African green monkey kidney epithelial (Vero) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, United States). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, United States), 2 mM l-alanyl-l-glutaminase dipeptide, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific).

Mouse hybridoma G3 secreting monoclonal antibody (mAbG3; IgG1-kappa isotype) that bound to the S1 protein of the PEDV was generated previously (Thavorasak et al., 2022). The cells were nurtured at 37°C in a humidified atmosphere containing 5% CO₂ in an incubator in a serum-free medium [CD medium supplemented with 8 mM l-alanyl-l-glutaminase dipeptide, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific)].

The PEDV (GII variant, strain P70, and isolated from PEDV-infected piglet in Thailand) was propagated in the Vero cells cultured in virus maintaining medium [DMEM supplemented with 2 μg/ml of N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich)]. Vero cell monolayers at ~80–100% confluent growth in the T175 culture flask were rinsed with sterile phosphate-buffered saline, pH 7.4 (PBS); the PEDV was added to the cells at MOI 0.001 and incubated at 37°C in a humidified atmosphere containing 5% CO₂ in an incubator for 1 h. The fluid was removed, the cells were rinsed twice with PBS, replenished with the virus maintaining medium, and incubated at 37°C in humidified 5% CO₂ atmosphere in an incubator. When the cytopathic effect (CPE; syncytial formation) reached the maximum level (approximately 48 h), the culture supernatant containing the PEDV was harvested, and centrifuged at 4500 × g and 4°C for 30 min to remove cells and debris. The clarified culture supernatant was filtered through a 0.2 μm Acrodisc^® syringe filter (Pall, Port Washington, NY, United States) and stored in small portions at −80°C until further use. The virus titer was determined by plaque-forming assay (PFA) and reported as plaque-forming units (pfu)/ml.

For the production of mouse monoclonal antibody G3 (mAbG3), log phase grown hybridoma G3 cells were seeded into a serum-free medium (CD medium, Thermo Fisher Scientific) at 3 × 10⁵ cells/ml and incubated further at 37°C in humidified 5% CO₂ atmosphere in an incubator for 96 h. The culture supernatant containing the mAbG3 was harvested, and centrifuged at 4500 × g and 4°C for 30 min to remove the cells and cell debris. The clarified supernatant was subjected to Hitrap protein G HP (Cytiva, Uppsala, Sweden) chromatography for the purification of the mAbG3. The protein content of the purified mAbG3 was quantified using the BCA assay (Thermo Fisher Scientific).

The neutralization assay was performed as described previously (Thavorasak et al., 2022). Purified mAbG3 (5–40 μg in 125 μl of PBS) was mixed with 100 pfu of PEDV P70 and incubated at 37°C for 1 h. Immune serum to PEDV S1 subunit (1:100), mAbA3 (Thavorasak et al., 2022), isotype-matched control mAb (IgG1-kappa; BioLegend, San Diego, CA, United States), and culture medium alone were included in the experiments as controls. The mixtures were then added to the confluent monolayers of Vero cells in a 24-well tissue culture plate. After 1 h of incubation, the supernatant was discarded, and the cells were rinsed for two times to remove the unbound virus. Carboxymethyl cellulose (2% CMC in DMEM supplemented with 2 μg of TPCK-treated trypsin) was added. At 48 h post-infection, the cells were fixed with 10% formalin, kept at room temperature (25 ± 2°C) for 1 h, and stained with 1% crystal violet in 10% ethanol. The CPE (syncytial formation) was counted under a light microscope at ×40 magnification. The percentage of mAbG3-mediated PEDV neutralization/enhancement was calculated: [1-(Average CPE count of test/Average CPE count of non-neutralization control)] × 100.

