RESEARCH ARTICLE INFECTIOUS DISEASE
Potent Neutralization of MERS-CoV by Human Neutralizing Monoclonal Antibodies to the Viral Spike Glycoprotein
The recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans. However, no prophylactic and therapeutic agents specifically against MERS-CoV are currently available. Entry of MERS-CoV into target cells depends on binding of the receptor binding domain (RBD) of the viral envelope spike glycoprotein to the cellular receptor dipeptidyl peptidase 4 (DPP4). We report the isolation and characterization of two potent human RBD-specific neutralizing monoclonal antibodies (MERS-4 and MERS-27) derived from single-chain variable region fragments of a nonimmune human antibody library. MERS-4 and MERS-27 inhibited infection of both pseudotyped and live MERS-CoV with IC50 (half-maximal inhibitory concentration) at nanomolar concentrations. MERS-4 also showed inhibitory activity against syncytia formation mediated by interaction between MERS-CoV spike glycoprotein and DPP4. Combination of MERS-4 and MERS-27 demonstrated a synergistic effect in neutralization against pseudotyped MERS-CoV. Biochemical analysis indicated that MERS-4 and MERS-27 blocked RBD interaction with DPP4 on the cell surface. MERS-4, in particular, bound soluble RBD with an about 45-fold higher affinity than DPP4. Mutagenesis analysis suggested that MERS-4 and MERS-27 recognized distinct regions in RBD. These results suggest that MERS-4 and MERS-27 are RBD-specific potent inhibitors and could serve as promising candidates for prophylactic and therapeutic interventions against MERS-CoV infection.
INTRODUCTION The recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans (1). Symptoms include fever, cough, and shortness of breath, similar to those seen during the outbreak of severe acute respiratory syndrome (SARS) in 2003, which was caused by the coronavirus SARS-CoV (2–8). MERS-CoV and related coronaviruses have been found in several animal species such as bats and dromedary camels (9–13). Genetic and phylogenetic characterization has shown that MERS-CoV belongs to lineage C of the genus of Betacoronavirus and is closely related to Tylonycteris bat coronavirus HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat coronavirus HKU5 (Pi-BatCoV HKU5), although the direct source and reservoirs of MERS-CoV remain uncertain (3, 5, 6, 9, 14–20). The high fatality rate (~40%) (http://www.who.int/csr/don/2014_01_09/en/index. html) and clusters of human-to-human transmission (21, 22) of this deadly virus have raised global concern about the potential for a MERS pandemic. Although treatment with interferon and ribavirin improves clinical outcome of MERS-CoV–infected rhesus macaques (23), and to a lesser extent in infected human (24), no MERS-CoV– specific treatment or vaccine is currently available (25–28). Clearly, 1 Comprehensive AIDS Research Center, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing 100084, China. 2Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China. 3 School of Life Sciences, Tsinghua University, Beijing 100084, China. 4Department of Microbiology, The University of Hong Kong, Hong Kong, China. 5Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China. *These authors contributed equally to this work. †Corresponding author. E-mail: [email protected] (L.Z.); xinquanwang@ mail.tsinghua.edu.cn (X.W.); [email protected] (B.-J.Z.)
there is an urgent need for an effective antiviral therapy for this emerging global threat. Like other coronaviruses, MERS-CoV uses its envelope spike (S) glycoprotein for interaction with a cellular receptor for entry into the target cell (4). The S glycoprotein consists of a globular S1 domain at the N-terminal region, followed by membrane-proximal S2 domain and transmembrane domain (7, 8, 29–31). Determinants of host range and cellular tropism are located in the receptor binding domain (RBD) within the S1 domain, whereas mediators of membrane fusion have been identified within the S2 domain (7, 8, 29, 30). Through copurification with the MERS-CoV S1 domain, Raj and colleagues identified that dipeptidyl peptidase 4 (DPP4; also called CD26) functions as a cellular receptor for MERS-CoV (4). We and others have recently determined the crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4 (32, 33). We showed that MERS-CoV RBD consists of a core and a receptor binding subdomain. The receptor binding subdomain directly interacts with blades 4 and 5 of DPP4 propeller but not its intrinsic hydrolase domain (32, 33). This finding suggests that agents capable of disrupting such interaction could serve as potential candidates for blocking entry of MERS-CoV into the target cell. Indeed, both polyclonal and monoclonal antibodies (mAbs) directed against DPP4 have been shown to inhibit MERS-CoV infection of primary human bronchial epithelia cells and Huh7 cells (4, 34). However, considering the important roles of DPP4 in immune regulation of T cell activation and chemokine function (35), antibodies against DPP4 may potentially lead to unwanted adverse effects when applied to human use. Antibodies directed to the viral receptor binding subdomain would therefore be more favored. Furthermore, RBDbased vaccine studies in experimental animals showed that induced
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Liwei Jiang,1* Nianshuang Wang,2* Teng Zuo,1,3 Xuanling Shi,1 Kwok-Man Vincent Poon,4 Yongkang Wu,5 Fei Gao,1 Danyang Li,1 Ruoke Wang,1 Jianying Guo,1 Lili Fu,1 Kwok-Yung Yuen,4 Bo-Jian Zheng,4† Xinquan Wang,2† Linqi Zhang1†
RESEARCH ARTICLE
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Fig. 1. Isolation and sequence analysis of MERS-CoV RBD–specific scFvs from nonimmune human antibody library displayed on the surface of yeast S. cerevisiae. (A) Flow cytometry analysis of sorted yeast population after second MACS (MACS2), first FACS (FACS1), and second FACS (FACS2). (B) Fourteen yeast clones with detectable binding specificity to MERS-CoV RBD. Negative controls include yeast clones either not induced in SG-CAA medium (Negative-1) or induced but no RBD was added to the medium (Negative-2). Cmyc is a protein tag used for monitoring scFv expression under induction condition. (C) Sequences of complementarity-determining region 3 (CDR3) for both VH and VL of the 14 yeast clones, together with their family designations and degree of similarity compared to their germline sequences.
