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Neutralizing Monoclonal Antibodies As Promising Therapeutics Against MERS Coronavirus

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Introduction

Middle east respiratory syndrome (MERS) emerged with the death of a man with pneumonia in Saudi Arabia in 2012, and the causative agent was subsequently identified as a novel coronavirus (MERS-CoV), which belongs to lineage C Betacoronaviruses. As a zoonoses, with dromedary camels as direct sources and bats as potential reservoirs, MERS-CoV is frequently introduced into humans. Moreover, once introduced into humans, human-to-human transmission can be responsible for approximately 40% of MERS cases globally, therefore, MERS-CoV has been a consistent threat to humans. As of October 2018, MERS-CoV has caused 2, 254 laboratory-confirmed cases, including 800 deaths, in 27 countries, with the fatality rate as high as 35%. Although MERS cases are primarily reported in the Middle East, with the facility of international travelling, MERS-CoV can also be a worldwide threat, which is well illustrated by the MERS outbreak in South Korea in 2015. Given the potential risk of causing worldwide public health emergencies and the absence of licensed vaccines and antiviral therapeutics, MERS-CoV has been added to the “List of Blueprint priority diseases” by WHO.

Although vaccines are the most important approach against viral infections, they generally take a long time to develop, and cannot provide immediate prophylactic protection or be used to treat ongoing viral infections. When combating emerging viruses, passive immunotherapeutics using neutralizing monoclonal antibodies (mAbs) have recently emerged as a powerful tool to provide prophylactic and therapeutic protection. Potent neutralizing mAbs can be achieved by using various technologies, such as hybridoma technology, humanized mouse, phage or yeast display of antibody library and single B cell isolation.

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Spike (S) protein of MERS-CoV as target for neutralizing mAbsMERS-CoV is a single, positive-stranded RNA virus of about 30kb, which encodes four major viral structural proteins, including spike (S), envelope (E), membrane (M) and nucleocapsid (N), as well as several accessory proteins. The S protein of MERS-CoV plays an important role in virus infection, and it is consisted of a receptor-binding subunit S1 and a membrane-fusion subunit S2, which mediates viral attachment to host cells and virus-cell membrane fusion, respectively. The S1 subunit contains a receptor-binding domain (RBD), which can bind to cell receptor dipeptidyl peptidase 4 (DPP4, also known as CD26), and mediates viral attachment to target cells [8]. Neutralizing mAbs binding to the S protein of MERS-CoV can prevent virus attachment and membrane fusion, therefore inhibit viral entry. The S protein of MERS-CoV is a key target for antivirals, and the RBD is the most popular focus. mAbs against the RBD of MERS-CoV derived from both human and mouse showed the most potent neutralizing activities. In this review, we summarized the current advances on neutralizing mAbs against MERS-CoV.

Mouse neutralizing mAbs against the RBD of MERS-CoV

4C2 and 2E6

Stable hybridoma cell lines were generated by immunizing mice with MERS-RBD protein, and screened for positive clones. Two neutralizing mAbs, 4C2 and 2E6, were identified. Both mAbs bound potently to the RBD of MERS-CoV and blocked both pseudoviruses and live MERS-CoV (strain HCoV-EmC/2012) entry in vitro with high efficacy. Humanized 4C2 showed similar neutralizing activity in vitro, and in vivo test also indicated that it could significantly reduce the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, highlighting its potential application in humans not only for preventing but also treating MERS-CoV infection. Crystallization of the 4C2 Fab/MERS-RBD complex showed that 4C2 recognized a conformational epitope that partially overlaps the receptor-binding footprint in the RBD of MERS-CoV, thereby interfering MERS-CoV binding to DPP4 by both steric hindrance and interface-residue competition. 2E6 competed with 4C2 for binding to MERS-RBD, indicating that they recognized proximate or overlapping epitopes.

Mersmab1

To generate neutralizing mAbs, mice were immunized with recombinant MERS-CoV S1 fused to a C-terminal Fc tag (S1-Fc), and stable hybridoma cell lines were generated for screening of positive clones. One neutralizing mAbs, Mersmab1, was developed, and it could effectively block the entry of pseudoviruses and live MERS-CoV (strain HCoV-EMC/2012) in vitro. Structure analysis showed that Mersmab1 bound to the RBD of MERS-CoV through recognizing conformational epitopes, and all of the residues critical for Mersmab1 binding were located on the left ridge of the receptor-binding motif (RBM). Mersmab1 could neutralize MERS-CoV by competitive blocking the binding of MERS-CoV RBD to its host receptor, DPP4. Based on escape mutation analysis of the key residues on RBD, neutralizing epitopes that were recognized by Mersmab1 were mapped. L506, D510, R511, E513 and W553 were critical for Mersmab1 binding to RBD, while D539, E536 or E565 did not affect the interaction of Mersmab1 and RBD at all.

