key: cord-0818586-ocdals9x
authors: Wang, Pan; Ding, Peiyang; Wei, Qiang; Wang, Aiping; Liu, Hongliang; Liu, Yunchao; Li, Qingmei; Xing, Yunrui; Li, Ge; Zhou, Enmin; Zhang, Gaiping
title: Precise location of two novel linear epitopes on the receptor-binding domain surface of MERS-CoV spike protein recognized by two different monoclonal antibodies
date: 2021-12-04
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2021.11.192
sha: afb71b6619778e119d1b14c1da7a754ac677d475
doc_id: 818586
cord_uid: ocdals9x

Middle East respiratory syndrome coronavirus (MERS-CoV) is a coronavirus which can cause severe human respiratory diseases with a fatality rate of almost 36%. In this study, we report the generation, characterization and epitope mapping of several monoclonal antibodies against the spike receptor-binding domain (RBD) of MERS-CoV. Two monoclonal antibodies (4C7 and 6E8) that can react with linearized RBD have been selected for subsequent identification of RBD mAb-binding epitopes. Two distinct novel linear epitopes, (423)FTCSQIS(429) and (546)SPLEGGGWL(554),were precisely located at the outermost surface of RBD by dot-blot hybridization and ELISAs. Multiple sequence alignment analysis showed that these two peptides were highly conserved. Alanine (A)-scanning mutagenesis demonstrated that residues 423F, 428I, and 429S are the crucial residues for the linear epitope (423)FTCSQIS(429) while residues 548L, 550G, 553W, 554L for epitope (546)SPLEGGGWL(554). These findings may be helpful for further understanding of the function of RBD protein and the development of subsequent diagnosis and detection methods.

m a male patient in Saudi Arabia in 2012 [9] , and was officia lly named MERS-CoV by the Coronavirus Research Group of t he World Health Organization in 2013 [10] . The first case of MERS imported from South Korea appeared in China in 2015 [9] , but it has not occurred since then [11] . Previous studies su ggested that MERS-CoV originated in bat [12, 13] . Evidence s uggests that a dromedary camel was the source of MERS-CoV that infected a patient who had close contact with the camel's nasal secretions. Camels may act as intermediate hosts that tra nsmit the virus from its reservoir to humans. Among dromedari es, seroprevalence field surveys have been conducted in a num ber of countries. To date, MERS-CoV RNA or MERS-CoV-spe cific antibodies have been identified in dromedaries in many co untries except Australia, Kazakhstan, and the Netherlands [14] . Genetic and phylogenetic characterization has shown that MERS-CoV belongs to lineage C of the genus of Betacoronavi rus [15] . It is currently the sixth known coronavirus that can c ause human diseases. Like other coronaviruses, MERS-CoV uses its envelope spi ke (S) glycoprotein for interaction with a cellular receptor for entry into the target cell [16] . The S protein is composed of a N-terminal globular S1 subunit, a membrane-proximal S2 subu nit and a transmembrane domain. The S1 subunit contains the determinants of host range and cellular tropism which are locat ed in the receptor binding domain (RBD), while the S2 subuni t contains mediators of membrane fusion [17] [18] [19] [20] . RBD consists of approximately 240 amino acids and has two subunits: core subdomain and receptor binding subdomain [21] [22] [23] .It is identifi ed that human CD26 (also known as human dipeptidyl peptidas til now. Mouse, camel, or human-derived neutralizing mAbs tar geting RBD have been developed [26] . Vaccines are the most i mportant approach against viral infections, but usually hard to develop. Neutralizing monoclonal antibodies (mAbs) have recent ly became a promising way to provide prophylactic and therap eutic protection of virus infections [27] .

In addition, the MERS-CoV RBD contains major neutraliza tion epitopes, induce most of the host immune responses, and could serve as the subunit vaccine against MERS-CoV infectio n [28] [29] [30] [31] . Therefore, studies that focus on identification of epit opes of MERS-CoV RBD, especially the conserved epitopes, w ould be of great importance for vaccine and diagnostic tools d evelopment.