polyclonal antibodies directed against MERS-CoV RBD can strongly inhibit the entry of MERS-CoV (36–39). These results provide the critical proof of concept that antibody directed against RBD is an effective way of blocking entry of MERS-CoV. Here, we report isolation and characterization of two potent human neutralizing mAbs against both pseudotyped and live MERS-CoV infection from nonimmune human antibody library. The mechanism of action of these neutralizing mAbs was to block interaction between the RBD on the S glycoprotein of MERS-CoV and cellular receptor DPP4. We believe the neutralizing mAbs identified here hold great promise for the development of prophylactic and therapeutic interventions against MERS-CoV infection.
RESULTS Isolation of MERS-CoV RBD–specific single-chain variable region fragments from nonimmune human antibody library We used purified and biotin-labeled soluble MERS-CoV RBD as antigen bait to select antibodies from nonimmune human single-chain variable region fragment (scFv) library displayed on the surface of yeast Saccharomyces cerevisiae (40, 41). The selection process consisted of two rounds of magnetic bead–activated cell sorting (MACS) followed by additional two rounds of florescence-activated cell sorting (FACS). Increasing proportion of RBD-specific yeast www.ScienceTranslationalMedicine.org
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Neutralizing activity of human immunoglobulin G1 form of selected scFv against pseudotyped and live MERS-CoV infection To evaluate neutralizing activity of selected 14 scFvs, we generated bivalent full-length human mAbs by fusing the variable region of each scFv with the constant region fragment of human immunoglobulin G1 (IgG1). Once confirmed by sequencing, 14 pairs of full-length heavy and light chain plasmids were cotransfected into 293T cells for the production and purification of mAbs. Neutralizing activity of these mAbs was then tested on susceptible Huh7 cells against pseudovirus infection expressing the MERS-CoV S glycoprotein. As shown in Fig. 2A, two mAbs (MERS-4 and MERS-27) demonstrated neutralizing activities with IC50 (half-maximal inhibitory concentration) less than 100 nM; MERS-4 was 0.37 nM, whereas MERS-27 was 63.96 nM. Furthermore, neutralizing activities of these two mAbs were specific against MERS-CoV because none of the pseudovirus bearing the envelope glycoprotein of SARS-CoV, HIV-1, or VSV-G was susceptible to their neutralization (Fig. 2A). Neutralizing activities of these two mAbs against live MERS-CoV were also studied using the clinical isolate hCoV-EMC from a MERS-CoV–infected patient (6). As shown in Fig. 2B, both MERS-4 and MERS-27 demonstrated potent inhibitory activity against MERS-CoV infection, with IC50 of 3.33 and 13.33 nM, respectively. Such inhibitory activities were comparable to that against pseudotyped MERS-CoV, although clear variations existed between the live and pseudotyped systems. In both systems, however, MERS-4 showed more potent neutralizing activity than MERS-27. In addition, we evaluated inhibitory activities of these two mAbs on syncytia formation between cells expressing the MERS-CoV S glycoprotein and those expressing the receptor DPP4. Specifically, the monkey kidney fibroblast cell line COS7 was transfected with plasmids encoding either full-length MERS-CoV S protein or human receptor DPP4. Twentyfour hours after transfection, transfected cells were trypsinized, washed, and mixed at a 1:1 ratio in the presence of 100 mg/ml of each of the two mAbs. Syncytia formation was observed under the microscope 36 hours afterward. Consistent with the neutralization results, MERS-4 was the most potent and completely inhibited syncytia formation at the indicated concentration, whereas MERS-27 was relatively inefficient (Fig. 2C). These results suggested that both MERS-4 and MERS-27 are potent inhibitors against MERS-CoV, and the potential reasons for their differences in inhibitory capacity are explained and discussed below.
