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. 2023 Jun 3;9(6):e16847. doi: 10.1016/j.heliyon.2023.e16847

Identification of conserved linear epitopes in the SARS-CoV-2 receptor-binding region using monoclonal antibodies

Yujie Yang a,1, Liling Zhou a,1, Chuncong Mo a,1, Longbo Hu b, Zhichao Zhou a, Ye Fan a, Wenkuan Liu a, Xiao Li a, Rong Zhou a,∗∗, Xingui Tian a,∗
PMCID: PMC10238280  PMID: 37292282

Abstract

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused millions of cases of infections, leading to a global health emergency. The SARS-CoV-2 spike (S) protein plays the most important role in viral infection, and S1 subunit and its receptor-binding domain (RBD) are widely considered the most attractive vaccine targets. The RBD is highly immunogenic and its linear epitopes are important for vaccine development and therapy, but linear epitopes on the RBD have rarely been reported. In this study, 151 mouse monoclonal antibodies (mAbs) against the SARS-CoV-2 S1 protein were characterized and used to identify epitopes. Fifty-one mAbs reacted with eukaryotic SARS-CoV-2 RBD. Sixty-nine mAbs reacted with the S proteins of Omicron variants B.1.1.529 and BA.5, indicating their potential as rapid diagnostic materials. Three novel linear epitopes of RBD, R6 (391CFTNVYADSFVIRGD405), R12 (463PFERDISTEIYQAGS477), and R16 (510VVVLSFELLHAPAT523), were identified; these were highly conserved in SARS-CoV-2 variants of concern and could be detected in the convalescent serum of COVID-19 patients. From pseudovirus neutralization assays, some mAbs including one detecting R12 were found to possess neutralizing activity. Together, from the reaction of mAbs with eukaryotic RBD (N501Y), RBD (E484K), and S1 (D614G), we found that a single amino acid mutation in the SARS-CoV-2 S protein may cause a structural alteration, exerting substantial impact on mAb recognition. Our results could, therefore, help us better understand the function of the SARS-CoV-2 S protein and develop diagnostic tools for COVID-19.

Keywords: SARS-CoV-2, Spike protein, Receptor-binding domain, Monoclonal antibody, Epitope

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has caused a pandemic. Patients infected with SARS-CoV-2 have common symptoms such as fever, cough, myalgia, and fatigue. Complications after admission include acute respiratory distress syndrome, acute heart injury, and secondary infections [1].

Phylogenetic analysis revealed that SARS-CoV-2 belongs to the subgenus Sarbecovirus of the genus Betacoronavirus [2]. SARS-CoV-2 is an enveloped, single-stranded, positive-sense RNA virus. Its viral genome encodes four structural proteins, the envelope (E), membrane (M), nucleocapsid (N), and spike (S) proteins [3].

Coronavirus infection is initiated by the binding of the S protein on the viral particle to host surface cellular receptors [4]. Structurally, the S protein is a clover-type trimer comprising three S1 subunits and one trimer S2 subunit [5]. SARS-CoV-2 uses the receptor-binding domain (RBD) in the S1 subunit to engage with the angiotensin-converting enzyme 2 (ACE2) receptor [6]. The RBD has become an important target for functional research and therapeutics development, and its linear epitope contributes to the study of humoral immune mechanisms and optimal vaccine development [7]. However, under native conditions RBD forms complex conformations and possesses a rare linear epitope [8,9]. Therefore, it is important to develop monoclonal antibodies (mAbs) that recognize the linear epitopes of RBD, which will facilitate RBD detection. Other than the RBD, other regions of the S1 subunit can also generate strong antibodies, and some antibodies have been shown to exhibit neutralizing activities [10,11]. Meanwhile, according to a WHO report, previously, there were four circulating variants of concern (VOCs), Alpha, Beta, Gamma and Delta, as well as the currently circulating VOC, Omicron variant. Some amino acid mutations in the S protein were reported to change the conformation of the RBD, many of which may be detrimental for antibody recognition [12].

In this study, we obtained 151 mouse-derived mAbs against the SARS-CoV-2 S1 protein and investigated the impacts of specific amino acid mutations (D614G in S1, N501Y, or E484K in the RBD) on mAb detection. We identified three new and highly conserved linear epitopes within the SARS-CoV-2 RBD using these mAbs. We also investigated the reaction of the mAbs with the S proteins of Omicron variants and established a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for Omicron antigen detection. This study aims to demonstrate the useful of these mAbs in the detection of SARS-CoV-2 and the treatment of COVID-19.

