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Emerging Microbes amp Infections

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Towards a solution to MERS protective humanmonoclonal antibodies targeting differentdomains and functions of the MERS-coronavirusspike glycoprotein

Ivy Widjaja Chunyan Wang Rien van Haperen Javier Gutieacuterrez-AacutelvarezBrenda van Dieren Nisreen MA Okba V Stalin Raj Wentao Li RaulFernandez-Delgado Frank Grosveld Frank J M van Kuppeveld Bart LHaagmans Luis Enjuanes Dubravka Drabek amp Berend-Jan Bosch

To cite this article Ivy Widjaja Chunyan Wang Rien van Haperen Javier Gutieacuterrez-AacutelvarezBrenda van Dieren Nisreen MA Okba V Stalin Raj Wentao Li Raul Fernandez-Delgado FrankGrosveld Frank J M van Kuppeveld Bart L Haagmans Luis Enjuanes Dubravka Drabek ampBerend-Jan Bosch (2019) Towards a solution to MERS protective human monoclonal antibodiestargeting different domains and functions of the MERS-coronavirus spike glycoprotein EmergingMicrobes amp Infections 81 516-530 DOI 1010802222175120191597644

To link to this article httpsdoiorg1010802222175120191597644

copy 2019 The Author(s) Published by InformaUK Limited trading as Taylor amp FrancisGroup on behalf of Shanghai ShangyixunCultural Communication Co Ltd

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Towards a solution to MERS protective human monoclonal antibodiestargeting different domains and functions of the MERS-coronavirus spikeglycoproteinIvy Widjajaa Chunyan Wanga Rien van Haperenbc Javier Gutieacuterrez-Aacutelvarez d Brenda van DierenaNisreen MA Okba e V Stalin Raj e Wentao Lia Raul Fernandez-Delgadod Frank GrosveldbcFrank J M van Kuppevelda Bart L Haagmans e Luis Enjuanesd Dubravka Drabekbc and Berend-Jan Boscha

aVirology Division Department of Infectious Diseases amp Immunology Faculty of Veterinary Medicine Utrecht University UtrechtNetherlands bDepartment of Cell Biology Erasmus MC Rotterdam Netherlands cHarbour Antibodies BV Rotterdam NetherlandsdDepartment of Molecular and Cell Biology National Center for Biotechnology-Spanish National Research Council (CNB-CSIC) MadridSpain eDepartment of Viroscience Erasmus Medical Center Rotterdam Netherlands

ABSTRACTThe Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatalrespiratory disease in humans Efforts to develop antibody-based therapies have focused on neutralizing antibodiesthat target the receptor binding domain of the viral spike protein thereby blocking receptor binding Here wedeveloped a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spikeprotein These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions ofthe MERS-CoV spike protein sialic acid binding receptor binding and membrane fusion Passive immunization withpotently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge Collectivelythese antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targetingdifferent spike protein epitopes and functions

ARTICLE HISTORY Received 12 November 2018 Revised 20 February 2019 Accepted 25 February 2019

KEYWORDS Coronavirus MERS antibodies spike protein

Introduction

Middle-East respiratory syndrome coronavirus (MERS-CoV) is an emerging zoonotic virus that causes severeand often fatal respiratory illness in humans Since itsfirst identification in Saudi Arabia in 2012 the MERS-CoV documented infections in humans steadilyincreased with 2298 cases as of February 2019 with anestimated 35 lethality [1] Dromedary camels are thenatural reservoir of MERS-CoV from which zoonotictransmission can occur A vast majority of dromedarycamels in the Arabian Peninsula appeared to be seropo-sitive for MERS-CoV and MERS-CoV strains found inepidemiologically-linked humans and dromedarycamels are nearly identical [2ndash5] Human-to-humantransmission is inefficient but can occur upon close con-tact such as in household or hospital settings amongpatients and from patients to health-care workers [2]The high rate ofMERS-CoV infection in the dromedarycamel reservoir poses a persistent threat for reintroduc-tion of MERS-CoV into humans

Despite its continuous threat to public health anti-viral therapies or vaccines to treat or prevent MERS-CoV infection are currently lacking The viral spikeson the surface of the enveloped MERS-CoV virionsare the primary antigenic target for the developmentof vaccines and antibody therapies [6] These spikesmediate virus entry into host cells and consist ofthree spike (S) glycoproteins each containing thereceptor binding subunit S1 and the membrane-anchored fusion-mediating subunit S2 Single-particlecryo-electron microscopic analyses of trimeric S ecto-domains of several coronaviruses including MERS-CoV [7ndash13] revealed a multi-domain architecture ofthe S1 subunit consisting of four core domains (desig-nated A though D) For MERS-CoV two of these spikedomains engage with host molecules to ensure entryinto target cells The S1B domain of the MERS-CoVspike protein binds the host receptor DPP4 which isessential for cell entry [1415] whereas the N-terminaldomain S1A binds to sialoglycans on mucins and on the

copy 2019 The Author(s) Published by Informa UK Limited trading as Taylor amp Francis Group on behalf of Shanghai Shangyixun Cultural Communication Co LtdThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby40) which permits unrestricteduse distribution and reproduction in any medium provided the original work is properly cited

CONTACT Berend-Jan Bosch bjboschuunlPresent address Merus NV Utrecht the NetherlandsPresent address School of Biology Indian Institute of Science Education and Research Thiruvananthapuram (IISER-TVM) Kerala India

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host cell surface which enhances infection ofMERS-CoV on human lung cells [16] Three S2 sub-units form the stem of the spikes which can undergoextensive conformational changes enabling membranefusion The surface of the S2 subunit ndash to which anti-bodies bind ndash shows a higher degree of sequence con-servation than the more variable S1 subunit [7]

Several potently neutralizing human monoclonalantibodies against MERS-CoV have been developedusing various approaches including single cell culturingmethods of memory B cells isolated from MERS-CoVpatients [17] hybridoma fusion of B-cells from immu-nized transgenic mice encoding human variable immu-noglobulin domains [18ndash20] and phage or yeast displayscreening of Fab fragments of non-immune humanantibody libraries [21ndash23] Some of these antibodiesshowed protection againstMERS-CoV challenge in ani-mal models [17 19ndash22 24ndash27] All of these antibodieswere selected based on their in vitro neutralizingcapacity and most of them targeted the MERS-CoVS1B receptor binding domain (RBD) Structural andfunctional studies indicate that the epitopes of thoseS1B-specific antibodies overlap with the DPP4 bindingsite explaining their potent neutralizing capacity [1719 20 28 29] So far only twomurine monoclonal anti-bodies have been described that target theMERS-CoV Soutside the RBD [29] Using a combination of DNA andprotein vaccination of mice Wang et al described S1-specific non-RBD and S2-specific neutralizing murineantibodies that showed potency for protection againstMERS-CoV in vivo [30] Although RBD-specific anti-bodies are undoubtly very potent in neutralizingMERS-CoV effective antibody therapies are likely torequire the combination of neutralizing and non-neu-tralizing antibodies targeting multiple epitopes andexhibiting diverse mechanisms of actions includingFc-mediated antibody effector functions [31ndash33] Inaddition such antibodies should preferably be humanto avoid an immune reaction against anti-MERS anti-bodies of a different species Identification of such pro-tective antibodies their targets and mechanisms ofactivity is important for developing antibody-basedtherapies against MERS-CoV

In this study we developed a set of human mono-clonal antibodies against MERS-CoV with diverse mech-anisms of action that show protective efficacy in vivo Weused the transgenic H2L2 mice encoding the humanimmunoglobulin variable regions to generate antibodiestargeting the MERS-CoV spike protein (MERS-S)From a large panel of MERS-S-specific H2L2 antibodieseight fully human monoclonal antibodies were generatedthat bind non-overlapping epitopes on MERS-S withhigh affinity and interfere with the three known func-tions of the viral protein sialic acid binding receptorbinding and membrane fusion These antibodies wereshown to protect mice from a lethal MERS-CoV infec-tion at low dosage These studies extend our knowledge

on the protective value of monoclonal antibodies target-ing ndash non-RBD ndash domains of MERS-S

Results

Isolation of H2L2 antibodies targeting differentdomains of the MERS-CoV spike protein

To further our understanding of antibodies that con-tribute to humoral immunity we generated andcharacterized a panel of human antibodies targetingdifferent functional domains of the MERS-CoV spikeprotein To develop human monoclonal antibodies(mAbs) we employed H2L2 transgenic mice carryingimmunoglobulin transgenes for human variable heavyand light chains and rodent constant regions (httpwwwharbourbiomedcom) In one immunizationexperiment H2L2 mice were immunized with purifiedMERS-S1 subunit (Figure 1(A) SI Appendix FigS1A) A second immunization experiment was doneto generate antibodies targeting the more conservedMERS-S2 subunit using the MERS-S ectodomainand MERS-S2 ectodomain as antigens following asequential immunization strategy (Figure 1(A) SIAppendix Figure S1A) Hybridoma cell lines weregenerated from spleen and lymph node derived B-cells from both immunization experiments and anti-body-containing hybridoma supernatants werescreened for MERS-S reactivity by ELISA The firstimmunization experiment provided 113 hybridomasupernatants (out of 4553) that reacted to MERS-S1To understand the immunogenicity landscape of theMERS-S1 subunit we mapped epitopes of these anti-bodies to S1 domains for which individual S1 domainswere expressed and used as antigens in ELISA (SIAppendix Figure S1B) The majority of S1-reactiveantibodies bound either to domain S1B (56) or S1A

(38) (Figure 1(B) SI Appendix Fig S2A) Four per-cent of the S1-reactive antibodies bound to eitherdomain S1C or S1D whereas some antibodies (2)did not bind to any of the S1 domains indicative ofbinding to interdomain epitopes (Figure 1(B) SIAppendix Figure S2A) The second immunizationwith MERS-S ectodomain and MERS-S2 ectodomainresulted in 50 hybridoma supernatants (out of 1158hybridomas) that were positive for binding in aMERS-S ectodomain ELISA Most of the MERS-Sreactive antibodies bound to S1 (84) and eight(16) were found to bind S2 (Figure 1(B)) Virus neu-tralization activity of hybridoma supernatants wasscreened using luciferase-encoding MERS-S pseudo-typed vesicular stomatitis virus Forty of the 113MERS-S1-specific antibodies from MERS-S1-immu-nized mice neutralized MERS-CoV infection (Figure1(B) SI Appendix Figure S2A) Notably all of theneutralizing antibody epitopes mapped to the receptorbinding domain S1B Two out of the eight identified

Emerging Microbes amp Infections 517

MERS-S2-specific antibodies were found to neutralizeMERS-S pseudovirus (Figure 1(B) SI AppendixFigure S2C) Screening the neutralizing antibodiesfor antigen binding competition identified six epitopegroups which was used for the selection of lead anti-bodies (SI Appendix Figure S2B)

Binding of lead human mAbs to the MERS-CoVspike protein

From the set of MERS-CoV-S specific H2L2 anti-bodies a panel of eight monoclonal antibodies(mAbs 110f3 77g6 16f9 12g5 18e5 46e10

Figure 1 Generation and characterization of monoclonal antibodies targeting the MERS-CoV spike protein (A) The MERS-CoV spike(S) protein and recombinant soluble MERS-CoV S antigens used for immunization of H2L2 transgenic mice to generate humanmonoclonal antibodies (mAbs) Upper panel Schematic representation of the MERS-CoV S protein indicated are S subunits (S1and S2) S1 domains (A through D) and known biological functions Middle panel Schematic representation of recombinant solubleMERS-CoV S antigens including the MERS-CoV S S1 subunit (MERS-S1) the ectodomain of its S2 subunit (MERS-S2ecto) or the entireMERS-S ectodomain (MERS-Secto) the latter containing a mutation at the furin cleavage site at the S1S2 junction and a C-terminallyfused T4 foldon trimerization tag to increase trimer stability (T4) Positions of signal peptides (SP) and StrepTag affinity tags (ST) areindicated Lower panel Immunization schedule H2L2 mice To generate monoclonal antibodies (mAbs) targeting the MERS-CoV Sprotein groups of H2L2 mice (six micegroup) were immunized with either MERS-S1 (6times) or sequentially immunized with MERS-Secto (3times) MERS-S2ecto (2times) and MERS-Secto (1x) Booster immunizations were done with two-week intervals and B-cells were har-vested from spleen and lymph nodes four days after the last immunization (B) Identified MERS-S1-reactive mAbs of hybridomasderived from B-cells of S1-immunized H2L2 mice were characterized for epitope location and virus neutralization using MERS-Spseudotyped VSV Pie charts show mAb frequencies relative to the total (indicated in the centre circle) Domain-level epitope map-ping was performed for MERS-S1-reactive mAbs and relative frequencies of mAbs binding to given S1 domains (S1A S1B or S1CD)are indicated The percentage of mAbs that was reactive to S1 but not to the S1A S1B or S1CD domains (S1other) is also shown Virusneutralization by S1-reactive mAbs was analysed using the luciferase-encoding MERS-CoV S pseudotyped VSV particles (C) Ident-ified MERS-Secto-reactive mAbs of hybridomas generated from SectoS2ecto immunized H2L2 mice were characterized for epitopelocation and virus neutralization as in (B)

518 I Widjaja et al

16c7 and 35g6) were selected with epitopes distribu-ted throughout different domains of the MERS-CoVspike protein for further detailed biophysical andfunctional characterization Selection of H2L2 mAbswas based on their unique variable heavy and lightchain sequences and on their capacity to neutralizeMERS-CoV relative to other mAbs within an epitopegroup (SI Appendix Figure S2C) We could notdetect MERS-S1A-specific neutralizing antibodies butwe nevertheless selected one non-neutralizing mAb(110f3) that recognizes this sialic acid-bindingdomain Fully human mAbs were generated by clon-ing the genes of the variable region of light and heavychain into human IgG1 expression vectors LikewiseIgG1 expression vectors were generated for theexpression of a previously reported potent MERS-CoV-neutralizing antibody (anti-MERS control) as abenchmark antibody [34] In addition we used anirrelevant antibody recognizing the Strep-tag affinitytag (isotype control) All reformatted antibodies

were expressed in human HEK-293 T cells and pur-ified using Protein-A affinity purification (SI Appen-dix Figure S3)

Epitope mapping of purified human mAbs to thedifferent domains of MERS-CoV S was done byELISA using soluble MERS-CoV Secto S1 S1A S1B

or S2ecto as antigens (Figure 2(A) SI AppendixFigure S1B) Domain-level epitope mappingconfirmed that mAb 110f3 bound to the sialic acidbinding domain S1A mAbs 77g6 16f9 12g5 18e5and 46e10 targeted the receptor binding domainS1B whereas mAbs 16c7 and 35g6 bound the ectodo-main of the membrane fusion subunit S2 (Figure 2(A)) Next competition for binding of the lead anti-bodies to the MERS-S ectodomain was tested usingbio-layer interferometry (Figure 2(B)) The bindingcompetition data indicated the existence of six epi-tope groups suggesting the presence of six distinctepitopes targeted by the eight lead mAbs on theMERS-CoV S protein group I (77g6 16f9 and

Figure 2 Human anti-MERS-S mAbs targeting six epitope groups distributed over multiple domains of the MERS-CoV spike protein(A) ELISA reactivity of the human anti-MERS-S mAbs to the indicated MERS-CoV spike glycoprotein domains (B) Binding compe-tition of anti-MERS-S mAbs analysed by bio-layer interferometry (BLI) Immobilized MERS-Secto antigen was saturated in bindingwith a given anti-MERS-S mAb (step 1) and then exposed to binding by a second mAb (step 2) Additional binding of the secondantibody indicates the presence of an unoccupied epitope whereas lack of binding indicates epitope blocking by the first antibodyAs a control the first mAb was also included in the second step to check for self-competition (C) Schematic distribution of epitopegroups of anti-MERS-S mAbs over the different MERS-S domains

Emerging Microbes amp Infections 519

12g5) group II (18e5) and group III (46e10) on theS1B domain group IV (110f3) on the S1A domainand groups V (16c7) and group VI (35g6) on theS2 ectodomain Antibodies of different groups com-peted minimally with each other indicating thattheir epitopes were largely distinct (Figure 2(B) and(C)) The binding kinetics of the eight human mono-clonal antibodies was determined by bio-layer inter-ferometry Strep-tagged MERS-S ectodomain wascaptured on the protein-A sensor via an anti-Streptagantibody and kinetic binding parameters of antibodieswere determined at 25degC and pH 74 All antibodiesdisplayed high affinity binding for the MERS-S ecto-domain with equilibrium dissociation constants (KD)in the nano- to the picomolar range (0081 to478 nM) (Table 1 SI Appendix Figure S4) Thebinding affinity of the MERS-CoV receptor DPP4 toMERS-S was measured in the same set up and waslower compared to the binding affinity of the mAbsthat target the receptor binding domain S1B (Table1 SI Appendix Figure S4)

Anti-MERS-S mAbs bind cell surface displayedMERS-CoV spike protein

To assess whether the lead mAbs can bind full-lengthMERS-CoV S expressed on the cell surface we trans-fected Huh-7 cells with plasmid encoding MERS-CoV S The spike gene was C-terminally extendedwith GFP to monitor MERS-S expression andmutated at the furin cleavage site to stabilize thespike protein in its native prefusion state and to pre-vent MERS-S-mediated cellndashcell fusion Binding oflead mAbs to cell-surface expressed MERS-S wasanalysed by flow cytometry and immunofluorescenceAll anti-MERS-S mAbs bound to non-permeabilizedMERS-S transfected (GFP-positive) Huh-7 cells inboth assays indicative for binding to cell surfacedisplayed MERS-CoV S (SI Appendix Figures S5and S6)

Neutralization activity of anti-MERS-S mAbs

The ability of human lead mAbs to neutralize MERS-CoV infection in vitro was tested on Vero cells with

luciferase-encoding MERS-S pseudotyped virus andwith authentic MERS-CoV using a plaque reductionneutralization test (PRNT) Levels of virus neutraliz-ation varied among the individual antibodies (Figure 3(A) and Table 2) The 77g6 16f9 and 12g5 mAbs alltargeting epitope group I on MERS-S1B showed themost potent neutralizing activity and displayed pico-molar half-maximal inhibitory concentrations againstMERS-S pseudotyped virus (IC50 = 7ndash30 pM) andauthentic MERS-CoV (PRNT50 = 53ndash200 pM) whichwas equivalent to or lower than our benchmarkMERS-S neutralizing monoclonal antibody that targetsthe same domain (Table 2) MERS-S1B mAbs from epi-tope group II (mAb 18e5) and III (mAb 46e10)

Table 1 Binding kinetics of mAbsMERS-Secto or DPP4MERS-Secto from bio-layer interferometry measurementsmAb KD (M) kon (M

minus1 secminus1) koff (secminus1)