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population was observed from 0.77% after the second MACS, to 1.75% after the first FACS, and up to 24.72% after the second FACS (Fig. 1A). Plasmid DNA containing the coding sequences for scFv was extracted from the yeast population sorted by the second FACS and then transformed into Escherichia coli for the production of sufficient quantity for sequencing and sequence analysis. Among the total 120 plasmid sequences analyzed, 23 distinct scFv sequences were identified and 14 of which were confirmed specifically binding to MERS-CoV RBD once transformed back and expressed on the surface of the yeast (Fig. 1B). Each of the 14 scFvs had distinct VH and VL sequences (fig. S1). Some of the scFvs, however, share identical CDR3 sequences in VH such as MERS-2, MERS-72, and MERS-97 (Fig. 1C). One clone (MERS-97) completely lacked a VL. The gene families of the 14 scFvs were quite divergent for both VH (3 VH1, 6 VH2, 4 VH3, 1 VH4, and 1 VH6) and VL (3 Vl1, 1 Vl2, 2 Vl3, 1 Vk1, 5 Vk3, and 2 Vk3) (Fig. 1C).
Fig. 2. Neutralizing activities of MERS-4 and MERS-27 against pseudotyped (A) and live (B) MERS-CoV, and against syncytia formation of COS7 cells mediated by the MERS-CoV S glycoprotein and the receptor DPP4 (C). Pseudoviruses bearing the envelope glycoprotein from SARS-CoV, HIV-1, or VSV-G were used as controls. Data shown are average values from four (A) and two (B) independent experiments. Dashed line indicates 50% inhibition, and two numbers (13.33 and 3.33 nM) above it are the IC50s for MERS-27 and MERS-4, respectively. VRC01 is a human neutralizing mAb against HIV-1 and used as a control. In (C), negative control represents COS7 cells transfected with mouse DPP4, whereas positive control represents those transfected with human DPP4 expression vector.
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RESEARCH ARTICLE mAbs to RBD measured by surface plasmon resonance (SPR) are associated with their neutralizing activities. As shown in Fig. 3 (B and C), MERS-4 had about 45-fold higher binding affinity (Kd = 0.98 nM) to RBD than soluble DPP4 (Kd = 44.5 nM). MERS-27, however, shared comparable binding affinity (Kd = 71.2 nM) to RBD with DPP4 (Kd = 44.5 nM). Collectively, these data suggest that the primary mechanism of the neutralizing activity of MERS-4 and MERS-27 was through blocking of RBD binding to cellular receptor DPP4, even though differential efficiency was observed between the two, perhaps due to their differences in binding affinity and/or epitope recognized.
Cooperativity of the two neutralizing mAbs for virus neutralization Because both MERS-4 and MERS-27 blocked binding of RBD to cellular receptor DPP4, we went further to determine whether the combination of the two mAbs in virus neutralization was synergistic, additive, or antagonistic using the median effect analysis approach (42–44). To start with, a constant ratio between MERS-4 and MERS-27 was set by their respective IC50 concentrations as determined above in Fig. 2A. Dose-dependent neutralization activity for MERS-4, MERS-27, or the combination thereof was then evaluated by serial threefold dilutions in concentrations from 27 times the IC50 to 1/81 of the IC50. Figure 4 shows the average results from four independent experiments to determine percent neutralization (Fig. 4A), fractional effect (Fig. 4B), and the combination index (CI) (Fig. 4C) using the CompuSyn program. Percent neutralization for combined MERS-4 and MERS-27 was about 5.26-fold reduction in IC50 and 4.89-fold reduction in IC90 compared to that for MERS-4 or MERS-27 alone (Fig. 4A). Furthermore, the CI values of combined MERS-4 and MERS-27 at fractional effect values of effective dose 25, 50, 75, 90, and 95% (ED25, ED50, ED75, ED90, and ED95) were 0.22, 0.28, 0.34, 0.42, and 0.50, respectively. As a CI value of 1 indicates an additive effect, 1 indicates antagonism, the combined MERS-4 and MERS-7 are clearly in strong synergism (0.1 to 0.3) at ED25 and ED50 and synergism (0.3 to 0.7) at ED75, ED90, and ED95 (42, 43). Figure 4C shows a logarithmic CI scale, values greater than 0 are indicative of antagonism, and values less than 0 are indicative of synergism. Similarly, combination of MERS-4 and MERS-27 demonstrated strong synergistic effect at lower antibody concentration and modest degree of synergism at higher antibody concentraFig. 3. MERS-4 and MERS-27 blocked the interaction between soluble RBD and receptor DPP4 tion, perhaps due to certain degree of steric on the Huh7 cell surface (A) and their binding kinetics and affinity to RBD (B and C). Flow cy- hindrance in the binding of these two antitometry analysis of soluble RBD binding to receptor DPP4 in the presence of varying concentrations bodies when they were in higher abundance.
of MERS-4 (A, left) or MERS-27 (A, right). VRC01 is a human neutralizing mAb against HIV-1 and used as a control. Overlay of binding of varying concentrations of MERS-4 and MERS-27 to RBD immobilized on the CM5 sensor chip (B). The black lines indicate the experimentally derived curves, whereas the red lines represent fitted curves based on the experimental data. Kon (association rate, in M−1 s−1), Koff (dissociation rate, in s−1), Ka (association constant, in M−1), and Kd (dissociation constant, in M) are indicated for each paired interaction (C).
