Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus

Abstract

Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a previously unidentified coronavirus (CoV), likely transmitted to humans by infected camels. There is no licensed vaccine or antiviral for MERS, therefore new prophylactic and therapeutic strategies to combat human infections are needed. In this study, we describe, for the first time, to our knowledge, the isolation of a potent MERS-CoV-neutralizing antibody from memory B cells of an infected individual. The antibody, named LCA60, binds to a novel site on the spike protein and potently neutralizes infection of multiple MERS-CoV isolates by interfering with the binding to the cellular receptor CD26. Importantly, using mice transduced with adenovirus expressing human CD26 and infected with MERS-CoV, we show that LCA60 can effectively protect in both prophylactic and postexposure settings. This antibody can be used for prophylaxis, for postexposure prophylaxis of individuals at risk, or for the treatment of human cases of MERS-CoV infection. The fact that it took only 4 mo from the initial screening of B cells derived from a convalescent patient for the development of a stable chinese hamster ovary (CHO) cell line producing neutralizing antibodies at more than 5 g/L provides an example of a rapid pathway toward the generation of effective antiviral therapies against emerging viruses.

Keywords: MERS-CoV; emerging viruses; neutralizing antibody; serotherapy.

Fig. 1.

Isolation of the LCA60 antibody from the memory B cells of a MERS-CoV–infected individual. MERS survivor plasma samples (two time points, T1 and T2, corresponding to 5 and 8 mo after infection, respectively) (A) and supernatants from EBV-immortalized B-cell cultures (B) were tested for the presence of MERS-CoV pseudovirus (London1/2012, abbreviated as LD1) neutralizing antibodies. LCA60 and LCA57 antibodies were tested for their ability to neutralize the homologous London1/2012-CoV (C) and bind to S protein (EMC/2012) (D). The LCA60 antibody was further tested against three infectious MERS-CoV isolates (E) and several EMC MERS-CoV MARMs (F).

Fig. 2.

LCA60 binds to S protein with high affinity. SPR sensograms showing the CoV S protein (EMC/2012) association and dissociation with the LCA57, LCA60, 3B11, and CD26 receptor. The antibodies and the receptor were immobilized on the sensor surface, followed by injection of S protein at the concentrations indicated in the figure. The line fitted to the experimental data and used to calculate the binding affinities is drawn in gray. KD values are reported in the table.

Fig. S1.

Structure of MERS-CoV RBD of the S protein and tested mutants. Residues important for binding of LCA60 antibody are shown in red on the white surface representation of the S protein RBD. Residues not affecting the binding of LCA60 when mutated are shown in blue.

Fig. S2.

Predicted structure of LCA60 antibody. Ten different antibody models were generated and used for docking. Antigen binding loops are shown in pink and violet on the cartoon representation of the antibody variable region. The main differences among the models involve the HCDR3 loop, whose structure does not follow known rules and thus cannot be predicted as accurately as other CDRs.

Fig. 3.

Computational models of MERS-CoV RBD in complex with antibody LCA60. (A and B) Structures of the complex between mAb LCA60 (light green and orange cartoons) and RBD–MERS (white surface) obtained by computational docking. S protein mutants that affect (red) or do not affect (blue) antibody binding are shown. Only model B is consistent with the mutagenesis data. (C) Experimental structure of the complex between CD26 (light blue cartoon) and MERS–RBD [Protein Data Bank (PDB) ID code 4KR0]. (D) Footprint of LCA60 antibody (light green) and CD26 (light blue) on the surface of MERS–RBD (white). Residues in contact with both LCA60 and CD26 are shown in violet.

Fig. S3.

Computational docking results, scoring chart. After an initial “global search” (not shown), selected docking models were subjected to a refinement stage, shown here for model A (Top) and model B (Bottom). In these charts, each diamond represents a docking model (∼10,000 were generated); the rmsd from a reference structure is shown on the x axis; the docking algorithm score is shown on the y axis. Lower values indicate more favorable conformations. The presence of scoring funnels (bottom left of the two charts) representing several models with the same structure and a good docking score is often considered an indication of reliable docking results.