77g6 3612 times 10minus10 3196 times 104 1154 times 10minus5

16f9 5298 times 10minus10 1175 times 104 6227 times 10minus6

12g5 8086 times 10minus11 7587 times 104 6134 times 10minus6

18e5 3178 times 10minus10 2442 times 104 4232 times 10minus6

46e10 3592 times 10minus10 3569 times 104 1134 times 10minus5

110f3 4784 times 10minus9 9550 times 103 4569 times 10minus5

16c7 5007 times 10minus10 5927 times 104 2968 times 10minus5

35g6 2246 times 10minus9 2107 times 104 4733 times 10minus5

α-MERS-CTRL 1289 times 10minus10 4576 times 104 5900 times 10minus6

DPP4 3416 times 10minus9 1423 times 104 4859 times 10minus5

Figure 3 Virus neutralization and receptor binding inhibitionby anti-MERS-S mAbs (A) Analysis of MERS-CoV neutralizingactivity by anti-MERS-S mAbs using MERS-S pseudotyped luci-ferase-encoding VSV A previously described RBD-specificMERS-CoV-neutralizing human monoclonal antibody (anti-MERS-CTRL) and irrelevant isotype monoclonal antibody (Iso-CTRL) were included as positive and negative control respect-ively Luciferase-expressing VSV particles pseudotyped with theMERS-CoV S protein or authentic MERS-CoV were incubatedwith antibodies at the indicated concentrations and the mixwas used to transduce Huh-7 cells At 24 h postinfection luci-ferase expression was measured and neutralization () was cal-culated as the ratio of luciferase signal relative to relative tonon-antibody-treated controls Data represent the mean (plusmnstandard deviation SD) of three independent experiments(B) Receptor binding inhibition by anti-MERS-S mAbs deter-mined by an ELISA-based assay Recombinant soluble MERS-Secto was preincubated with serially diluted anti-MERS-SmAbs and added to ELISA plates coated with soluble theMERS-CoV S ectodomain Binding of MERS-Secto to DPP4 wasmeasured using HRP-conjugated antibody recognizing theStreptag affinity tag on DPP4 Data represent the mean (plusmn stan-dard deviation SD) of three independent experiments

520 I Widjaja et al

neutralized MERS-S pseudovirus infection at nanomo-lar concentrations (IC50 = 10 and 032 nM resp) andexhibited no detectable or moderate neutralizingactivity against authentic MERS-CoV (PRNT50 = gt667 and 667 nM resp) The MERS-S1A-specificmAb 110f3 lacked MERS-CoV neutralization activityin both virus neutralization assays The anti-MERS-S2 mAbs 16c7 and 35g6 were able to neutralizeMERS-S pseudovirus (IC50 = 245 and 166 nMresp) albeit at higher concentrations than the mostpotent neutralizing MERS-S1B mAbs (about 100-foldhigher) The isotype control did not show any neutral-ization in both assays Collectively our data demon-strate that antibodies targeting the receptor bindingdomain S1B of the MERS-CoV spike protein displaythe highest potential for neutralization of MERS-CoVinfection in vitro

Anti-MERS-S1B mAbs neutralize MERS-CoV byblocking receptor binding

To understand the mechanism of action of lead mAbswe set up assays to assess antibody interference withthe diverse functions of the MERS-CoV S domainsTo assess whether antibodies can compete with viralbinding to the host receptor DPP4 we developed anELISA-based receptor binding inhibition assay inwhich binding of MERS-S ectodomain to DPP4-coatedELISA plates is quantified and interference with recep-tor binding by antibodies is measured as a reduction inbinding signal In the absence of antibodies the MERS-S ectodomain showed stable binding to DPP4 (Figure 3(B)) Whereas anti-MERS-S1B mAb 18e5 showed weakinterference with binding of MERS-S ectodomain toDPP4 all other MERS-S1B-specific mAbs (mAbs77g6 16f9 12g5 46e10 and anti-MERS-CTRL)potently inhibited binding of MERS-S ectodomain toDPP4 in a concentration-dependent manner (Figure 3(B)) The data indicate that these antibodies partlyoverlap with or bind sufficiently close to the receptor-binding site on S1B to compete with receptor bindingNone of the antibodies that bind outside the RBDdomain (MERS-S1A and -S2) could block receptor

binding The potency of the S1B-specific mAbs to inhi-bit receptor binding corresponds with the ability ofthese antibodies to neutralize virus infection(Table 2) indicating that the inhibition of virusndashrecep-tor interaction by these antibodies is their main mech-anism of neutralization in vitro

Anti-MERS-S1A mAb 110f3 blocks binding ofMERS-S1A to sialoglycoconjugates

Recently we demonstrated that the MERS-S1A domainfacilitates viral binding to cell-surface sialoglycoconju-gates which can serve as a cell attachment factor forMERS-CoV [16] We assessed whether the MERS-S1A-targeting mAb 110f3 can interfere with bindingof MERS-S1A to sialoglycoconjugates on the surface oferythrocytes using the hemagglutination inhibitionassay To this end we used lumazine synthase (LS)nanoparticles multivalently displaying MERS-S1A

(S1A-LS) which were earlier employed to demonstratethe sialic-acid dependent hemagglutination by theMERS-S1A domain [16] Hemagglutination wasobserved when S1A-LS was mixed with erythrocytes(Figure 4(A)) S1A-LS mediated hemaglutination wasabrogated upon addition of the MERS-S1A mAb110f3 but not upon addition of the isotype controlNext we assessed whether interference of 110f3 withbinding to sialylated receptors could inhibit Sia-depen-dentMERS-CoV infection Binding to sialoglycansmayaidMERS-CoV entry into DPP4-positive cells depend-ing on the cell type Infection of Vero cells does notdepend on cell surface sialic acids concurrent with alow abundancy of the MERS-CoV S1A glycotopes onthose cells [16] Correspondingly infection of Verocells with MERS-S pseudovirus could not be inhibitedby 110f3 (Figure 4(B)) By contrast infection ofhuman lung Calu-3 cells was shown to rely on cell sur-face sialic acids which correlated with the abundance ofMERS-CoV S1A receptors [16] Contrary to Vero cellsinfection of Calu-3 cells could be inhibited by 110f3(Figure 4(B)) suggesting that antibody binding toMERS-S1A can neutralize MERS-CoV infection viainhibition of virus binding to cell surface sialoglycans

Table 2 Virus neutralization and receptor binding inhibition by anti-MERS-S mAbs

mAb MERS-S target

IC50 MERS-S VSVpp PRNT50 MERS-CoV RBI50

μgml nM μgml nM μgml nM

77g6 S1B 0001 0007 0008 0053 0007 004716f9 0006 004 003 0200 0013 008712g5 0002 0013 003 0200 0014 009318e5 1500 10 gt1 gt667 gt10 gt66746e10 0048 0320 1 6667 0137 0913110f3 S1A gt10 gt 667 gt1 gt667 gt10 gt66716c7 S2A 0367 2447 1 667 gt10 gt66735g6 2488 166 gt1 gt66 gt10 gt667α-MERS-CTRL S1B 0005 0033 003 0200 0022 0147Iso-CTRL ndash gt10 gt 667 gt1 gt667 ndash ndash

IC50 mAb concentration resulting in half-maximal infection of MERS-S VSV pseudovirus (MERS-S VSVpp) on Vero cells PRNT50 highest mAb dilution resulting in gt 50 reduction in the number of MERS-CoV infected Vero cells RBI50 mAb concentration of that gives half-maximal receptor binding

Emerging Microbes amp Infections 521

Anti-MERS-S2 mAbs interfere with MERS-Smediated membrane fusion

The coronavirus S2 subunit encompasses the machin-ery for fusion of viral and host cell membranes a pro-cess that is driven by extensive refolding of themetastable prefusion S2 into a stable postfusion state[35] We hypothesized that antibodies targeting theMERS-S2 subunit might neutralize MERS-CoV infec-tion by inhibiting this fusion process To test this wedeveloped a MERS-CoV-S driven cellndashcell fusionassay using a modified MERS-CoV spike protein Tomonitor expression of MERS-CoV S in cells weextended the viral fusion protein C-terminally withGFP In addition we mutated the furin cleavage siteat the S1S2 junction to increase the dependency ofMERS-CoV S fusion activation on exogenous addition

of trypsin Expression of this MERS-S variant upontransfection of DPP4-expressing Huh-7 cells could bereadily observed by the GFP signal (Figure 4(C))Upon addition of trypsin large GFP-fluorescent syncy-tia were detected indicating MERS-S mediated cellndashcellfusion (Figure 4(C)) As expected the addition of anti-MERS-S1B (77g6) blocked the formation of syncytiasince cellndashcell fusion is dependent on receptor inter-action No effect on syncytium formation was seenfor the MERS-S1A mAb 110f3 In contrast theMERS-S2-specific mAbs 16c7 and 35g6 both blockedsyncytium formation Since both neutralizing anti-bodies did not interfere with receptor binding (Figure 3(B)) we surmise that binding of these S2-specific anti-bodies inhibits infection by preventing conformationalchanges in the S2 subunit of the MERS-CoV spikeprotein that are required for fusion

Figure 4 Anti-MERS-S mAbs targeting MERS-S1A and -S2 domains block domain-specific functions (A) The anti-MERS-S1A mAb110f3 interferes with MERS-S1A-mediated sialic acid binding determined by a hemagglutination inhibition assay [16] The sia-lic-acid binding domain S1A of MERS-S was fused to lumazine synthase (LS) protein that can self-assemble to form 60-meric nano-particle (S1A-LS) which enables multivalent high affinity binding of the MERS-S1A domain to sialic acid ligands such as onerythocytes Human red blood cells were mixed with S1A-LS in the absence or presence of 2-fold dilutions of the MERS-S1A-specificmAb 110f3 Isotype control antibody was included as a negative control Hemagglutination was scored after 2 h of incubation at 4degC The hemagglutination inhibition assay was performed three times a representative experiment is shown (B) Neutralization ofMERS-S pseudotyped VSV by anti-MERS-S mAb 110f3 on Vero and Calu-3 cells Data represent the mean (plusmn standard deviation SD)of three independent experiments (C) The anti-MERS-S2 mAbs 16c7 and 35g6 block MERS-S-mediated cellndashcell fusion Huh-7 cellswere transfected with plasmid expressing MERS-CoV S C-terminally fused to GFP Two days after transfection cells were treatedwith trypsin to activate membrane the fusion function of the MERS-CoV S protein and incubated in the presence or absence of anti-MERS-S2 mAbs 16c7 and 35g6 or the anti-MERS-S1B mAb 77g6 and anti-MERS-S1A 110f3 all at 10 μgml Formation of MERS-Smediated cellndashcell fusion was visualized by fluorescence microscopy Merged images of MERS-S-GFP expressing cells (green) andDAPI-stained cell nuclei (blue) are shown Experiment was repeated two times and representative images are shown

522 I Widjaja et al

Protective activity of mAbs from lethal MERS-CoV challenge

To assess the prophylactic efficacy of our lead mAbsagainst MERS-CoV infection in vivo we used trans-genic K18-hDPP4 mice expressing human DPP4 [36]Six hours prior to MERS-CoV infection mice (5micegroup) were injected intraperitoneally with a50 μg dose of each mAb (equivalent to 18 mg mAbper kg body weight) The percentage of survival andweight change following challenge was monitored for12 days MERS-CoV infection was consistently lethalas all mice that received the monoclonal isotype controlshowed significant weight loss and had succumbed tothe infection between 7 and 8 days post-challenge(Figure 5(A) and (B)) Contrarily all MERS-S1B bind-ing mAbs showed high levels of protection againstlethal MERS-CoV challenge (80ndash100 Figure 5)Anti-MERS-S1B mAbs 77g6 12g5 and the benchmarkanti-MERS control mAb uniformly protected animalsfrom death whereas the MERS-S1B mAbs 16f9

18e5 and 46e10 protected 4 out of 5 animals (80)in this model The MERS-S1A binding mAb 110f3afforded partial protection frommortality (40) Nota-bly the anti-MERS-S2 mAbs 16c7 and 35g6 protectedall five animals from lethal infection Relative to theisotype control treated mice mice treated withMERS-S specific antibodies showed reduced weightloss (Figure 5(B)) These results highlight that anti-bodies targeting non-RBD domains (ic S1A and S2)of the MERS-CoV spike protein can contribute tohumoral immunity against MERS-CoV infection

Discussion

The recurring spillover infections of MERS-CoV inhumans from its dromedary camel reservoir the highmortality and person-to-person transmission pose asignificant threat to public health As there are cur-rently no licensed vaccines or treatments for combat-ting MERS-CoV infections the development of

Figure 5 Human anti-MERS-S mAbs protect mice against lethal MERS-CoV challenge (AndashB) Fifty microgram of antibody (equivalentto 18 mg mAbkg body weight) was infused intraperitoneally in K18-hDPP4-transgenic mice 6 h before challenge with 5 times 103 pfumice of MERS-CoV Five mice per group were used in the experiment Survival rates (A) and weight loss (B) (expressed as a per-centage of the initial weight) was monitored daily until 12 days post-inoculation

Emerging Microbes amp Infections 523

effective counter measures is a critical need andrecently prioritized by the World Health Organizationin the research and development Blueprint for action toprevent epidemics [37] Antibody therapies targetingcritical entry functions of viral glycoproteins areincreasingly recognized as promising antiviral strat-egies to protect humans from lethal disease [38] ForMERS-CoV passive immunization studies with neu-tralizing antibodies in small animals support the ideathat antibody therapies may hold promise to protecthumans from lethal MERS-CoV mediated diseaseMost of these protective antibodies neutralize virusinfection by occupying the receptor binding domain(RBD) and compete with the host receptor Here wedescribe the protective activity of individual humanantibodies targeting RBD as well as non-RBD spikedomains and their interference with the three knownfunctions of the spike glycoprotein Collectively thearsenal of protective antibodies that bind to and func-tionally inhibit the activity of multiple spike proteindomains can reveal new ways to gain humoral protec-tion against the emerging MERS coronavirus

Potent MERS-CoV neutralizing antibodies as ourstudy and that of others have shown commonly targetthe S1B receptor binding domain [17ndash23] The anti-S1B

antibodies physically prevent binding to the host receptorDPP4 attributed to their higher binding affinity forMERS-S thereby potently neutralizing MERS-CoVinfection The five S1B-specific antibodies identified inthis study were found to target three non-overlappingepitope groups on the MERS-CoV S1B domain consist-ent with the three epitope groups at the spike-DPP4receptor interface reported by Tang et al [22] TheseS1B antibodies displayed varying neutralization potencywith 100-1000-fold differences in IC50 values with anti-bodies targeting epitope group I showing ultrapotentneutralizing activity at sub-nanomolar levels Irrespectiveof the neutralizing potency all S1B antibodies displayedsignificant protective activity (80ndash100 survival rates)in mice from lethal MERS-CoV challenge

Apart from engaging the host receptor DPP4 weearlier demonstrated that the MERS-CoV spike proteinbinds sialoglycoconjugates (Siarsquos) on mucins and thehost cell surface via an independently functionaldomain the N-terminal domain S1A This Sia-bindingactivity serves as an attachment factor for MERS-CoVinfection in a cell-type dependent manner [16] Wenow show that binding of the isolated mAb 110f3 tothis MERS-S1A domain abrogates Sia-binding andcan inhibit the Sia-dependent infection of humanlung Calu-3 cells by MERS-CoV [16] Moreover pas-sive immunization of mice with this MERS-S1A-specific mAb resulted in 40 protection frommortalityfollowing MERS-CoV infection These findings under-line the role of sialic acid binding for MERS-CoV infec-tion in vivo and demonstrate the importance of anti-MERS-S1A antibodies in protection

From all MERS-CoV mAbs described only twomAbs ndash G2 and G4 ndash targeted epitopes outside theRBD [29] G2 binds an epitope in S1 outside theRBD whereas G4 targets a variable loop in S2 (Wang2015) Both murine antibodies were shown to protectmice from a lethal infection of MERS-CoV in vivoBoth antibodies were neutralizing though the interfer-ence of these antibodies with spike functions was notdelineated Using a tailored immunization scheme toboost antibody responses to the MERS-CoV fusion-mediating S2 subunit we were able to recover MERS-S2-specific mAbs two of which demonstrated neutra-lizing activity and fully protected mice from a lethalMERS-CoV challenge These two S2 antibodies didnot interfere with receptor binding yet abrogatedcellndashcell fusion implying their interference withspike-mediated membrane fusion by preventing con-formational changes required for fusion The highlyprotective S2 mAbs only displayed modest in vitro neu-tralizing activity indicating that strong neutralizingactivity is not a prerequisite for protection

Apart from interfering with functions of viral pro-teins antibodies are able to employ a broad range ofantiviral activities through the innate immune systemBinding of antibodies to glycoproteins on the surface ofinfected cells or on viruses may decorate them fordestruction through Fc-mediated antiviral activitiesincluding antibody-dependent cellular cytotoxicity(ADCC) antibody-dependent cell-mediated phagocy-tosis (ADCP) or complement-dependent cytotoxicity(CDC) [39] These functions may be irrespective ofthe neutralization capacity of antibodies as long asthey can bind surface antigens on the infected cellCongruent with this prerequisite all of the anti-MERS-S antibodies were able to bind cell surface dis-played spikes In addition our human antibodieswere of the IgG1 isotype which was shown to be themost potent human IgG isotype in mice showingefficient binding to all activating mouse Fcγ receptorsand induction of ADCCADCP with mouse naturalkiller cells and mouse macrophages [40] Furtherresearch is needed to define the contribution of Fc-functions to antibody-mediated protection fromMERS-CoV infection

We identified human antibodies targeting six dis-tinct epitope classes across different domains of theMERS-CoV spike protein each of which showing pro-tective activity in mice against lethal MERS-CoV chal-lenge This discovery holds promise for thedevelopment of antibody therapeutics against MERS-CoV infection Passive immunization with a combi-nation of antibodies targeting different domains andfunctions of the viral glycoprotein might be more pro-tective against virus infection than single epitope mAbtherapy as was shown for other enveloped RNAviruses [31 32 41ndash43] Which combination of anti-bodies provides synergistic protective activity against

524 I Widjaja et al

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

16c7 and 35g6) were selected with epitopes distribu-ted throughout different domains of the MERS-CoVspike protein for further detailed biophysical andfunctional characterization Selection of H2L2 mAbswas based on their unique variable heavy and lightchain sequences and on their capacity to neutralizeMERS-CoV relative to other mAbs within an epitopegroup (SI Appendix Figure S2C) We could notdetect MERS-S1A-specific neutralizing antibodies butwe nevertheless selected one non-neutralizing mAb(110f3) that recognizes this sialic acid-bindingdomain Fully human mAbs were generated by clon-ing the genes of the variable region of light and heavychain into human IgG1 expression vectors LikewiseIgG1 expression vectors were generated for theexpression of a previously reported potent MERS-CoV-neutralizing antibody (anti-MERS control) as abenchmark antibody [34] In addition we used anirrelevant antibody recognizing the Strep-tag affinitytag (isotype control) All reformatted antibodies

were expressed in human HEK-293 T cells and pur-ified using Protein-A affinity purification (SI Appen-dix Figure S3)

Epitope mapping of purified human mAbs to thedifferent domains of MERS-CoV S was done byELISA using soluble MERS-CoV Secto S1 S1A S1B

or S2ecto as antigens (Figure 2(A) SI AppendixFigure S1B) Domain-level epitope mappingconfirmed that mAb 110f3 bound to the sialic acidbinding domain S1A mAbs 77g6 16f9 12g5 18e5and 46e10 targeted the receptor binding domainS1B whereas mAbs 16c7 and 35g6 bound the ectodo-main of the membrane fusion subunit S2 (Figure 2(A)) Next competition for binding of the lead anti-bodies to the MERS-S ectodomain was tested usingbio-layer interferometry (Figure 2(B)) The bindingcompetition data indicated the existence of six epi-tope groups suggesting the presence of six distinctepitopes targeted by the eight lead mAbs on theMERS-CoV S protein group I (77g6 16f9 and