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Epitope mapping of MERS-4 and MERS-27 Because MERS-4 and MERS-27 appeared to be synergistic in neutralizing activity, 30 April 2014
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Blocking of RBD binding to receptor DPP4 by neutralizing mAbs Because all mAbs reported here were initially isolated on the basis of their recognition of RBD, observed neutralizing activity of MERS-4 and MERS-27 might be mediated through their competitive binding to RBD, thereby interfering or disrupting interaction between RBD and cellular receptor DPP4. To test this hypothesis, we first examined whether the two mAbs could inhibit the binding of soluble RBD to DPP4-expressing susceptible Huh7 cells. As shown in Fig. 3A, when Huh7 cells were incubated with soluble RBD in the presence of varying concentrations of the two mAbs and analyzed by flow cytometry, differential inhibitory effects were observed. Consistent with the neutralization results against live and pseudotyped MERS-CoV and inhibitory effect on syncytia formation, MERS-4 was more potent than MERS-27 in inhibiting interaction between soluble RBD and receptor DPP4 on the surface of Huh7 (Fig. 3A). At a concentration of 333 nM (about two times greater molar concentration than soluble RBD), MERS-4 completely blocked the binding of soluble RBD to Huh7 cells, and clear dose effect was also identified (Fig. 3A, left). In contrast, MERS-27 demonstrated relatively modest inhibitory activity under the same experimental conditions (Fig. 3A, right). Second, we studied whether the binding kinetics and affinities of the two
epitopes recognized by the two mAbs were likely to be different. To test this hypothesis, we generated a total of 14 mutant MERS-CoV S glycoproteins with single- or multiple-residue substitutions at the interface between RBD and DPP4 (Fig. 5A). All but two (13 and 14) pseudotyped viruses bearing the mutant S glycoprotein were able to infect Huh7 cells and therefore used to test for their relative resistance to MERS-4 and MERS-27 neutralization and compared to those bearing the wild-type S glycoprotein. As shown in Fig. 5 (A and B), several mutations resulted in substantial increases in resistance against neutralization of MERS-4. For instance, a single-residue substitution of D455A, E513A, and R542A increased resistant levels about 6-, 13-, and 3-fold against MERS-4, respectively, whereas the same mutation had little impact on resistance to MERS-27. Some residue substitutions such as Y499A, R511A, and, in particular, W553A increased sensitivity of MERS-CoV to MERS-4 and MERS-27 neutralization, presumably by affecting RBD structure in such a way allowing anti-
body easier to access and bind with higher affinity. Lastly, because none of the mutations had significantly increased resistance to MERS-27, its epitope or major residues recognized were likely located beyond the residues studied here. These findings suggest that the epitopes recognized by MERS-4 and MERS-27 are rather distinct, which is consistent with the coaction between the two mAbs in neutralizing activity against pseudotyped MERS-CoV. To understand the spatial relationship of the abovementioned residues and their differences in conferring resistance to MERS-4 and MERS-27, we highlighted their positions on the crystal structure of RBD previously resolved by our group (33). As shown in Fig. 5C, critical residues conferring resistance to MERS-4 (D455A, E513A, and R542A) constitute a wide triangle area (red) located right at the center of the interface between RBD and receptor DPP4. If the red triangle could indeed represent the footprint of MERS-4 on the RBD, it may help to explain why MERS-4 is such a potent mAb capable of inhibiting live and
Fig. 4. Effects of combined MERS-4 and MERS-27 in neutralizing pseudotyped MERS-CoV. (A) Percent neutralization was calculated for serial threefold dilutions of each antibody alone and in combination at constant ratios in a range of concentrations from 27 times to 1/81 of IC50s. The constant ratios of the combined antibodies were their IC50s. On the x axis, a dose of 1 was at the IC50 con-
centration. (B) Fractional effect (FA) plots generated by the CompuSyn program for MERS-4, MERS-27, and their combination showing dosage versus effect. (C) Median effect plot of calculated CI values (logarithmic) versus FA values, in which a log CI of 0 is antagonism. Data shown are average values from four independent experiments.
