www.sciencemag.org/cgi/content/full/science.aad5224/DC1

Supplementary Materials for

Protective monotherapy against lethal Ebola virus infection by a

potently neutralizing antibody

Davide Corti, John Misasi, Sabue Mulangu, Daphne A. Stanley, Masaru Kanekiyo, Suzanne Wollen, Aurélie Ploquin, Nicole A. Doria-Rose, Ryan P. Staupe,

Michael Bailey, Wei Shi, Misook Choe, Hadar Marcus, Emily A. Thompson, Alberto Cagigi, Chiara Silacci, Blanca Fernandez-Rodriguez, Laurent Perez,

Federica Sallusto, Fabrizia Vanzetta, Gloria Agatic, Elisabetta Cameroni, Neville Kisalu, Ingelise Gordon, Julie E. Ledgerwood, John R. Mascola, Barney S. Graham,

Jean-Jacques Muyembe-Tamfun, John C. Trefry, Antonio Lanzavecchia, Nancy J. Sullivan*

*Corresponding author. E-mail: njsull@mail.nih.gov

Published 25 February 2016 on Science First Release DOI: 10.1126/science.aad5224

This PDF file includes: Materials and Methods

Figs. S1 to S16

References

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Materials and Methods Isolation of monoclonal antibodies from EBOV survivors

Two subjects who survived the 1995 EBOV Kikwit variant outbreak in the Democratic Republic of Congo were identified and enrolled in VRC200 clinical trial #NCT00067054 after giving signed informed consent. These subjects were the sole survivors of a family of 15 people who were infected during the outbreak. At the time of infection, subject 1 (S1) was a male 28-year-old who had severe laboratory-confirmed illness and, following recovery, worked for several months in the EVD ward caring for other patients. His sister (S2) was 20-years old and had moderate disease severity that was clinically diagnosed based on contact history and symptoms. Peripheral blood mononuclear cells (PBMCs) were obtained, stained with directly labeled antibodies to CD22 (Pharmingen) and to immunoglobulin IgM, IgD, and IgA. CD22+IgM–IgD–IgA– B cells were isolated using FACS Aria, pulsed with Epstein-Barr Virus (50% B958 supernatant) and seeded at 30 cells/well (for a total of 2.7 × 105 purified cells) in replicate cultures in medium supplemented with CpG 2006 and irradiated allogeneic PBMCs, as previously described (10). Culture supernatants were collected after 2 weeks and tested for binding to ELISA plates coated with EBOV GP (Mayinga variant), their specificity was confirmed using an unrelated antigen (tetanus toxoid) and positive cultures were further tested for their ability to neutralize EBOV pseudoviruses. Cultures that scored positive in the EBOV neutralization assay were sub-cloned by limiting dilution.

Antibody purification, labeling, genetic analysis, and reversion to germline

The usage of VH and VL gene segments was determined by sequencing, and analysis for homology to known human V, D, and J genes was performed using the IMGT database (http://www.imgt.org/). Human antibodies were affinity purified by protein A chromatography (GE Healthcare) and dialyzed against PBS. Selected antibodies were biotinylated using the EZ-Link NHS-PEO Solid Phase Biotinylation Kit (Pierce). Antibodies were also produced recombinantly by cloning VH and VL genes via PCR into human Igγ1, Igκ (mAb114, 165, 166), and Igλ (mAb100) expression vectors using gene-specific primers (21). Antibodies used for animal studies were produced by transient transfection of suspension cultured 293FreeStyle cells (Invitrogen) with PEI or Expi cells with Expifectamine293 (Invitrogen). Supernatants from transfected cells were collected after 6-10 days of culture and IgGs were affinity purified by Protein A chromatography (GE Healthcare) and dialyzed against PBS. Purified mAbs were then concentrated with Amicon Ultra centrifugal filters and sterilized by 0.22 µm filtration. The purity was assessed by SEC-HPLC and SDS-PAGE. Endotoxin content was measured with the Endpoint Chromogenic LAL assay (QCL-1000 TM assay, Lonza) according to manufacturing instructions and shown to be below 0.25 EU/ml. Antibody concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific) using Rituximab (Roche) as internal standard or A280 using an Nanodrop (Thermo Scientific). Germlined VH and VL nucleotide sequences were synthesized by Genscript, and their accuracy was confirmed by sequencing.

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Antibodies KZ52 and 13C6 was purchased from IBT Bioservices. Unless otherwise noted

isotype control antibody was an anti-HIV gp120 IgG1.

ELISA for serum antibody titer and GP-binding Binding of EVD survivor’s polyclonal sera, monoclonal antibodies and antibody in

non-human primates to EBOV GP was evaluated by enzyme-linked immunosorbent assay (ELISA) as described previously (22). Titers for survivor and non-human primates were calculated as reciprocal EC90 values (22).