2. Materials and methods

2.1. MAbs and recombinant proteins

In total, ascites from 151 mouse-derived mAbs against the SARS-CoV-2 S1 subunit protein were produced by Guangzhou Ruida Co., Ltd, which was pretreated using filtration centrifugation. The eukaryotic SARS-CoV-2-RBD [SARS-CoV-2-RBD(e)] was provided by Hexin Co., Ltd (Guangzhou, China). The following proteins were provided by Yiqiao Shenzhou Biological Co., Ltd (Beijing, China): SARS-CoV-2 S protein (S1 subunit, his tag) (Cat:40591-V08B1), SARS-CoV-2 S1(D614G)-His recombinant protein (Cat:40591-V08H3), SARS-CoV-2 RBD(N501Y)-His recombinant protein (Cat:40592-V08H82), and SARS-CoV-2 RBD(E484K)-His recombinant protein (Cat:40592-V08H84), expressed with a polyhistidine tag at the C-terminus in HEK293 cells. The SARS-CoV-2 variant B.1.1.529 (Omicron) spike trimer protein (S1+S2 ECD) (Cat: 40589-V08H26) and BA.5 Spike trimer protein (S1+S2 ECD) (Cat:40589-V08H33) were provided by Yiqiao Shenzhou. Sixteen peptides covering the SARS-CoV-2-RBD were synthesized by Shanghai Gill Biochemical Company. Each peptide was 14–15 amino acids in length with an overlap of three amino acids (Table 1).

Table 1.

Synthesized SARS-CoV-2 RBD peptides.

Peptide Amino acid sequence and position in the S protein
nCoV-R1 330NITNLCPFGEVFNAT344
nCoV-R2 342NATRFASVYAWNRKR356
nCoV-R3 354RKRISNCVADYSVLY368
nCoV-R4 366VLYNSASFSTFKCYG381
nCoV-R5 379CYGVSPTKLNDLCFT393
nCoV-R6 391CFTNVYADSFVIRGD405
nCoV-R7 403RGDEVRQIAPGQTGK417
nCoV-R8 415TGKIADYNYKLPDDF429
nCoV-R9 427DDFTGCVIAWNSNNL441
nCoV-R10 439NNLDSKVGGNYNYLY453
nCoV-R11 451YLYRLFRKSNLKPFE465
nCoV-R12 463PFERDISTEIYQAGS477
nCoV-R13 475AGSTPCNGVEGFNCY489
nCoV-R14 487NCYFPLQSYGFQPTN501
nCoV-R15 499PTNGVGYQPYRVVV512
nCoV-R16 510VVVLSFELLHAPAT523
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2.2. Serum samples

Convalescent serum samples were collected from patients with SARS-CoV-2 infection. The SARS-CoV-2 antibody content in serum was determined via indirect ELISA, which indicated that these samples were specifically bound to the SARS-CoV-2 S protein; this is considered the reference test in coronavirus serology. Leftover serum samples collected from Guangzhou Women and Children's Medical Center from five healthy individuals before 2019 were used as controls. Specimens were stored at −20 °C until use, and written informed consent was provided by study participants. The study protocol was reviewed and approved by The First Affiliated Hospital of Guangzhou Medical University Ethics Committee (No. 2020–77). Parts of the study included quality improvement activities and, therefore, were exempt from review.

2.3. ELISA

Indirect ELISA was performed as previously described by coating the plates with recombinant proteins or synthesized peptides [13]. Briefly, the SARS-CoV-2 S1 protein was diluted to 1 μg/mL with coating solution (0.05 mol/L, pH 9.6 carbonate buffer), and coated overnight in an ELISA plate (100 μL per well) at 4 °C. The plates were then washed once with phosphate-buffered saline (PBS) with Tween 20 (PBST), blocked at 37 °C for 2 h with 3% bovine serum albumin (BSA; 200 μL per well) in PBST, and washed with PBST once more. mAbs were diluted at 1:10000, 1:100,000, or 1:1,000,000 with PBST containing 3% BSA (PBS as negative control). Exactly 100 μL of diluent was added to each well with duplicate wells for each mAb at 37 °C for 1 h. The plates were then washed trice with PBST. HRP-conjugated goat anti-mouse IgG + IgM + IgA H&L (Abcam, Cambridge, UK) was used as the secondary antibody and added to each well (100 μL) for incubation at 37 °C for 1 h. The plate was washed trice with PBST. In total, 100 μL of TMB single-component substrate solution (Solarbio, Beijing, China) was added in the dark at about 25 °C for 5 min to develop color and the reaction was terminated with 2 M H2SO4. Optical density (OD) was measured at 450 nm. Mouse antisera against SARS-CoV-2-RBD were used as the positive control, and mouse sera immunized with PBS were used as the negative control. When the OD of a sample fails to reach 2.1 times the OD of a negative control, it is considered to be 0 even though its titer may be lower than the initial dilution [14].