Alternatively, the PEDV RNAs recovered from the treated infected cells were determined by qRT-PCR. The virus neutralization assay was performed as described above with a slight modification. The DMEM supplemented with 2 μg of TPCK-treated trypsin was used instead of 2% CMC in DMEM supplemented with 2 μg of TPCK-treated trypsin. After 6 h post-infection, total RNA from the mAbG3/control-treated infected cells was extracted separately using TRIzol^® reagent (Thermo Fisher Scientific). The amount of viral RNA was quantified by qRT-PCR using a one-step brilliant III SYBR green RT-qPCR master mix (Agilent Technologies, Santa Clara, CA, United States). The qRT-PCR primers for the PEDV nucleocapsid (N) gene and house-keeping gene control are listed in Supplementary Table S1. The PCR reaction mixture consisted of 6 μl of nuclease-free water, 10 μl of 2× SYBR Green QPCR master mix, 0.4 μl of 10 μM of each specific primer, 0.2 μl of 100 mM dithiothreitol (DTT), 1 μl of RT/RNase block, and 2 μl of RNA template. The conditions for PCR thermal cycles were 50°C for 10 min, 95°C for 3 min, 40 cycles at 95°C for 5 s, and 58°C for 10s, followed by melt curve analysis protocol. The copy numbers of viruses were calculated from Cq values and compared.

The full-length S1 subunit of the PEDV was prepared as described previously (Thavorasak et al., 2022). For preparing the S1 subunit truncated polypeptides, i.e., S1-0 (residues M1-V217), S1-A (residues T218-N508), and S1-BCD (residues D509-T728; Li et al., 2017), total RNA was isolated from culture supernatant containing PEDV using TRIzol reagent (Thermo Fisher Scientific). The isolated RNA was subjected to RevertAid First Strand cDNA Synthesis Kit using a reverse primer specific to the PEDV E gene for synthesizing the cDNA. The cDNA was then used as a template for the amplification of DNA sequences coding for the S1-0, S1-A, and S1-BCD polypeptides using specific primers listed in Supplementary Table S2. The amplified DNA sequences were ligated separately to pJET1.2 plasmids, and the recombinant plasmids were transformed into DH5α E. coli. The transformed DH5α E. coli colonies with the respective DNA inserts were cultured in Luria-Bertani (LB) broth supplemented with 100 μg/ml ampicillin (LB-A) at 37°C with shaking aeration (250 rpm) and incubated overnight. The recombinant plasmids were then extracted from the DH5α E. coli and subcloned into pTriEx1.1 Hygro DNA (Merck KGaA, Darmstadt, Germany) via BamHI and XhoI restriction sites. The ligated plasmids were transformed into NiCo21 (DE3) E. coli. Transformed NiCo21 (DE3) E. coli clones that carried the correct DNA inserted sequences (verified by Sanger sequencing) were grown in LB-A broth at 37°C with shaking aeration until OD at 600 nm was 0.4–0.5. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to individual cultures to 0.1 mM final concentration, and the cultures were incubated further for 3 h. The 8× His-tagged recombinant polypeptides were purified from the bacterial homogenates by using Ni-NTA affinity resin (Thermo Fisher Scientific) under denaturing conditions.

One microgram each of the full-length recombinant S1 (rS1), S1-0, S1-A, and S1-BCD were prepared in protein loading buffer and subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were blotted onto the nitrocellulose membrane (NC). The empty sites of blotted NC were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h. After washing with TBST, the NC was immersed into a solution of either mouse anti-6× His antibody or purified mAbG3 solution (5 μg in 6 ml TBST) and kept at room temperature for 1 h. The membrane was washed with the TBST and placed in a solution of goat anti-mouse Ig-alkaline phosphatase (AP) conjugate (1:3,000; Southern Biotech, Birmingham, AL, United States) in TBST at room temperature for 1 h. After washing, BCIP/NBT substrate (KPL, Gaithersburg, MD, United States) was used for the visualization of the antigen–antibody reactive bands. The enzymatic reaction was stopped by rinsing the NC with distilled water.

For indirect ELISA, individual wells of 96-well microplate (Nunc MaxiSorp^™, Thermo Fisher Scientific) were coated with 1 μg of purified S1, S1-0, S1-A, and S1-BCD in 100 μl of PBS. The plate was kept at 4°C overnight. The supernatants were discarded, and the empty sites of the well surface were blocked with 1% BSA in PBS containing 0.1% Tween-20 (PBST) at 37°C for 1 h. One hundred microliters of PBST containing 1 μg of mAbG3 was added to each antigen-coated well and incubated at 37°C for 1 h. The wells were washed with PBST, added 100 μl of goat anti-mouse Ig-horseradish peroxidase conjugate (HRP; 1:5,000; Southern Biotech) in PBST, and kept at 37°C for 1 h. The fluids were discarded; all wells were washed with PBST, added 100 μl of ABTS substrate (KPL) individually, and the plate was kept at room temperature in darkness for 30 min. The OD of the content of each well was measured at 405 nm against blank (reactants without antibody) by spectrophotometry (BioTek, Winooski, VT, United States). Mouse anti-6× His antibody and PBS (instead of mAbG3) were included as positive and negative controls, respectively.