Ebola virus GP vectors

Plasmid vector WT GP (Z) has been described previously (23). A vector expressing a soluble mucin deleted (ΔMuc) GP, GPΔMucΔTM-GCN4-HisSA (Δ309-505, Δ657-676), was made using codon optimization and then synthesized and directly cloned in frame to a GCN4 trimerization domain-His-Strep Tactin domains (MKQIEDKIEEILSKIYHIENEIARIKKLIGEVASSSIEGRGSHHHHHHSAWSHPQFEK) and sequence verified by Genscript. EBOV GP variant Makona-C05 (Acc#KJ660348) was codon optimized, synthesized and sequence verified by Genscript.

Pseudotyped virus neutralization assay

Supernatants or purified mAbs from immortalized B cell clones isolated from EVD survivor donors were assessed for neutralization potency using a single-round infection assay with EBOV GP-pseudotyped lentiviruses particles which express a luciferase reporter gene following entry (22). Unless indicated, all experiments utilized particles bearing GP from the EBOV Mayinga variant. In brief, HEK293T cells were used as infection targets and incubated in a 96-well plate 1 day before infection with pseudovirus in the presence of serially diluted supernatant or purified mAbs. Infected target cells were lysed 72 hours after infection and assayed with the Luciferase Assay System or Bright Glo (Promega), using a Victor X3 Plate Reader (PerkinElmer) to detect luciferase activity.

Plaque Reduction Neutralization Test (PRNT) assay

Antibodies were randomized and coded to blind the study team to the identity of each group. The antibodies were diluted in α-MEM (Gibco). An equal volume of α-MEM containing 1,000 pfu/mL challenge virus was added to an equal volume of media containing antibody to yield a concentration of 0.2 mg/ml antibody and a target dose of 500 pfu/mL virus within the final neutralization mixture. A mock sample using media only treated virus was used as a negative control. The mixtures were incubated together at 37°C for one hour. After the incubation, 200 µL of the mixture was added to each well of a 6 well plate containing confluent Vero E6 cells for a target of 100 pfu/well. A plaque assay was then conducted as described previously (24). After the incubation period, the monolayer was overlaid with 0.5 % agarose in MEM. Plaques developed for one week and were stained with a neutral red overlay. The following day plaques were counted and neutralization was assessed by comparing plaques numbers in the mAb treated versus mock treated samples.

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Antibody-dependent cell-mediated cytotoxicity (ADCC)

rAd5 EBOV GP-transduced and non-transduced HEK293T cells were double labeled with membrane-bound and intracellular stains in order to detect ADCC activity. Specifically, cells were incubated with 8 µM Plum stain (Plum cell labeling kit M.T.T.I. CellVue) followed by FBS. The cells were then washed with RPMI 1640, incubated with 5 µM Carboxyflourescein Succinimidyl ester (CFSE) (Vybrant CFDA SE cell tracer kit, Invitrogen), incubated with FBS and washed again with RPMI 1640. Doubly labeled EBOV GP expressing cells were plated in a V-bottomed 96-well plate at 5,000 cells/well. Antibodies were added to duplicate samples at 31.6 ng/ml to the target cells for 20 minutes at room temperature. Anti-RSV antibody (palivizumab) was used as a control antibody. Effector cells resuspended in RPMI were then added to the target cells at the effector-to-target cell (ET) ratio 1:50 which was found to give the best signal to noise ratio. Each plate was incubated for 4 hr at 37°C/5% CO2. After 4 hr, plates were centrifuged at 250 x g and cells were fixed with 1% Paraformaldehyde (PFA) and analyzed via flow cytometry. As a control, labeled non-transduced HEK293T cells were also used as targets for ADCC activity. Thirty thousand non-gated events were acquired within 6 hr of the start of the ADCC assay using an LSRII cytometer (Becton Dickinson). The CFSE emission channel was read in B515 using a neutral density filter and Plum emission was read in R660. Following acquisition, analysis was performed using FlowJo software (Tree Star). Percent killing was obtained by quantifying dead cells (Plum+,CFSE-) out of the total Plum positive population. For mAbs, ADCC killing was measured by subtracting percent killing of non-transduced cells from percent killing of transduced cells.

Antibody variants

UCA sequences of the isolated antibodies were determined with reference to the IMGT database (http://www.imgt.org/). Antibody variants in which single or multiple mutations were reverted to the germline sequence were produced by gene synthesis (Genscript) and used to produce a large set of mAb114 and mAb100 antibody variants.