Cross-reactivities between mAbs and SARS-CoV-2 RBD(e), as well as SARS-CoV-2 variant-related proteins, were also detected via indirect ELISA. The recombinant protein, SARS-CoV-2-RBD(e), SARS-CoV-2-RBD(N501Y), SARS-CoV-2-RBD(E484K), or SARS-CoV-2 S1(D614G), was diluted to 1 μg/mL and used to coat the ELISA plate. Next, the appropriate dilution of primary mAb was added to the wells. Antibody incubation was established as described above. Mouse antisera against SARS-CoV-2-RBD were used as the positive control, and mouse sera immunized with PBS were used as the negative control. OD450 was used to determine whether there was a cross-reaction between the mAb and the protein.

A double-antibody sandwich ELISA was established to detect Omicron variants B.1.1.529 and BA.5 using the mAbs. Briefly, ELISA plates were coated with mAb (2 μg/ml) in PBS (pH 7.4) overnight at 4 °C and then blocked with a blocking buffer. Increasing concentrations (0, 0.01, 0.1, 1, or 5 μg/mL) of purified SARS-CoV-2 Omicron variant S proteins were added to the respective wells in the blocking buffer and incubated at 37 °C for 1 h. HRP-conjugated secondary mAbs (1:1000 dilution) were then added to the plate after washing. Further investigations were performed as described above.

2.4. Epitope mapping via competitive ELISA

The coating solution was used to dilute the SARS-CoV-2 S1 to 1 μg/mL. Exactly 51 mAbs against SARS-CoV-2-RBD(e) were diluted 105 folds. Preincubation of synthesized peptides (1 μg/mL) with mAbs at 37 °C for 1 h and added to the SARS-CoV-2 S1-coated ELISA plate at 37 °C for 1 h. The plate was washed with PBST and incubated with goat anti-mouse IgG + IgM + IgA (HRP). Finally, TMB single-component substrate solution was added to develop color and the reaction was terminated with 2 M H2SO4. OD at 450 nm was determined to compare the binding of the same mAb with 16 different peptides. If the peptides had no reactivity with mAb, the OD value would be high.

2.5. SARS-CoV-2 pseudovirus neutralization assay

SARS-CoV-2 pseudovirus neutralization assay was performed as previously described [14]. Briefly, hACE2-expressing cells, hACE2-293T, were seeded onto 96-well plates 18 h before infection. mAbs were serially diluted and co-incubated with the same volume of pseudotype particles at 37 °C for 1 h. MEM was added as the negative control. The mixtures were added to the monolayers of hACE2-293T. Next, the plates were incubated at 37 °C in 5% CO2. Forty-eight hours later, the cells were lysed and analyzed using Promega GloMax Explorer (Promega, Madison, USA). Inhibition rates of mAbs at different dilutions were calculated and compared to those of the negative control. Anti-SARS-CoV-2-RBD mouse serum samples were used as the positive control, and mouse anti-PBS serum samples were used as the negative control. For each experiment, the positive convalescent serum from a patient with COVID-19 was used as a standard experimental control.

2.6. Multiple sequence alignments of spike proteins from HCoVs and SARS-related coronaviruses

SnapGene 4.2.4 was used to conduct routine sequence management. MAFFT was used to perform sequence alignment with default parameters. Protein sequences were retrieved from the National Center for Biotechnology Information (Bethesda, MD, USA) database. The virus strains and the GenBank numbers were as follows: HCoV-HKU1 (NC_006577), HCoV-OC43 (NC_003045.1), HCoV-229E (NC_002645), HCoV-NL63 (NC_005831), SARS-CoV-2 (NC_045512), SARS-CoV-1 (NC_004718), MERS-CoV (NC_019843), SARSr-CoV RaTG13 (MN996532.2), SARSr-CoV BtKY72 (KY352407.1), SARSr-CoV PC4-227 (AY613950.1); the SARS-CoV-2 variants, Alpha (B.1.1.7, MZ310552.1), Beta (B.1.351, MZ202314.1), Gamma (P.1, MZ169911.1), Delta (B.1.617.2, MA318159.1), and Omicron (B.1.1.529, OM678335.1).