Figure 2 Human anti-MERS-S mAbs targeting six epitope groups distributed over multiple domains of the MERS-CoV spike protein(A) ELISA reactivity of the human anti-MERS-S mAbs to the indicated MERS-CoV spike glycoprotein domains (B) Binding compe-tition of anti-MERS-S mAbs analysed by bio-layer interferometry (BLI) Immobilized MERS-Secto antigen was saturated in bindingwith a given anti-MERS-S mAb (step 1) and then exposed to binding by a second mAb (step 2) Additional binding of the secondantibody indicates the presence of an unoccupied epitope whereas lack of binding indicates epitope blocking by the first antibodyAs a control the first mAb was also included in the second step to check for self-competition (C) Schematic distribution of epitopegroups of anti-MERS-S mAbs over the different MERS-S domains

Emerging Microbes amp Infections 519

12g5) group II (18e5) and group III (46e10) on theS1B domain group IV (110f3) on the S1A domainand groups V (16c7) and group VI (35g6) on theS2 ectodomain Antibodies of different groups com-peted minimally with each other indicating thattheir epitopes were largely distinct (Figure 2(B) and(C)) The binding kinetics of the eight human mono-clonal antibodies was determined by bio-layer inter-ferometry Strep-tagged MERS-S ectodomain wascaptured on the protein-A sensor via an anti-Streptagantibody and kinetic binding parameters of antibodieswere determined at 25degC and pH 74 All antibodiesdisplayed high affinity binding for the MERS-S ecto-domain with equilibrium dissociation constants (KD)in the nano- to the picomolar range (0081 to478 nM) (Table 1 SI Appendix Figure S4) Thebinding affinity of the MERS-CoV receptor DPP4 toMERS-S was measured in the same set up and waslower compared to the binding affinity of the mAbsthat target the receptor binding domain S1B (Table1 SI Appendix Figure S4)

Anti-MERS-S mAbs bind cell surface displayedMERS-CoV spike protein

To assess whether the lead mAbs can bind full-lengthMERS-CoV S expressed on the cell surface we trans-fected Huh-7 cells with plasmid encoding MERS-CoV S The spike gene was C-terminally extendedwith GFP to monitor MERS-S expression andmutated at the furin cleavage site to stabilize thespike protein in its native prefusion state and to pre-vent MERS-S-mediated cellndashcell fusion Binding oflead mAbs to cell-surface expressed MERS-S wasanalysed by flow cytometry and immunofluorescenceAll anti-MERS-S mAbs bound to non-permeabilizedMERS-S transfected (GFP-positive) Huh-7 cells inboth assays indicative for binding to cell surfacedisplayed MERS-CoV S (SI Appendix Figures S5and S6)

Neutralization activity of anti-MERS-S mAbs

The ability of human lead mAbs to neutralize MERS-CoV infection in vitro was tested on Vero cells with

luciferase-encoding MERS-S pseudotyped virus andwith authentic MERS-CoV using a plaque reductionneutralization test (PRNT) Levels of virus neutraliz-ation varied among the individual antibodies (Figure 3(A) and Table 2) The 77g6 16f9 and 12g5 mAbs alltargeting epitope group I on MERS-S1B showed themost potent neutralizing activity and displayed pico-molar half-maximal inhibitory concentrations againstMERS-S pseudotyped virus (IC50 = 7ndash30 pM) andauthentic MERS-CoV (PRNT50 = 53ndash200 pM) whichwas equivalent to or lower than our benchmarkMERS-S neutralizing monoclonal antibody that targetsthe same domain (Table 2) MERS-S1B mAbs from epi-tope group II (mAb 18e5) and III (mAb 46e10)

Table 1 Binding kinetics of mAbsMERS-Secto or DPP4MERS-Secto from bio-layer interferometry measurementsmAb KD (M) kon (M

minus1 secminus1) koff (secminus1)

77g6 3612 times 10minus10 3196 times 104 1154 times 10minus5

16f9 5298 times 10minus10 1175 times 104 6227 times 10minus6

12g5 8086 times 10minus11 7587 times 104 6134 times 10minus6

18e5 3178 times 10minus10 2442 times 104 4232 times 10minus6

46e10 3592 times 10minus10 3569 times 104 1134 times 10minus5

110f3 4784 times 10minus9 9550 times 103 4569 times 10minus5

16c7 5007 times 10minus10 5927 times 104 2968 times 10minus5

35g6 2246 times 10minus9 2107 times 104 4733 times 10minus5

α-MERS-CTRL 1289 times 10minus10 4576 times 104 5900 times 10minus6

DPP4 3416 times 10minus9 1423 times 104 4859 times 10minus5

Figure 3 Virus neutralization and receptor binding inhibitionby anti-MERS-S mAbs (A) Analysis of MERS-CoV neutralizingactivity by anti-MERS-S mAbs using MERS-S pseudotyped luci-ferase-encoding VSV A previously described RBD-specificMERS-CoV-neutralizing human monoclonal antibody (anti-MERS-CTRL) and irrelevant isotype monoclonal antibody (Iso-CTRL) were included as positive and negative control respect-ively Luciferase-expressing VSV particles pseudotyped with theMERS-CoV S protein or authentic MERS-CoV were incubatedwith antibodies at the indicated concentrations and the mixwas used to transduce Huh-7 cells At 24 h postinfection luci-ferase expression was measured and neutralization () was cal-culated as the ratio of luciferase signal relative to relative tonon-antibody-treated controls Data represent the mean (plusmnstandard deviation SD) of three independent experiments(B) Receptor binding inhibition by anti-MERS-S mAbs deter-mined by an ELISA-based assay Recombinant soluble MERS-Secto was preincubated with serially diluted anti-MERS-SmAbs and added to ELISA plates coated with soluble theMERS-CoV S ectodomain Binding of MERS-Secto to DPP4 wasmeasured using HRP-conjugated antibody recognizing theStreptag affinity tag on DPP4 Data represent the mean (plusmn stan-dard deviation SD) of three independent experiments

520 I Widjaja et al

neutralized MERS-S pseudovirus infection at nanomo-lar concentrations (IC50 = 10 and 032 nM resp) andexhibited no detectable or moderate neutralizingactivity against authentic MERS-CoV (PRNT50 = gt667 and 667 nM resp) The MERS-S1A-specificmAb 110f3 lacked MERS-CoV neutralization activityin both virus neutralization assays The anti-MERS-S2 mAbs 16c7 and 35g6 were able to neutralizeMERS-S pseudovirus (IC50 = 245 and 166 nMresp) albeit at higher concentrations than the mostpotent neutralizing MERS-S1B mAbs (about 100-foldhigher) The isotype control did not show any neutral-ization in both assays Collectively our data demon-strate that antibodies targeting the receptor bindingdomain S1B of the MERS-CoV spike protein displaythe highest potential for neutralization of MERS-CoVinfection in vitro

Anti-MERS-S1B mAbs neutralize MERS-CoV byblocking receptor binding

To understand the mechanism of action of lead mAbswe set up assays to assess antibody interference withthe diverse functions of the MERS-CoV S domainsTo assess whether antibodies can compete with viralbinding to the host receptor DPP4 we developed anELISA-based receptor binding inhibition assay inwhich binding of MERS-S ectodomain to DPP4-coatedELISA plates is quantified and interference with recep-tor binding by antibodies is measured as a reduction inbinding signal In the absence of antibodies the MERS-S ectodomain showed stable binding to DPP4 (Figure 3(B)) Whereas anti-MERS-S1B mAb 18e5 showed weakinterference with binding of MERS-S ectodomain toDPP4 all other MERS-S1B-specific mAbs (mAbs77g6 16f9 12g5 46e10 and anti-MERS-CTRL)potently inhibited binding of MERS-S ectodomain toDPP4 in a concentration-dependent manner (Figure 3(B)) The data indicate that these antibodies partlyoverlap with or bind sufficiently close to the receptor-binding site on S1B to compete with receptor bindingNone of the antibodies that bind outside the RBDdomain (MERS-S1A and -S2) could block receptor

binding The potency of the S1B-specific mAbs to inhi-bit receptor binding corresponds with the ability ofthese antibodies to neutralize virus infection(Table 2) indicating that the inhibition of virusndashrecep-tor interaction by these antibodies is their main mech-anism of neutralization in vitro

Anti-MERS-S1A mAb 110f3 blocks binding ofMERS-S1A to sialoglycoconjugates

Recently we demonstrated that the MERS-S1A domainfacilitates viral binding to cell-surface sialoglycoconju-gates which can serve as a cell attachment factor forMERS-CoV [16] We assessed whether the MERS-S1A-targeting mAb 110f3 can interfere with bindingof MERS-S1A to sialoglycoconjugates on the surface oferythrocytes using the hemagglutination inhibitionassay To this end we used lumazine synthase (LS)nanoparticles multivalently displaying MERS-S1A

(S1A-LS) which were earlier employed to demonstratethe sialic-acid dependent hemagglutination by theMERS-S1A domain [16] Hemagglutination wasobserved when S1A-LS was mixed with erythrocytes(Figure 4(A)) S1A-LS mediated hemaglutination wasabrogated upon addition of the MERS-S1A mAb110f3 but not upon addition of the isotype controlNext we assessed whether interference of 110f3 withbinding to sialylated receptors could inhibit Sia-depen-dentMERS-CoV infection Binding to sialoglycansmayaidMERS-CoV entry into DPP4-positive cells depend-ing on the cell type Infection of Vero cells does notdepend on cell surface sialic acids concurrent with alow abundancy of the MERS-CoV S1A glycotopes onthose cells [16] Correspondingly infection of Verocells with MERS-S pseudovirus could not be inhibitedby 110f3 (Figure 4(B)) By contrast infection ofhuman lung Calu-3 cells was shown to rely on cell sur-face sialic acids which correlated with the abundance ofMERS-CoV S1A receptors [16] Contrary to Vero cellsinfection of Calu-3 cells could be inhibited by 110f3(Figure 4(B)) suggesting that antibody binding toMERS-S1A can neutralize MERS-CoV infection viainhibition of virus binding to cell surface sialoglycans

Table 2 Virus neutralization and receptor binding inhibition by anti-MERS-S mAbs

mAb MERS-S target

IC50 MERS-S VSVpp PRNT50 MERS-CoV RBI50

μgml nM μgml nM μgml nM

77g6 S1B 0001 0007 0008 0053 0007 004716f9 0006 004 003 0200 0013 008712g5 0002 0013 003 0200 0014 009318e5 1500 10 gt1 gt667 gt10 gt66746e10 0048 0320 1 6667 0137 0913110f3 S1A gt10 gt 667 gt1 gt667 gt10 gt66716c7 S2A 0367 2447 1 667 gt10 gt66735g6 2488 166 gt1 gt66 gt10 gt667α-MERS-CTRL S1B 0005 0033 003 0200 0022 0147Iso-CTRL ndash gt10 gt 667 gt1 gt667 ndash ndash

IC50 mAb concentration resulting in half-maximal infection of MERS-S VSV pseudovirus (MERS-S VSVpp) on Vero cells PRNT50 highest mAb dilution resulting in gt 50 reduction in the number of MERS-CoV infected Vero cells RBI50 mAb concentration of that gives half-maximal receptor binding

Emerging Microbes amp Infections 521

Anti-MERS-S2 mAbs interfere with MERS-Smediated membrane fusion

The coronavirus S2 subunit encompasses the machin-ery for fusion of viral and host cell membranes a pro-cess that is driven by extensive refolding of themetastable prefusion S2 into a stable postfusion state[35] We hypothesized that antibodies targeting theMERS-S2 subunit might neutralize MERS-CoV infec-tion by inhibiting this fusion process To test this wedeveloped a MERS-CoV-S driven cellndashcell fusionassay using a modified MERS-CoV spike protein Tomonitor expression of MERS-CoV S in cells weextended the viral fusion protein C-terminally withGFP In addition we mutated the furin cleavage siteat the S1S2 junction to increase the dependency ofMERS-CoV S fusion activation on exogenous addition

of trypsin Expression of this MERS-S variant upontransfection of DPP4-expressing Huh-7 cells could bereadily observed by the GFP signal (Figure 4(C))Upon addition of trypsin large GFP-fluorescent syncy-tia were detected indicating MERS-S mediated cellndashcellfusion (Figure 4(C)) As expected the addition of anti-MERS-S1B (77g6) blocked the formation of syncytiasince cellndashcell fusion is dependent on receptor inter-action No effect on syncytium formation was seenfor the MERS-S1A mAb 110f3 In contrast theMERS-S2-specific mAbs 16c7 and 35g6 both blockedsyncytium formation Since both neutralizing anti-bodies did not interfere with receptor binding (Figure 3(B)) we surmise that binding of these S2-specific anti-bodies inhibits infection by preventing conformationalchanges in the S2 subunit of the MERS-CoV spikeprotein that are required for fusion

Figure 4 Anti-MERS-S mAbs targeting MERS-S1A and -S2 domains block domain-specific functions (A) The anti-MERS-S1A mAb110f3 interferes with MERS-S1A-mediated sialic acid binding determined by a hemagglutination inhibition assay [16] The sia-lic-acid binding domain S1A of MERS-S was fused to lumazine synthase (LS) protein that can self-assemble to form 60-meric nano-particle (S1A-LS) which enables multivalent high affinity binding of the MERS-S1A domain to sialic acid ligands such as onerythocytes Human red blood cells were mixed with S1A-LS in the absence or presence of 2-fold dilutions of the MERS-S1A-specificmAb 110f3 Isotype control antibody was included as a negative control Hemagglutination was scored after 2 h of incubation at 4degC The hemagglutination inhibition assay was performed three times a representative experiment is shown (B) Neutralization ofMERS-S pseudotyped VSV by anti-MERS-S mAb 110f3 on Vero and Calu-3 cells Data represent the mean (plusmn standard deviation SD)of three independent experiments (C) The anti-MERS-S2 mAbs 16c7 and 35g6 block MERS-S-mediated cellndashcell fusion Huh-7 cellswere transfected with plasmid expressing MERS-CoV S C-terminally fused to GFP Two days after transfection cells were treatedwith trypsin to activate membrane the fusion function of the MERS-CoV S protein and incubated in the presence or absence of anti-MERS-S2 mAbs 16c7 and 35g6 or the anti-MERS-S1B mAb 77g6 and anti-MERS-S1A 110f3 all at 10 μgml Formation of MERS-Smediated cellndashcell fusion was visualized by fluorescence microscopy Merged images of MERS-S-GFP expressing cells (green) andDAPI-stained cell nuclei (blue) are shown Experiment was repeated two times and representative images are shown

522 I Widjaja et al

Protective activity of mAbs from lethal MERS-CoV challenge

To assess the prophylactic efficacy of our lead mAbsagainst MERS-CoV infection in vivo we used trans-genic K18-hDPP4 mice expressing human DPP4 [36]Six hours prior to MERS-CoV infection mice (5micegroup) were injected intraperitoneally with a50 μg dose of each mAb (equivalent to 18 mg mAbper kg body weight) The percentage of survival andweight change following challenge was monitored for12 days MERS-CoV infection was consistently lethalas all mice that received the monoclonal isotype controlshowed significant weight loss and had succumbed tothe infection between 7 and 8 days post-challenge(Figure 5(A) and (B)) Contrarily all MERS-S1B bind-ing mAbs showed high levels of protection againstlethal MERS-CoV challenge (80ndash100 Figure 5)Anti-MERS-S1B mAbs 77g6 12g5 and the benchmarkanti-MERS control mAb uniformly protected animalsfrom death whereas the MERS-S1B mAbs 16f9

18e5 and 46e10 protected 4 out of 5 animals (80)in this model The MERS-S1A binding mAb 110f3afforded partial protection frommortality (40) Nota-bly the anti-MERS-S2 mAbs 16c7 and 35g6 protectedall five animals from lethal infection Relative to theisotype control treated mice mice treated withMERS-S specific antibodies showed reduced weightloss (Figure 5(B)) These results highlight that anti-bodies targeting non-RBD domains (ic S1A and S2)of the MERS-CoV spike protein can contribute tohumoral immunity against MERS-CoV infection

Discussion

The recurring spillover infections of MERS-CoV inhumans from its dromedary camel reservoir the highmortality and person-to-person transmission pose asignificant threat to public health As there are cur-rently no licensed vaccines or treatments for combat-ting MERS-CoV infections the development of

Figure 5 Human anti-MERS-S mAbs protect mice against lethal MERS-CoV challenge (AndashB) Fifty microgram of antibody (equivalentto 18 mg mAbkg body weight) was infused intraperitoneally in K18-hDPP4-transgenic mice 6 h before challenge with 5 times 103 pfumice of MERS-CoV Five mice per group were used in the experiment Survival rates (A) and weight loss (B) (expressed as a per-centage of the initial weight) was monitored daily until 12 days post-inoculation

Emerging Microbes amp Infections 523

effective counter measures is a critical need andrecently prioritized by the World Health Organizationin the research and development Blueprint for action toprevent epidemics [37] Antibody therapies targetingcritical entry functions of viral glycoproteins areincreasingly recognized as promising antiviral strat-egies to protect humans from lethal disease [38] ForMERS-CoV passive immunization studies with neu-tralizing antibodies in small animals support the ideathat antibody therapies may hold promise to protecthumans from lethal MERS-CoV mediated diseaseMost of these protective antibodies neutralize virusinfection by occupying the receptor binding domain(RBD) and compete with the host receptor Here wedescribe the protective activity of individual humanantibodies targeting RBD as well as non-RBD spikedomains and their interference with the three knownfunctions of the spike glycoprotein Collectively thearsenal of protective antibodies that bind to and func-tionally inhibit the activity of multiple spike proteindomains can reveal new ways to gain humoral protec-tion against the emerging MERS coronavirus

Potent MERS-CoV neutralizing antibodies as ourstudy and that of others have shown commonly targetthe S1B receptor binding domain [17ndash23] The anti-S1B

antibodies physically prevent binding to the host receptorDPP4 attributed to their higher binding affinity forMERS-S thereby potently neutralizing MERS-CoVinfection The five S1B-specific antibodies identified inthis study were found to target three non-overlappingepitope groups on the MERS-CoV S1B domain consist-ent with the three epitope groups at the spike-DPP4receptor interface reported by Tang et al [22] TheseS1B antibodies displayed varying neutralization potencywith 100-1000-fold differences in IC50 values with anti-bodies targeting epitope group I showing ultrapotentneutralizing activity at sub-nanomolar levels Irrespectiveof the neutralizing potency all S1B antibodies displayedsignificant protective activity (80ndash100 survival rates)in mice from lethal MERS-CoV challenge

Apart from engaging the host receptor DPP4 weearlier demonstrated that the MERS-CoV spike proteinbinds sialoglycoconjugates (Siarsquos) on mucins and thehost cell surface via an independently functionaldomain the N-terminal domain S1A This Sia-bindingactivity serves as an attachment factor for MERS-CoVinfection in a cell-type dependent manner [16] Wenow show that binding of the isolated mAb 110f3 tothis MERS-S1A domain abrogates Sia-binding andcan inhibit the Sia-dependent infection of humanlung Calu-3 cells by MERS-CoV [16] Moreover pas-sive immunization of mice with this MERS-S1A-specific mAb resulted in 40 protection frommortalityfollowing MERS-CoV infection These findings under-line the role of sialic acid binding for MERS-CoV infec-tion in vivo and demonstrate the importance of anti-MERS-S1A antibodies in protection