Binding of antibody variants to transfected cells

mAb114 and mAb100 antibody variants were used to stain MDCK-SIAT1 cell lines transduced to express EBOV GP as a stable membrane protein (Makona variant). Binding of antibodies was analysed using a Becton Dickinson FACS Canto2 (BD Biosciences) with FlowJo software (TreeStar). The relative affinities of antibody binding to surface GP were determined by interpolating the concentration of antibody required to achieve 50% maximal binding (EC50) from the plotted binding curves using the mean-fluorescence intensity (MFI) fitted with a 4-parameter nonlinear regression with a variable slope.

Inhibition of binding assay on GP-expressing cells

mAb100 and mAb114 were biotinylated using the EZ-Link NHS-PEO solid phase biotinylation kit (Pierce). Labeled antibodies were tested for binding to GP-expressing MDCK-SIAT-1 cells to determine the optimal concentration of each antibody to achieve 70–80% maximal binding. The biotin-labelled antibodies were then used as probes to assess, by flow cytometry, whether their binding (measured using fluorophore-conjugated

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streptavidin) was inhibited by pre-incubation of GP cells with homologous or heterologous unlabelled antibodies.

Production of purified GP

Expi293 (Invitrogen) cells were transfected with GP ΔMucΔTM-GCN4 HisSA and pCMV-Sport Furin (7:3 ratio) using 293Fection (Invitrogen) at a ratio of 2 mL 293Fectin:1mg total DNA. 18-24 hours following transfection, 1/10th volume of AbBooster (ABI Scientific) was added and culture media collected 5 days later. Supernatant was filtered and protein purified as described previously (12).

Biolayer interferometry antibody cross-competition assay

Antibody cross-competition was determined based on biolayer interferometry using a fortéBio Octet HTX instrument. EBOV GP ΔMuc protein was loaded onto HIS biosensors (AR2G, fortéBio) through amine coupling for 600 s. Biosensors were equilibrated for 120 s in 1% BSA in PBS (BSA-PBS) prior to capturing competitor mAbs. GP proteins were diluted to 10 µg/mL; mAbs KZ52, mAb100, mAb114, 13C6, and IgG1 isotype control Ab were diluted to 35 µg/mL in BSA-PBS. Binding of competitor mAbs was assessed for 300 s followed by a brief equilibration for 60 s prior to binding assessment of probing mAbs. Binding of probing mAbs was assessed for 300 s. Percent inhibition (PI) of probing mAbs binding to GP by competitor mAbs was carried out by an equation: PI = 100 − [(probing mAb binding in the presence competitor mAb) ⁄ (probing mAb binding in the absence of competitor mAb)] × 100. All the assays were performed in duplicate and with agitation set to 1,000 rpm at 30°C.

Animal study and safety

Research was conducted under an IACUC-approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facilities where this research was conducted are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adhere to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. Animal study protocols were approved by both the Vaccine Research Center and United States Army Medical Research Institute of Infectious Diseases (USAMRIID) IACUCs. All animals were Indian-origin rhesus macaques (Macaca mulatta), female, approximately 2–5 years of age and were obtained from Covance. Animals were randomly assigned to treatment groups based on sequential selection from a population inventory. USAMRIID investigators were blinded to investigational antibodies but not treatment status. Challenge studies included a single untreated animal (control); the use of historical control (n>50) allows for one untreated control to be used in each challenge experiment Sample sizes of three animals per Bio-Safety Level-4 (BSL4) EBOV challenge group provide 80% power to detect a difference in survival rates assuming 100% survival (3/3 treated survive) vs. 0% survival in negative controls at the 95% confidence level (1-tailed Fisher exact test). Animals were transferred one week prior to challenge to the BSL-4 facility for exposure to a lethal (1000 PFU) i.m. EBOV Kikwit variant challenge. While at USAMRIID the monkeys were fed and checked daily. During the EBOV challenge study, blood was collected from the NHP for hematological, biochemical and virological

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analyses. Prior to blood sampling or treatment, animals were anesthetized with ketamine or telazol. Following the development of clinical signs, animals were checked multiple times daily. Institute scoring criteria were used to determine timing of humane euthanasia under anesthesia.