Human neutralizing mAbs against the RBD of MERS-CoV

3B11

Using an ultra-large nonimmune human Ab-phage display library and a unique spanning strategy, seven human neutralizing mAbs with varying neutralization efficacy to MERS-CoV were identified. Binding detection demonstrated that the epitopes of these mAbs lay within the aa 349–590 of the S protein, which overlapped a large part of the RBD (aa 377-662) of MERS-CoV. Binding competition assays showed that these mAbs recognized at least three distinct epitope groups, which was further confirmed by the escape studies. With no cross-epitope resistance, these mAbs neutralized MERS-CoV by competitively blocking the binding of the RBD of MERS-CoV with its cell receptor, DPP4. Escape mutant assays showed that five amino acid residues were critical for neutralization escape of these mAbs, namely L506, T512, Y540, R542 and P547. Of the seven mAbs, 3B11 exhibited the best neutralization activity against both pseudovirus and live MERS-CoV (strain HCoV-EMC/2012) in vitro. Moreover, 3B11 IgG did not induce neutralization escape. In vivo test demonstrated that 3B11 could reduce lung pathology in rhesus monkeys infected with MERS-CoV (strain Jordan-n3/2012). With its high neutralizing activity and lack of mutant escape, the 3B11 IgG is a promising therapeutic mAbs.

m336

Three human mAbs, m336, m337 and m338 were isolated from a large phage display library, and their IgG1 formats were converted. The binding sties of the three mAbs were within the RBD of MERS-CoV (aa 377-588), therefore they neutralized MERS-CoV by competing with DPP4 binding to the RBD. The three mAbs also competed with each other for binding to the RBD of MERS-CoV, and mutant analysis showed that the three mAbs possessed overlapping but distinct epitopes. M336 was the most potent mAb, and it neutralized both pseudotyped and live MERS-CoV with exceptional potency in vitro. Residues crucial for m336 binding in the RBD of MERS-CoV were L506, D510, E536, D539, W553 and V555. In vivo study demonstrated that prophylaxis with m336 could reduce the viral titers in the lung of rabbits infected with MERS-CoV (strain HCoV-EMC/2012), and m336 could also provide transgenic mice expressing human DPP4 with fully prophylactic and therapeutic protection from MERS-CoV infection. However, a recent study with non-human primate, common marmoset, showed that 336 could alleviate the severity of the diseases but not provide full protection against MERS-CoV.

LCA60

IgG+ memory B cells were isolated from a MERS patient, and were subsequently immortalized with EBV. A neutralizing mAbs, LCA60, was identified, which was the first fully human neutralizing mAb isolated from memory B cells of an MERS patient. LCA60 could efficiently neutralize MERS-CoV strains from London, Saudi Arabia and Jordan in vitro. In vivo study showed that LCA60 could provide BALB/c mice transducted with adenoviral vectors expressing human DPP4 with both prophylactic and post-exposure protection against MERS-CoV. Furthermore, the neutralizing efficacy of LCA60 was evaluated in IFN-α/β receptor-deficient mice (IFNAR-KO), which were a more stringent model of MERS-CoV infection. Administration of LCA60 after infection could also reduce lung viral titers in these mice. With its native heavy and light chain pairs, LCA60 is more potent than 3B11 and comparable to m336. Cross-competition experiment demonstrated that LCA60 competed with 3B11 for binding to RBD. LCA60 interacted with the RBD residues around K493, and the LCA60 footprint on the RBD partially overlaps with that of its cell receptor DPP4. Four positions in the RBD could affected with the LCA60 binding, namely T489, K493, E565 and E536, which are conserved in all MERS-CoV isolates to date. Moreover, the binding affinity of LCA60 to the RBD of MERS-CoV was significantly higher (~500-fold) compared with DPP4. Therefore, one major mechanism of LCA60 neutralization was to competitively inhibit the interaction of the RBD of MERS-CoV with its cell receptor DPP4. So far, a GMP-approved cell line expressing the purified and highly potent antibody in high concentration has been established (LCA60. 273. 1), highlighting its application as promising therapeutics against MERS-CoV.

REGN3051 and REGN3048

By immunizing humanized transgenic mice, VelocImmune mice, with DNA encoding the S protein of MERS-CoV and recombinant purified S protein, hybridoma B cells producing neutralizing mAbs against the S protein of MERS-CoV were generated. Two fully human neutralizing mAb that could cobind to the RBD of MERS-CoV with high affinity were subsequently selected, namely REGN3051 and REGN3048. The two mAbs bound to distinct epitopes on RBD, focusing on aa 367-606, which are conserved during the natural evolution of MERS-CoV. Mutation as a result of selective pressure by one mAb should not affect the binding of the other mAb. In vitro test showed that REGN3051 could neutralize the prototype EMC/2012 strain and all clinical mutant isolates (A431P, S457G, S460F, A482V, L506F, D509G and V534A). With the exception of V534A variant, REGN3048 achieved similar neutralizing activity. In vivo study demonstrated that REGN3051 and REGN3048 could reduced virus replication in mice expressing human DPP4 in both prophylactic and therapeutic settings. When evaluated in common marmoset, both mAbs seemed to be more effective in prophylaxis rather than treatment of MERS-CoV infection.

