The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes

Abstract

The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids [aa] 744-759, 747-774 and/or 756-771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371-1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499-600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV.

Keywords: Neutralizing antibodies; Porcine epidemic diarrhea virus (PEDV); Spike.

Fig. 1

Immunoreactivity of PEDV specific monoclonal antibodies with PEDV and ORFV-PEDV-S infected cells. Vero cells were infected with PEDV strain CO13 or S-INDEL variant strain USA/IOWA/106/2013. Primary ovine fetal turbinate (OFTu) cells were infected with the recombinant ORFV-PEDV-S. Cells were fixed with 80% acetone (Vero) or 3.7% formaldehyde (OFTu) and incubated with cell culture supernatants of hybridoma cultures secreting PEDV S-specific mAbs. Goat anti-mouse IgG+IgM+IgA secondary antibodies (FITC or DyLight594) were added and cells evaluated under a fluorescence microscope (200 X).

Fig. 2

Immunoreactivity of PEDV specific monoclonal antibodies for linear epitopes of the S protein. Purified whole PEDV preparations (WV, full length S: ~150 kDa, cleaved S1/S2: ~80 kDa), recombinant S (S1-S2; aa 630–800, S: ~24 kDa) and recombinant nucleocapsid (N, ~49 kDa) proteins were subjected to SDS-PAGE in 4–20% gradient acrylamide gels. Proteins were blotted into nitrocellulose membranes and probed with each PEDV S-specific mAb. Swine convalescent serum (α-PEDV) was used as positive control. Blots were developed with a chemiluminescent method.

Fig. 3

Reactivity of PEDV specific monoclonal antibodies to prototype and S-INDEL PEDV strains and truncated regions of S protein. The reactivity of each PEDV-S mAb with whole PEDV preparations (WV; strains CO13 [A] and S-INDEL [B]) and truncated recombinant S proteins (COE; aa 499–600 [C]; and aa 630–800 [D]) were evaluated by indirect ELISAs. Swine convalescent serum (α-PEDV) was used as positive control and anti-PRRSV mAb SDOW-17 was used as a negative control. Each mAb was subjected to 2-fold dilutions (1:20–1:160) and tested in three independent experiments. All antibodies were initially diluted to a working solution of 3 mg/mL. Optical densities (OD) shown represent the average of three independent experiments.

Fig. 4

Neutralizing activity of PEDV S-specific mAbs. The neutralizing activity of each PEDV-S mAb was determined by fluorescent focus neutralization assay (FFN; 90% reduction cutoff) and plaque reduction neutralization test (PRNT, 80% reduction cutoff). Swine convalescent serum (α-PEDV) was used as positive control. Each mAb was tested in three independent experiments.

Fig. 5

Epitope mapping ELISA of PEDV S-specific monoclonal antibodies. The epitope specificity of each neutralizing mAb was assessed by pepscan ELISAs (A-D). Swine convalescent serum (α-PEDV) was used as positive control and anti-PRRSV mAb SDOW17 was used as a negative control. Each mAb was subjected to 2-fold dilutions (1:20–1:160) and tested in three independent experiments. All antibodies were initially diluted to a working solution of 3 mg/mL. Optical densities (OD) shown represent the average of three independent experiments.

Fig. 6

Schematic representation of PEDV S and its immunodominant neutralizing epitopes (PEDV strain CO13, GenBank accession no. KF272920). (A) Diagram depicting the main features of the PEDV S protein, including: putative cleavage site between S1 and S2 subunits at aa 729, signal peptide (aa 1–18), N-terminal domain (aa 19–233), fusion peptide (aa 891–908), heptad repeat region 1 (HR1, aa 978–1117), heptad repeat region 2 (HR2, aa 1274–1313), and the transmembrane domain (aa 1328–1350). (B) Diagram depicting the regions containing B-cell epitopes (N-terminal domain [NTD]/S0; aa 19–220; collagenase equivalent [COE] aa 499–638; and aa 638–789) and targeted by neutralizing antibodies (NTD/S0, aa 19–220; COE, aa 499–638, and aa 1371–1377). (C) Spike immunodominant neutralizing regions (aa 499-600, aa 744–759, aa 747–774, aa 756–771, and aa 1371–1377) and mAb reactivity with neutralizing epitopes.

Fig. 7

Neutralizing activity of PEDV S-specific mAbs correlates with endpoint ELISA titers against recombinant S1-S2 recombinant protein. (A) Endpoint neutralizing antibody titers as determined by FFN assay were transformed to log 2 and compared to endpoint S ELISA (aa 630–800) titers. End point neutralizing titers were considered the reciprocal of the highest dilution capable of inhibiting virus infectivity by 90%, whereas endpoint ELISA titers were considered as the highest mAb dilution above the assay cutoff OD value of 0.16. Each antibody was tested in three independent experiment and the average endpoint titer is presented. (B) Correlation plot between neutralizing and S ELISA endpoint titers for PEDV S mAbs.