocytes (ATCC) were added at 2.5 × 104 cells per well and incubated for ∼18 hr at 37°C. Cells were fixed with 4% paraformaldehyde and analyzed on a BD LSRII flow cytometer, and a phagocytic score was determined using the percentage of FITC+ cells and the median fluorescence intensity of the FITC+ cells. The glycan cap-specific mAb c13C6 (IBT Bioservices) was used as a positive control, and the HIV-specific mAb 2G12 (Polymun Scientifics) was used as a negative control.

Antibody-mediated neutrophil phagocytosis (ADNP) Recombinant EBOV GP ΔTM (IBT Bioservices) was biotinylated and coupled to Alexa Fluor 488 Neutravidin beads (Life Technologies). Antibodies were diluted to 5 μg/mL in cell culture medium and incubated with beads for 2 hr at 37°C. White blood cells were isolated from donor peripheral blood by lysis of red blood cells, followed by three washes with PBS. Cells were added at a concentration of 5.0 × 104 cells/well and incubated for 1 hr at 37°C. Cells were stained with CD66b (Pacific Blue, Clone G10F5; BioLegend), CD3 (Alexa 700, Clone UCHT1; BD Biosciences), and CD14 (APC-Cy7, Clone MφP9; BD Biosciences), and fixed with 4% paraformaldehyde, and analyzed by flow cytometry on a BD LSR II flow cytometer. Neutrophils were defined as SSC-Ahigh CD66b+, CD3-, CD14-. A phagocytic score was determined using the percentage of FITC+ cells and the median fluorescence intensity of the FITC+ cells. The glycan cap-specific mAb c13C6 (IBT Bioservices) was used as a positive control, and the HIV-specific mAb 2G12 (Polymun Scientifics) was used as a negative control.

Antibody-dependent NK cell degranulation Recombinant EBOV GP ΔTM (IBT Bioservices) was coated onto a MaxiSorp 96 well plates (Nunc) at 300 ng/well at 4°C for 18 hr. Wells were washed three times with PBS and blocked with 5% bovine serum albumin in PBS. Antibodies were diluted to 5 μg/mL in PBS, and added to the plates, and were incubated for an additional 2 hr at 37°C. Unbound antibodies were removed by washing three times with PBS, and human NK cells freshly isolated from peripheral blood of human donors by negative selection (Stem Cell Technologies, Canada) were added at 5 × 104 cells/well in the presence of 4 μg/mL brefeldin A (Sigma Aldrich) and 5 μg/mL GolgiStop (Life Technologies) and anti-CD107a antibody (PE-Cy5, Clone H4A3, BD Biosciences). Plates were incubated for 5 hr at 37°C. Cells were stained for NK cell markers (CD56 PE-Cy7, clone B159, BD Biosciences; CD16 APC-Cy7, clone 3G8, BD Biosciences; CD3 Alexa Fluor700, clone UCHT1, BD Biosciences), followed by fixation and permeabilization with Fix and Perm (Life Technologies) according to the manufacturer’s instructions to stain for intracellular IFNγ (APC, Clone B27, BD Biosciences) and MIP-1β (PE, Clone D21-1351, BD Biosciences). Cells were analyzed on a BD LSRII flow cytometer. The glycan cap-specific mAb c13C6 (IBT Bioservices) was used as a positive control, and the HIV-specific mAb 2G12 (Polymun Scientifics, Austria) was used as a negative control.

Antibody-mediated complement deposition (ADCD) Recombinant EBOV GP (IBT Bioservices) was biotinylated and coupled to red fluorescent Neutravidin beads (Life Technologies). Antibodies were diluted to 5μg/mL in RPMI-1640, and incubated with GP-coated beads for 2 hr at 37°C. Freshly reconstituted guinea pig complement (Cedarlane Labs) was diluted in veronal buffer with 0.1% fish gelatin (Boston Bioproducts), added to the antibody-bead complexes, and incubated for 20 min at 37°C. Beads were washed twice with phosphate buffered saline containing 15 mM EDTA, and stained with an anti-guinea pig C3 antibody conjugated to FITC (MP Biomedicals) for 15 min at ambient temperature. Beads were washed twice more with PBS, and C3 deposition onto beads was analyzed on a BD LSRII flow cytometer and the median fluorescence intensity of the FITC+ of all beads was measured.