For dot ELISA, 1 μg of purified S1-0, S1-A, and S1-BCD were dotted separately onto the NC strip by using the Bio-Dot^® microfiltration apparatus (Bio-Rad). The membranes were dried for 30 min and blocked with 5% skim milk in TBST for 1 h at room temperature. After washing with TBST, 5 μg of mAbG3 in 6 ml of TBST was added and kept at room temperature for 1 h. Mouse anti-6× His antibody (1:1,000; Bio-Rad) and PBS were included as positive and negative controls, respectively. After washing, the membranes were immersed in goat anti-mouse Ig-alkaline phosphatase conjugate (1:3,000; Southern Biotech) in TBST at room temperature for 1 h. After washing, BCIP/NBT substrate (KPL) was used to visualize the reactive dots on membranes.

To identify the region (sub-domains) of the PEDV S1 subunit that was bound by the mAbG3, Ph.D.^™- 12 Phage Display Peptide Library (New England Biolab, Ipswich, MA, United States) was used for panning with the mAbG3. For this experiment, 1 μg of purified mAbG3 was added to a well of 96-well microplate (Nunc MaxiSorp^™) and kept at 4°C overnight. The empty sites of the well surface were blocked with 1% BSA in TBST. After washing, a diluted phage library (~2 × 10¹¹ peptide displaying phages in 100 μl of 0.5% BSA in TBST) was added to the mAbG3 coated well and incubated at room temperature for 1 h. The unbound phages were removed by washing thoroughly with TBST. The bound phages were eluted with 0.2 M glycine HCl (pH 2.2) for 10 min and immediately neutralized with 1 M Tris–HCl (pH 9.1). The phages were added to an early log phase grown ER2738 E. coil to amplify the phages and kept at 37°C for 4.5 h. The culture supernatant was harvested after centrifugation at 12,000× g and 4°C for 15 min The clear supernatant was mixed with 1/6 volume of 20% (w/v) PEG8000 and 2.5 M NaCl to precipitate and concentrate the phages. The concentrated phages were titrated on LB/IPTG/X-gal plates and used for the second- and third-round phage panning. The eluted phages of the third-round panning were diluted in LB broth (10¹–10⁴), inoculated into mid-log phase grown ER2738 E. coli, mixed with prewarmed (45°C) top agar, and poured onto LB/IPTG/X-gal agar plates. The plates were incubated at 37°C for 16 h. Ten single blue colonies were randomly picked and inoculated into mid-log phase grown ER2738 E. coli for phage amplification. The E. coli culture supernatants were collected for phage DNA isolation and sequencing. The DNA sequences coding for the peptides displayed by the mAbG3-bound phages were deduced by using QIAGEN CLC workbench program (Version 20.0.4¹; accessed on 11 March 2022). The phage mimotopes (M types) were then multiply aligned with the monomeric S1 sequence of PEDV GI classical strain CV777 and GII P70 strain by using Jalview software (Version 2.11.2.2; Waterhouse et al., 2009) to determine the region of the S1 subunit that matched with the mimotope sequences, i.e., the mAbG3 epitope.