3. Results

3.1. mAb preparation

A total of 151 mAb ascites against SARS-CoV-2 S1 were characterized and used to identify epitopes. Indirect ELISA was performed using serial dilutions of mAbs. Fig. 1A shows the ELISA result of mAbs diluted 104 folds to react with SARS-CoV-2 S1. Among 151 mAbs, 51 specifically recognized the eukaryotic SARS-CoV-2-RBD (Fig. 1B), as follows: K7-62, K7-64, K7-65, K7-67, K7-80, K7-82, K7-83, K7-84, K7-85, K7-87, K7-90, K7-101, K7-103, K7-105, K7-106, K7-108, K7-112, K7-119, K7-123, K7-125, K7-129, K7-132, K7-138, K7-140, K7-141, K7-144, K7-148, K7-149, K7-150, K7-154, K7-155, K7-156, K7-159, K7-166, K7-168, K7-174, K7-183, K7-184, K7-185, K7-186, K7-187, K7-198, K7-200, K7-201, K7- 202, K7-209, K7-210, K7-212, K7-214, K7-215, and K7-216. Mouse antisera against trimeric prokaryotic SARS-CoV-2 RBD were used as the positive control. Mouse antisera immunized with PBS were used as the negative control.

Fig. 1.

Fig. 1

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The titer and neutralizing effect of mAbs. (A) The reaction of 151 mAbs with recombinant SARS-CoV-2 S1 protein at serial dilutions of 1:104. The dot-and-dash grid line shows the threshold value. (B) Detection of the mAbs with SARS-CoV-2-RBD(e). (C) Neutralization inhibition rate of mAbs diluted 40 folds against SARS-CoV-2 pseudovirus. (D) Heat map of the neutralization inhibition of monoclonal antibodies against pseudoviruses at different dilutions. The selected mAbs with an inhibition rate of more than 30% at a certain dilution are shown. Mouse antisera against SARS-CoV-2-RBD were used as the positive control, and mouse sera immunized with PBS were used as the negative control.

The neutralizing activity of mAbs against the SARS-CoV-2 pseudovirus was determined using the pseudovirus neutralization assay. K7-87, K7-90, K7-155, and K7-210 were found to have a low neutralization efficacy, which was dose dependent, as shown in Fig. 1C and D.

3.2. Epitope mapping of SARS-CoV-2 RBD

The 51 mAbs that recognized eukaryotic SARS-CoV-2 RBD were further used to identify the epitopes using 16 synthesized overlapping SARS-CoV-2 RBD peptides via competitive ELISA. Competition ELISA revealed that three peptides inhibited the binding of mAbs to SARS-CoV-2 S1, indicating that R6 (391CFTNVYADSFVIRGD405), R12 (463PFERDISTEIYQAGS477), and R16 (510VVVLSFELLHAPAT523) are epitopes (Fig. 2A). K7-159 recognized R6; K7-62, K7-82, K7-84, K7-90, K7-101, K7-108, K7-119, K7-148, K7-183, K7-184, K7-185, K7-212, and K7-216 recognized R12; and K7-149 and K7-150 recognized R16 (Table 2). Furthermore, serum samples from five convalescent serum samples from COVID-19 patients and four healthy individuals were used to react with the three peptides, R6, R12, and R16, coupled to HSA. The presence of anti-SARS-CoV-2 antibodies in serum samples of COVID-19 patients was identified by serum antibody binding to the SARS-CoV-2 S1 protein (Fig. 2B). However, substantial amounts of antibodies against R6, R12, or R16 were detected in the convalescent serum of COVID-19 patients compared to that from healthy donors.

Fig. 2.