Fig. 5. Epitope mapping of MERS-4 and MERS-27 by mutagenesis (A and B) and spatial analysis of the key residues on the crystal structure of RBD (C). The residue substitutions and their locations on the RBD are indicated (A). Numbers in the column under MERS-4 and MERS-27 represent the fold changes in IC50 of mutant viruses relative to the wildtype (WT) (>1: increase resistance and
Potent Neutralization of MERS-CoV by Human Neutralizing Monoclonal Antibodies to the Viral Spike Glycoprotein
The recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans. However, no prophylactic and therapeutic agents specifically against MERS-CoV are currently available. Entry of MERS-CoV into target cells depends on binding of the receptor binding domain (RBD) of the viral envelope spike glycoprotein to the cellular receptor dipeptidyl peptidase 4 (DPP4). We report the isolation and characterization of two potent human RBD-specific neutralizing monoclonal antibodies (MERS-4 and MERS-27) derived from single-chain variable region fragments of a nonimmune human antibody library. MERS-4 and MERS-27 inhibited infection of both pseudotyped and live MERS-CoV with IC50 (half-maximal inhibitory concentration) at nanomolar concentrations. MERS-4 also showed inhibitory activity against syncytia formation mediated by interaction between MERS-CoV spike glycoprotein and DPP4. Combination of MERS-4 and MERS-27 demonstrated a synergistic effect in neutralization against pseudotyped MERS-CoV. Biochemical analysis indicated that MERS-4 and MERS-27 blocked RBD interaction with DPP4 on the cell surface. MERS-4, in particular, bound soluble RBD with an about 45-fold higher affinity than DPP4. Mutagenesis analysis suggested that MERS-4 and MERS-27 recognized distinct regions in RBD. These results suggest that MERS-4 and MERS-27 are RBD-specific potent inhibitors and could serve as promising candidates for prophylactic and therapeutic interventions against MERS-CoV infection.
INTRODUCTION The recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans (1). Symptoms include fever, cough, and shortness of breath, similar to those seen during the outbreak of severe acute respiratory syndrome (SARS) in 2003, which was caused by the coronavirus SARS-CoV (2–8). MERS-CoV and related coronaviruses have been found in several animal species such as bats and dromedary camels (9–13). Genetic and phylogenetic characterization has shown that MERS-CoV belongs to lineage C of the genus of Betacoronavirus and is closely related to Tylonycteris bat coronavirus HKU4 (Ty-BatCoV HKU4) and Pipistrellus bat coronavirus HKU5 (Pi-BatCoV HKU5), although the direct source and reservoirs of MERS-CoV remain uncertain (3, 5, 6, 9, 14–20). The high fatality rate (~40%) (http://www.who.int/csr/don/2014_01_09/en/index. html) and clusters of human-to-human transmission (21, 22) of this deadly virus have raised global concern about the potential for a MERS pandemic. Although treatment with interferon and ribavirin improves clinical outcome of MERS-CoV–infected rhesus macaques (23), and to a lesser extent in infected human (24), no MERS-CoV– specific treatment or vaccine is currently available (25–28). Clearly, 1 Comprehensive AIDS Research Center, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing 100084, China. 2Ministry of Education Key Laboratory of Protein Science, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China. 3 School of Life Sciences, Tsinghua University, Beijing 100084, China. 4Department of Microbiology, The University of Hong Kong, Hong Kong, China. 5Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China. *These authors contributed equally to this work. †Corresponding author. E-mail: [email protected] (L.Z.); xinquanwang@ mail.tsinghua.edu.cn (X.W.); [email protected] (B.-J.Z.)
there is an urgent need for an effective antiviral therapy for this emerging global threat. Like other coronaviruses, MERS-CoV uses its envelope spike (S) glycoprotein for interaction with a cellular receptor for entry into the target cell (4). The S glycoprotein consists of a globular S1 domain at the N-terminal region, followed by membrane-proximal S2 domain and transmembrane domain (7, 8, 29–31). Determinants of host range and cellular tropism are located in the receptor binding domain (RBD) within the S1 domain, whereas mediators of membrane fusion have been identified within the S2 domain (7, 8, 29, 30). Through copurification with the MERS-CoV S1 domain, Raj and colleagues identified that dipeptidyl peptidase 4 (DPP4; also called CD26) functions as a cellular receptor for MERS-CoV (4). We and others have recently determined the crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4 (32, 33). We showed that MERS-CoV RBD consists of a core and a receptor binding subdomain. The receptor binding subdomain directly interacts with blades 4 and 5 of DPP4 propeller but not its intrinsic hydrolase domain (32, 33). This finding suggests that agents capable of disrupting such interaction could serve as potential candidates for blocking entry of MERS-CoV into the target cell. Indeed, both polyclonal and monoclonal antibodies (mAbs) directed against DPP4 have been shown to inhibit MERS-CoV infection of primary human bronchial epithelia cells and Huh7 cells (4, 34). However, considering the important roles of DPP4 in immune regulation of T cell activation and chemokine function (35), antibodies against DPP4 may potentially lead to unwanted adverse effects when applied to human use. Antibodies directed to the viral receptor binding subdomain would therefore be more favored. Furthermore, RBDbased vaccine studies in experimental animals showed that induced
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Liwei Jiang,1* Nianshuang Wang,2* Teng Zuo,1,3 Xuanling Shi,1 Kwok-Man Vincent Poon,4 Yongkang Wu,5 Fei Gao,1 Danyang Li,1 Ruoke Wang,1 Jianying Guo,1 Lili Fu,1 Kwok-Yung Yuen,4 Bo-Jian Zheng,4† Xinquan Wang,2† Linqi Zhang1†
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Fig. 1. Isolation and sequence analysis of MERS-CoV RBD–specific scFvs from nonimmune human antibody library displayed on the surface of yeast S. cerevisiae. (A) Flow cytometry analysis of sorted yeast population after second MACS (MACS2), first FACS (FACS1), and second FACS (FACS2). (B) Fourteen yeast clones with detectable binding specificity to MERS-CoV RBD. Negative controls include yeast clones either not induced in SG-CAA medium (Negative-1) or induced but no RBD was added to the medium (Negative-2). Cmyc is a protein tag used for monitoring scFv expression under induction condition. (C) Sequences of complementarity-determining region 3 (CDR3) for both VH and VL of the 14 yeast clones, together with their family designations and degree of similarity compared to their germline sequences.