In this study, we immunized the BALB/c mice with RBD protein and prepared five mouse monoclonal antibodies, two of which(4C7 and 6E8)can recognize the linearized RBD prote in. Among the five mAbs , only 12G9 have neutralization acti vity by using MERS-CoV pseudotyped virus-based assay, while cisely. Alanine-scanning mutagenesis revealed that residues 423F, 428I, 429S, and 548L, 549E,550G, 553W, 554L were the core binding sites involved in antibody recognition. The B cell line ar epitopes information obtained in this study might be valuabl e for further research of the function of RBD protein and in d ifferent aspects of the diagnosis of MERS-CoV.

Human embryonic kidney 293F cells (HEK 293F) were obta 

To identify the linear epitope of MERS-CoV RBD, the trunc ated RBD constructs were designed as a series of overlapping fra gments such that each fragment overlapped with the next fragme nt in a 5-or 8-amino-acid overlap zone ( Fig. 3A and B). The pri mers used to clone each truncated construct are shown in Table 1 . were substituted one by one with alanine (A) as shown in Table   6 . Mutant peptides were firstly coupled with BSA as described a bove, and then served as antigens. Two synthetic peptides, N1-4 and C2-1, which were renamed P1( 412 TKLLSLFSVNDFTCSQISP AAI 433 ) and P2( 542 RKQLSPLEGGGWLVASGSTV 561 ) respectively.

MRBD-rFc was used as the positive control. BSA was used as t he negative control. Mutant peptides were analyzed by using 6E8 or 4C7 as the primary antibody.

The spatial distribution and structural property of the identifi body titers from MRBD-His immunized group reached 1:4096,00 0, which were significantly higher than PBS group, indicating tha t the MRBD-His protein induced intensive antibody response in mice.

The mouse with highest antibody titer in Fig.1C was selecte d to generate mAbs against MERS-CoV RBD. MRBD-rFc-based ELISA was performed to screen for the positive hybridoma cells.

Through several rounds of subcloning, five hybridoma cell lines secreting desired mAbs were obtained. We named them 4C7, 6E 8, 7D11, 5F8, 12G9, separately. The subtype of the five mAbs w as all IgG1, and the light chain of all mAbs was Kappa ( Table   2 ). Results from western blotting showed that 4C7 and 6E8 react ed with the denatured MERS-CoV RBD, suggesting that these tw o mAbs recognize the linear epitopes (Fig.3A) . Meanwhile, the re maining three mAbs supposed to bind with conformational epitop es, which did not recognize the denatured MERS-CoV RBD prot e extranuclear area of the HEK293T cells which were transfected with the plasmid pEGFP-C1-MRBD.

Following the methods in 2.6 , we used the formula describ (Fig.4) .

On the basis of the above results, 4C7 and 6E8 were chosen for further mapping of specific linear B-cell epitopes. A total o f six truncated MERS-CoV RBD overlapping fragments were con structed and expressed in HEK-293T cells (Fig. 5A) . Dot blot res ults showed that truncated fragments N and N1 could be recogni zed by mAb 6E8 while C and C2 could be recognized by mAb 4C7 (Fig. 5B) . Eight peptides based on the amino acid sequence of N1 and C2 namely N1-1, N1-2, N1-3, N1-4, C2-1, C2-2, C2-3, C2-4, were designed and synthesized. Dot blot and ELISA re sults showed that only N1-4 and C2-1 could be recognized by th J o u r n a l P r e -p r o o f e mAb 6E8 and 4C7, respectively (Fig. 5C ). These two synthetic peptides, N1-4 and C2-1, were renamed P1 ( 412 TKLLSLFSVND FTCSQISPAAI 433 ) and P2 ( 542 RKQLSPLEGGGWLVASGSTV 561 ), respectively. In order to determine the precise recognition epitope s, peptide P1 and P2 were further truncated and characterized by removing the amino acids one by one from the N-terminus and C-terminus until the accurate position of the epitope was determi ned (Table 4, 5). As shown in Fig. 6A and 6C, the mAb 6E8 co uld well recognize the N-truncated peptides until the 423F residu e was removed, while the mAb could recognize the C-truncated peptides until the 429S residue was deleted (Fig.6A) . Thus, the minimal motif of P1 recognized by the mAb 6E8 was 423 FTCSQI S 429 . As shown in Fig. 6B and 6D , the mAb 4C7 could recogniz e the N-truncated peptides of P2 until the 546S residue was remo ved. Meanwhile, mAb 4C7 could not react to C-truncated peptide s of P2 when 554L was substituted. These results suggest that th e minimal motif of P2 recognized by the mAb 4C7 was 546 SPLE GGGWL 554 .