Fig. 2

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Epitope identification and similarity analysis. (A) The epitopes recognized by the monoclonal antibody are shown in the three-dimensional structure of the S-trimer, with different epitopes labeled in different colors. The three linear epitopes R6, R12, and R16 are depicted in red, green, and blue, respectively. The image was edited with Chimera X and the PDB registration number of the S protein is 6zp7. (B) Epitope recognition with the convalescent plasma of patients with COVID-19. The cross-reaction of the convalescent sera of five patients with COVID-19 and the sera of four healthy persons were detected with SARS-CoV-2 S1 protein and the three peptides of SARS-CoV-2 RBD using indirect ELISA. The lines show the mean and SD, statistical significance was determined using a two-way ANOVA multiple comparisions test. *, P < 0.05. ***, P < 0.001. (C, D) Multiple amino acid sequence alignments of spike proteins of seven human coronaviruses, severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs), and SARS-CoV-2 VOCs, showing the epitope regions here. Different colors represent physico-chemical properties and the conservation of residues among the sequences. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Table 2.

Epitope mapping and characterization of mAbs.

MAb (K7-) Total number S1 (D614G) eRBD eRBD (N501Y) eRBD (E484K) S B.1.1.529 S
BA.5		 Epitope
84, 90, 101, 108, 119, 148, 183, 184, 185 9 + + + + + + R12
212, 216 2 + + + + - - R12
62, 82 2 - + + + - - R12
159 1 + + - - + + R6
149 1 + + - - + + R16
150 1 + + - - - - R16
64, 65, 67, 80, 83, 85, 87, 103, 105, 106, 112, 125, 129, 132, 138, 140, 141, 144, 154, 155, 156, 168, 174, 186, 198, 200, 201, 202, 209, 210, 215 31 + + - - + + un
166 1 + + - - + - un
187 1 + + - - - - un
63, 68, 69, 72, 73, 74, 76, 78, 86, 88, 89, 91, 94, 97,99, 102, 104, 107, 109, 113, 115, 121, 124, 126, 127, 130, 131, 134, 135, 136, 145, 146, 151, 161, 164, 165, 167, 171, 176, 178, 179, 180, 188, 190, 191, 192, 195, 196, 197, 205, 206, 208, 211 53 + - - - + - un
61, 66, 70, 77, 79, 92, 93, 98, 100, 111, 114, 116, 117, 133, 137, 139, 142, 143, 147, 153, 157, 181, 182, 189, 199, 204, 213 27 + - - - + + un
81, 118, 152, 158, 160, 162, 169, 172, 193, 194, 217 11 + - - - - - un
123, 214 2 - + - - - - un
71, 75, 120, 122, 170, 173, 175, 177, 207 9 - - - - - - un
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S1(D614G), mutant SARS-CoV-2 S1(D614G); eRBD, eukaryotic SARS-CoV-2 RBD; eRBD(N501Y), mutant eukaryotic SARS-CoV-2 RBD(N501Y); eRBD(E484K), mutant eukaryotic SARS-CoV-2 RBD(E484K); +, detectable reaction; -, undetectable reaction; R6, 391CFTNVYADSFVIRGD405. R12, 463PFERDISTEIYQAGS477; R16, 510VVVLSFELLHAPAT523; Un, undefined.

Multiple alignments of S proteins derived from seven HCoVs and two other SARSr-CoV strains were carried out to gain insights into the distribution of the epitope regions. All three epitopes, SARS-CoV-2-R6, -R12, and -R16, were unique to the other five HCoVs (HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, and MERS-CoV) (Fig. 2C). The R16 epitope differed from those of SARS-CoV-1, SARSr-CoV RaTG13, and SARSr-CoV BtKY72 by only one amino acid. There was no difference between SARS-CoV-1, SARSr-CoV RaTG13, and SARSr-CoV BtKY72 with respect to the corresponding regions of the R16 epitope (Fig. 2C). Interestingly, in the corresponding region R12, there was no difference between SARS-CoV-2 and SARSr-CoV BtKY72, and there were six amino acids in the corresponding R12 region of SARSr-CoV RaTG13, which were not consistent with those of SARS-CoV-2 (Fig. 2C). On the contrary, the R6 epitope differed from SARSr-CoV RaTG13 by only one amino acid and differed from SARSr-CoV BtKY72 by five amino acids (Fig. 2C). It is noteworthy that these three epitopes were highly conserved in the current circulating variants of SARS-CoV-2, including Alpha, Beta, Delta, Gamma, and Omicron, and only one amino acid mutation was found in the corresponding R12 region of Omicron (S→N), and one in R6 of Omicron BA.5 variant (D→N) (Fig. 2D).