polyclonal antibodies directed against MERS-CoV RBD can strongly inhibit the entry of MERS-CoV (36–39). These results provide the critical proof of concept that antibody directed against RBD is an effective way of blocking entry of MERS-CoV. Here, we report isolation and characterization of two potent human neutralizing mAbs against both pseudotyped and live MERS-CoV infection from nonimmune human antibody library. The mechanism of action of these neutralizing mAbs was to block interaction between the RBD on the S glycoprotein of MERS-CoV and cellular receptor DPP4. We believe the neutralizing mAbs identified here hold great promise for the development of prophylactic and therapeutic interventions against MERS-CoV infection.
RESULTS Isolation of MERS-CoV RBD–specific single-chain variable region fragments from nonimmune human antibody library We used purified and biotin-labeled soluble MERS-CoV RBD as antigen bait to select antibodies from nonimmune human single-chain variable region fragment (scFv) library displayed on the surface of yeast Saccharomyces cerevisiae (40, 41). The selection process consisted of two rounds of magnetic bead–activated cell sorting (MACS) followed by additional two rounds of florescence-activated cell sorting (FACS). Increasing proportion of RBD-specific yeast www.ScienceTranslationalMedicine.org
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Neutralizing activity of human immunoglobulin G1 form of selected scFv against pseudotyped and live MERS-CoV infection To evaluate neutralizing activity of selected 14 scFvs, we generated bivalent full-length human mAbs by fusing the variable region of each scFv with the constant region fragment of human immunoglobulin G1 (IgG1). Once confirmed by sequencing, 14 pairs of full-length heavy and light chain plasmids were cotransfected into 293T cells for the production and purification of mAbs. Neutralizing activity of these mAbs was then tested on susceptible Huh7 cells against pseudovirus infection expressing the MERS-CoV S glycoprotein. As shown in Fig. 2A, two mAbs (MERS-4 and MERS-27) demonstrated neutralizing activities with IC50 (half-maximal inhibitory concentration) less than 100 nM; MERS-4 was 0.37 nM, whereas MERS-27 was 63.96 nM. Furthermore, neutralizing activities of these two mAbs were specific against MERS-CoV because none of the pseudovirus bearing the envelope glycoprotein of SARS-CoV, HIV-1, or VSV-G was susceptible to their neutralization (Fig. 2A). Neutralizing activities of these two mAbs against live MERS-CoV were also studied using the clinical isolate hCoV-EMC from a MERS-CoV–infected patient (6). As shown in Fig. 2B, both MERS-4 and MERS-27 demonstrated potent inhibitory activity against MERS-CoV infection, with IC50 of 3.33 and 13.33 nM, respectively. Such inhibitory activities were comparable to that against pseudotyped MERS-CoV, although clear variations existed between the live and pseudotyped systems. In both systems, however, MERS-4 showed more potent neutralizing activity than MERS-27. In addition, we evaluated inhibitory activities of these two mAbs on syncytia formation between cells expressing the MERS-CoV S glycoprotein and those expressing the receptor DPP4. Specifically, the monkey kidney fibroblast cell line COS7 was transfected with plasmids encoding either full-length MERS-CoV S protein or human receptor DPP4. Twentyfour hours after transfection, transfected cells were trypsinized, washed, and mixed at a 1:1 ratio in the presence of 100 mg/ml of each of the two mAbs. Syncytia formation was observed under the microscope 36 hours afterward. Consistent with the neutralization results, MERS-4 was the most potent and completely inhibited syncytia formation at the indicated concentration, whereas MERS-27 was relatively inefficient (Fig. 2C). These results suggested that both MERS-4 and MERS-27 are potent inhibitors against MERS-CoV, and the potential reasons for their differences in inhibitory capacity are explained and discussed below.