From all MERS-CoV mAbs described only twomAbs ndash G2 and G4 ndash targeted epitopes outside theRBD [29] G2 binds an epitope in S1 outside theRBD whereas G4 targets a variable loop in S2 (Wang2015) Both murine antibodies were shown to protectmice from a lethal infection of MERS-CoV in vivoBoth antibodies were neutralizing though the interfer-ence of these antibodies with spike functions was notdelineated Using a tailored immunization scheme toboost antibody responses to the MERS-CoV fusion-mediating S2 subunit we were able to recover MERS-S2-specific mAbs two of which demonstrated neutra-lizing activity and fully protected mice from a lethalMERS-CoV challenge These two S2 antibodies didnot interfere with receptor binding yet abrogatedcellndashcell fusion implying their interference withspike-mediated membrane fusion by preventing con-formational changes required for fusion The highlyprotective S2 mAbs only displayed modest in vitro neu-tralizing activity indicating that strong neutralizingactivity is not a prerequisite for protection

Apart from interfering with functions of viral pro-teins antibodies are able to employ a broad range ofantiviral activities through the innate immune systemBinding of antibodies to glycoproteins on the surface ofinfected cells or on viruses may decorate them fordestruction through Fc-mediated antiviral activitiesincluding antibody-dependent cellular cytotoxicity(ADCC) antibody-dependent cell-mediated phagocy-tosis (ADCP) or complement-dependent cytotoxicity(CDC) [39] These functions may be irrespective ofthe neutralization capacity of antibodies as long asthey can bind surface antigens on the infected cellCongruent with this prerequisite all of the anti-MERS-S antibodies were able to bind cell surface dis-played spikes In addition our human antibodieswere of the IgG1 isotype which was shown to be themost potent human IgG isotype in mice showingefficient binding to all activating mouse Fcγ receptorsand induction of ADCCADCP with mouse naturalkiller cells and mouse macrophages [40] Furtherresearch is needed to define the contribution of Fc-functions to antibody-mediated protection fromMERS-CoV infection

We identified human antibodies targeting six dis-tinct epitope classes across different domains of theMERS-CoV spike protein each of which showing pro-tective activity in mice against lethal MERS-CoV chal-lenge This discovery holds promise for thedevelopment of antibody therapeutics against MERS-CoV infection Passive immunization with a combi-nation of antibodies targeting different domains andfunctions of the viral glycoprotein might be more pro-tective against virus infection than single epitope mAbtherapy as was shown for other enveloped RNAviruses [31 32 41ndash43] Which combination of anti-bodies provides synergistic protective activity against

524 I Widjaja et al

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

12g5) group II (18e5) and group III (46e10) on theS1B domain group IV (110f3) on the S1A domainand groups V (16c7) and group VI (35g6) on theS2 ectodomain Antibodies of different groups com-peted minimally with each other indicating thattheir epitopes were largely distinct (Figure 2(B) and(C)) The binding kinetics of the eight human mono-clonal antibodies was determined by bio-layer inter-ferometry Strep-tagged MERS-S ectodomain wascaptured on the protein-A sensor via an anti-Streptagantibody and kinetic binding parameters of antibodieswere determined at 25degC and pH 74 All antibodiesdisplayed high affinity binding for the MERS-S ecto-domain with equilibrium dissociation constants (KD)in the nano- to the picomolar range (0081 to478 nM) (Table 1 SI Appendix Figure S4) Thebinding affinity of the MERS-CoV receptor DPP4 toMERS-S was measured in the same set up and waslower compared to the binding affinity of the mAbsthat target the receptor binding domain S1B (Table1 SI Appendix Figure S4)

Anti-MERS-S mAbs bind cell surface displayedMERS-CoV spike protein

To assess whether the lead mAbs can bind full-lengthMERS-CoV S expressed on the cell surface we trans-fected Huh-7 cells with plasmid encoding MERS-CoV S The spike gene was C-terminally extendedwith GFP to monitor MERS-S expression andmutated at the furin cleavage site to stabilize thespike protein in its native prefusion state and to pre-vent MERS-S-mediated cellndashcell fusion Binding oflead mAbs to cell-surface expressed MERS-S wasanalysed by flow cytometry and immunofluorescenceAll anti-MERS-S mAbs bound to non-permeabilizedMERS-S transfected (GFP-positive) Huh-7 cells inboth assays indicative for binding to cell surfacedisplayed MERS-CoV S (SI Appendix Figures S5and S6)

Neutralization activity of anti-MERS-S mAbs

The ability of human lead mAbs to neutralize MERS-CoV infection in vitro was tested on Vero cells with

luciferase-encoding MERS-S pseudotyped virus andwith authentic MERS-CoV using a plaque reductionneutralization test (PRNT) Levels of virus neutraliz-ation varied among the individual antibodies (Figure 3(A) and Table 2) The 77g6 16f9 and 12g5 mAbs alltargeting epitope group I on MERS-S1B showed themost potent neutralizing activity and displayed pico-molar half-maximal inhibitory concentrations againstMERS-S pseudotyped virus (IC50 = 7ndash30 pM) andauthentic MERS-CoV (PRNT50 = 53ndash200 pM) whichwas equivalent to or lower than our benchmarkMERS-S neutralizing monoclonal antibody that targetsthe same domain (Table 2) MERS-S1B mAbs from epi-tope group II (mAb 18e5) and III (mAb 46e10)

Table 1 Binding kinetics of mAbsMERS-Secto or DPP4MERS-Secto from bio-layer interferometry measurementsmAb KD (M) kon (M

minus1 secminus1) koff (secminus1)

77g6 3612 times 10minus10 3196 times 104 1154 times 10minus5

16f9 5298 times 10minus10 1175 times 104 6227 times 10minus6

12g5 8086 times 10minus11 7587 times 104 6134 times 10minus6

18e5 3178 times 10minus10 2442 times 104 4232 times 10minus6

46e10 3592 times 10minus10 3569 times 104 1134 times 10minus5

110f3 4784 times 10minus9 9550 times 103 4569 times 10minus5

16c7 5007 times 10minus10 5927 times 104 2968 times 10minus5

35g6 2246 times 10minus9 2107 times 104 4733 times 10minus5

α-MERS-CTRL 1289 times 10minus10 4576 times 104 5900 times 10minus6

DPP4 3416 times 10minus9 1423 times 104 4859 times 10minus5

Figure 3 Virus neutralization and receptor binding inhibitionby anti-MERS-S mAbs (A) Analysis of MERS-CoV neutralizingactivity by anti-MERS-S mAbs using MERS-S pseudotyped luci-ferase-encoding VSV A previously described RBD-specificMERS-CoV-neutralizing human monoclonal antibody (anti-MERS-CTRL) and irrelevant isotype monoclonal antibody (Iso-CTRL) were included as positive and negative control respect-ively Luciferase-expressing VSV particles pseudotyped with theMERS-CoV S protein or authentic MERS-CoV were incubatedwith antibodies at the indicated concentrations and the mixwas used to transduce Huh-7 cells At 24 h postinfection luci-ferase expression was measured and neutralization () was cal-culated as the ratio of luciferase signal relative to relative tonon-antibody-treated controls Data represent the mean (plusmnstandard deviation SD) of three independent experiments(B) Receptor binding inhibition by anti-MERS-S mAbs deter-mined by an ELISA-based assay Recombinant soluble MERS-Secto was preincubated with serially diluted anti-MERS-SmAbs and added to ELISA plates coated with soluble theMERS-CoV S ectodomain Binding of MERS-Secto to DPP4 wasmeasured using HRP-conjugated antibody recognizing theStreptag affinity tag on DPP4 Data represent the mean (plusmn stan-dard deviation SD) of three independent experiments

520 I Widjaja et al

neutralized MERS-S pseudovirus infection at nanomo-lar concentrations (IC50 = 10 and 032 nM resp) andexhibited no detectable or moderate neutralizingactivity against authentic MERS-CoV (PRNT50 = gt667 and 667 nM resp) The MERS-S1A-specificmAb 110f3 lacked MERS-CoV neutralization activityin both virus neutralization assays The anti-MERS-S2 mAbs 16c7 and 35g6 were able to neutralizeMERS-S pseudovirus (IC50 = 245 and 166 nMresp) albeit at higher concentrations than the mostpotent neutralizing MERS-S1B mAbs (about 100-foldhigher) The isotype control did not show any neutral-ization in both assays Collectively our data demon-strate that antibodies targeting the receptor bindingdomain S1B of the MERS-CoV spike protein displaythe highest potential for neutralization of MERS-CoVinfection in vitro

Anti-MERS-S1B mAbs neutralize MERS-CoV byblocking receptor binding

To understand the mechanism of action of lead mAbswe set up assays to assess antibody interference withthe diverse functions of the MERS-CoV S domainsTo assess whether antibodies can compete with viralbinding to the host receptor DPP4 we developed anELISA-based receptor binding inhibition assay inwhich binding of MERS-S ectodomain to DPP4-coatedELISA plates is quantified and interference with recep-tor binding by antibodies is measured as a reduction inbinding signal In the absence of antibodies the MERS-S ectodomain showed stable binding to DPP4 (Figure 3(B)) Whereas anti-MERS-S1B mAb 18e5 showed weakinterference with binding of MERS-S ectodomain toDPP4 all other MERS-S1B-specific mAbs (mAbs77g6 16f9 12g5 46e10 and anti-MERS-CTRL)potently inhibited binding of MERS-S ectodomain toDPP4 in a concentration-dependent manner (Figure 3(B)) The data indicate that these antibodies partlyoverlap with or bind sufficiently close to the receptor-binding site on S1B to compete with receptor bindingNone of the antibodies that bind outside the RBDdomain (MERS-S1A and -S2) could block receptor

binding The potency of the S1B-specific mAbs to inhi-bit receptor binding corresponds with the ability ofthese antibodies to neutralize virus infection(Table 2) indicating that the inhibition of virusndashrecep-tor interaction by these antibodies is their main mech-anism of neutralization in vitro

Anti-MERS-S1A mAb 110f3 blocks binding ofMERS-S1A to sialoglycoconjugates

Recently we demonstrated that the MERS-S1A domainfacilitates viral binding to cell-surface sialoglycoconju-gates which can serve as a cell attachment factor forMERS-CoV [16] We assessed whether the MERS-S1A-targeting mAb 110f3 can interfere with bindingof MERS-S1A to sialoglycoconjugates on the surface oferythrocytes using the hemagglutination inhibitionassay To this end we used lumazine synthase (LS)nanoparticles multivalently displaying MERS-S1A

(S1A-LS) which were earlier employed to demonstratethe sialic-acid dependent hemagglutination by theMERS-S1A domain [16] Hemagglutination wasobserved when S1A-LS was mixed with erythrocytes(Figure 4(A)) S1A-LS mediated hemaglutination wasabrogated upon addition of the MERS-S1A mAb110f3 but not upon addition of the isotype controlNext we assessed whether interference of 110f3 withbinding to sialylated receptors could inhibit Sia-depen-dentMERS-CoV infection Binding to sialoglycansmayaidMERS-CoV entry into DPP4-positive cells depend-ing on the cell type Infection of Vero cells does notdepend on cell surface sialic acids concurrent with alow abundancy of the MERS-CoV S1A glycotopes onthose cells [16] Correspondingly infection of Verocells with MERS-S pseudovirus could not be inhibitedby 110f3 (Figure 4(B)) By contrast infection ofhuman lung Calu-3 cells was shown to rely on cell sur-face sialic acids which correlated with the abundance ofMERS-CoV S1A receptors [16] Contrary to Vero cellsinfection of Calu-3 cells could be inhibited by 110f3(Figure 4(B)) suggesting that antibody binding toMERS-S1A can neutralize MERS-CoV infection viainhibition of virus binding to cell surface sialoglycans

Table 2 Virus neutralization and receptor binding inhibition by anti-MERS-S mAbs

mAb MERS-S target

IC50 MERS-S VSVpp PRNT50 MERS-CoV RBI50

μgml nM μgml nM μgml nM

77g6 S1B 0001 0007 0008 0053 0007 004716f9 0006 004 003 0200 0013 008712g5 0002 0013 003 0200 0014 009318e5 1500 10 gt1 gt667 gt10 gt66746e10 0048 0320 1 6667 0137 0913110f3 S1A gt10 gt 667 gt1 gt667 gt10 gt66716c7 S2A 0367 2447 1 667 gt10 gt66735g6 2488 166 gt1 gt66 gt10 gt667α-MERS-CTRL S1B 0005 0033 003 0200 0022 0147Iso-CTRL ndash gt10 gt 667 gt1 gt667 ndash ndash

IC50 mAb concentration resulting in half-maximal infection of MERS-S VSV pseudovirus (MERS-S VSVpp) on Vero cells PRNT50 highest mAb dilution resulting in gt 50 reduction in the number of MERS-CoV infected Vero cells RBI50 mAb concentration of that gives half-maximal receptor binding

Emerging Microbes amp Infections 521

Anti-MERS-S2 mAbs interfere with MERS-Smediated membrane fusion

The coronavirus S2 subunit encompasses the machin-ery for fusion of viral and host cell membranes a pro-cess that is driven by extensive refolding of themetastable prefusion S2 into a stable postfusion state[35] We hypothesized that antibodies targeting theMERS-S2 subunit might neutralize MERS-CoV infec-tion by inhibiting this fusion process To test this wedeveloped a MERS-CoV-S driven cellndashcell fusionassay using a modified MERS-CoV spike protein Tomonitor expression of MERS-CoV S in cells weextended the viral fusion protein C-terminally withGFP In addition we mutated the furin cleavage siteat the S1S2 junction to increase the dependency ofMERS-CoV S fusion activation on exogenous addition

of trypsin Expression of this MERS-S variant upontransfection of DPP4-expressing Huh-7 cells could bereadily observed by the GFP signal (Figure 4(C))Upon addition of trypsin large GFP-fluorescent syncy-tia were detected indicating MERS-S mediated cellndashcellfusion (Figure 4(C)) As expected the addition of anti-MERS-S1B (77g6) blocked the formation of syncytiasince cellndashcell fusion is dependent on receptor inter-action No effect on syncytium formation was seenfor the MERS-S1A mAb 110f3 In contrast theMERS-S2-specific mAbs 16c7 and 35g6 both blockedsyncytium formation Since both neutralizing anti-bodies did not interfere with receptor binding (Figure 3(B)) we surmise that binding of these S2-specific anti-bodies inhibits infection by preventing conformationalchanges in the S2 subunit of the MERS-CoV spikeprotein that are required for fusion

Figure 4 Anti-MERS-S mAbs targeting MERS-S1A and -S2 domains block domain-specific functions (A) The anti-MERS-S1A mAb110f3 interferes with MERS-S1A-mediated sialic acid binding determined by a hemagglutination inhibition assay [16] The sia-lic-acid binding domain S1A of MERS-S was fused to lumazine synthase (LS) protein that can self-assemble to form 60-meric nano-particle (S1A-LS) which enables multivalent high affinity binding of the MERS-S1A domain to sialic acid ligands such as onerythocytes Human red blood cells were mixed with S1A-LS in the absence or presence of 2-fold dilutions of the MERS-S1A-specificmAb 110f3 Isotype control antibody was included as a negative control Hemagglutination was scored after 2 h of incubation at 4degC The hemagglutination inhibition assay was performed three times a representative experiment is shown (B) Neutralization ofMERS-S pseudotyped VSV by anti-MERS-S mAb 110f3 on Vero and Calu-3 cells Data represent the mean (plusmn standard deviation SD)of three independent experiments (C) The anti-MERS-S2 mAbs 16c7 and 35g6 block MERS-S-mediated cellndashcell fusion Huh-7 cellswere transfected with plasmid expressing MERS-CoV S C-terminally fused to GFP Two days after transfection cells were treatedwith trypsin to activate membrane the fusion function of the MERS-CoV S protein and incubated in the presence or absence of anti-MERS-S2 mAbs 16c7 and 35g6 or the anti-MERS-S1B mAb 77g6 and anti-MERS-S1A 110f3 all at 10 μgml Formation of MERS-Smediated cellndashcell fusion was visualized by fluorescence microscopy Merged images of MERS-S-GFP expressing cells (green) andDAPI-stained cell nuclei (blue) are shown Experiment was repeated two times and representative images are shown

522 I Widjaja et al

Protective activity of mAbs from lethal MERS-CoV challenge

To assess the prophylactic efficacy of our lead mAbsagainst MERS-CoV infection in vivo we used trans-genic K18-hDPP4 mice expressing human DPP4 [36]Six hours prior to MERS-CoV infection mice (5micegroup) were injected intraperitoneally with a50 μg dose of each mAb (equivalent to 18 mg mAbper kg body weight) The percentage of survival andweight change following challenge was monitored for12 days MERS-CoV infection was consistently lethalas all mice that received the monoclonal isotype controlshowed significant weight loss and had succumbed tothe infection between 7 and 8 days post-challenge(Figure 5(A) and (B)) Contrarily all MERS-S1B bind-ing mAbs showed high levels of protection againstlethal MERS-CoV challenge (80ndash100 Figure 5)Anti-MERS-S1B mAbs 77g6 12g5 and the benchmarkanti-MERS control mAb uniformly protected animalsfrom death whereas the MERS-S1B mAbs 16f9

18e5 and 46e10 protected 4 out of 5 animals (80)in this model The MERS-S1A binding mAb 110f3afforded partial protection frommortality (40) Nota-bly the anti-MERS-S2 mAbs 16c7 and 35g6 protectedall five animals from lethal infection Relative to theisotype control treated mice mice treated withMERS-S specific antibodies showed reduced weightloss (Figure 5(B)) These results highlight that anti-bodies targeting non-RBD domains (ic S1A and S2)of the MERS-CoV spike protein can contribute tohumoral immunity against MERS-CoV infection

Discussion

The recurring spillover infections of MERS-CoV inhumans from its dromedary camel reservoir the highmortality and person-to-person transmission pose asignificant threat to public health As there are cur-rently no licensed vaccines or treatments for combat-ting MERS-CoV infections the development of

Figure 5 Human anti-MERS-S mAbs protect mice against lethal MERS-CoV challenge (AndashB) Fifty microgram of antibody (equivalentto 18 mg mAbkg body weight) was infused intraperitoneally in K18-hDPP4-transgenic mice 6 h before challenge with 5 times 103 pfumice of MERS-CoV Five mice per group were used in the experiment Survival rates (A) and weight loss (B) (expressed as a per-centage of the initial weight) was monitored daily until 12 days post-inoculation

Emerging Microbes amp Infections 523

effective counter measures is a critical need andrecently prioritized by the World Health Organizationin the research and development Blueprint for action toprevent epidemics [37] Antibody therapies targetingcritical entry functions of viral glycoproteins areincreasingly recognized as promising antiviral strat-egies to protect humans from lethal disease [38] ForMERS-CoV passive immunization studies with neu-tralizing antibodies in small animals support the ideathat antibody therapies may hold promise to protecthumans from lethal MERS-CoV mediated diseaseMost of these protective antibodies neutralize virusinfection by occupying the receptor binding domain(RBD) and compete with the host receptor Here wedescribe the protective activity of individual humanantibodies targeting RBD as well as non-RBD spikedomains and their interference with the three knownfunctions of the spike glycoprotein Collectively thearsenal of protective antibodies that bind to and func-tionally inhibit the activity of multiple spike proteindomains can reveal new ways to gain humoral protec-tion against the emerging MERS coronavirus

