Single-dose treatment with a humanized neutralizing antibody affords full protection of a human transgenic mouse model from lethal Middle East respiratory syndrome (MERS)-coronavirus infection

2.2. Preparation of recombinant proteins

MERS-CoV RBD proteins were prepared as previously described (Ma et al., 2014). Briefly, MERS-CoV RBDs (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"AFS88936.1","term_id":"407076737","term_text":"AFS88936.1"}}AFS88936.1, residues 377–588 of the spike protein) fused with a C-terminal human Fc or His₆ tag were transiently expressed in a 293T cell culture system and purified using HiTrap Protein A FF (Fc-fused proteins) or HisTrap HP (His₆-tagged proteins) (GE Healthcare, Little Chalfont, UK). Mutant RBD proteins were generated by site-specific polymerase chain reaction (PCR) mutagenesis and confirmed by sequencing analysis. Mutant RBDs containing a C-terminal His₆ tag were transiently expressed in 293T cell supernatants and purified by HisTrap HP as described above.

2.3. Generation of humanized mAb hMS-1

The hMS-1 mAb was generated by humanizing the murine mAb Mersmab1 using a previously described protocol (Boulianne et al., 1984). Briefly, total RNA was isolated from the Mersmab1 hybridoma cells and used to synthesize cDNA. Primers mVH-f (5′ ATG GRA TGG AGC TGG ATC TT 3′) and mVH-r (5′ ATA GAC AGA TGG GGG TGT CGT TTT GGC 3′) were used to amplify the variable region of the heavy chain (V _H) gene, and mVK-f (5′ ATG GAG WCA GAC ACA CTC CT 3′) and mVK-r (5′ GGA TACA GTT GGT GCA GCA TC 3′) (Du et al., 2013a) were used for the variable region of the light chain (V _L) gene. Sequence-confirmed V _H and V _L genes were inserted into the pRBO/Ig expression vector (containing human IgG1-and human immunoglobulin kappa chain constant regions). Recombinant plasmid was transfected into 293F cells and humanized mAb was purified from the cell culture supernatants. Purified hMS-1 mAbs were resolved by reducing 12% sodium dodecyl sulphate-polyacrylamide electrophoresis (SDS-PAGE) and non-reducing 8% SDS-PAGE, respectively.

2.4. Enzyme-linked immunosorbent assay (ELISA)

The reactivity of hMS-1 and MERS-CoV RBD was detected by ELISA as described (Du et al., 2014). Briefly, ELISA plates were coated overnight at 4 °C with His₆-tagged RBD protein (1 μg/ml) and blocked with 2% no-fat milk at 37 °C for 2 h. Serial diluted hMS-1 or trastuzumab control (Roche, Roswell, GA, USA) was added to the plates and incubated at 37 °C for 1 h. Horseradish peroxidase-conjugated goat anti-human IgG (1:5,000, Santa Cruz Biotechnology, Dallas, TX, USA) was then added and further incubated at 37 °C for 1 h. The reaction was visualized by TMB substrate addition (Invitrogen, Carlsbad, CA, USA) and stopped by 1N H₂SO₄. The absorbance at 450 nm was measured using an ELISA plate reader (BioTek, Winooski, VT, USA). The half-maximal effective concentration (EC₅₀) of hMS-1 was calculated using GraphPad Prism 5.01 software (La Jolla, CA, USA). hMS-1 epitope mapping was performed by ELISA as described above except that the ELISA plates were coated with a series of mutant RBD proteins (2.5 μg/ml).

2.5. Surface plasmon resonance (SPR)

The affinity of hMS-1 binding to the MERS-CoV RBD was assayed using a Biacore T100 instrument (GE Healthcare, Little Chalfont, UK). hMS-1 (5 μg/ml) was immobilized on a CM5 sensor chip with an amine coupling kit. The reference flow cell was treated with amine coupling reagent without exposure to hMS-1. The running buffer was HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20). His₆-tagged RBD protein at gradient concentrations (2, 4, 8, 16, and 32 nM) was flowed over the chip surface. The chip was regenerated with 100 mM H₃PO₄. The sensorgram was analyzed using BIAevaluation software, and the data were fitted to a 1:1 binding model.

2.6. MERS-CoV neutralization assay

The MERS-CoV neutralization assay was performed as described (Du et al., 2013b, Tao et al., 2013). Briefly, serially 2-fold-diluted hMS-1 or a trastuzumab control was incubated with 100 50% tissue culture infective doses (TCID₅₀) of MERS-CoV at 37 °C for 1 h followed by incubation with Vero cells at 37 °C for 72 h. The cytopathic effect (CPE) was observed daily and recorded on day 3 following inoculation. The 50% neutralizing dose (ND₅₀) was calculated by the Reed-Muench method (Reed and Muench, 1938) and determined as the antibody concentration that completely inhibited virus-induced CPE in at least 50% of the wells.