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population was observed from 0.77% after the second MACS, to 1.75% after the first FACS, and up to 24.72% after the second FACS (Fig. 1A). Plasmid DNA containing the coding sequences for scFv was extracted from the yeast population sorted by the second FACS and then transformed into Escherichia coli for the production of sufficient quantity for sequencing and sequence analysis. Among the total 120 plasmid sequences analyzed, 23 distinct scFv sequences were identified and 14 of which were confirmed specifically binding to MERS-CoV RBD once transformed back and expressed on the surface of the yeast (Fig. 1B). Each of the 14 scFvs had distinct VH and VL sequences (fig. S1). Some of the scFvs, however, share identical CDR3 sequences in VH such as MERS-2, MERS-72, and MERS-97 (Fig. 1C). One clone (MERS-97) completely lacked a VL. The gene families of the 14 scFvs were quite divergent for both VH (3 VH1, 6 VH2, 4 VH3, 1 VH4, and 1 VH6) and VL (3 Vl1, 1 Vl2, 2 Vl3, 1 Vk1, 5 Vk3, and 2 Vk3) (Fig. 1C).
Fig. 2. Neutralizing activities of MERS-4 and MERS-27 against pseudotyped (A) and live (B) MERS-CoV, and against syncytia formation of COS7 cells mediated by the MERS-CoV S glycoprotein and the receptor DPP4 (C). Pseudoviruses bearing the envelope glycoprotein from SARS-CoV, HIV-1, or VSV-G were used as controls. Data shown are average values from four (A) and two (B) independent experiments. Dashed line indicates 50% inhibition, and two numbers (13.33 and 3.33 nM) above it are the IC50s for MERS-27 and MERS-4, respectively. VRC01 is a human neutralizing mAb against HIV-1 and used as a control. In (C), negative control represents COS7 cells transfected with mouse DPP4, whereas positive control represents those transfected with human DPP4 expression vector.
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RESEARCH ARTICLE mAbs to RBD measured by surface plasmon resonance (SPR) are associated with their neutralizing activities. As shown in Fig. 3 (B and C), MERS-4 had about 45-fold higher binding affinity (Kd = 0.98 nM) to RBD than soluble DPP4 (Kd = 44.5 nM). MERS-27, however, shared comparable binding affinity (Kd = 71.2 nM) to RBD with DPP4 (Kd = 44.5 nM). Collectively, these data suggest that the primary mechanism of the neutralizing activity of MERS-4 and MERS-27 was through blocking of RBD binding to cellular receptor DPP4, even though differential efficiency was observed between the two, perhaps due to their differences in binding affinity and/or epitope recognized.
Cooperativity of the two neutralizing mAbs for virus neutralization Because both MERS-4 and MERS-27 blocked binding of RBD to cellular receptor DPP4, we went further to determine whether the combination of the two mAbs in virus neutralization was synergistic, additive, or antagonistic using the median effect analysis approach (42–44). To start with, a constant ratio between MERS-4 and MERS-27 was set by their respective IC50 concentrations as determined above in Fig. 2A. Dose-dependent neutralization activity for MERS-4, MERS-27, or the combination thereof was then evaluated by serial threefold dilutions in concentrations from 27 times the IC50 to 1/81 of the IC50. Figure 4 shows the average results from four independent experiments to determine percent neutralization (Fig. 4A), fractional effect (Fig. 4B), and the combination index (CI) (Fig. 4C) using the CompuSyn program. Percent neutralization for combined MERS-4 and MERS-27 was about 5.26-fold reduction in IC50 and 4.89-fold reduction in IC90 compared to that for MERS-4 or MERS-27 alone (Fig. 4A). Furthermore, the CI values of combined MERS-4 and MERS-27 at fractional effect values of effective dose 25, 50, 75, 90, and 95% (ED25, ED50, ED75, ED90, and ED95) were 0.22, 0.28, 0.34, 0.42, and 0.50, respectively. As a CI value of 1 indicates an additive effect, 1 indicates antagonism, the combined MERS-4 and MERS-7 are clearly in strong synergism (0.1 to 0.3) at ED25 and ED50 and synergism (0.3 to 0.7) at ED75, ED90, and ED95 (42, 43). Figure 4C shows a logarithmic CI scale, values greater than 0 are indicative of antagonism, and values less than 0 are indicative of synergism. Similarly, combination of MERS-4 and MERS-27 demonstrated strong synergistic effect at lower antibody concentration and modest degree of synergism at higher antibody concentraFig. 3. MERS-4 and MERS-27 blocked the interaction between soluble RBD and receptor DPP4 tion, perhaps due to certain degree of steric on the Huh7 cell surface (A) and their binding kinetics and affinity to RBD (B and C). Flow cy- hindrance in the binding of these two antitometry analysis of soluble RBD binding to receptor DPP4 in the presence of varying concentrations bodies when they were in higher abundance.
of MERS-4 (A, left) or MERS-27 (A, right). VRC01 is a human neutralizing mAb against HIV-1 and used as a control. Overlay of binding of varying concentrations of MERS-4 and MERS-27 to RBD immobilized on the CM5 sensor chip (B). The black lines indicate the experimentally derived curves, whereas the red lines represent fitted curves based on the experimental data. Kon (association rate, in M−1 s−1), Koff (dissociation rate, in s−1), Ka (association constant, in M−1), and Kd (dissociation constant, in M) are indicated for each paired interaction (C).
