Enhancing epitope of PEDV spike protein

Abstract

Porcine epidemic diarrhea virus (PEDV) is the causative agent of a highly contagious enteric disease of pigs characterized by diarrhea, vomiting, and severe dehydration. PEDV infects pigs of all ages, but neonatal pigs during the first week of life are highly susceptible; the mortality rates among newborn piglets may reach 80-100%. Thus, PEDV is regarded as one of the most devastating pig viruses that cause huge economic damage to pig industries worldwide. Vaccination of sows and gilts at the pre-fertilization or pre-farrowing stage is a good strategy for the protection of suckling piglets against PEDV through the acquisition of the lactating immunity. However, vaccination of the mother pigs for inducing a high level of virus-neutralizing antibodies is complicated with unstandardized immunization protocol and unreliable outcomes. Besides, the vaccine may also induce enhancing antibodies that promote virus entry and replication, so-called antibody-dependent enhancement (ADE), which aggravates the disease upon new virus exposure. Recognition of the virus epitope that induces the production of the enhancing antibodies is an existential necessity for safe and effective PEDV vaccine design. In this study, the enhancing epitope of the PEDV spike (S) protein was revealed for the first time, by using phage display technology and mouse monoclonal antibody (mAbG3) that bound to the PEDV S1 subunit of the S protein and enhanced PEDV entry into permissive Vero cells that lack Fc receptor. The phages displaying mAbG3-bound peptides derived from the phage library by panning with the mAbG3 matched with several regions in the S1-0 sub-domain of the PEDV S1 subunit, indicating that the epitope is discontinuous (conformational). The mAbG3-bound phage sequence also matched with a linear sequence of the S1-BCD sub-domains. Immunological assays verified the phage mimotope results. Although the molecular mechanism of ADE caused by the mAbG3 via binding to the newly identified S1 enhancing epitope awaits investigation, the data obtained from this study are helpful and useful in designing a safe and effective PEDV protein subunit/DNA vaccine devoid of the enhancing epitope.

Keywords: antibody-dependent enhancement; enhancing epitope; phage display technology; phage mimotope; phage panning; porcine epidemic diarrhea; porcine epidemic diarrhea virus; spike protein.

Figure 1

Enhancement of the PEDV infectivity mediated by the mAbG3. (A) The mAbG3 at the concentrations of 5, 10, 20, and 40 μg was mixed with 100 pfu of PEDV GII strain P70 and incubated for 1 h before adding to the confluent monolayers of Vero cells. Immune serum to PEDV S1 subunit (1:100) and mAbA3 (40 μg) served as positive neutralization controls, medium alone served as negative neutralization/enhancement control, and isotype-matched IgG1-kappa mAb served as irrelevant mAb control. After 1 h of incubation, the fluid in each well was removed; the cells were rinsed and covered with 2% CMC in DMEM supplemented with 2 μg/ml of TPCK-treated trypsin. Two days post-infection, cells were fixed, and the number of syncytial formations was counted and calculated to % enhancement of PEDV infectivity compared to the medium alone. Data of each bar graph are presented as the mean and standard deviation of the % mAbG3-mediated enhancement of PEDV infectivity from triplicate wells of each treatment. (B) Results of qRT- PCR for determination of PEDV RNA recovered from different treatment groups. Y-axis, fold changes of PEDV RNA in different treatment groups compared to medium alone; X-axis, various treatment groups. By one-way ANOVA: ns, not significantly different; ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001; ^****, p < 0.0001. (C) Examples of the PEDV-mediated Vero cell CPE of different treatments. (D) Appearance of syncytial formation of PEDV-infected Vero cells (upper panel) compared with normal Vero cells (lower panel) at ×100 magnification of a light microscope.

Figure 2

Linear amino acid sequences of S1-0 (residues 154–218/221) and S1-BCD (residues 602–666/669) sub-domains of S1 proteins of PEDV strains CV777 (GI; GenBank: AEX92968.1) and P70 (GII) showing regions matched with the phage mimotope types 1–4 (M1-M4), i.e., the S1-0 residues 165GITWDNDRVTVF176 matched with M2 (M2S1–0), 179KIYHFYFKNDWS190 matched with M3 (M3S1–0), 191RVATKCYNSGGC202 matched with M4 (M4S1–0), 203AMQYVYEPTYYM214 matched with M1 (M1S1–0), and S1-BCD sub-domain residues 646LDVCTKYTIYGF657 matched with M4 (M4S1-BCD). The amino acid residues were colored according to the Zappos scheme that is based on physicochemical properties. *, identical amino acids; :, conserved amino acids.

Figure 3

Production of recombinant polypeptides of PEDV S1 subunit (S1-0, S1-A, and S1-BCD). (A) PCR amplificons of the DNA sequences coding for the S1-0 (651 bp), S1-A (873 bp), and S1-BCD (660 bp). Lane M, 1 kb DNA ladder. The numbers on the left are DNA sizes in bp. (B) SDS-PAGE-separated patterns of the purified S1-0 (26 kDa), S1-A (32 kDa), and S1-BCD (24 kDa) after staining with CBB dye. (C) Western blot patterns of the purified S1-0, S1-A, and S1-BCD as detected by mouse anti-6× His antibody. Lanes M of (B) and (C) are protein molecular mass markers. The numbers on the left of (B) and (C) are protein molecular masses in kDa.

Figure 4

Binding of mAbG3 to recombinant polypeptides S1-0, S1-A, and S1-BCD of the PEDV S1 subunit. (A) Indirect ELISA (OD 405 nm) of mAbG3 binding to the full-length recombinant S1 subunit and the S1 polypeptides S1-0, S1-A, and S1-BCD. Mouse anti-6× His antibody and mouse immune serum (1:10,000) served as positive binding controls, and PBS served as a negative binding control. (B) Binding of mAbG3 to S1-0, S1-A, and S1-BCD by dot ELISA. Mouse anti-6× His antibody (1:1,000) and PBS served as positive and negative binding controls, respectively. (C) Binding of mAbG3 to SDS-PAGE-separated S1-0, S1-A, and S1-BCD by Western blotting (left panel). Mouse anti-6× His antibody (1:1,000) was used as a positive control (right panel). (D) Binding of mAbG3 to M1S1–0, M2S1–0, M3S1–0, M4S1–0, M4S1-BCD, and control peptides by peptide binding ELISA. Y-axis, OD 405 nm; X-axis, peptides used as the test antigens; PBS served as control.

Figure 5

Multiple sequence alignment of amino acid sequences of spike proteins of various PEDV strains including PEDV P70 (a strain that was used in this study), CV777 (classical strain), USA/Colorado/2013 (prototype strain that was used for producing baculovirus-derived S subunit vaccine that caused severe enhancement; GenBank: KF272920.1), 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). The M3S1–0 sequences (red box) are highly conserved, and M4S1-BCD sequences (blue box) are identical among these PEDV strains. *, identical amino acids; :, conserved amino acids.

Figure 6

(A) PEDV trimeric spike protein (side view) showing locations of S1-0 (red, purple, and yellow), S1-A (green), S1-BCD (cyan and blue), and S2 subunits (gray). (B) Monomeric S1-0 showing the regions matched with phage mimotopes M1-M4 (purple and yellow); the yellow area is the M3S1–0. (C) S1-BCD monomer (cyan) showing the region bound by the mAbG3 (M4S1-BCD in blue).