3.3. Reaction of mAbs with the SARS-CoV-2 Omicron variants

Indirect ELISA was performed to detect the Omicron variants using the mAbs which currently circulate worldwide. Fig. 3A shows the mutation sites of the S proteins of Omicron B.1.1.529 and BA.5. E484K. N501Y and D614G mutations were conserved in Omicron variants. Among 151 mAbs, 69 reacted with both S proteins of Omicron B.1.1.529 and BA.5 (Fig. 3B); 28 mAbs did not react with the Omicron B.1.1.529 S protein, and 82 mAbs did not react with the Omicron BA.5 S protein. Strikingly, 54 mAbs recognized Omicron B.1.1.529 but not BA.5 (Fig. 3C; Table 2). Some mAbs reacted with both Omicron B.1.1.529 and BA.5 S resulting in high OD values. Interestingly, there were 34 mAbs which recognized both eRBD and Omicron B.1.1.529 S, but not eukaryotic mutants RBD-N501Y and RBD-E484K (Table 2). However, the mutations N501Y and E484A are kept in Omicron B.1.1.529 S. Therefore, a double-antibody sandwich ELISA was established with the optimal mAbs K7-155 and K7-157 to detect Omicron variants (Fig. 3D).

Fig. 3.

Fig. 3

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Reaction of mAbs with SARS-CoV-2 Omicron variants. (A) Reaction of mAbs with S proteins of SARS-CoV-2 Omicron variants B.1.1.529 and BA.5. (B) Common and specific reactions of mAbs with different proteins of Omicron B.1.1.529 S, Omicron BA.5 S and SARS CoV-2 S1. Different colored circles represent different proteins; the “69″ of all color overlapping areas in the figure represents that among 151 mAbs which reacted with SARS CoV-2 S1, sixty-nine mAbs recognized Omicron B.1.1.529 S, Omicron BA 5 S; the “54″ in the blue green overlapping area indicates that fifty-four mAbs reacted with Omicron B.1.1.529 S and SARS- CoV-2 S1, but not Omicron BA.5 S protein. (C) Schematic diagram of amino acid variation sites of SARS-CoV-2 Omicron variants B.1.1.529 and BA.5 S proteins. The regions of SARS-CoV-2 prototype S1-NTD, RBD, S1, and S2 are presented. (D) Double-antibody sandwich ELISA to detect S proteins of SARS-CoV-2 Omicron variants B.1.1.529 and BA.5. K7-155 reacted with eRBD, Omicron B.1.1.529 S, BA.5 S, RBD-N501Y and RBD-E484K; K7-157 reacted with both S proteins of Omicron B.1.1.529 and BA.5, eRBD but not RBD-N501Y and RBD-E484K. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Marked impact of a single amino acid mutation on mAb recognition

Of the 151 mAbs that recognized SARS-CoV-2 S1, 138 also recognized SARS-CoV-2 S1(D614G) (Fig. 4A). Forty-seven mAbs recognized both SARS-CoV-2-RBD(e) and SARS-CoV-2 S1(D614G), and four mAbs, K7-62, K7-82, K7-123, K7-214, recognized SARS-CoV-2-RBD(e) but not SARS-CoV-2 S1(D614G) (Fig. 4C; Table 2). Among 151 mAbs, 13 recognized eukaryotic SARS-CoV-2-RBD(N501Y) and SARS-CoV-2-RBD(E484K), as follows: K7-62, K7-82, K7-84, K7-90, K7-101, K7-108, K7-119, K7-148, K7-183, K7-184, K7-185, K7-212, and K7-216 (Fig. 4B). We found that all 13 mAbs recognized eukaryotic SARS-CoV-2 RBD also recognized R12 (463PFERDISTEIYQAGS477) (Fig. 4C).

Fig. 4.

Fig. 4

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Reaction of the mAbs with single amino acid mutant proteins. (A) Reaction of the mAbs with mutant eukaryotic SARS-CoV-2-S1(D614G) detected using ELISA. Anti-SARS-CoV-2-RBD mouse sera were used as the positive control, and mouse anti-PBS sera were used as the negative control. The dot-and-dash grid line shows the threshold value. (B) Cross-reaction of the mAbs with mutant eukaryotic SARS-CoV-2-RBD(N501Y) and SARS-CoV-2-RBD(E484K). (C) Common and specific reactions of mAbs with different proteins of SARS- CoV-2 S1(D614G), Omicron B.1.1.529 S, BA.5 S, SARS- CoV-2-RBD(e), RBD(E484K), RBD(N501Y) and epitopes R12 (463PFERDISTEIYQAGS477). Different colored parts represent different proteins, the entire circle represents 151 strains of monoclonal antibody that react with SARS-CoV-2 S1.