3.6 Core binding sites of the linear epitopes for the mAbs 6E8 and

In order to define the crucial residues involved in peptide bi nding by mAbs 6E8 and 4C7, each residue of the two identified J o u r n a l P r e -p r o o f linear epitopes ( 423 FTCSQIS 429 and 546 SPLEGGGWL 554 ) was sequ entially substituted by alanine ( Table 6 ). As shown in Fig.7A , the alanine-scanning mutagenesis of epitope 423 FTCSQIS 429 revealed that no binding of mAbs was observed to peptides with alanine s ubstitutions of 423F, 428I, and 429S, implying that residues 423F, 428I, and 429S were the crucial residues for the linear epitope 4 23 FTCSQIS 429 . Meanwhile, for epitope 546 SPLEGGGWL 554 , the su bstitution of residues 548L, 549E, 550G, 553W, 554L with alanin e resulted in the loss of antigenicity (Fig. 7B) , indicating that the five residues were the core binding sites of epitope 546 SPLEGG GWL 554 .

Based on the structure of the receptor binding domain (RBD) of MERS-CoV spike protein (PDB: 4KQZ), the location of the t wo linear epitopes were defined. As shown in Fig. 8A , the epito pe 423 FTCSQIS 429 was located on β sheet and loop in secondary s tructure. The epitope 546 SPLEGGGWL 554 was partly located on β sheet while the rest of the sequence located on αhelix and loo p in secondary structure (Fig. 8A) . Besides, both two epitopes w ere exposed on the MERS CoV RBD surface (Fig. 8B ). Moreove r, both epitopes showed high antigenic index but weak hydrophili city in the analysis of the secondary structure prediction ( 3, or HCoV-HKU1 cause only the common cold [33] . And they are not directly connected to MERS-CoV.

Corona viruses are enveloped viruses, which are further di vided into four different subtypes, alpha-, beta-, gamma-, and d elta-CoV [34, 35] . So far, seven CoVs are confirmed can cause human diseases [36, 37] . Three of beta-CoVs ,SARS-CoV, M ERS-CoV, and SARS-CoV-2 are life-threatening [38] . Therefore, since the discovery of MERS-CoV in 2012, MERS-CoV has a ttracted great attention in biomedical research [9, 39] .

Anti-MERS-CoV mAbs from immunized mice have isolate d from several research groups [40] , most reported MERS-Co V mAbs with neutralizing activities are RBD specific [41] .Gene rally, neutralizing mAbs targeting RBD are more potent than th ose targeting other regions of S protein [42] . The reported mou se neutralizing mAbs including Mersmab1, 4C2 and 2E6 were all bound to the RBD of MERS-CoV through recognizing conf ormational epitopes [26] . MERS-CoV S protein trimer frequentl y performs conformational changes, switching between a lying down and a standing up conformation, which represent recepto J o u r n a l P r e -p r o o f r-inaccessible and receptor-accessible conformation [43] . When neutralizing antibodies bind to RBD, they can cause conformati onal change in S protein, similar to receptor-virus interaction [44] . Yang According to the former studies, 10% of B cell-recognizing e pitopes are linear epitopes, this is due to most of the B-cells e pitopes have conformational changes in three dimensional struct ure, the antigen internalizing process and antigen recognizing a used. Although structural method like X-ray crystallography can alsoprecisely locate the epitope position , its widespread use limited by time consuming and huge cost [50] .

