Identification of Conserved B and T Cell Epitopes in Glycoprotein S of Mexican Porcine Epidemic Diarrhea Virus (PEDV) Strains via Immunoinformatics Analysis, Molecular Docking, and Immunofluorescence

Figures

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  Figure 1. Neighbor-Joining phylogenetic tree of 54 Mexican PEDV strains. The tree indicates four main groups: G1a (blue), G1b INDEL (green), G2a (red), and G2b (orange). Mexican strains are highlighted in bold.

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  Figure 3. Immunofluorescence analysis of PK-15 cells transfected with the recombinant plasmid PEDVV5. (A) The recombinant protein PEDVV5 was constructed containing a signal peptide (SP) followed by the conserved B cell epitopes S02, S03, S04, S06, and S07 that were placed at the N-terminus, while the transmembrane domain and the epitopes PADRE, c-Myc, and polihistidine (6Xhis) were placed at the C-terminus. (B) Non-transfected PK-15 cells were treated via specific immunofluorescence against PEDV. (C) PK-15 cells transfected with the plasmid PEDVV5 were processed via PEDV-specific immunofluorescence using anti-PEDV sera from infected pigs and FITC-conjugated protein A; the nuclei were stained with Hoechst. All microscopy images are of the same scale (20 μm). PC indicates phase contrast. (D) Quantification of the anti-PEDV fluorescence intensity expressed in arbitrary units (AU) for non-transfected and transfected cells. Each symbol represents the total fluorescence intensity measured from a single cell. All cells are plotted using a box plot showing the lower limit (25th percentile), center (50th percentile), and upper limit (95th percentile) of fluorescence intensity. Statistical analysis…

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  Figure 2. Location of B cell epitopes in a crystal structure of the PEDV S glycoprotein with high homology (PDB 7W6M) and in the modeling of the S glycoprotein from Mexican PEDV strains. (a) Visualization of B cell epitopes of the S glycoproteins from Mexican PEDV strains in a protein crystal with high homology (KY929406.1). (b) Modeling of the S glycoprotein of PEDV isolated in Michoacan (GenBank access number: MH006965.1), (c) Tlaxcala (GenBank access number: MN091346.1), (d) Veracruz (GenBank access number: MH013464.1), (e) Puebla (GenBank access number: MH006963.1), (f) Jalisco (GenBank access number: MH004420.1), and (g) Estado de Mexico (GenBank access number: MT490316.1). The conserved B cell epitopes were visualized to know their location on the surface of the PEDV S glycoprotein using Chimera X software. From left to the right: top view, side view, and bottom view. Colors indicate the different epitopes found: epitope S01 and epitope S05 are located within a pore of the glycoprotein and are represented in blue (hidden), epitope S02 is highlighted in red, epitope S03 is highlighted in yellow, epitope S04 is highlighted in purple, the epitope S06 is highlighted in sky blue, and…

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  Figure 4. Molecular docking shows the binding of HTL epitopes derived from PEDV S glycoprotein to the pockets of the peptide-binding groove of SLA-II (SLA-DRB1*1101). (A) HTL1 interacts through four pockets (P4, P6, P7, and P9); (B) HTL09 interacts with two pockets (P4 and P9); (C) HTL12 interacts with three pockets (P4, P7, and P9); (D) HTL13 interacts with two pockets (P4 and P9); (E) HTL17 interacts with three pockets (P4, P6, and P7); (F) HTL27 interacts with two pockets (P6 and P9); (G) HTL41 interacts with two pockets (P4 and P7); and (H) HTL49 interacts with three pockets (P4, P6, and P7) of the SLA-II beta chain. The HTL epitopes are observed in cyan, whereas the pockets belonging to the beta chain, P1, P4, P6, P7, and P9, are observed as sticks in green, yellow, hot pink, orange, and purple blue, respectively.

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  Figure 5. Molecular docking of the predicted CTL epitopes and SLA-I (SLA-I*0401). (A) Molecular docking analysis between the AY9 peptide (ATAAATEAY) derived from the Ebola virus VP35 and SLA-I*0401 obtained by performing a co-crystallization experiment (PDB accession number: 3QQ4). The interaction of the AY9 peptide with the six SLA-I pockets (pockets (A–F)) is observed, as in the original experiment [33]. (B) CTL04 interacts with three pockets (B,C,E); (C) CTL05 interacts with four pockets (B–E); (D) CTL06 interacts with five pockets (A–E); (E) CTL12 interacts with two pockets (D,E); (F) CTL03 interacts with four pockets (A,C–E); (G) CTL17 interacts with five pockets (A–D,F); (H) CTL18 interacts with four pockets (C–F); (I) CTL20 interacts with three pockets (A,C,D); and (J) CTL22 interacts with five pockets (A,C–F) of the SLA-I. The PEDV S glycoprotein-derived CTL epitopes are represented in cyan, and the six pockets (A–F) of the SLA-I heavy chain are represented by green, yellow, hot pink, orange, wheat, and purple blue sticks, respectively.