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Epitope mapping of MERS-4 and MERS-27 Because MERS-4 and MERS-27 appeared to be synergistic in neutralizing activity, 30 April 2014
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Blocking of RBD binding to receptor DPP4 by neutralizing mAbs Because all mAbs reported here were initially isolated on the basis of their recognition of RBD, observed neutralizing activity of MERS-4 and MERS-27 might be mediated through their competitive binding to RBD, thereby interfering or disrupting interaction between RBD and cellular receptor DPP4. To test this hypothesis, we first examined whether the two mAbs could inhibit the binding of soluble RBD to DPP4-expressing susceptible Huh7 cells. As shown in Fig. 3A, when Huh7 cells were incubated with soluble RBD in the presence of varying concentrations of the two mAbs and analyzed by flow cytometry, differential inhibitory effects were observed. Consistent with the neutralization results against live and pseudotyped MERS-CoV and inhibitory effect on syncytia formation, MERS-4 was more potent than MERS-27 in inhibiting interaction between soluble RBD and receptor DPP4 on the surface of Huh7 (Fig. 3A). At a concentration of 333 nM (about two times greater molar concentration than soluble RBD), MERS-4 completely blocked the binding of soluble RBD to Huh7 cells, and clear dose effect was also identified (Fig. 3A, left). In contrast, MERS-27 demonstrated relatively modest inhibitory activity under the same experimental conditions (Fig. 3A, right). Second, we studied whether the binding kinetics and affinities of the two
epitopes recognized by the two mAbs were likely to be different. To test this hypothesis, we generated a total of 14 mutant MERS-CoV S glycoproteins with single- or multiple-residue substitutions at the interface between RBD and DPP4 (Fig. 5A). All but two (13 and 14) pseudotyped viruses bearing the mutant S glycoprotein were able to infect Huh7 cells and therefore used to test for their relative resistance to MERS-4 and MERS-27 neutralization and compared to those bearing the wild-type S glycoprotein. As shown in Fig. 5 (A and B), several mutations resulted in substantial increases in resistance against neutralization of MERS-4. For instance, a single-residue substitution of D455A, E513A, and R542A increased resistant levels about 6-, 13-, and 3-fold against MERS-4, respectively, whereas the same mutation had little impact on resistance to MERS-27. Some residue substitutions such as Y499A, R511A, and, in particular, W553A increased sensitivity of MERS-CoV to MERS-4 and MERS-27 neutralization, presumably by affecting RBD structure in such a way allowing anti-
body easier to access and bind with higher affinity. Lastly, because none of the mutations had significantly increased resistance to MERS-27, its epitope or major residues recognized were likely located beyond the residues studied here. These findings suggest that the epitopes recognized by MERS-4 and MERS-27 are rather distinct, which is consistent with the coaction between the two mAbs in neutralizing activity against pseudotyped MERS-CoV. To understand the spatial relationship of the abovementioned residues and their differences in conferring resistance to MERS-4 and MERS-27, we highlighted their positions on the crystal structure of RBD previously resolved by our group (33). As shown in Fig. 5C, critical residues conferring resistance to MERS-4 (D455A, E513A, and R542A) constitute a wide triangle area (red) located right at the center of the interface between RBD and receptor DPP4. If the red triangle could indeed represent the footprint of MERS-4 on the RBD, it may help to explain why MERS-4 is such a potent mAb capable of inhibiting live and
Fig. 4. Effects of combined MERS-4 and MERS-27 in neutralizing pseudotyped MERS-CoV. (A) Percent neutralization was calculated for serial threefold dilutions of each antibody alone and in combination at constant ratios in a range of concentrations from 27 times to 1/81 of IC50s. The constant ratios of the combined antibodies were their IC50s. On the x axis, a dose of 1 was at the IC50 con-
centration. (B) Fractional effect (FA) plots generated by the CompuSyn program for MERS-4, MERS-27, and their combination showing dosage versus effect. (C) Median effect plot of calculated CI values (logarithmic) versus FA values, in which a log CI of 0 is antagonism. Data shown are average values from four independent experiments.
Fig. 5. Epitope mapping of MERS-4 and MERS-27 by mutagenesis (A and B) and spatial analysis of the key residues on the crystal structure of RBD (C). The residue substitutions and their locations on the RBD are indicated (A). Numbers in the column under MERS-4 and MERS-27 represent the fold changes in IC50 of mutant viruses relative to the wildtype (WT) (>1: increase resistance and