Antibody administration

In the mAb100/mAb114 cocktail challenge, antibodies were diluted in PBS and administered at 4 mg/kg/dose of mAb100 and 46 mg/kg/dose of mAb114 for a total antibody concentration of 50 mg/kg. For the mAb114 monotherapy challenge experiments, animals received 50 mg/kg/dose of mAb114. Antibodies were administered via intravenous injection in peripheral veins using ≤20 gauge butterfly needles over a period ≥ 15 minutes in a single bolus via syringe pump. Detection of EBOV

RNA was isolated from plasma of EBOV-exposed NHP and quantified by real time qPCR as described previously (25). EDTA plasma was added to TriReagent LS (Sigma), 1 part to 3 parts, in preparation for qRT-PCR. Inactivated samples were extracted and eluted with AVE Buffer (QIAGEN, Valencia, CA) using a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA). All samples were run on an Applied Biosystems 7500 Fast Dx Real-Time PCR instrument (Life Technologies, Grand Island, NY). Reactions were performed with SuperScript II One-Step RT-PCR System (Life Technologies, Grand Island, NY) with additional MgSO4 added to a final concentration of 3.0 mM. All samples were run in triplicate 5 µL each. The average of the triplicates was multiplied by 200 to obtain genomes equivalents per mL, then multiplied by a dilution factor of 4 for the final reported value. The sequence of the primer and probes for the EBOV glycoprotein are described below. The genomic equivalents (ge) were determined using a synthetic RNA standard curve of known concentration. Forward primer: 5′ - TTT TCA ATC CTC AAC CGT AAG GC - 3′; REVERSE PRIMER : 5′ - CAG TCC GGT CCC AGA ATG TG - 3′; PROBE: 6FAM - CAT GTG CCG CCC CAT CGC TGC – TAMRA.

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Fig. S1. Second screening of immortalized memory B-cells from Survivor 1 14 million PBMCs were used to isolate 59,500 IgG memory B cells which were immortalized as in Fig. 1A-D. Shown are ELISA A450 values for undiluted (Und.) and 1:27 dilutions (1/27) for the 21 supernatants that specifically bound to GP (n=1). Amongst the 21 supernatants, only 2 B cell clones (mAb165, mAb166) were rescued for further analysis.

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Fig. S2. Pseudotype virus neutralization by isolated monoclonal antibodies (A) Summary of pseudotyped lentivirus EBOV GP Mayinga variant virus neutralization assays were performed as in Fig. 2B. IC50, IC90, and IC99 were determined using non-linear regression-variable slope (Graph Pad). 95% Confidence Interval (CI) and number of replicates (n) for each mAb. (B) Pseudotype EBOV Makona variant neutralization by mAb100 and mAb114. Lentivirus particles bearing GPs from EBOV Makona variant were incubated with serially diluted mAb100, mAb114 or isotype control. Infection measured as in Fig. 2B, mean ± s.d. (n=1).

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Fig. S3. Inhibition of wild type EBOV by isolated mAbs Native EBOV neutralization by the indicated mAbs at 0.2 µg/mL or media alone was performed under BSL-4 conditions using PRNT as described in the Materials and Methods. Inhibition is calculated relative to virus incubated with media alone (Mock), mean ± s.d. (n=1).

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Fig. S4. mAb100 and mAb114 variants Amino acid sequences of mAb100 (A, B), mAb114 (C, D) and variants descended from a putative unmutated common ancestor (UCA) for heavy and light chains. Shaded regions represent complementary determination regions 1-3. Subsets of somatic mutations in the somatic wild type heavy (sH) or light (sL) chains were reverted to the germline sequence. gH or gL, germline V-gene revertants of sH or sL in which the HCDR or LCDR3 are mature; gH-FR or gL-FR, germline V-gene revertants of sH or sL in which the HCDRs or LCDRs are mature; gH-FR1-2-4, germline V-gene revertants of sH in which the HCDRs and HFR3 are mature; gH-FR3, germline V-gene revertants of sH in which the HCDRs and HFR1, HFR2 and HFR4 are mature.

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Fig. S5. Additional clinical data from passive transfer of mAb114/mAb100

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Fig. S6. Additional hematology data from passive transfer of mAb114/mAb100

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Fig. S7. Additional serum chemistries from passive transfer of mAb114/mAb100

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Fig. S8. Clinical observation scoring from passive transfer of mAb114/mAb100 Data from multiple observations each day post-exposure are presented as the maximal level observed for that day.

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Fig. S9. Additional clinical data from passive transfer of mAb114

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Fig. S10. Additional hematology data from passive transfer of mAb114

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Fig. S11. Additional serum chemistries from passive transfer of mAb114

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Fig. S12. Clinical observation scoring from passive transfer of mAb114 Data from multiple observations each day post-exposure are presented as the maximal level observed for that day.

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Fig. S13. Additional clinical data from delayed mAb114 passive transfer

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Fig. S14. Additional hematology data from delayed mAb114 passive transfer

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Fig. S15. Additional serum chemistries from delayed mAb114 passive transfer

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Fig. S16. Clinical observation scoring from delayed mAb114 passive transfer Data from multiple observations each day post-exposure are presented as the maximal level observed for that day.

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