Potent MERS-CoV neutralizing antibodies as ourstudy and that of others have shown commonly targetthe S1B receptor binding domain [17ndash23] The anti-S1B

antibodies physically prevent binding to the host receptorDPP4 attributed to their higher binding affinity forMERS-S thereby potently neutralizing MERS-CoVinfection The five S1B-specific antibodies identified inthis study were found to target three non-overlappingepitope groups on the MERS-CoV S1B domain consist-ent with the three epitope groups at the spike-DPP4receptor interface reported by Tang et al [22] TheseS1B antibodies displayed varying neutralization potencywith 100-1000-fold differences in IC50 values with anti-bodies targeting epitope group I showing ultrapotentneutralizing activity at sub-nanomolar levels Irrespectiveof the neutralizing potency all S1B antibodies displayedsignificant protective activity (80ndash100 survival rates)in mice from lethal MERS-CoV challenge

Apart from engaging the host receptor DPP4 weearlier demonstrated that the MERS-CoV spike proteinbinds sialoglycoconjugates (Siarsquos) on mucins and thehost cell surface via an independently functionaldomain the N-terminal domain S1A This Sia-bindingactivity serves as an attachment factor for MERS-CoVinfection in a cell-type dependent manner [16] Wenow show that binding of the isolated mAb 110f3 tothis MERS-S1A domain abrogates Sia-binding andcan inhibit the Sia-dependent infection of humanlung Calu-3 cells by MERS-CoV [16] Moreover pas-sive immunization of mice with this MERS-S1A-specific mAb resulted in 40 protection frommortalityfollowing MERS-CoV infection These findings under-line the role of sialic acid binding for MERS-CoV infec-tion in vivo and demonstrate the importance of anti-MERS-S1A antibodies in protection

From all MERS-CoV mAbs described only twomAbs ndash G2 and G4 ndash targeted epitopes outside theRBD [29] G2 binds an epitope in S1 outside theRBD whereas G4 targets a variable loop in S2 (Wang2015) Both murine antibodies were shown to protectmice from a lethal infection of MERS-CoV in vivoBoth antibodies were neutralizing though the interfer-ence of these antibodies with spike functions was notdelineated Using a tailored immunization scheme toboost antibody responses to the MERS-CoV fusion-mediating S2 subunit we were able to recover MERS-S2-specific mAbs two of which demonstrated neutra-lizing activity and fully protected mice from a lethalMERS-CoV challenge These two S2 antibodies didnot interfere with receptor binding yet abrogatedcellndashcell fusion implying their interference withspike-mediated membrane fusion by preventing con-formational changes required for fusion The highlyprotective S2 mAbs only displayed modest in vitro neu-tralizing activity indicating that strong neutralizingactivity is not a prerequisite for protection

Apart from interfering with functions of viral pro-teins antibodies are able to employ a broad range ofantiviral activities through the innate immune systemBinding of antibodies to glycoproteins on the surface ofinfected cells or on viruses may decorate them fordestruction through Fc-mediated antiviral activitiesincluding antibody-dependent cellular cytotoxicity(ADCC) antibody-dependent cell-mediated phagocy-tosis (ADCP) or complement-dependent cytotoxicity(CDC) [39] These functions may be irrespective ofthe neutralization capacity of antibodies as long asthey can bind surface antigens on the infected cellCongruent with this prerequisite all of the anti-MERS-S antibodies were able to bind cell surface dis-played spikes In addition our human antibodieswere of the IgG1 isotype which was shown to be themost potent human IgG isotype in mice showingefficient binding to all activating mouse Fcγ receptorsand induction of ADCCADCP with mouse naturalkiller cells and mouse macrophages [40] Furtherresearch is needed to define the contribution of Fc-functions to antibody-mediated protection fromMERS-CoV infection

We identified human antibodies targeting six dis-tinct epitope classes across different domains of theMERS-CoV spike protein each of which showing pro-tective activity in mice against lethal MERS-CoV chal-lenge This discovery holds promise for thedevelopment of antibody therapeutics against MERS-CoV infection Passive immunization with a combi-nation of antibodies targeting different domains andfunctions of the viral glycoprotein might be more pro-tective against virus infection than single epitope mAbtherapy as was shown for other enveloped RNAviruses [31 32 41ndash43] Which combination of anti-bodies provides synergistic protective activity against

524 I Widjaja et al

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

neutralized MERS-S pseudovirus infection at nanomo-lar concentrations (IC50 = 10 and 032 nM resp) andexhibited no detectable or moderate neutralizingactivity against authentic MERS-CoV (PRNT50 = gt667 and 667 nM resp) The MERS-S1A-specificmAb 110f3 lacked MERS-CoV neutralization activityin both virus neutralization assays The anti-MERS-S2 mAbs 16c7 and 35g6 were able to neutralizeMERS-S pseudovirus (IC50 = 245 and 166 nMresp) albeit at higher concentrations than the mostpotent neutralizing MERS-S1B mAbs (about 100-foldhigher) The isotype control did not show any neutral-ization in both assays Collectively our data demon-strate that antibodies targeting the receptor bindingdomain S1B of the MERS-CoV spike protein displaythe highest potential for neutralization of MERS-CoVinfection in vitro

Anti-MERS-S1B mAbs neutralize MERS-CoV byblocking receptor binding

To understand the mechanism of action of lead mAbswe set up assays to assess antibody interference withthe diverse functions of the MERS-CoV S domainsTo assess whether antibodies can compete with viralbinding to the host receptor DPP4 we developed anELISA-based receptor binding inhibition assay inwhich binding of MERS-S ectodomain to DPP4-coatedELISA plates is quantified and interference with recep-tor binding by antibodies is measured as a reduction inbinding signal In the absence of antibodies the MERS-S ectodomain showed stable binding to DPP4 (Figure 3(B)) Whereas anti-MERS-S1B mAb 18e5 showed weakinterference with binding of MERS-S ectodomain toDPP4 all other MERS-S1B-specific mAbs (mAbs77g6 16f9 12g5 46e10 and anti-MERS-CTRL)potently inhibited binding of MERS-S ectodomain toDPP4 in a concentration-dependent manner (Figure 3(B)) The data indicate that these antibodies partlyoverlap with or bind sufficiently close to the receptor-binding site on S1B to compete with receptor bindingNone of the antibodies that bind outside the RBDdomain (MERS-S1A and -S2) could block receptor

binding The potency of the S1B-specific mAbs to inhi-bit receptor binding corresponds with the ability ofthese antibodies to neutralize virus infection(Table 2) indicating that the inhibition of virusndashrecep-tor interaction by these antibodies is their main mech-anism of neutralization in vitro

Anti-MERS-S1A mAb 110f3 blocks binding ofMERS-S1A to sialoglycoconjugates

Recently we demonstrated that the MERS-S1A domainfacilitates viral binding to cell-surface sialoglycoconju-gates which can serve as a cell attachment factor forMERS-CoV [16] We assessed whether the MERS-S1A-targeting mAb 110f3 can interfere with bindingof MERS-S1A to sialoglycoconjugates on the surface oferythrocytes using the hemagglutination inhibitionassay To this end we used lumazine synthase (LS)nanoparticles multivalently displaying MERS-S1A

(S1A-LS) which were earlier employed to demonstratethe sialic-acid dependent hemagglutination by theMERS-S1A domain [16] Hemagglutination wasobserved when S1A-LS was mixed with erythrocytes(Figure 4(A)) S1A-LS mediated hemaglutination wasabrogated upon addition of the MERS-S1A mAb110f3 but not upon addition of the isotype controlNext we assessed whether interference of 110f3 withbinding to sialylated receptors could inhibit Sia-depen-dentMERS-CoV infection Binding to sialoglycansmayaidMERS-CoV entry into DPP4-positive cells depend-ing on the cell type Infection of Vero cells does notdepend on cell surface sialic acids concurrent with alow abundancy of the MERS-CoV S1A glycotopes onthose cells [16] Correspondingly infection of Verocells with MERS-S pseudovirus could not be inhibitedby 110f3 (Figure 4(B)) By contrast infection ofhuman lung Calu-3 cells was shown to rely on cell sur-face sialic acids which correlated with the abundance ofMERS-CoV S1A receptors [16] Contrary to Vero cellsinfection of Calu-3 cells could be inhibited by 110f3(Figure 4(B)) suggesting that antibody binding toMERS-S1A can neutralize MERS-CoV infection viainhibition of virus binding to cell surface sialoglycans

Table 2 Virus neutralization and receptor binding inhibition by anti-MERS-S mAbs

mAb MERS-S target

IC50 MERS-S VSVpp PRNT50 MERS-CoV RBI50

μgml nM μgml nM μgml nM

77g6 S1B 0001 0007 0008 0053 0007 004716f9 0006 004 003 0200 0013 008712g5 0002 0013 003 0200 0014 009318e5 1500 10 gt1 gt667 gt10 gt66746e10 0048 0320 1 6667 0137 0913110f3 S1A gt10 gt 667 gt1 gt667 gt10 gt66716c7 S2A 0367 2447 1 667 gt10 gt66735g6 2488 166 gt1 gt66 gt10 gt667α-MERS-CTRL S1B 0005 0033 003 0200 0022 0147Iso-CTRL ndash gt10 gt 667 gt1 gt667 ndash ndash

IC50 mAb concentration resulting in half-maximal infection of MERS-S VSV pseudovirus (MERS-S VSVpp) on Vero cells PRNT50 highest mAb dilution resulting in gt 50 reduction in the number of MERS-CoV infected Vero cells RBI50 mAb concentration of that gives half-maximal receptor binding

Emerging Microbes amp Infections 521

Anti-MERS-S2 mAbs interfere with MERS-Smediated membrane fusion

The coronavirus S2 subunit encompasses the machin-ery for fusion of viral and host cell membranes a pro-cess that is driven by extensive refolding of themetastable prefusion S2 into a stable postfusion state[35] We hypothesized that antibodies targeting theMERS-S2 subunit might neutralize MERS-CoV infec-tion by inhibiting this fusion process To test this wedeveloped a MERS-CoV-S driven cellndashcell fusionassay using a modified MERS-CoV spike protein Tomonitor expression of MERS-CoV S in cells weextended the viral fusion protein C-terminally withGFP In addition we mutated the furin cleavage siteat the S1S2 junction to increase the dependency ofMERS-CoV S fusion activation on exogenous addition

of trypsin Expression of this MERS-S variant upontransfection of DPP4-expressing Huh-7 cells could bereadily observed by the GFP signal (Figure 4(C))Upon addition of trypsin large GFP-fluorescent syncy-tia were detected indicating MERS-S mediated cellndashcellfusion (Figure 4(C)) As expected the addition of anti-MERS-S1B (77g6) blocked the formation of syncytiasince cellndashcell fusion is dependent on receptor inter-action No effect on syncytium formation was seenfor the MERS-S1A mAb 110f3 In contrast theMERS-S2-specific mAbs 16c7 and 35g6 both blockedsyncytium formation Since both neutralizing anti-bodies did not interfere with receptor binding (Figure 3(B)) we surmise that binding of these S2-specific anti-bodies inhibits infection by preventing conformationalchanges in the S2 subunit of the MERS-CoV spikeprotein that are required for fusion

Figure 4 Anti-MERS-S mAbs targeting MERS-S1A and -S2 domains block domain-specific functions (A) The anti-MERS-S1A mAb110f3 interferes with MERS-S1A-mediated sialic acid binding determined by a hemagglutination inhibition assay [16] The sia-lic-acid binding domain S1A of MERS-S was fused to lumazine synthase (LS) protein that can self-assemble to form 60-meric nano-particle (S1A-LS) which enables multivalent high affinity binding of the MERS-S1A domain to sialic acid ligands such as onerythocytes Human red blood cells were mixed with S1A-LS in the absence or presence of 2-fold dilutions of the MERS-S1A-specificmAb 110f3 Isotype control antibody was included as a negative control Hemagglutination was scored after 2 h of incubation at 4degC The hemagglutination inhibition assay was performed three times a representative experiment is shown (B) Neutralization ofMERS-S pseudotyped VSV by anti-MERS-S mAb 110f3 on Vero and Calu-3 cells Data represent the mean (plusmn standard deviation SD)of three independent experiments (C) The anti-MERS-S2 mAbs 16c7 and 35g6 block MERS-S-mediated cellndashcell fusion Huh-7 cellswere transfected with plasmid expressing MERS-CoV S C-terminally fused to GFP Two days after transfection cells were treatedwith trypsin to activate membrane the fusion function of the MERS-CoV S protein and incubated in the presence or absence of anti-MERS-S2 mAbs 16c7 and 35g6 or the anti-MERS-S1B mAb 77g6 and anti-MERS-S1A 110f3 all at 10 μgml Formation of MERS-Smediated cellndashcell fusion was visualized by fluorescence microscopy Merged images of MERS-S-GFP expressing cells (green) andDAPI-stained cell nuclei (blue) are shown Experiment was repeated two times and representative images are shown

522 I Widjaja et al

Protective activity of mAbs from lethal MERS-CoV challenge

To assess the prophylactic efficacy of our lead mAbsagainst MERS-CoV infection in vivo we used trans-genic K18-hDPP4 mice expressing human DPP4 [36]Six hours prior to MERS-CoV infection mice (5micegroup) were injected intraperitoneally with a50 μg dose of each mAb (equivalent to 18 mg mAbper kg body weight) The percentage of survival andweight change following challenge was monitored for12 days MERS-CoV infection was consistently lethalas all mice that received the monoclonal isotype controlshowed significant weight loss and had succumbed tothe infection between 7 and 8 days post-challenge(Figure 5(A) and (B)) Contrarily all MERS-S1B bind-ing mAbs showed high levels of protection againstlethal MERS-CoV challenge (80ndash100 Figure 5)Anti-MERS-S1B mAbs 77g6 12g5 and the benchmarkanti-MERS control mAb uniformly protected animalsfrom death whereas the MERS-S1B mAbs 16f9

18e5 and 46e10 protected 4 out of 5 animals (80)in this model The MERS-S1A binding mAb 110f3afforded partial protection frommortality (40) Nota-bly the anti-MERS-S2 mAbs 16c7 and 35g6 protectedall five animals from lethal infection Relative to theisotype control treated mice mice treated withMERS-S specific antibodies showed reduced weightloss (Figure 5(B)) These results highlight that anti-bodies targeting non-RBD domains (ic S1A and S2)of the MERS-CoV spike protein can contribute tohumoral immunity against MERS-CoV infection

Discussion

The recurring spillover infections of MERS-CoV inhumans from its dromedary camel reservoir the highmortality and person-to-person transmission pose asignificant threat to public health As there are cur-rently no licensed vaccines or treatments for combat-ting MERS-CoV infections the development of

Figure 5 Human anti-MERS-S mAbs protect mice against lethal MERS-CoV challenge (AndashB) Fifty microgram of antibody (equivalentto 18 mg mAbkg body weight) was infused intraperitoneally in K18-hDPP4-transgenic mice 6 h before challenge with 5 times 103 pfumice of MERS-CoV Five mice per group were used in the experiment Survival rates (A) and weight loss (B) (expressed as a per-centage of the initial weight) was monitored daily until 12 days post-inoculation

Emerging Microbes amp Infections 523

effective counter measures is a critical need andrecently prioritized by the World Health Organizationin the research and development Blueprint for action toprevent epidemics [37] Antibody therapies targetingcritical entry functions of viral glycoproteins areincreasingly recognized as promising antiviral strat-egies to protect humans from lethal disease [38] ForMERS-CoV passive immunization studies with neu-tralizing antibodies in small animals support the ideathat antibody therapies may hold promise to protecthumans from lethal MERS-CoV mediated diseaseMost of these protective antibodies neutralize virusinfection by occupying the receptor binding domain(RBD) and compete with the host receptor Here wedescribe the protective activity of individual humanantibodies targeting RBD as well as non-RBD spikedomains and their interference with the three knownfunctions of the spike glycoprotein Collectively thearsenal of protective antibodies that bind to and func-tionally inhibit the activity of multiple spike proteindomains can reveal new ways to gain humoral protec-tion against the emerging MERS coronavirus

Potent MERS-CoV neutralizing antibodies as ourstudy and that of others have shown commonly targetthe S1B receptor binding domain [17ndash23] The anti-S1B

antibodies physically prevent binding to the host receptorDPP4 attributed to their higher binding affinity forMERS-S thereby potently neutralizing MERS-CoVinfection The five S1B-specific antibodies identified inthis study were found to target three non-overlappingepitope groups on the MERS-CoV S1B domain consist-ent with the three epitope groups at the spike-DPP4receptor interface reported by Tang et al [22] TheseS1B antibodies displayed varying neutralization potencywith 100-1000-fold differences in IC50 values with anti-bodies targeting epitope group I showing ultrapotentneutralizing activity at sub-nanomolar levels Irrespectiveof the neutralizing potency all S1B antibodies displayedsignificant protective activity (80ndash100 survival rates)in mice from lethal MERS-CoV challenge

Apart from engaging the host receptor DPP4 weearlier demonstrated that the MERS-CoV spike proteinbinds sialoglycoconjugates (Siarsquos) on mucins and thehost cell surface via an independently functionaldomain the N-terminal domain S1A This Sia-bindingactivity serves as an attachment factor for MERS-CoVinfection in a cell-type dependent manner [16] Wenow show that binding of the isolated mAb 110f3 tothis MERS-S1A domain abrogates Sia-binding andcan inhibit the Sia-dependent infection of humanlung Calu-3 cells by MERS-CoV [16] Moreover pas-sive immunization of mice with this MERS-S1A-specific mAb resulted in 40 protection frommortalityfollowing MERS-CoV infection These findings under-line the role of sialic acid binding for MERS-CoV infec-tion in vivo and demonstrate the importance of anti-MERS-S1A antibodies in protection

From all MERS-CoV mAbs described only twomAbs ndash G2 and G4 ndash targeted epitopes outside theRBD [29] G2 binds an epitope in S1 outside theRBD whereas G4 targets a variable loop in S2 (Wang2015) Both murine antibodies were shown to protectmice from a lethal infection of MERS-CoV in vivoBoth antibodies were neutralizing though the interfer-ence of these antibodies with spike functions was notdelineated Using a tailored immunization scheme toboost antibody responses to the MERS-CoV fusion-mediating S2 subunit we were able to recover MERS-S2-specific mAbs two of which demonstrated neutra-lizing activity and fully protected mice from a lethalMERS-CoV challenge These two S2 antibodies didnot interfere with receptor binding yet abrogatedcellndashcell fusion implying their interference withspike-mediated membrane fusion by preventing con-formational changes required for fusion The highlyprotective S2 mAbs only displayed modest in vitro neu-tralizing activity indicating that strong neutralizingactivity is not a prerequisite for protection

Apart from interfering with functions of viral pro-teins antibodies are able to employ a broad range ofantiviral activities through the innate immune systemBinding of antibodies to glycoproteins on the surface ofinfected cells or on viruses may decorate them fordestruction through Fc-mediated antiviral activitiesincluding antibody-dependent cellular cytotoxicity(ADCC) antibody-dependent cell-mediated phagocy-tosis (ADCP) or complement-dependent cytotoxicity(CDC) [39] These functions may be irrespective ofthe neutralization capacity of antibodies as long asthey can bind surface antigens on the infected cellCongruent with this prerequisite all of the anti-MERS-S antibodies were able to bind cell surface dis-played spikes In addition our human antibodieswere of the IgG1 isotype which was shown to be themost potent human IgG isotype in mice showingefficient binding to all activating mouse Fcγ receptorsand induction of ADCCADCP with mouse naturalkiller cells and mouse macrophages [40] Furtherresearch is needed to define the contribution of Fc-functions to antibody-mediated protection fromMERS-CoV infection

