A New Neutralization Epitope in the Spike Protein of Porcine Epidemic Diarrhea Virus

Abstract

Porcine epidemic diarrhea virus (PEDV) infects piglets and causes serious diarrhea as well as vomiting, dehydration, and death. The trimeric S protein plays a crucial role in the induction of neutralizing antibodies, and many neutralizing monoclonal antibodies (mAbs) against PEDV S protein have been developed. However, these mAbs exclusively target the S1 protein. In this study, we obtained a neutralizing mAb, 5F7, against the S2 protein of PEDV, and this mAb could neutralize new variant genotype 2 PEDV strains (LNCT2), as well as a genotype 1 PEDV strain (CV777), in vitro. The core sequence of the epitope was found in amino acid sequence 1261 aa~1337 aa. These findings confirm that the S2 protein possessed neutralizing epitopes and provided knowledge to aid further research on this virus.

Keywords: PEDV; neutralizing monoclonal antibody; spike protein.

Figure 1

Reaction of mAb 5F7 with PEDV-positive cells, as detected by IFA. The cells infected by PEDV strains LNCT2 and CV777 yielded positive signals, and Vero E6 cells were used as negative controls. The heavy chain of mAb 5F7 was IgG2a, whereas the light chain was kappa, as determined by the SBA Clonotyping System-HRP (Southern Biotech). Bar, 400 μm.

Figure 2

Neutralization of distinct PEDV strains by mAb 5F7. PEDV strains LNCT2 and CV777 were used to test the neutralization features of mAb 5F7. The IC₅₀ of the mAb 5F7 in neutralizing strains LNCT2 and CV777 were 54.44 μg/mL and 66.17 μg/mL, respectively. The neutralization tests were repeated three times. The five points of antibody concentration from low to high are 10 μg/mL, 31.25 μg/mL, 62.5 μg/mL 125 μg/mL, 250 μg/mL, 500 μg/mL. The figures were generated with GraphPad Prism version 9.0 (GraphPad Software, La Jolla, CA, USA).

Figure 3

Identification of mAb 5F7 to the PEDV S protein expressed in Vero E6 cells in Western blot analysis. Lane M, protein marker. The Vero E6 cells in lane 1 were transfected with pcDNA3.1(−)–LNCT2–S, expressing the PEDV S protein, whereas the Vero E6 cells in lanes 2 and 3 were infected with PEDV CV777 and LNCT2. Vere E6 cells were used as negative controls (Lane 4). mAb 5F7 reacted well with the PEDV S protein expressed in Vero E6 cells and the S protein of PEDV CV777 and LNCT2. The mAb 5F7 did not react with the Vero E6 cells.

Figure 4

Schematic diagram of the PEDV S fragments used for B-cell epitope mapping. Four rounds of S peptides were conducted to investigate epitopes of the generated mAb.

Figure 5

Identification of the epitope recognized by mAb 5F7 using IFA. Recombinant plasmids pcDNA3.1(−)–LNCT2–S1 and –S2, as well as pcDNA3.1(−)–LNCT2–S2-a, -b, -c, -d, -e, -f, and -g, were used to transfect 293T cells. The results show that the cells transfected with pcDNA3.1(−)–LNCT2-S2, S2-b, -d, and f plasmids reacted with mAb 5F7, while the cells transfected with pcDNA3.1(−)–LNCT2-S1, S2-a, -c, -e, -g, -h, -i, and -j plasmids did not react with mAb 5F7. Bar, 200 μm.

Figure 6

Comparison of the mAb 5F7 epitope amino acid sequence among the different PEDV strains. The mAb 5F7 epitope sequences from different strains of PEDV are shown.