Rapid fluorimetric antibody-mediated cytotoxicity assay (RFADCC) Domi et al., 2018 Domi A.

Feldmann F.

Basu R.

McCurley N.

Shifflett K.

Emanuel J.

Hellerstein M.S.

Guirakhoo F.

Orlandi C.

Flinko R.

et al. A single dose of modified Vaccinia Ankara expressing Ebola virus like particles protects nonhuman primates from lethal Ebola virus challenge. Orlandi et al., 2016 Orlandi C.

Flinko R.

Lewis G.K. A new cell line for high throughput HIV-specific antibody-dependent cellular cytotoxicity (ADCC) and cell-to-cell virus transmission studies. Domi et al., 2018 Domi A.

Feldmann F.

Basu R.

McCurley N.

Shifflett K.

Emanuel J.

Hellerstein M.S.

Guirakhoo F.

Orlandi C.

Flinko R.

et al. A single dose of modified Vaccinia Ankara expressing Ebola virus like particles protects nonhuman primates from lethal Ebola virus challenge. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EBOV GP-reactive IgG or Fab was quantified with an EBOV-adapted modification of the RFADCC assay (). Briefly, a target cell line was made by transfecting 293F cells with a full-length DNA expressing GP from the EBOV-Kikwit isolate followed by transfecting with two separate DNA constructs expressing EGFP and the chimeric CCR5-SNAP tag protein. The new cell line, designated EBOV GPkik-293FS EGFP CCR5-SNAP, expresses EBOV-Kikwit GP on the plasma membrane, EGFP in the cytoplasm and the SNAP-tag CCR5, which can be specifically labeled with SNAP-Surface Alexa Fluor-647 (NEB), on the cell surface (). A human anti-EBOV GP mAb KZ52 (a neutralizing antibody) (IBT) were used as positive control and the unrelated human mAb DENV 2D22 as a negative control. The ADCC activity was quantified by incubating three-fold serial dilutions of mAbs with EBOV GPkik-293FS EGFP CCR5-SNAP target cells for 15 min at ambient temperature and then adding human PBMC as effector cells for 2 hr at 37°C, after which cells were washed once with PBS, fixed with 2% PFA, stained and analyzed with an LSRII Fortessa flow cytometer (BD Biosciences). Data analysis was performed with FlowJo software (Tree Star Inc.). The percentage cytotoxicity of the mAb was determined as the number of target cells losing EGFP (by virtue of ADCC) but retaining the surface expression of CCR5-SNAP.

Analysis of viremia by plaque assay Ilinykh et al., 2016 Ilinykh P.A.

Shen X.

Flyak A.I.

Kuzmina N.

Ksiazek T.G.

Crowe Jr., J.E.

Bukreyev A. Chimeric filoviruses for identification and characterization of monoclonal antibodies. Virus titration was performed in Vero-E6 cells by plaque assay on serum samples collected from ferrets, as previously described () with some modifications. Briefly, duplicate 10-fold serial dilutions of sera were applied to Vero-E6 cell monolayers in 96 well plates for 1 hr, covered with 100 μL of 0.9% methylcellulose (Sigma) overlay and incubated at 37°C for 6 days. The overlay was removed, cell monolayers were fixed with formalin, washed three times with PBS, and blocked for 1 hr with 5% non-fat dry milk in PBS-T. Plaques were immunostained with rabbit anti-GP primary antibodies (IBT Bioservices) at a 1:5,000 followed by goat-anti rabbit secondary IgG polyclonal HRP-labeled antibody (KPL) at a 1:1,000 dilution in PBS-T. Virus plaques were visualized by staining with a 4CN two component peroxidase substrate system (KPL). The limit of detection was 100 PFU/mL.

Serum chemistry markers Serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase, total bilirubin, alkaline phosphatase, glucose, total protein, blood urea nitrogen, creatinine, phosphorus, calcium (Ca2+), sodium (Na+), potassium (K+), and globulin by using a VetScan VS2 Chemistry Analyzer with comprehensive Diagnostic Profile Reagent Rotor Package (Abaxis).