We identified human antibodies targeting six dis-tinct epitope classes across different domains of theMERS-CoV spike protein each of which showing pro-tective activity in mice against lethal MERS-CoV chal-lenge This discovery holds promise for thedevelopment of antibody therapeutics against MERS-CoV infection Passive immunization with a combi-nation of antibodies targeting different domains andfunctions of the viral glycoprotein might be more pro-tective against virus infection than single epitope mAbtherapy as was shown for other enveloped RNAviruses [31 32 41ndash43] Which combination of anti-bodies provides synergistic protective activity against

524 I Widjaja et al

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

Anti-MERS-S2 mAbs interfere with MERS-Smediated membrane fusion

The coronavirus S2 subunit encompasses the machin-ery for fusion of viral and host cell membranes a pro-cess that is driven by extensive refolding of themetastable prefusion S2 into a stable postfusion state[35] We hypothesized that antibodies targeting theMERS-S2 subunit might neutralize MERS-CoV infec-tion by inhibiting this fusion process To test this wedeveloped a MERS-CoV-S driven cellndashcell fusionassay using a modified MERS-CoV spike protein Tomonitor expression of MERS-CoV S in cells weextended the viral fusion protein C-terminally withGFP In addition we mutated the furin cleavage siteat the S1S2 junction to increase the dependency ofMERS-CoV S fusion activation on exogenous addition

of trypsin Expression of this MERS-S variant upontransfection of DPP4-expressing Huh-7 cells could bereadily observed by the GFP signal (Figure 4(C))Upon addition of trypsin large GFP-fluorescent syncy-tia were detected indicating MERS-S mediated cellndashcellfusion (Figure 4(C)) As expected the addition of anti-MERS-S1B (77g6) blocked the formation of syncytiasince cellndashcell fusion is dependent on receptor inter-action No effect on syncytium formation was seenfor the MERS-S1A mAb 110f3 In contrast theMERS-S2-specific mAbs 16c7 and 35g6 both blockedsyncytium formation Since both neutralizing anti-bodies did not interfere with receptor binding (Figure 3(B)) we surmise that binding of these S2-specific anti-bodies inhibits infection by preventing conformationalchanges in the S2 subunit of the MERS-CoV spikeprotein that are required for fusion

Figure 4 Anti-MERS-S mAbs targeting MERS-S1A and -S2 domains block domain-specific functions (A) The anti-MERS-S1A mAb110f3 interferes with MERS-S1A-mediated sialic acid binding determined by a hemagglutination inhibition assay [16] The sia-lic-acid binding domain S1A of MERS-S was fused to lumazine synthase (LS) protein that can self-assemble to form 60-meric nano-particle (S1A-LS) which enables multivalent high affinity binding of the MERS-S1A domain to sialic acid ligands such as onerythocytes Human red blood cells were mixed with S1A-LS in the absence or presence of 2-fold dilutions of the MERS-S1A-specificmAb 110f3 Isotype control antibody was included as a negative control Hemagglutination was scored after 2 h of incubation at 4degC The hemagglutination inhibition assay was performed three times a representative experiment is shown (B) Neutralization ofMERS-S pseudotyped VSV by anti-MERS-S mAb 110f3 on Vero and Calu-3 cells Data represent the mean (plusmn standard deviation SD)of three independent experiments (C) The anti-MERS-S2 mAbs 16c7 and 35g6 block MERS-S-mediated cellndashcell fusion Huh-7 cellswere transfected with plasmid expressing MERS-CoV S C-terminally fused to GFP Two days after transfection cells were treatedwith trypsin to activate membrane the fusion function of the MERS-CoV S protein and incubated in the presence or absence of anti-MERS-S2 mAbs 16c7 and 35g6 or the anti-MERS-S1B mAb 77g6 and anti-MERS-S1A 110f3 all at 10 μgml Formation of MERS-Smediated cellndashcell fusion was visualized by fluorescence microscopy Merged images of MERS-S-GFP expressing cells (green) andDAPI-stained cell nuclei (blue) are shown Experiment was repeated two times and representative images are shown

522 I Widjaja et al

Protective activity of mAbs from lethal MERS-CoV challenge

To assess the prophylactic efficacy of our lead mAbsagainst MERS-CoV infection in vivo we used trans-genic K18-hDPP4 mice expressing human DPP4 [36]Six hours prior to MERS-CoV infection mice (5micegroup) were injected intraperitoneally with a50 μg dose of each mAb (equivalent to 18 mg mAbper kg body weight) The percentage of survival andweight change following challenge was monitored for12 days MERS-CoV infection was consistently lethalas all mice that received the monoclonal isotype controlshowed significant weight loss and had succumbed tothe infection between 7 and 8 days post-challenge(Figure 5(A) and (B)) Contrarily all MERS-S1B bind-ing mAbs showed high levels of protection againstlethal MERS-CoV challenge (80ndash100 Figure 5)Anti-MERS-S1B mAbs 77g6 12g5 and the benchmarkanti-MERS control mAb uniformly protected animalsfrom death whereas the MERS-S1B mAbs 16f9

18e5 and 46e10 protected 4 out of 5 animals (80)in this model The MERS-S1A binding mAb 110f3afforded partial protection frommortality (40) Nota-bly the anti-MERS-S2 mAbs 16c7 and 35g6 protectedall five animals from lethal infection Relative to theisotype control treated mice mice treated withMERS-S specific antibodies showed reduced weightloss (Figure 5(B)) These results highlight that anti-bodies targeting non-RBD domains (ic S1A and S2)of the MERS-CoV spike protein can contribute tohumoral immunity against MERS-CoV infection

Discussion

The recurring spillover infections of MERS-CoV inhumans from its dromedary camel reservoir the highmortality and person-to-person transmission pose asignificant threat to public health As there are cur-rently no licensed vaccines or treatments for combat-ting MERS-CoV infections the development of

Figure 5 Human anti-MERS-S mAbs protect mice against lethal MERS-CoV challenge (AndashB) Fifty microgram of antibody (equivalentto 18 mg mAbkg body weight) was infused intraperitoneally in K18-hDPP4-transgenic mice 6 h before challenge with 5 times 103 pfumice of MERS-CoV Five mice per group were used in the experiment Survival rates (A) and weight loss (B) (expressed as a per-centage of the initial weight) was monitored daily until 12 days post-inoculation

Emerging Microbes amp Infections 523

effective counter measures is a critical need andrecently prioritized by the World Health Organizationin the research and development Blueprint for action toprevent epidemics [37] Antibody therapies targetingcritical entry functions of viral glycoproteins areincreasingly recognized as promising antiviral strat-egies to protect humans from lethal disease [38] ForMERS-CoV passive immunization studies with neu-tralizing antibodies in small animals support the ideathat antibody therapies may hold promise to protecthumans from lethal MERS-CoV mediated diseaseMost of these protective antibodies neutralize virusinfection by occupying the receptor binding domain(RBD) and compete with the host receptor Here wedescribe the protective activity of individual humanantibodies targeting RBD as well as non-RBD spikedomains and their interference with the three knownfunctions of the spike glycoprotein Collectively thearsenal of protective antibodies that bind to and func-tionally inhibit the activity of multiple spike proteindomains can reveal new ways to gain humoral protec-tion against the emerging MERS coronavirus

Potent MERS-CoV neutralizing antibodies as ourstudy and that of others have shown commonly targetthe S1B receptor binding domain [17ndash23] The anti-S1B

antibodies physically prevent binding to the host receptorDPP4 attributed to their higher binding affinity forMERS-S thereby potently neutralizing MERS-CoVinfection The five S1B-specific antibodies identified inthis study were found to target three non-overlappingepitope groups on the MERS-CoV S1B domain consist-ent with the three epitope groups at the spike-DPP4receptor interface reported by Tang et al [22] TheseS1B antibodies displayed varying neutralization potencywith 100-1000-fold differences in IC50 values with anti-bodies targeting epitope group I showing ultrapotentneutralizing activity at sub-nanomolar levels Irrespectiveof the neutralizing potency all S1B antibodies displayedsignificant protective activity (80ndash100 survival rates)in mice from lethal MERS-CoV challenge

Apart from engaging the host receptor DPP4 weearlier demonstrated that the MERS-CoV spike proteinbinds sialoglycoconjugates (Siarsquos) on mucins and thehost cell surface via an independently functionaldomain the N-terminal domain S1A This Sia-bindingactivity serves as an attachment factor for MERS-CoVinfection in a cell-type dependent manner [16] Wenow show that binding of the isolated mAb 110f3 tothis MERS-S1A domain abrogates Sia-binding andcan inhibit the Sia-dependent infection of humanlung Calu-3 cells by MERS-CoV [16] Moreover pas-sive immunization of mice with this MERS-S1A-specific mAb resulted in 40 protection frommortalityfollowing MERS-CoV infection These findings under-line the role of sialic acid binding for MERS-CoV infec-tion in vivo and demonstrate the importance of anti-MERS-S1A antibodies in protection

From all MERS-CoV mAbs described only twomAbs ndash G2 and G4 ndash targeted epitopes outside theRBD [29] G2 binds an epitope in S1 outside theRBD whereas G4 targets a variable loop in S2 (Wang2015) Both murine antibodies were shown to protectmice from a lethal infection of MERS-CoV in vivoBoth antibodies were neutralizing though the interfer-ence of these antibodies with spike functions was notdelineated Using a tailored immunization scheme toboost antibody responses to the MERS-CoV fusion-mediating S2 subunit we were able to recover MERS-S2-specific mAbs two of which demonstrated neutra-lizing activity and fully protected mice from a lethalMERS-CoV challenge These two S2 antibodies didnot interfere with receptor binding yet abrogatedcellndashcell fusion implying their interference withspike-mediated membrane fusion by preventing con-formational changes required for fusion The highlyprotective S2 mAbs only displayed modest in vitro neu-tralizing activity indicating that strong neutralizingactivity is not a prerequisite for protection

Apart from interfering with functions of viral pro-teins antibodies are able to employ a broad range ofantiviral activities through the innate immune systemBinding of antibodies to glycoproteins on the surface ofinfected cells or on viruses may decorate them fordestruction through Fc-mediated antiviral activitiesincluding antibody-dependent cellular cytotoxicity(ADCC) antibody-dependent cell-mediated phagocy-tosis (ADCP) or complement-dependent cytotoxicity(CDC) [39] These functions may be irrespective ofthe neutralization capacity of antibodies as long asthey can bind surface antigens on the infected cellCongruent with this prerequisite all of the anti-MERS-S antibodies were able to bind cell surface dis-played spikes In addition our human antibodieswere of the IgG1 isotype which was shown to be themost potent human IgG isotype in mice showingefficient binding to all activating mouse Fcγ receptorsand induction of ADCCADCP with mouse naturalkiller cells and mouse macrophages [40] Furtherresearch is needed to define the contribution of Fc-functions to antibody-mediated protection fromMERS-CoV infection

We identified human antibodies targeting six dis-tinct epitope classes across different domains of theMERS-CoV spike protein each of which showing pro-tective activity in mice against lethal MERS-CoV chal-lenge This discovery holds promise for thedevelopment of antibody therapeutics against MERS-CoV infection Passive immunization with a combi-nation of antibodies targeting different domains andfunctions of the viral glycoprotein might be more pro-tective against virus infection than single epitope mAbtherapy as was shown for other enveloped RNAviruses [31 32 41ndash43] Which combination of anti-bodies provides synergistic protective activity against

524 I Widjaja et al

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

Protective activity of mAbs from lethal MERS-CoV challenge

To assess the prophylactic efficacy of our lead mAbsagainst MERS-CoV infection in vivo we used trans-genic K18-hDPP4 mice expressing human DPP4 [36]Six hours prior to MERS-CoV infection mice (5micegroup) were injected intraperitoneally with a50 μg dose of each mAb (equivalent to 18 mg mAbper kg body weight) The percentage of survival andweight change following challenge was monitored for12 days MERS-CoV infection was consistently lethalas all mice that received the monoclonal isotype controlshowed significant weight loss and had succumbed tothe infection between 7 and 8 days post-challenge(Figure 5(A) and (B)) Contrarily all MERS-S1B bind-ing mAbs showed high levels of protection againstlethal MERS-CoV challenge (80ndash100 Figure 5)Anti-MERS-S1B mAbs 77g6 12g5 and the benchmarkanti-MERS control mAb uniformly protected animalsfrom death whereas the MERS-S1B mAbs 16f9

18e5 and 46e10 protected 4 out of 5 animals (80)in this model The MERS-S1A binding mAb 110f3afforded partial protection frommortality (40) Nota-bly the anti-MERS-S2 mAbs 16c7 and 35g6 protectedall five animals from lethal infection Relative to theisotype control treated mice mice treated withMERS-S specific antibodies showed reduced weightloss (Figure 5(B)) These results highlight that anti-bodies targeting non-RBD domains (ic S1A and S2)of the MERS-CoV spike protein can contribute tohumoral immunity against MERS-CoV infection

Discussion

The recurring spillover infections of MERS-CoV inhumans from its dromedary camel reservoir the highmortality and person-to-person transmission pose asignificant threat to public health As there are cur-rently no licensed vaccines or treatments for combat-ting MERS-CoV infections the development of

Figure 5 Human anti-MERS-S mAbs protect mice against lethal MERS-CoV challenge (AndashB) Fifty microgram of antibody (equivalentto 18 mg mAbkg body weight) was infused intraperitoneally in K18-hDPP4-transgenic mice 6 h before challenge with 5 times 103 pfumice of MERS-CoV Five mice per group were used in the experiment Survival rates (A) and weight loss (B) (expressed as a per-centage of the initial weight) was monitored daily until 12 days post-inoculation

Emerging Microbes amp Infections 523

effective counter measures is a critical need andrecently prioritized by the World Health Organizationin the research and development Blueprint for action toprevent epidemics [37] Antibody therapies targetingcritical entry functions of viral glycoproteins areincreasingly recognized as promising antiviral strat-egies to protect humans from lethal disease [38] ForMERS-CoV passive immunization studies with neu-tralizing antibodies in small animals support the ideathat antibody therapies may hold promise to protecthumans from lethal MERS-CoV mediated diseaseMost of these protective antibodies neutralize virusinfection by occupying the receptor binding domain(RBD) and compete with the host receptor Here wedescribe the protective activity of individual humanantibodies targeting RBD as well as non-RBD spikedomains and their interference with the three knownfunctions of the spike glycoprotein Collectively thearsenal of protective antibodies that bind to and func-tionally inhibit the activity of multiple spike proteindomains can reveal new ways to gain humoral protec-tion against the emerging MERS coronavirus

Potent MERS-CoV neutralizing antibodies as ourstudy and that of others have shown commonly targetthe S1B receptor binding domain [17ndash23] The anti-S1B

antibodies physically prevent binding to the host receptorDPP4 attributed to their higher binding affinity forMERS-S thereby potently neutralizing MERS-CoVinfection The five S1B-specific antibodies identified inthis study were found to target three non-overlappingepitope groups on the MERS-CoV S1B domain consist-ent with the three epitope groups at the spike-DPP4receptor interface reported by Tang et al [22] TheseS1B antibodies displayed varying neutralization potencywith 100-1000-fold differences in IC50 values with anti-bodies targeting epitope group I showing ultrapotentneutralizing activity at sub-nanomolar levels Irrespectiveof the neutralizing potency all S1B antibodies displayedsignificant protective activity (80ndash100 survival rates)in mice from lethal MERS-CoV challenge

Apart from engaging the host receptor DPP4 weearlier demonstrated that the MERS-CoV spike proteinbinds sialoglycoconjugates (Siarsquos) on mucins and thehost cell surface via an independently functionaldomain the N-terminal domain S1A This Sia-bindingactivity serves as an attachment factor for MERS-CoVinfection in a cell-type dependent manner [16] Wenow show that binding of the isolated mAb 110f3 tothis MERS-S1A domain abrogates Sia-binding andcan inhibit the Sia-dependent infection of humanlung Calu-3 cells by MERS-CoV [16] Moreover pas-sive immunization of mice with this MERS-S1A-specific mAb resulted in 40 protection frommortalityfollowing MERS-CoV infection These findings under-line the role of sialic acid binding for MERS-CoV infec-tion in vivo and demonstrate the importance of anti-MERS-S1A antibodies in protection

From all MERS-CoV mAbs described only twomAbs ndash G2 and G4 ndash targeted epitopes outside theRBD [29] G2 binds an epitope in S1 outside theRBD whereas G4 targets a variable loop in S2 (Wang2015) Both murine antibodies were shown to protectmice from a lethal infection of MERS-CoV in vivoBoth antibodies were neutralizing though the interfer-ence of these antibodies with spike functions was notdelineated Using a tailored immunization scheme toboost antibody responses to the MERS-CoV fusion-mediating S2 subunit we were able to recover MERS-S2-specific mAbs two of which demonstrated neutra-lizing activity and fully protected mice from a lethalMERS-CoV challenge These two S2 antibodies didnot interfere with receptor binding yet abrogatedcellndashcell fusion implying their interference withspike-mediated membrane fusion by preventing con-formational changes required for fusion The highlyprotective S2 mAbs only displayed modest in vitro neu-tralizing activity indicating that strong neutralizingactivity is not a prerequisite for protection

Apart from interfering with functions of viral pro-teins antibodies are able to employ a broad range ofantiviral activities through the innate immune systemBinding of antibodies to glycoproteins on the surface ofinfected cells or on viruses may decorate them fordestruction through Fc-mediated antiviral activitiesincluding antibody-dependent cellular cytotoxicity(ADCC) antibody-dependent cell-mediated phagocy-tosis (ADCP) or complement-dependent cytotoxicity(CDC) [39] These functions may be irrespective ofthe neutralization capacity of antibodies as long asthey can bind surface antigens on the infected cellCongruent with this prerequisite all of the anti-MERS-S antibodies were able to bind cell surface dis-played spikes In addition our human antibodieswere of the IgG1 isotype which was shown to be themost potent human IgG isotype in mice showingefficient binding to all activating mouse Fcγ receptorsand induction of ADCCADCP with mouse naturalkiller cells and mouse macrophages [40] Furtherresearch is needed to define the contribution of Fc-functions to antibody-mediated protection fromMERS-CoV infection

We identified human antibodies targeting six dis-tinct epitope classes across different domains of theMERS-CoV spike protein each of which showing pro-tective activity in mice against lethal MERS-CoV chal-lenge This discovery holds promise for thedevelopment of antibody therapeutics against MERS-CoV infection Passive immunization with a combi-nation of antibodies targeting different domains andfunctions of the viral glycoprotein might be more pro-tective against virus infection than single epitope mAbtherapy as was shown for other enveloped RNAviruses [31 32 41ndash43] Which combination of anti-bodies provides synergistic protective activity against