Consequently, the mapping of conformational epitopes is c omplicated and usually requires other ways to complete, such a s computational tools and phage display techniques. These met hods are much more difficult than traditional linear epitope ide ntification methods [51, 52] . Functional methods are used to de tect the binding efficiency of antibody with protein fragments, synthetic peptides or recombinant proteins, and more convenient to conduct [50] .Thus, the linear epitopes were chosen to be f urther determined. Besides, for the development of epitope-base d vaccines, the specificity can be enhanced by only selecting t he antigenic domains of proteins exposed on the surface in ord er to elicit strong immune responses [53] . Precise location of B-cell epitopes was related to antigenicity, accessibility of surfa ce, flexibility, hydrophilicity and predictions of linear epitope bs (7D11, 5F8, 12G9) did not react with the denatured RBD p rotein, so we speculated that they recognize the conformational epitopes. (Fig. 3) . The result of pseudotyped virus-based neutr alization assay showed that only mAb 12G9, which recognized the conformational epitopes, had the neutralization activity.

Subsequently, the linear epitopes recognized by mAbs 6E8 and 4C7 were scanned by truncated recombinant RBD protein and overlapping synthetic peptides. As shown in Fig. 5 and 6 , the precise linear epitopes recognized by mAbs 6E8 and 4C7 were determined to be 423 FTCSQIS 429 and 546 SPLEGGGWL 554 , r espectively. Furthermore, we assessed the interaction between t he epitope and the monoclonal antibody through alanine-scannin g mutagenesis ( Table 6 ). As shown in Fig.7A , the 423F, 428I, and 429S mutants basically eliminated the reactivity between th e peptide and monoclonal antibody 6E8. On the contrary, 424T, 425C, 426S, and 427Q mutants hardly affect the binding abili ty to monoclonal antibodies. Thus, the results shows that 423F, 428I, and 429S are the key amino acids of epitope 423 FTCSQI S 429 . For epitope 546 SPLEGGGWL 554 , when 548L, 549E, 550G, J o u r n a l P r e -p r o o f 553W, and 554L were mutated, the reactivity of the peptides w ith monoclonal antibody 4C7 was drastically reduced, and only light or blurry blots could be observed on the nitrocellulose membrane (Fig.7B) . Besides, the corresponding ELISA results s howed that the OD 450 values of the five mutants were significa ntly lower than that of the 546S, 547P, 551G, and 552G muta nts. Thus, the core amino acids of polypeptide 546 SPLEGGGW L 554 were 548L, 549E, 550G, 553W, and 554L. Notably, it is r eported that the residue 553W was the key site of mouse neu tralizing antibodies may inhibit DPP4 binding at , and potently neutralize infection of pseudotyped or live MERS-CoV [41, 55] .

Mabs targeting the RBD could recognize key residues tha t are crucial for DPP4 binding. Also, changing of one or sever al of these critical residues in RBD may lead to the emergenc e of escape mutant virus strains, in which the mAbs targeting the unmutant residues in RBD will reduce or lose neutralizing ability against the mutant strains [42] . In summary, a total of five monoclonal antibodies were id entified in this study, two of which could recognize linear epit opes. Epitopes 423 FTCSQIS 429 and 546 SPLEGGGWL 554 were prec ise located by mAbs 6E8 and 4C7, respectively. The crucial re sidues were further determined . The results of structural analy sis showed that these two epitopes were exposed on the surfac e of the RBD. Multiple sequence alignment analysis showed th at the epitopes were highly conserved. To precise locate the su rface-exposed peptides, especially those B cell epitopes that are antigenic and conserved, rather than focus on the whole patho The ELISA data has been tested in three independent replicates, and the OD 450 values were presented as means and SD with error bars. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Table 6 Alanine-scanning mutagenesis of the minimal motifs of peptides 423 J o u r n a l P r e -p r o o f

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