To identify the enhancing epitope of the mAbG3 on the PEDV S1 subunit, peptide binding ELISA was performed. Five biotin-labeled 12 amino acid peptides of the PEDV S1 subunit that matched with the mAbG3-bound phage mimotopes and a control peptide were synthesized (Genscript, Piscataway, NJ, United States). Individual peptides were diluted in PBS to 10 μg/ml, and 100 μl of the diluted peptides were added to the separate wells (triplicate) of the streptavidin-coated microplate (Pierce^™ Streptavidin Coated HighCapacity Plates; Thermo Fisher Scientific), and the plate was kept at 4°C overnight. The plate was washed three times with TBST, and the empty sites of the well surface were blocked with 5% skim milk in TBST. After 1 h at 37°C, the fluids were discarded. The wells were washed again, and 1 μg of purified mAbG3 in 100 μl of TBST was added to the individual wells. Buffer was included as a negative antibody control. The plate was incubated at 37°C for 1 h. After washing, 100 μl of HRP-labeled mouse IgG-kappa binding protein (Santa Cruz Biotechnology, Dallas, TX, United States; 1:1,000 in TBST) was added to each well, and the plate was incubated further at 37°C for 1 h. After washing, ABTS substrate (KPL; 100 μl) was added to each well, and the plate was kept in darkness at room temperature for 30 min. The OD of individual wells was measured at 405 nm (BioTek spectrometer).

Amino acid sequences of the S1 subunits of the PEDV strains that have been submitted to the National Center of Biotechnology Information (NCBI) database within the past 5 years, including PEDV SD2021 (GenBank: UJZ92254.1), PEDV TRS2021 (GenBank: UJZ92268.1), TW/PT01/2020 (GenBank: UOK15705.1), SC-YB73 (GenBank: QNL15619.1), XJ1904-34 (GenBank: UGN13709.1), PEDV-H18-Barcelona-Vic (GenBank: QKV43853.1), PEDV-2118-1-Orense-Covelas (GenBank: QKV43823.1), CT P10 (GenBank: QHB92364.1), EdoMex/205/2018 (GenBank: QQK84868.1); and 0100/5P (GenBank: UAJ21031.1), as well as CV777 (classical strain, GenBank: AEX92968.1), USA/Colorado/2013 (prototypic strain used for producing baculovirus-derived S subunit vaccine that caused severe PEDV enhancement in immunized piglets in a challenge study, Yu et al., 2021; GenBank: KF272920.1), and our P70 strain, were multiply aligned and compared.

To locate the S1 epitope that bound to mAbG3, a cryoelectron structure of PEDV spike protein (PDB: 6vv5; Kirchdoerfer et al., 2021) was subjected to PyMOL software (Version 4.6; The PyMOL Molecular Graphics System, Schrödinger LLC, New York, NY, United States) for generating the spike protein structure, and the locations of the mAbG3 epitope were mapped on the S protein structure.

Statistical analysis of data was performed using GraphPad Prism software (Version 9.2; San Diego). Data were analyzed by using one-way ANOVA and were considered significantly different when p < 0.05.

About 5, 10, 20, and 40 μg concentrations of purified mAbG3 were mixed with PEDV GII field isolated P70 strain and incubated at 37°C for 1 h before adding to the confluent monolayers of Vero cells. After allowing the virus adsorption for 1 h, the fluids were removed, and the cells were rinsed two times before adding 2% CMC in 500 μl of DMEM supplemented with 2 μg/ml of TPCK-treated trypsin. Two days post-infection, the cells were fixed and stained with 10% formalin and 1% crystal violet in 10% ethanol, respectively. Mouse immune serum to PEDV S1 subunit (1:100), 40 μg of mAbA3 (Thavorasak et al., 2022), 40 μg of isotype-matched control mAb (IgG1-kappa), and culture medium alone were included in the experiments as controls. Cytopathic effect (CPE) was determined under light microscopy at ×40 magnification. It was found that instead of mediating neutralization of the PEDV infectivity, on contrary, the mAbG3 at 40 μg concentration significantly enhanced the PEDV infectivity by increasing CPE (the numbers of syncytia) compared to the medium (p < 0.0001) and the isotype control (p < 0.01), while the immune serum and the mAbA3 neutralized the PEDV infectivity (Figure 1A). The mAbG3 at 5, 10, and 20 μg concentrations showed a trend toward the enhancement of infectivity, but the CPE counts were not significantly different from the medium alone (p > 0.05). The results of qRT-PCR conformed to the CPE results (Figure 1B). Examples of the PEDV-mediated CPE in the Vero cell monolayer of different virus treatments are shown in Figure 1C. A comparison of the Vero syncytium to normal Vero cells is shown in Figure 1D. Three independent experiments were performed with reproducibility.