524 I Widjaja et al

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

effective counter measures is a critical need andrecently prioritized by the World Health Organizationin the research and development Blueprint for action toprevent epidemics [37] Antibody therapies targetingcritical entry functions of viral glycoproteins areincreasingly recognized as promising antiviral strat-egies to protect humans from lethal disease [38] ForMERS-CoV passive immunization studies with neu-tralizing antibodies in small animals support the ideathat antibody therapies may hold promise to protecthumans from lethal MERS-CoV mediated diseaseMost of these protective antibodies neutralize virusinfection by occupying the receptor binding domain(RBD) and compete with the host receptor Here wedescribe the protective activity of individual humanantibodies targeting RBD as well as non-RBD spikedomains and their interference with the three knownfunctions of the spike glycoprotein Collectively thearsenal of protective antibodies that bind to and func-tionally inhibit the activity of multiple spike proteindomains can reveal new ways to gain humoral protec-tion against the emerging MERS coronavirus

Potent MERS-CoV neutralizing antibodies as ourstudy and that of others have shown commonly targetthe S1B receptor binding domain [17ndash23] The anti-S1B

antibodies physically prevent binding to the host receptorDPP4 attributed to their higher binding affinity forMERS-S thereby potently neutralizing MERS-CoVinfection The five S1B-specific antibodies identified inthis study were found to target three non-overlappingepitope groups on the MERS-CoV S1B domain consist-ent with the three epitope groups at the spike-DPP4receptor interface reported by Tang et al [22] TheseS1B antibodies displayed varying neutralization potencywith 100-1000-fold differences in IC50 values with anti-bodies targeting epitope group I showing ultrapotentneutralizing activity at sub-nanomolar levels Irrespectiveof the neutralizing potency all S1B antibodies displayedsignificant protective activity (80ndash100 survival rates)in mice from lethal MERS-CoV challenge

Apart from engaging the host receptor DPP4 weearlier demonstrated that the MERS-CoV spike proteinbinds sialoglycoconjugates (Siarsquos) on mucins and thehost cell surface via an independently functionaldomain the N-terminal domain S1A This Sia-bindingactivity serves as an attachment factor for MERS-CoVinfection in a cell-type dependent manner [16] Wenow show that binding of the isolated mAb 110f3 tothis MERS-S1A domain abrogates Sia-binding andcan inhibit the Sia-dependent infection of humanlung Calu-3 cells by MERS-CoV [16] Moreover pas-sive immunization of mice with this MERS-S1A-specific mAb resulted in 40 protection frommortalityfollowing MERS-CoV infection These findings under-line the role of sialic acid binding for MERS-CoV infec-tion in vivo and demonstrate the importance of anti-MERS-S1A antibodies in protection

From all MERS-CoV mAbs described only twomAbs ndash G2 and G4 ndash targeted epitopes outside theRBD [29] G2 binds an epitope in S1 outside theRBD whereas G4 targets a variable loop in S2 (Wang2015) Both murine antibodies were shown to protectmice from a lethal infection of MERS-CoV in vivoBoth antibodies were neutralizing though the interfer-ence of these antibodies with spike functions was notdelineated Using a tailored immunization scheme toboost antibody responses to the MERS-CoV fusion-mediating S2 subunit we were able to recover MERS-S2-specific mAbs two of which demonstrated neutra-lizing activity and fully protected mice from a lethalMERS-CoV challenge These two S2 antibodies didnot interfere with receptor binding yet abrogatedcellndashcell fusion implying their interference withspike-mediated membrane fusion by preventing con-formational changes required for fusion The highlyprotective S2 mAbs only displayed modest in vitro neu-tralizing activity indicating that strong neutralizingactivity is not a prerequisite for protection

Apart from interfering with functions of viral pro-teins antibodies are able to employ a broad range ofantiviral activities through the innate immune systemBinding of antibodies to glycoproteins on the surface ofinfected cells or on viruses may decorate them fordestruction through Fc-mediated antiviral activitiesincluding antibody-dependent cellular cytotoxicity(ADCC) antibody-dependent cell-mediated phagocy-tosis (ADCP) or complement-dependent cytotoxicity(CDC) [39] These functions may be irrespective ofthe neutralization capacity of antibodies as long asthey can bind surface antigens on the infected cellCongruent with this prerequisite all of the anti-MERS-S antibodies were able to bind cell surface dis-played spikes In addition our human antibodieswere of the IgG1 isotype which was shown to be themost potent human IgG isotype in mice showingefficient binding to all activating mouse Fcγ receptorsand induction of ADCCADCP with mouse naturalkiller cells and mouse macrophages [40] Furtherresearch is needed to define the contribution of Fc-functions to antibody-mediated protection fromMERS-CoV infection

We identified human antibodies targeting six dis-tinct epitope classes across different domains of theMERS-CoV spike protein each of which showing pro-tective activity in mice against lethal MERS-CoV chal-lenge This discovery holds promise for thedevelopment of antibody therapeutics against MERS-CoV infection Passive immunization with a combi-nation of antibodies targeting different domains andfunctions of the viral glycoprotein might be more pro-tective against virus infection than single epitope mAbtherapy as was shown for other enveloped RNAviruses [31 32 41ndash43] Which combination of anti-bodies provides synergistic protective activity against

524 I Widjaja et al

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

MERS-CoV and which combination of epitopes is thebest to target needs to be further evaluated In additioncombinations of antibodies targeting distinct epitopescan mitigate viral antigenic escape as has been demon-strated for a number of viruses including MERS-CoV[30 43ndash45] Particularly the use of (combinations of)antibodies targeting conserved epitopes in the spikeglycoprotein that are critical for viral entry may furtherdecrease chances of escape mutants Such antibodiesmay as well offer cross-protection against relatedviruses Antibodies targeting conserved epitopes inthe stem regions of glycoproteins of the envelopedvirus including influenza virus and ebolavirus offerprotection against a wide range of antigenically distinctvariants [46ndash48] Likewise the generation of antibodiestargeting the conserved S2 stem of the coronavirusspike protein that can cross-bind spikes of related cor-onaviruses may even allow the development of mAbswith broad protection against related future emergingcoronaviruses Furthermore it is important that theantibodies we describe are completely human whichhas the important advantage that they can be used sev-eral times in the same host without invoking animmune response against the antibody as may berequired for people working with camels or infectedpatients

Finally the presence of protective antibody epitopesin multiple spike domains suggests that multi-domainapproaches of spike-based vaccines may provide abroader repertoire of immune responses compared toRBD-focused vaccine antigen and reduce the risk ofantigenic escape This study also defines different cor-relates of humoral protection which may need to beconsidered in the evaluation of vaccine-inducedimmune responses

Materials and methods

Production of recombinant MERS-CoV S proteins Agene encoding the MERS-CoV spike glycoprotein(EMC isolate GenBank YP_0090472041) was syn-thesized by GenScript USA Inc and the sequence wascodon-optimized tomaximize expression in the baculo-virus expression system To produce soluble MERS-Sectodomain the gene fragment encoding the MERS-CoV S ectodomain (amino acid 19ndash1262) was clonedin-frame between honeybee melittin (HBM) secretionsignal peptide and T4 fibritin (foldon) trimerizationdomain followed by Strep-tag purification tag in thepFastbac transfer vector (Invitrogen) The furin clea-vage site R747SVR751 at the S1S2 junction was mutatedto KSVR to prevent cleavage by furin at this positionThe genes encoding MERS-S1 subunit (amino acid19-748) MERS-S2 ectodomain (amino acid 752-1262) MERS-S1A (amino acid 19-357) and MERS-S1B [358ndash588] were cloned in-frame between theHBM secretion signal peptide and a triple StrepTag

purification tag in the pFastbac transfer vector Gener-ation of bacmid DNA and recombinant baculoviruswas performed according to protocols from the Bac-to-Bac system (Invitrogen) and expression of MERS-CoV S variants was performed by infection of recombi-nant baculovirus of Sf-9 cells Recombinant proteinswere harvested from cell culture supernatants 3 dayspost infection and purified using StrepTactin sepharoseaffinity chromatography (IBA) The soluble MERS-Sectodomain used for immunization was produced inthe Drosophila expression system as described before[7] by cloning the gene insert from the pFastbac transfervector into the pMT expression vector (InvitrogenThermo Fisher Scientific the Netherlands) Productionof recombinant MERS-S1 (amino acid 1-747) used forimmunization and of soluble DPP4 was described pre-viously [15] In brief the MERS-S1 (amino acid 1-747)encoding sequence was C-terminally fused to a genefragment encoding the Fc region of human IgG andcloned into the pCAGGS mammalian expression vec-tor expressed by plasmid transfection in HEK-293 Tcells and affinity purified from the culture supernatantusing Protein-A affinity chromatography The Fc part ofS1-Fc fusion protein was proteolytically removed bythrombin following Protein-A affinity purificationusing the thrombin cleavage site present at the S1-Fcjunction The sequence encoding the human DPP4ectodomain (amino acid 39ndash766) N-terminally fusedto the Streptag purification tag was cloned into thepCAGGS vector expressed by plasmid transfection ofHEK-293 T cells and purified from the cell culturesupernatant using StrepTactin sepharose affinitychromatography Production of lumazine synthase(LS) nanoparticles displaying the MERS-CoV spikedomain S1A (S1A-LS) has been described previously[16] In brief the MERS-S1A encoding sequence (resi-dues 19ndash357) was N-terminally fused to a CD5 signalpeptide sequence followed by a Streptag purificationtag sequence (IBA) and C-terminally fused to the luma-zine synthase-encoding sequence fromAquifex aeolicus(GenBank WP_0108800271) via a Gly-Ser linker andsubsequent cloned into the pCAGGS vector expressedin HEK-293 T cells and purified from the cell culturesupernatant using StrepTactin affinity chromatography

Production of recombinant monoclonal antibodiesFor recombinant mAb production cDNArsquos encodingthe variable heavy (VH) and light (VL) chain regionsof anti-MERS-S H2L2 mAbs were cloned intoexpression plasmids containing the human IgG1heavy chain and Ig kappa light chain constant regionsrespectively (Invivogen) Both plasmids contain theinterleukin-2 signal sequence to enable efficientsecretion of recombinant antibodies Synthetic VHand VL gene fragments of the benchmark antibody(MERS-CTRL) were synthezised based on previouslydescribed sequences for the MERS-S monoclonal anti-body ldquoH1H15211Prdquo [34] Recombinant human anti-

Emerging Microbes amp Infections 525

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

MERS-S antibodies were produced in HEK-293 T cellsfollowing transfection with pairs of the IgG1 heavy andlight chain expression plasmids according to protocolsfrom Invivogen Antibodies were purified from tissueculture supernatants using Protein-A affinity chrom-atography Purified antibodies were stored at 4degCuntil use

Generation of anti-MERS-S H2L2 mAbs Twogroups of six H2L2 mice were immunized in twoweeks intervals six times with purified MERS-S1(group I) and MERS-S ectodomain followed byMERS-S2 ectodomain (group II) as outlined in Figure1(A) Antigens were injected at 20 μgmouse using Sti-mune Adjuvant (Prionics) freshly prepared accordingto the manufacturer instruction for the first injectionwhile boosting was done using Ribi (Sigma) adjuvantInjections were done subcutaneously into the left andright groin each (50 μl) and 100 μl intraperitoneallyFour days after the last injection spleen and lymphnodes are harvested and hybridomas made by a stan-dard method using SP 20 myeloma cell line(ATCCCRL-1581) as a fusion partner Hybridomaswere screened in antigen-specific ELISA and thoseselected for further development subcloned and pro-duced on a small scale (100 ml of medium) For thispurpose hybrydomas are cultured in serum- andprotein-free medium for hybridoma culturing(PFHM-II (1X) Gibco) with the addition of non-essen-tial amino acids (100times NEAA Biowhittaker Lonza CatBE13-114E) Antibodies were purified from the cellsupernatant using Protein-G affinity chromatographyPurified antibodies were stored at 4degC until use

MERS-S pseudotyped virus neutralization assayProduction of VSV pseudotyped with MERS-S wasperformed as described previously with some adap-tations [49] Briefly HEK-293 T cells were transfectedwith a pCAGGS expression vector encoding MERS-Scarrying a 16-aa cytoplasmic tail truncation Oneday post transfection cells were infected with theVSV-G pseudotyped VSVΔG bearing the firefly (Photi-nus pyralis) luciferase reporter gene [49] Twenty fourhours later MERS-S-VSVΔG pseudotypes were har-vested and titrated on African green monkey kidneyVero cells In the virus neutralization assay MERS-SmAbs were serially diluted at two times the desiredfinal concentration in DMEM supplemented with 1foetal calf serum (Bodinco) 100 Uml Penicillin and100 microgml Streptomycin Diluted mAbs were incubatedwith an equal volume of MERS-S-VSVΔG pseudotypesfor 1 h at room temperature inoculated on confluentVero monolayers in 96-well plated and further incu-bated at 37degC for 24 h Luciferase activity wasmeasured on a Berthold Centro LB 960 plate lumin-ometer using D-luciferin as a substrate (Promega)The percentage of infectivity was calculated as theratio of luciferase readout in the presence of mAbs nor-malized to luciferase readout in the absence of mAb

The half maximal inhibitory concentrations (IC50)were determined using 4-parameter logistic regression(GraphPad Prism v70)

MERS-CoV neutralization assay Neutralization ofauthentic MERS-CoV was performed using a plaquereduction neutralization test (PRNT) as described ear-lier [50] In brief mAbs were two-fold serially dilutedand mixed with MERS-CoV for 1 h The mixture wasthen added to Huh-7 cells and incubated for 1 hrafter which the cells were washed and further incubatedin medium for 8 h Subsequently the cells werewashed fixed permeabilized and the infection wasdetected using immunofluorescent staining ThePRNT titre was determined as the highest mAbdilution resulting in a gt 50 reduction in the numberof infected cells (PRNT50)

ELISA analysis of MERS-CoV S binding by anti-bodies NUNC Maxisorp plates (Thermo Scientific)were coated with the indicated MERS-CoV antigen at100 ng well at 4degC overnight Plates were washedthree times with Phosphate Saline Buffer (PBS) con-taining 005 Tween-20 and blocked with 3 BovineSerum Albumin (BSA) in PBS containing 01Tween-20 at room temperature for 2 h Four-foldsserial dilutions of mAbs starting at 10 microgml (dilutedin blocking buffer) were added and plates were incu-bated for 1 h at room temperature Plates were washedthree times and incubated with HRP-conjugated goatanti-human secondary antibody (ITK Southern Bio-tech) diluted 12000 in blocking buffer for one hourat room temperature HRP activity was measured at450 nm using tetramethylbenzidine substrate (BioFX)and an ELISA plate reader (EL-808 Biotek)

ELISA analysis of receptor binding inhibition byantibodies Recombinant soluble DPP4 was coatedon NUNC Maxisorp plates (Thermo Scientific) at 4degC overnight Plates were washed three times withPBS containing 005 Tween-20 and blocked with3 BSA in PBS containing 01 Tween-20 at roomtemperature for 2 h Recombinant MERS-CoV S ecto-domain and serially diluted anti-MERS mAbs weremixed for 1 h at RT added to the plate for 1 h atroom temperature after which plates were washedthree times Binding of MERS-CoV S ectodomain toDPP4 was detected using HRP-conjugated anti-Strep-MAb (IBA) that recognizes the Streptag affinity tagon the MERS-CoV S ectodomain Detection of HRPactivity was performed as described above

Antibody competition assay Competition amongmAbs for binding to the same epitope on MERS-CoVS was determined using Bio-Layer Interferometry(BLI) on Octet QK (Pall ForteBio) at 25degC All reagentswere diluted in PBS The assay was performed followingthese steps (1) anti-Strep mAb (50 μgml) was coatedon Protein A biosensor (Pall ForteBio) for 30 mins(2) blocking of sensor with rabbit IgG (50 μgml) for30 mins (3) Recombinant Strep-tagged MERS-CoV S

526 I Widjaja et al

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

ectodomain (50 μgml) was immobilized to the sensorfor 15 mins (4) Addition of mAb 1 (50 μgml) for15 min to allow saturation of binding to the immobi-lized antigen (5) Addition of a mAb 2 (50 μgml) for15 mins The first antibody (mAb 1) was taken alongto verify the saturation of binding A 5-minutes washingstep in PBS was included in between steps

Binding kinetics and affinity measurements Bind-ing kinetics and affinity of mAbs to the MERS-S ecto-domain was measured by BLI using the Octet QK at25degC The optimal loading concentration of anti-MERS-S mAbs onto anti-human Fc biosensors (PallForteBio) was predetermined to avoid saturation ofthe sensor The kinetic binding assay was performedby loading anti-MERS mAb at optimal concentration(42 nM) on anti-human Fc biosensor for 10 minsAntigen association step was performed by incubatingthe sensor with a range of concentrations of the recom-binant MERS-S ectodomain (200ndash67 to 22ndash74 nM) for10 min followed by a dissociation step in PBS for60 min The kinetics constants were calculated using11 Langmuir binding model on Fortebio Data Analysis70 software

Hemagglutination inhibition assay The potency ofmAbs to inhibit hemagglutination by MERS-S1A-dis-playing lumazine synthase nanoparticles (S1A-LS)was performed as described previously [16] with slightmodification Two-folds serial dilutions of S1A-LS inPBS containing 01 bovine serum albumin (BSA)were mixed with 05 human erythrocyte in V-bottom96-well plate (Greiner Bio-One) and incubated at 4degCfor 2 h The hemagglutination titre was scored and theconcentration of S1A-LS that resulted in 8 hemaggluti-nation units was determined Subsequently two-foldserial dilutions of anti-MERS-S mAbs in PBS contain-ing 01 BSA were mixed with S1A-LS (8 hemaggluti-nation units) in a V-bottom 96-well plate After30 mins incubation at room temperature humanerythrocytes were added to a final concentration of05 (vv) The mixture was incubated at 4degC for 2 hand the hemagglutination inhibition activity by anti-MERS-S mAbs was scored

Fusion inhibition assay Huh-7 cells were seededwith a density of 105 cells per ml After reaching 70ndash80 confluency cells were transfected with expressionplasmid encoding full length MERS-CoV S fused toGreen Fluorescence Protein (GFP) using jetPRIMEreg(Polyplus transfection New York USA cat no 114-07) The furin recognition site in the MERS-CoV Swas mutated to inhibit the cleavage of protein Twodays post transfection cells were treated with 10 μgml trypsin (to activate MERS-CoV spike fusion func-tion) in the presence or absence of 10 μgml anti-MERS-S mAbs After incubation at 37degC for 2 h thecells were fixed by incubation with 4 paraformalde-hyde in PBS for 20 min at room temperature andstained for nuclei with 46-diamidino-2-phenylindole

(DAPI) Cells expressing MERS-CoV S were detectedby fluorescence microscopy using the C-terminallyappended GFP and MERS-CoV S-mediated cellndashcellfusion was observed by the formation of (fluorescent)multi-nucleated syncytia The fluorescence imageswere recorded using the EVOS FL fluorescence micro-scope (Thermo Fisher Scientific the Netherlands)

Antibody-mediated protection of mice challengedwith MERS-CoV In vivo efficacy of mAbs specific forthe S protein of MERS-CoV and of an isotype matchednegative controlmAbswas evaluated in the protection ofthe transgenic mouse model K18 TghDpp4 expressingthe receptor for the humanMERS-CoV [36] susceptibleto the virulent virus MERS-CoV intranasal infection ofthese transgenic mice expressing human DPP4 causes alethal disease associated with encephalitis lung mono-nuclear cell infiltration alveolar oedema and microvas-cular thrombosis with airways generally unaffected [36]To test the prophylactic efficacy of mAbs in vivo groupsof 5mice 20ndash30weeks old were given 18 mgof the anti-body per kg mouse by intraperitoneal injection 6 hbefore intranasal infection with a lethal dose of MERS-CoV (EMC isolate 5 times 103 pfumouse challenge doseresulting in consistently lethal infection in untreatedmice) Whereas the administered dose was consistentlylethal for all mice that received the isotype control anti-body mice that received the best virus neutralizing anti-bodies were fully protected