Fig. S4.

Alignment of 21 sequences of the S protein RBD representative of 123 isolates from 2012 to 2015. Highlighted in red are the four positions at which mutations confer loss of LCA60 binding. Shown are the accession numbers for each sequence and in parentheses the number of isolates with identical sequence in the analyzed amino acid region.

Fig. 4.

LCA60 blocks MERS-CoV RBD binding to its cognate cellular receptor CD26. Cross-competition SPR assay was used to investigate the epitopes region; LCA57, LCA60, 3B11 antibodies and CD26 receptor (ligands) were first immobilized on different channels (vertical column) of the SPR chip. The spike protein was subsequently injected to form the complexes; finally, the three antibodies and the receptor (analytes) were injected one in each horizontal row. If a binding event is detected (red borders rectangle), the analyte has a different epitope from the ligand immobilized in the first step; if no binding is detected, ligand and analyte share overlapping epitopes.

Fig. 5.

Passive transfer of LCA60 confers protection to mice prophylactically and therapeutically. We transferred 200 µL of diluted antibodies in PBS into Ad5-hDPP4–transduced BALB/c mice (6 wk, female) intraperitoneally 1 d before (A and C) or after (B and D) MERS-CoV EMC/2012 (EMC) (A and B) or London1/2012 (LD1) (C and D) infection. Mice were then infected with 1 × 10⁵ pfu MERS-CoV intranasally. Virus titers in the lungs were measured at the indicated time points. (E) Intranasally delivered LCA60 protected MERS-CoV–infected mice. We transferred 50 µL of diluted antibodies in PBS intranasally into Ad5-hDPP4–transduced BALB/c mice (6 wk, female) 1 d after infection with 1 × 10⁵ pfu of the EMC/2012 strain of MERS-CoV. Virus titers in the lungs were measured on day 3 after infection. Titers are expressed as pfu/g tissue. n = 3–4 mice per group per time point. *P < 0.05 compared with hIgG1 group.

Fig. S5.

LCA60 antibody does not prevent infection. We transferred 200 µL of diluted antibodies in PBS into Ad5-hDPP4–transduced BALB/c mice (6 wk, female) at a dose of 15 mg/kg intraperitoneally 1 d before intranasal infection with MERS-CoV EMC (A and B) or EMC-LCA60 MARM (mutant) strains. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as pfu/g tissue. n = 3 mice per group per time point. Of note, plaques of the LCA escape mutant strain are much smaller than those of the EMC strain.

Fig. 6.

LCA60 protected MERS-CoV–infected IFNAR-KO mice. We transferred 200 µL of diluted antibodies (15 mg/kg) in PBS into Ad5-hDPP4–transduced IFNAR-KO mice (6–12 wk) intraperitoneally 1 d after MERS-CoV infection. Mice were then infected with 1 × 10⁵ pfu MERS-CoV London1/2012 strain (A) or EMC/2012 strain (B–E) intranasally. Virus titers in the lungs were measured at the indicated time points. Additional groups of animals were monitored daily for morbidity, mortality, and body weight loss up to 14 d after infection (C). The histologic analysis was performed on animals killed on day 7 after infection (D and E). Mice were anesthetized and transcardially perfused with PBS followed by zinc formalin. Lungs were removed, fixed in zinc formalin, and paraffin embedded. Sections were stained with hematoxylin and eosin for histological analysis. (F) Intranasally delivered LCA60-protected MERS-CoV–infected mice. We transferred 50 µL of diluted antibody (10 mg/kg) in PBS intranasally into Ad5-hDPP4–transduced IFNAR-KO mice (6 wk, female) 1 d after infection with 1 × 10⁵ pfu of the EMC/2012 strain of MERS-CoV. Virus titers in the lungs were measured at the indicated time points. Titers are expressed as pfu/g tissue. n = 3 mice per group per time point. *P < 0.05 compared with hIgG1 or PBS groups.