All nine mAbs that could react with all mutants; the S proteins of Omicron B.1.1.529 and BA.5, S1(D614G), RBD(N501Y), and RBD(E484K), recognized the epitope R12. As expected, all 13 mAbs that did not react with the mutant SARS-CoV-2 S1-D614G protein did not recognize these two Omicron S proteins (Fig. 4C; Table 2). The figure represents the overlapping area that has reaction with SARS CoV-2 S1 (D614G) and SARS CoV-2-RBD (e), with “2"; “2"; “1"; “9"; “33″, 47 in total, that is, 47 mAbs recognized both SARS-CoV-2-RBD(e) and SARS-CoV-2 S1(D614G); the “2″ and “2″ in the “SARS CoV-2-RBD (e)" circle are four, that is, four mAbs reacted with SARS CoV-2-RBD (e) but not with SARS CoV-2 S1 (D614G). The “2″, “2″ and “9″ in the center of the figure indicate that 13 mAbs all recognized SARS CoV-2-RBD (e) and RBD (E484K), RBD (N501Y) and epitope R12; among them, “9″ can identify all mutations.

4. Discussion

The global outbreak of the coronavirus pandemic has highlighted the need to rapidly develop effective methods for the detection and treatment of SARS-CoV-2 infection. SARS-CoV-2 infection is initiated by the binding of the S protein on the viral particle to host surface cellular receptors [15]. Within the S protein region, the S1 structural domain induces high levels of IgG and IgA antibodies [16], and its RBD is the key region for binding with ACE2 receptor and neutralizing antibody recognition s [17,18]. MAbs are potential diagnostic and therapeutic agents for viral infections, while mAbs targeting SARS-CoV-2 RBD show novel applications for development of potential epitope-specific vaccines [19]. In particular, mouse hybridoma mAbs are most commonly used for immunoassay analysis and early screening for disease because of their stable source, easy preparation, and high yield [20]. In this study, S1 protein derived from the SARS-CoV-2 virus were used for mouse immunization to generate a panel of mouse mAbs. In total, 151 mouse mAbs were characterized, and three novel linear epitopes of RBD, R6 (391CFTNVYADSFVIRGD405), R12 (463PFERDISTEIYQAGS477), and R16 (510VVVLSFELLHAPAT523) were identified (Table 2). Thirteen of 16 mAbs recognized R12 (463PFERDISTEIYQAGS477), which was superior to other epitopes. Of these, the mAb K7-90 had a weak neutralization effect. In addition, the effect of specific amino acid mutations (D614G in S1, N501Y, or E484K in RBD) and the S proteins of Omicron sublineages BA.1 and BA.5 on the detection of mAbs was investigated.

SARS-CoV-2 variants with multiple spike mutations are either resistant to antibodies produced in response to infection or vaccination or they increase transmissibility or disease severity [21]. The D614G mutation changes the conformation of the RBD and the stability of the S protein [22]. Thirteen mAbs did not react with SARS-CoV-2 S1(D614G). Importantly, four mAbs recognized SARS-CoV-2-RBD(e) but not SARS-CoV-2 S1(D614G); of these, K7-62 and K7-82 recognized the epitope R12 (463PFERDISTEIYQAGS477). Our results here indicate that the D614G mutation may cause a conformational change in S1, mask the linear epitope, and affect the accurate binding of the mAbs.