Acknowledgements

We thank dr Yoshiharu Matsuura (Osaka University Japan)for the provision of the luciferase-encoding VSV-G pseudo-typed VSVΔG-luc virus and Alejandra Tortorici (Institut Pas-teur France) for providing MERS-S ectodomain The miceused in this study were provided by Harbour Antibodies BVa daughter company of Harbour Biomed (httpwwwharbourbiomedcom) I W and B-J B designed and coordi-nated the study I W C W Rv H J G Aacute Bv D V S RW L R F D B L H D D and B-J B performed researchI W C W Rv H J G Aacute Bv D V S R W L R F DF G F Jv K B L H L E D D and B-J B analyseddata and I W and B-J B wrote the paper with commentsfrom all authors

Disclosure statement

VSR BLH and BJB are inventors on a patent appli-cation on MERS-CoV held by Erasmus MC (applicationno 61704531 filed 23 September 2012 publication noWO 2014045254 A2) All authors are inventors on a patentapplication on coronavirus antibodies (application noEP193821238) filed 20 February 2019

Funding

This study was financed by a grant from the IMI-ZAPI (Zoo-notic Anticipation and Preparedness Initiative (ZAPI) pro-ject Innovative Medicines Initiative (IMI)) [grantagreement no 115760] with the assistance and financial

Emerging Microbes amp Infections 527

support of IMI and the European Commission and in-kindcontributions from European Federation of PharmaceuticalIndustries and Associations partners This study was alsopartially financed by grants from the Ministry of Scienceand Innovation of Spain (BIO2016-75549-R AEIFEDERUE) and NIH (2PO1AIO6O699) This work was supportedby Center for Scientific Review China Scholarship Council

ORCID

Javier Gutieacuterrez-Aacutelvarez httporcidorg0000-0003-0690-9496Nisreen MA Okba httporcidorg0000-0002-2394-1079V Stalin Raj httporcidorg0000-0003-2250-8481Bart L Haagmans httporcidorg0000-0001-6221-2015

References

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[2] Omrani A S J A Al-Tawfiq and Z A Memish2015 Middle east respiratory syndrome coronavirus(MERS-CoV) animal to human interaction PathogGlob Health 109354ndash362 doi 1010802047772420151122852

[3] Azhar E I S A El-Kafrawy S A Farraj A MHassan M S Al-Saeed A M Hashem and T AMadani 2014 Evidence for camel-to-human trans-mission of MERS coronavirus N Engl J Med3702499ndash2505 doi 101056NEJMoa1401505

[4] Haagmans B L S H Al Dhahiry C B Reusken V SRaj M Galiano R Myers G J Godeke M Jonges EFarag A Diab H Ghobashy F Alhajri M Al-ThaniS A Al-Marri H E Al Romaihi A Al Khal ABermingham A D Osterhaus M M AlHajri andM P Koopmans 2014 Middle East respiratory syn-drome coronavirus in dromedary camels an outbreakinvestigation Lancet Infect Dis 14140ndash145 doi 101016S1473-3099(13)70690-X

[5] Memish Z A M Cotten B Meyer S J Watson A JAlsahafi A A Al Rabeeah V M Corman A SiebergH Q Makhdoom A Assiri M Al Masri SAldabbagh B J Bosch M Beer M A Muller PKellam and C Drosten 2014 Human infection withMERS coronavirus after exposure to infected camelsSaudi Arabia 2013 Emerg Infect Dis 201012ndash1015doi 103201eid2006140402

[6] Du L Y Yang Y Zhou L Lu F Li and S Jiang 2017MERS-CoV spike protein a key target for antiviralsExpert Opin Ther Targets 21131ndash143 doi 1010801472822220171271415

[7] Walls A C M A Tortorici B J Bosch B FrenzP J M Rottier F DiMaio F A Rey and D Veesler2016 Cryo-electron microscopy structure of a corona-virus spike glycoprotein trimer Nature 531114ndash117doi 101038nature16988

[8] Walls A C M A Tortorici B Frenz J Snijder W LiF A Rey F DiMaio B J Bosch and D Veesler 2016Glycan shield and epitope masking of a coronavirusspike protein observed by cryo-electron microscopyNat Struct Mol Biol 23899ndash905 doi 101038nsmb3293

[9] Xiong X M A Tortorici J Snijder C Yoshioka A CWalls W Li A T McGuire F A Rey B J Bosch andD Veesler 2017 Glycan shield and fusion activation of

a deltacoronavirus spike glycoprotein fine-tuned forenteric infections J Virol doi JVI01628-17 [pii]

[10] Kirchdoerfer R N C A Cottrell N Wang J PallesenHM Yassine H L Turner K S Corbett B S GrahamJ S McLellan and A B Ward 2016 Pre-fusion struc-ture of a human coronavirus spike protein Nature531118ndash121 doi 101038nature17200

[11] Shang J Y Zheng Y Yang C Liu Q Geng W Tai LDu Y Zhou W Zhang and F Li 2018 Cryo-Electronmicroscopy Structure of Porcine Deltacoronavirusspike protein in the prefusion state J Virol 92 doi101128JVI01556-17 JVI01556-17 [pii]

[12] Shang J Y Zheng Y Yang C Liu Q Geng C LuoW Zhang and F Li 2018 Cryo-EM structure of infec-tious bronchitis coronavirus spike protein revealsstructural and functional evolution of coronavirusspike proteins PLoS Pathog 14e1007009 doi 101371journalppat1007009

[13] Yuan Y D Cao Y Zhang J Ma J Qi Q Wang GLu Y Wu J Yan Y Shi X Zhang and G F Gao2017 Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptorbinding domains Nat Commun 815092 doi 101038ncomms15092

[14] Raj V S H Mou S L Smits D H Dekkers M AMuller R Dijkman D Muth J A Demmers AZaki R A Fouchier V Thiel C Drosten P JRottier A D Osterhaus B J Bosch and B LHaagmans 2013 Dipeptidyl peptidase 4 is a functionalreceptor for the emerging human coronavirus-EMCNature 495251ndash254 doi 101038nature12005

[15] Mou H V S Raj F J van Kuppeveld P J Rottier BL Haagmans and B J Bosch 2013 The receptor bind-ing domain of the new Middle East respiratory syn-drome coronavirus maps to a 231-residue region inthe spike protein that efficiently elicits neutralizingantibodies J Virol 879379ndash9383 doi 101128JVI01277-13

[16] Li W R J G Hulswit I Widjaja V S Raj RMcBride W Peng W Widagdo M A Tortorici Bvan Dieren Y Lang J W M van Lent J CPaulson C A M de Haan R J de Groot F J Mvan Kuppeveld B L Haagmans and B J Bosch2017 Identification of sialic acid-binding function forthe Middle East respiratory syndrome coronavirusspike glycoprotein Proc Natl Acad Sci U S A114E8508ndashE8517 doi 101073pnas1712592114

[17] Corti D J Zhao M Pedotti L Simonelli SAgnihothram C Fett B Fernandez-Rodriguez MFoglierini G Agatic F Vanzetta R Gopal C JLangrish N A Barrett F Sallusto R S Baric LVarani M Zambon S Perlman and A Lanzavecchia2015 Prophylactic and postexposure efficacy of a potenthuman monoclonal antibody against MERS corona-virus Proc Natl Acad Sci U S A 11210473ndash10478doi 101073pnas1510199112

[18] Du L G Zhao Y Yang H Qiu L Wang Z Kou XTao H Yu S Sun C T Tseng S Jiang F Li and YZhou 2014 A conformation-dependent neutralizingmonoclonal antibody specifically targeting receptor-binding domain in Middle East respiratory syndromecoronavirus spike protein J Virol 887045ndash7053doi 101128JVI00433-14

[19] Li Y Y Wan P Liu J Zhao G Lu J Qi Q Wang XLu Y Wu W Liu B Zhang K Y Yuen S PerlmanG F Gao and J Yan 2015 A humanized neutralizingantibody against MERS-CoV targeting the receptor-

528 I Widjaja et al

binding domain of the spike protein Cell Res251237ndash1249 doi 101038cr2015113

[20] Pascal K E C M Coleman A O Mujica V KamatA Badithe J Fairhurst C Hunt J Strein A Berrebi JM Sisk K L Matthews R Babb G Chen K M Lai TT Huang W Olson G D Yancopoulos N Stahl MB Frieman and C A Kyratsous 2015 Pre- and post-exposure efficacy of fully human antibodies againstspike protein in a novel humanized mouse model ofMERS-CoV infection Proc Natl Acad Sci U S A1128738ndash8743 doi 101073pnas1510830112

[21] Jiang L N Wang T Zuo X Shi K M Poon Y WuF Gao D Li R Wang J Guo L Fu K Y Yuen B JZheng X Wang and L Zhang 2014 Potent neutraliz-ation of MERS-CoV by human neutralizing mono-clonal antibodies to the viral spike glycoprotein SciTransl Med 6234ra59 doi 101126scitranslmed3008140

[22] Tang X C S S Agnihothram Y Jiao J Stanhope RL Graham E C Peterson Y Avnir A S Tallarico JSheehan Q Zhu R S Baric and W A Marasco 2014Identification of human neutralizing antibodies againstMERS-CoV and their role in virus adaptive evolutionProc Natl Acad Sci U S A 111E2018ndashE2026 doi101073pnas1402074111

[23] Ying T L Du T W Ju P Prabakaran C C Lau LLu Q Liu L Wang Y Feng Y Wang B J Zheng KY Yuen S Jiang and D S Dimitrov 2014Exceptionally potent neutralization of Middle East res-piratory syndrome coronavirus by human monoclonalantibodies J Virol 887796ndash7805 doi 101128JVI00912-14

[24] Johnson R F U Bagci L Keith X Tang D JMollura L Zeitlin J Qin L Huzella C J Bartos NBohorova O Bohorov C Goodman D H Kim MH Paulty J Velasco K J Whaley J C Johnson JPettitt B L Ork J Solomon N Oberlander Q ZhuJ Sun M R Holbrook G G Olinger R S Baric LE Hensley P B Jahrling and W A Marasco 20163B11-N a monoclonal antibody against MERS-CoVreduces lung pathology in rhesus monkeys followingintratracheal inoculation of MERS-CoV Jordan-n32012 Virology 49049ndash58 doi 101016jvirol201601004

[25] Qiu H S Sun H Xiao J Feng Y Guo W Tai YWang L Du G Zhao and Y Zhou 2016 Single-dose treatment with a humanized neutralizing anti-body affords full protection of a human transgenicmouse model from lethal Middle East respiratory syn-drome (MERS)-coronavirus infection Antiviral Res132141ndash148 doi 101016jantiviral201606003

[26] Agrawal A S T Ying X Tao T Garron A AlgaissiY Wang L Wang B H Peng S Jiang D S Dimitrovand C T Tseng 2016 Passive transfer of A Germline-like neutralizing human monoclonal antibody Protectstransgenic mice against lethal Middle East respiratorysyndrome coronavirus infection Sci Rep 631629doi 101038srep31629

[27] Houser K V L Gretebeck T Ying Y Wang LVogel E W Lamirande K W Bock I N Moore DS Dimitrov and K Subbarao 2016 ProphylaxisWith a Middle East respiratory syndrome coronavirus(MERS-CoV)-specific human monoclonal antibodyProtects Rabbits from MERS-CoV infection J InfectDis 2131557ndash1561 doi 101093infdisjiw080

[28] Yu X S Zhang L Jiang Y Cui D Li D Wang NWang L Fu X Shi Z Li L Zhang and X Wang

2015 Structural basis for the neutralization of MERS-CoV by a human monoclonal antibody MERS-27Sci Rep 513133 doi 101038srep13133

[29] Wang L W Shi M G Joyce K Modjarrad Y ZhangK Leung C R Lees T Zhou H M Yassine MKanekiyo Z Y Yang X Chen M M Becker MFreeman L Vogel J C Johnson G Olinger J PTodd U Bagci J Solomon D J Mollura LHensley P Jahrling M R Denison S S Rao KSubbarao P D Kwong J R Mascola W P Kongand B S Graham 2015 Evaluation of candidate vac-cine approaches for MERS-CoV Nat Commun67712 doi 101038ncomms8712

[30] Wang L W Shi J D Chappell M G Joyce Y ZhangM Kanekiyo M M Becker N van Doremalen RFischer N Wang K S Corbett M Choe R D MasonJ G Van Galen T Zhou K O Saunders K M TattiL M Haynes P D Kwong K Modjarrad W P KongJ S McLellan M R Denison V J Munster J RMascola and B S Graham 2018 Importance of neutra-lizing monoclonal antibodies targeting multiple anti-genic sites on MERS-CoV spike to avoid neutralizationescape J Virol doi JVI02002-17 [pii]

[31] Qiu X G Wong J Audet A Bello L Fernando J BAlimonti H Fausther-Bovendo H Wei J Aviles EHiatt A Johnson J Morton K Swope O BohorovN Bohorova C Goodman D Kim M H Pauly JVelasco J Pettitt G G Olinger K Whaley B Xu JE Strong L Zeitlin and G P Kobinger 2014Reversion of advanced Ebola virus disease in nonhu-man primates with ZMapp Nature 51447ndash53 doi101038nature13777

[32] Saphire E O and M J Aman 2016 Feverish Questfor Ebola Immunotherapy Straight or CocktailTrends Microbiol 24684ndash686 doi S0966-842X(16)30049-X [pii]

[33] Saphire E O S L Schendel M L Fusco KGangavarapu B M Gunn A Z Wec P J HalfmannJ M Brannan A S Herbert X Qiu K Wagh S HeE E Giorgi J Theiler K B J Pommert T B KrauseH L Turner C D Murin J Pallesen E Davidson RAhmed M J Aman A Bukreyev D R Burton J ECrowe Jr C W Davis G Georgiou F Krammer CA Kyratsous J R Lai C Nykiforuk M H Pauly PRijal A Takada A R Townsend V Volchkov L MWalker C I Wang L Zeitlin B J Doranz A BWard B Korber G P Kobinger K G Andersen YKawaoka G Alter K Chandran J M Dye and ViralHemorrhagic Fever Immunotherapeutic Consortium2018 Systematic analysis of monoclonal antibodiesagainst Ebola virus GP defines Features that contributeto protection Cell 174938ndash952 e13 doi S0092-8674(18)30960-7 [pii]

[34] Kyratsous C N Stahl and S Sfvapalasingam 2015Regeneron Pharmaceuticals Inc International publi-cation number WO2015179535

[35] Walls A C M A Tortorici J Snijder X Xiong B JBosch F A Rey and D Veesler 2017 Tectonic con-formational changes of a coronavirus spike glyco-protein promote membrane fusion Proc Natl AcadSci U S A 11411157ndash11162 doi 101073pnas1708727114

[36] Li K C Wohlford-Lenane S Perlman J Zhao A KJewell L R Reznikov K N Gibson-Corley D KMeyerholz and P B McCray Jr 2016 Middle East res-piratory syndrome coronavirus causes multiple OrganDamage and lethal disease in mice transgenic for

Emerging Microbes amp Infections 529

human Dipeptidyl Peptidase 4 J Infect Dis 213712ndash722 doi 101093infdisjiv499

[37] World Health Organization httpwwwwhointblueprintwhatresearch-developmentmeeting-report-prioritizationpdfua = 1

[38] Bossart K N and C C Broder 2006 Developmentstowards effective treatments for Nipah and Hendravirus infection Expert Rev Anti Infect Ther 443ndash55 doi 101586147872104143

[39] Lu L L T J Suscovich S M Fortune and G Alter2017 Beyond binding antibody effector functions ininfectious diseases Nat Rev Immunol 1846ndash61doi 101038nri2017106

[40] Overdijk M B S Verploegen A Ortiz Buijsse TVink J H Leusen W K Bleeker and P W Parren2012 Crosstalk between human IgG isotypes and mur-ine effector cells J Immunol 1893430ndash3438 doijimmunol1200356 [pii]

[41] Audet J G Wong H Wang G Lu G F Gao GKobinger and X Qiu 2015 Molecular characteriz-ation of the monoclonal antibodies composingZMAb a protective cocktail against Ebola virus SciRep 46881 doi 101038srep06881

[42] Qiu X J Audet G Wong S Pillet A Bello T CabralJ E Strong F Plummer C R Corbett J B Alimontiand G P Kobinger 2012 Successful treatment of ebolavirus-infected cynomolgus macaques with monoclonalantibodies Sci Transl Med 4138ra81 doi 101126scitranslmed3003876

[43] Pal P K A Dowd J D Brien M A Edeling SGorlatov S Johnson I Lee W Akahata G J NabelM K Richter J M Smit D H Fremont T C PiersonM T Heise and M S Diamond 2013 Developmentof a highly protective combinationmonoclonal antibodytherapy against Chikungunya virus PLoS Pathog 9e1003312 doi 101371journalppat1003312

[44] Bakker A B W E Marissen R A Kramer A B RiceW C Weldon M Niezgoda C A Hanlon S ThijsseH H Backus J de Kruif B Dietzschold C ERupprecht and J Goudsmit 2005 Novel humanmonoclonal antibody combination effectively neutra-lizing natural rabies virus variants and individual invitro escape mutants J Virol 799062ndash9068 doi 79149062 [pii]

[45] Enjuanes L S Zuniga C Castano-Rodriguez JGutierrez-Alvarez J Canton and I Sola 2016Molecular Basis of coronavirus Virulence and vaccinedevelopment Adv Virus Res 96245ndash286 doiS0065-3527(16)30042-2 [pii]

[46] Corti D J Voss S J Gamblin G Codoni A MacagnoD Jarrossay S G Vachieri D Pinna A Minola FVanzetta C Silacci B M Fernandez-Rodriguez GAgatic S Bianchi I Giacchetto-Sasselli L Calder FSallusto P Collins L F Haire N Temperton J PLangedijk J J Skehel andA Lanzavecchia 2011 A neu-tralizing antibody selected fromplasma cells that binds togroup 1 and group 2 influenza A hemagglutininsScience 333850ndash856 doi 101126science1205669

[47] Furuyama W A Marzi A Nanbo E Haddock JMaruyama H Miyamoto M Igarashi R YoshidaO Noyori H Feldmann and A Takada 2016Discovery of an antibody for pan-ebolavirus therapySci Rep 620514 doi 101038srep20514

[48] Gilchuk P N Kuzmina P A Ilinykh K Huang B MGunn A Bryan E Davidson B J DoranzH L TurnerM L Fusco M S Bramble N A Hoff E Binshtein NKose A I Flyak R Flinko C Orlandi R Carnahan EH Parrish A M Sevy R G Bombardi P K Singh PMukadi J J Muyembe-Tamfum M D Ohi E OSaphire G K Lewis G Alter A B Ward A WRimoin A Bukreyev and J E Crowe Jr 2018Multifunctional Pan-ebolavirus antibody recognizes asite of broad Vulnerability on the ebolavirus glyco-protein Immunity doi S1074-7613(18)30300-5 [pii]

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530 I Widjaja et al