2.7. Flow cytometric analysis

Determination of the hMS-1 inhibitory ability against the binding between MERS-CoV RBD and hDPP4-expressing Huh-7 cells was performed by flow cytometry as described (Du et al., 2013b). Briefly, Huh-7 cells (5 × 10⁵) were incubated with Fc-fused RBD protein (2 μg/ml) in the presence of serially diluted hMS-1 or with the trastuzumab control at room temperature for 30 min, followed by incubation with DyLight 488-labeled goat anti-human IgG antibody at room temperature for 30 min. The fluorescence signals were analyzed by flow cytometry (Guava, EMD Millipore, Germany).

2.8. Pseudovirus neutralization assay

The pseudovirus neutralization assay was performed as described (Zhao et al., 2013). Briefly, 293T cells were co-transfected with plasmids encoding an Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and encoding the MERS-CoV spike protein (EMC2012 strain) or MERS-CoV emergent strains with the corresponding RBD mutations, to package pseudotyped MERS-CoVs. Pseudoviruses were incubated with serially diluted hMS-1 at 37 °C for 1 h and the mixture was then added to Huh-7 cells pre-plated in 96-well tissue culture plates (10⁴/well) 6 h before inoculation. After 72 h, the cells were lysed with cell lysis buffer (Promega, Madison, WI, USA) and transferred into 96-well luminometer plates. The luciferase activity was determined using an Infinite M1000 luminometer (Tecan, San Jose, CA, USA). ND₅₀ was calculated using the dose-response inhibition model in GraphPad Prism.

2.9. Multiple sequence alignment of MERS-CoV RBDs

A total of 278 full-length spike protein sequences were collected from the MERS Coronavirus Database of The Virus Variation Resource (Brister et al., 2014) for multiple sequence alignment using the MUSCLE program (Edgar, 2004). GenBank accession numbers of selected MERS-CoV isolates are listed in Supplementary materials and methods.

2.10. Animal infection and antiviral therapy

We used 8–10-week-old female hDPP4-Tg mice to evaluate the hMS-1 passive therapeutic effect against lethal MERS-CoV infection (Zhao et al., 2015). Following intraperitoneal anesthetization with sodium pentobarbital (5 mg/kg of body weight), mice under lethal virus infection were intranasally inoculated with 10^4.6 TCID₅₀ MERS-CoV (EMC2012 strain) in 20 μl Dulbecco’s modified Eagle’s medium (DMEM). After 24 h, the mice were intravenously injected with 2 mg/kg purified hMS-1 or the trastuzumab mAb control. Infected mice were observed for 23 days to calculate the body weight change and survival rate. Additionally, 5 mice were sacrificed from each group on day 3 post-infection and lung tissues were collected for virological detection and histopathological evaluation.

2.11. Viral titer detection

Viral titer was detected as described (Zhao et al., 2015). Briefly, mouse lung tissues were homogenized in DMEM plus antibiotics to achieve 10% (w/v) suspensions, which were then titrated on monolayers of Vero cells in quadruplicate. The CPE was observed daily under phase-contrast microscopy for 3 days. The viral titer was calculated by the Reed-Muench method and expressed as log₁₀TCID₅₀/g of tissue.

2.12. Histopathology analysis

Collected tissues were immediately fixed in 10% neutral buffered formalin and then embedded in paraffin. Sections (4 μm in thickness) were generated, mounted onto slides, and stained by hematoxylin and eosin (H&E). Histopathological changes were observed using light microscopy. The extent of lung injury was scored according to the levels of degeneration and necrosis of the bronchi and bronchiolar epithelium, infiltration of inflammatory cells, alveoli degeneration and collapse, expansion of the parenchymal wall, hemorrhage, and interstitial edema (Sun et al., 2013).

3.2. Humanized mAb hMS-1 neutralizes MERS-CoV infection by blocking the binding of MERS-CoV RBD to the hDPP4 receptor

The neutralizing activity of hMS-1 against live MERS-CoV infection was determined. hMS-1 could potently neutralize infection of live MERS-CoV (EMC2012 strain) in permissive Vero cells with an ND₅₀ of 3.34 μg/ml (Table 1) whereas trastuzumab did not exhibit antiviral activity against MERS-CoV infection. These results suggest that humanized hMS-1 maintained the potent ability to neutralize MERS-CoV infection.