The N501Y mutation causes the formation of additional hydrogen bonds between RBD and ACE2 [23,24], reduces the compactness of the protein and causes significant changes in the secondary structure and folding of the S protein [25]. E484K is a charge reversal mutation, which generates additional salt bridges and hydrogen bonds to enhance the binding to ACE2, it will block the binding of the RBD-directed SARS-CoV-2 antibody to this domain [13,26]. We found that a single amino acid mutation, such as N501Y, E484K, may have a great impact on antibody-RBD protein binding. In the present study, K7-159 recognized the epitope R6 (391CFTNVYADSFVIRGD405) and K7-149 and K7-150 recognized the epitope R16 (510VVVLSFELLHAPAT523). The sites recognized by these mAbs did not contain any mutations; N501 and E484 mutations might cause changes in protein conformation and affect the binding of these mAbs, leading to immune escape and antibody off-target effects. Study of the mutant protein has revealed that the SARS-CoV-2 mutant strain still exerts a certain influence on the accurate binding of mAbs. Interestingly, we also found 33 mAbs that recognized both eRBD and Omicron B.1.1.529 S but not the eukaryotic mutants RBD-N501Y and RBD-E484K. For example, K7-159 and recognized the epitopes R6 (391CFTNVYADSFVIRGD405) and R16 (510VVVLSFELLHAPAT523), respectively. However, the mutations N501Y and E484A are kept in Omicron B.1.1.529 S. This result indicated that the N501Y and E484K mutations have greater impact on RBD than on the full-length S protein, suggesting the more stable structure of S than RBD. This discovery may be helpful to understand the mechanism of immune escape by SARS-CoV-2 variants.

In this study, we found no mAb with high neutralization activity. Only mAbs K7-87, K7-90, K7-155, and K7-210 showed weak neutralization activities. These mAbs were obtained from S1-immunized mice. These results are consistent with the previous study in which the quality and quantity of antibodies obtained from mice immunized with S1 were low [27]. In addition, linear peptide-directed immunization might not effectively generate “valid” antibodies in vivo because the conformation of the peptide might differ from that in the native spike protein. The conformation of the recombinant S1 protein immunized in mice might differ from that in the native S protein [28]. Only K7-90 recognized the RBD linear epitope 463PFERDISTEIYQAGS477, and K7-87, K7-155, and K7-210 might recognize the conformational epitope of RBD. All four of these mAbs reacted with Omicron variant B.1.1.529 and BA.5, indicating the epitope is highly conserved. Excessive concentrations of non-neutralizing antibodies may lead to antibody-dependent effects [29], which may be why the inhibition rates of some mAbs shown in this study were negative (Fig. 1B). These linear peptides may be candidates for a cytotoxic T cell response but not for protective humoral immunity, which should be further investigated.

Strikingly, the BA.1-specific antibodies are largely evaded by BA.2 and BA.4/BA.5 owing to D405 N and F486V mutations [30]. Our results also support this finding; 54 mAbs recognized Omicron B.1.1.529 but not BA.5, suggesting that BA.4/BA.5 display increased evasion of humoral immunity compared with that seen with BA.1. Importantly, 69 mAbs could recognize the current circulating Omicron variants B.1.1.529 and BA.5, which could be used to develop rapid diagnostic tools. We established a sandwich ELISA method using antibodies K7-155 and K7-157, which should be investigated in future studies with the SARS-CoV-2 virus and clinical samples. Due to the lack of a P3 laboratory, the current neutralization test was performed with a pseudovirus and not wild-type viruses.

In summary, several mouse-derived mAbs were obtained that bound to the SARS-CoV-2-RBD and S proteins of the mutant and had good reactivity in ELISA. The three novel linear epitopes recognized by the mAbs were identified as 391–405, 463–477, 510–523 amino acid residues of the S protein. Additionally, K7-90, which recognized the RBD linear epitope 463PFERDISTEIYQAGS477, was found to have a low neutralization efficacy. This study suggests that these mAbs may be useful tools for studying the function of the SARS-CoV-2 S protein, as well as for some clinical testing applications.

Funding

This study was supported by the National Natural Science Foundation of China (82072264), Guangzhou Science and Technology Plan Project (Joint Project of Municipal Colleges and Universities) (202102010364-ZNSA-2020003), the Natural Science Foundation of Guangdong Province of China (2021A1515011071; 2019A1515011681), and Guangzhou Institute of Respiratory Health Open Project (2020GIRHHMS01).

Author contribution statement

Yujie Yang: Performed the experiments; Analyzed and interpreted the data.

Liling Zhou, Chuncong Mo: Performed the experiments.

Longbo Hu, Zhichao Zhou, Ye Fan, Wenkuan Liu, Xiao Li, Rong Zhou: Contributed reagents, materials, analysis tools or data.

Xingui Tian: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Data availability statement

Data included in article/supp. material/referenced in article.

Additional information

No additional information is available for this paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank Prof. Chenyang Li for preparing the mAbs and Rong Liu for help in animal experiments.

Contributor Information

Rong Zhou, Email: zhourong@gird.cn.

Xingui Tian, Email: xgtian@gzhmu.edu.cn.

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