MCA1

An anti-MERS-CoV phage display library was constructed with the peripheral B cells of a MERS survivor, and a fully human neutralizing mAb against MERS-CoV, MCA1, was isolated. MCA1 showed potent neutralizing activity against MERS-CoV (strain HCoV-EMC/2012) in vitro. Moreover, MCA1 could completely inhibit MERS-CoV replication in common marmosets as both prophylactic and therapeutic. Structure analysis of the complex of MERS-CoV RBD and MCA1 Fab showed that MCA1 formed direct contacts with the receptor-binding site (RBS) subdomain on the RBD, which was largely overlapped with the DPP4-binding site. In addition, the MCA1 Fab epitope could also generate steric clashes with the RBS. Therefore, the neutralizing mechanism of MCA1 was achieved by steric clashes as well as by competing with DPP4 for binding to the RBD.

MERS-4 and MERS-27

Two potent human neutralizing mAbs, MERS-4 and MERS-27, were derived from a nonimmune human antibody library. MERS-4 and MERS-27 could inhibit the infection of both pseudotyped and live MERS-CoV (hCoV-EMC/2012) in vitro. Mutagenesis analysis suggested that MERS-4 and MERS-27 recognized distinct regions in the RBD of MERS-CoV, and the epitope of MERS-27 might be located away from that recognized by MERS-4. Mutagenesis analysis demonstrated that aa D455, L507, E513, R542, L545, S546, P547, G549 and S508 in the RBD were important for MERS-4 binding, while only aa S508 was important for MERS-27. The combination of MERS-4 and MERS-27 demonstrated a synergistic effect against pseudotyped MERS-CoV. MERS-4 bound to RBD with a much higher affinity than DPP4. The primary mechanism of the neutralizing activity of MERS-4 and MERS-27 was through blocking of RBD binding to DPP4. Further structure analysis showed that MERS-4 bound to a unique epitope and caused conformational changes in the RBD interface critical for accommodating DPP4, therefore indirectly disrupted the interaction between RBD and DPP4. Furthermore, MERS-4 demonstrated synergistic effects with m336 and 5F9, which is a NTD-specific mAb. The special neutralizing mechanism made MERS-4 a valuable addition for the combined use of mAbs against MERS-CoV infection.

MERS-GD27 and MERS-GD33

Thirteen ultrapotent neutralizing mAbs, which all targeted the RBD of MERS-CoV were generated following a protocol for the rapid production of antigen-specific human mAbs. Briefly, antibody-secreting cells were isolated from the whole blood of a MERS patient, and the antibody genes were amplified and cloned into vectors to transfect human cell lines to produce mAbs. Of the 13 mAbs, MERS-GD27 and MERS-GD33 exhibited the strongest neutralizing activities against both pseudovirus and live MERS-CoV (strain HCoV-EMC/2012) in vitro. MERS-GD27 directly competed the binding of RBD to DPP4, and the structural basis of MERS-GD27 neutralization and recognition revealed its epitope almost completely overlapped with the RBD. MERS-GD27 and MERS-GD33 recognized distinct epitopes on RBD, and had a low level of competing activity. The combined use of the two mAbs demonstrated synergistic effects in neutralization against MERS-CoV. Mutagenesis analysis demonstrated that aa L506, D509, V534, E536 and A556 were important for the neutralization of MERS-GD27, and aa R511was critical for MERS-GD33. Moreover, in vivo study found that MERS-GD27 could provide both prophylactic and therapeutic protection for transgenic mice expressing human DPP4 against MERS-CoV infection.

Camel neutralizing mAbs against the RBD of MERS-CoV

Dromedary camels exposed to MERS-CoV showed only very mild symptoms but develop exceptionally potent neutralizing antibodies. Camelid species naturally produced heavy chain-only antibodies (HCAbs), which are dimeric and devoid of light chains, and their antigen recognition region is solely formed by the variable heavy chains (VHHs), which have long complementarity-determining region 3 (CDR3) loops and are capable of binding to unique epitopes not accessible to conventional antibodies. Notably, camelid VHHs is relatively stable and can be produced with high yields in prokaryotic system. Because of these beneficial properties, camel VHHs have be gaining more attention as promising therapeutics against MERS-CoV infection.

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