Because the mAbG3 was found to enhance the PEDV infectivity, it was interesting to identify the epitope of this monoclonal antibody for the future design of a safe PEDV subunit/DNA vaccine. The Ph.D.^™-12 Phage Display Peptide Library was used as a tool for the identification of the phage peptides that were bound by the mAbG3, and the mAbG3-bound phage peptides were aligned with the PEDV S1 linear sequence to identify the mAbG3-bound S1 sub-domain region, i.e., mAbG3 epitope. After panning the phage library with the mAbG3, the mAbG3-bound phages were propagated; the phage DNAs were isolated, sequenced, and deduced. The deduced amino acid sequences, designated mimotope (M) types, were aligned with monomeric S1 segments of CV777 classical strain (GenBank: AEX92968.1) and PEDV GII variant P70 strain. The results revealed that there were four phage mimotope (M) types (M1-M4) that matched with the residues in the linear S1 subunit, including M1: FFDYMYLPGFAA, M2: SFDWPTHKMFNL, M3: NLYNYMFADLYT, and M4: TDLCHLYNMHGC. The M1 phage mimotope sequence matched with 203AMQYVYEPTYYM214 of S1-0 (designated M1S1–0), M2 sequence matched with 165GITWDNDRVTVF176 of S1-0 (designated M2S1–0), M3 sequence matched with 179KIYHFYFKNDWS190 of S1-0 (designated M3S1–0), and M4 sequence matched with 191RVATKCYNSGGC202 of S1-0 (designated M4S1–0) and 646LDVCTKYTIYGF657 of S1-BCD (designated M4S1-BCD; Figure 2).

The truncated polypeptides of the PEDV S1 subunit (S1-0, S1-A, and S1-BCD) were prepared for verification of the S1 regions bound by the mAbG3. Amplicons of the DNA sequences coding for the S1-0, S1-A, and S1-BCD (651, 873, and 660 bp, respectively) are shown in Figure 3A. The recombinant S1 polypeptides with 8× His tag expressed by the transformed NiCo21(DE3) E. coli clones were then purified by Ni-NTA affinity resin under denaturing conditions with 8 M urea buffer. The purified polyproteins were then refolded by drop-wise dialysis against PBS. The polypeptides, i.e., S1-0 (residues 1–217; ~26 kDa), S1-A (residues 218–508; ~32 kDa), and S1-BCD (residues 509–728; ~24 kDa) after SDS-PAGE and staining with Coomassie Brilliant Blue G-250 (CBB) dye are shown in Figure 3B. Figure 3C shows Western blot patterns of the SDS-PAGE-separated polypeptides probed with mouse anti-6× His antibody.

The mAbG3 was tested for binding to the purified full-length S1 and S1-0, S1-A, and S1-BCD polypeptides in the indirect ELISA, dot ELISA, and Western blot analysis. The indirect and dot ELISA results showed that the mAbG3 bound to the full-length S1 subunit and the S1-0 and S1-BCD polypeptides but not the S1-A polypeptide (Figures 4A,B). When the mAbG3 was tested for binding to the S1 polypeptides by Western blot analysis under reducing conditions using mouse anti-6× His antibody as positive binding control, the mAbG3 bound to all three S1 sub-domain polypeptides (Figure 4C).

Biotin-labeled M1S1–0, M2S1–0, M3S1–0, M4S1–0, M4S1-BCD, and control peptides were commercially synthesized and used as antigens in the indirect ELISA for testing the mAbG3 binding. In the indirect ELISA process, the synthesized peptides were used to coat separate wells of a microplate, and the coated wells were added with the mAbG3. The mAbG3 yielded a strong binding signal to M3S1–0, a significant signal to the M4S1-BCD, negligible signals to M1S1–0, M2S1–0, and M4S1–0, and no signal to control peptide (Figure 4D).

The results revealed that the M3S1–0 and M4S1-BCD that were bound by the mAbG3 are highly conserved (Figure 5). Locations of the mAbG3 epitope on the PEDV trimeric S, monomeric S1-0, and monomeric S1-BCD proteins are shown in Figures 6A–C, respectively.