Since hMS-1 targeted the MERS-CoV RBD, its neutralization mechanism was thus investigated by measuring its ability to block the binding between MERS-CoV RBD and the hDPP4 receptor by flow cytometric analysis (Du et al., 2014). Whereas high fluorescence intensity was detected in Huh-7 cells incubated with RBD protein alone, only background fluorescence levels were observed in the RBD-Huh-7 binding samples in the presence of hMS-1 at 5 μg/ml. This suggested that hMS-1 was able to strongly block the binding between MERS-CoV RBD and cell-associated hDPP4 (Fig. 2 A) and that the blocking effect occurred in a dose-dependent manner (Fig. 2B). In contrast, trastuzumab did not affect MERS-CoV RBD binding to the DPP4-expressing Huh-7 cells (Fig. 2A and B). These results strongly suggest that hMS-1 neutralized MERS-CoV infection by inhibiting the binding of MERS-CoV RBD to hDPP4.

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Fig. 2

hMS-1 blocks the binding between MERS-CoV RBD and the hDPP4 receptor. The blocking effect of hMS-1 on the cell-binding activity of MERS-CoV RBD was determined by flow cytometric analysis. (A) Blockage of Fc-fused MERS-CoV RBD protein binding to Huh-7 cells by 20 μg/ml hMS-1. Black line, Huh-7 cell control; purple line, binding of Fc-fused RBD to Huh-7 cells in the absence of antibody; blue line, irrelevant Trastuzumab mAb control; red line, MERS-CoV RBD binding of Huh-7 cells in the presence of hMS-1. (B) Flow cytometric analysis shows that hMS-1 inhibited the binding between the Fc-fused RBD protein and Huh-7 cells in a dose-dependent manner. The data are presented as the mean percentages of inhibition ± SEM (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. Humanized mAb hMS-1 recognizes conserved RBD epitopes and cross-neutralizes MERS-CoV evolutionary strains

To identify the epitopes on the MERS-CoV RBD recognized by hMS-1, we utilized a panel of mutant RBD proteins generated by mutating the key residues at the MERS-CoV RBD and the hDPP4 binding interface to alanine (Du et al., 2014); the binding between hMS-1 and the respective mutant RBD proteins was then measured by ELISA as described (Gao et al., 2013, Lu et al., 2013). hMS-1 was not capable of recognizing the RBD of the R511A mutant and reduced its binding to RBDs with mutations at D510A and W553A, respectively (Fig. 3 A). In comparison, the remaining mutations on the RBD did not affect the binding between hMS-1 and RBD with the exception of L506A, which exhibited a slight loss of binding activity. A pseudovirus neutralization assay further revealed that pseudotyped MERS-CoV expressing the spike protein containing R511A, D510A, or W553A mutants completely abolished (R511A), or reduced (D510A or W553A) the inhibitory effect of hMS-1 on pseudovirus infection. In contrast, L506A did not change the ability of hMS-1 to inhibit MERS pseudovirus infection (Fig. 3B). These results indicate that the R511 residue of RBD is a crucial neutralizing epitope recognized by hMS-1.

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Fig. 3

Mapping of epitopes recognized by hMS-1 in MERS-CoV RBD. (A) Binding of hMS-1 to RBD mutant proteins as detected by ELISA. (B) Neutralizing activity of hMS-1 to pseudovirus mutants in a pseudovirus-based neutralization assay. Data are presented as the means ± SEM (n = 2). Protein and pseudovirus without mutations served as the wild type (WT) controls.

Alignment of 278 full-length spike protein sequences deposited in the NCBI MERS-CoV database illustrated that the RBD epitope recognized by hMS-1 is highly conserved among MERS-CoV emergent isolates (Supplementary Fig. 1). In particular, the R511 residue was not naturally mutated during MERS-CoV evolution, indicating the ability of hMS-1 to cross-neutralize MERS-CoV emergent strains. To further understand whether the natural RBD mutations of the evolved strains of MERS-CoV isolated from the 2012 to 2015 outbreaks affected the hMS-1 neutralizing activity, we constructed a series of pseudoviruses expressing MERS-CoV spike proteins with these mutated RBD residues. hMS-1 exhibited potent neutralizing activities toward all MERS mutant pseudoviruses tested, with ND₅₀ values ranging from 0.009 to 0.091 μg/ml (Table 2 ). These results suggest that hMS-1 exhibits the ability to cross-neutralize emergent MERS-CoV isolates.

This work was supported by the China National Program of Infectious Disease fund 2014ZX10004001004, an NIH grant (R21AI109094), and an intramural fund of the New York Blood Center (NYB000348). The authors declared no conflict of interest.