Identification of a novel B-cell epitope in the spike protein of porcine epidemic diarrhea virus

Abstract

Background: Porcine epidemic diarrhea virus (PEDV) infection causes an acute enteric tract infectious disease characterized by vomiting, anorexia, dehydration, weight loss and high mortality in neonatal piglets. During PEDV infection, the spike protein (S) is a major virion structural protein interacting with receptors and inducing neutralizing antibodies. However, the neutralizing B-cell epitopes within PEDV S protein have not been well studied.Methods: To accurately identify the important immunodominant region of S1, the purified truncated S1 proteins (SA, SB, SC, SD and SE) were used to immunize BALB/c mice to prepare polyclonal antibodies. The antisera titers were determined by indirect ELISA, western blot and IFA after four immunizations to find the important immunodominant region of S1, and then purified the immunodominant region of S1 protein and immunized mice to generate the special antibodies, and then used recombinant peptides to determine the B-cell epitopes of monoclonal antibodies.Results: Five antisera of recombinant proteins of the spike protein region of PEDV were generated and we found that only the polyclonal antibody against part of the S1 region (signed as SE protein, residues 666-789) could recognize the native PEDV. Purified SE protein was used to immunize BALB/c mice and generate mAb 2E10. Pepscan of the SE protein demonstrated that SE16 ( 722 SSTFNSTREL 731 ) is the minimal linear epitope required for reactivity with the mAb 2E10. Further investigation indicated that the epitope SE16 was localized on the surface of PEDV S protein in the 3D structure.Conclusions: A mAb 2E10 that is specifically bound to PEDV was generated and identified a specific linear B-cell epitope (SE16, 722 SSTFNSTREL 731 ) of the mAb. The epitope region of PEDV S1 localized in the different regions in comparison with the earlier identified epitopes. These findings enhance the understanding of the PEDV spike protein structure for vaccine design and provide a potential use for developing diagnostic methods to detect PEDV.

Targeting HLA-F suppresses the proliferation of glioma cells via a reduction in hexokinase 2-dependent glycolysis

“…Six- to eight-week-old female BALB/c mice were immunized with 50 μg purified HLA-F protein emulsified in the same volume of complete Freund's adjuvant by intraperitoneal injection 28 . Three additional injections were administered to the mice at 2-week intervals with the same dose of antigen emulsified in incomplete Freund's adjuvant 28 .…”

“…Six- to eight-week-old female BALB/c mice were immunized with 50 μg purified HLA-F protein emulsified in the same volume of complete Freund's adjuvant by intraperitoneal injection 28 . Three additional injections were administered to the mice at 2-week intervals with the same dose of antigen emulsified in incomplete Freund's adjuvant 28 . At the end of the experiments, immunized mice were sacrificed, and single spleen cells were fused with SP2/0 myeloma cells utilizing polyethylene glycol 1450 (PEG1450, Sigma-Aldrich, USA) 28 , 29 .…”

Characteristics of the spike and ORF3 genes of porcine epidemic diarrhea virus in Henan and Shanxi provinces of China

“…This suggests that these aa changes might have been involved in immune escape, reducing the effectiveness of the CV777-based vaccine and ultimately leading to outbreaks of severe diarrhea on Chinese swine farms. Elsewhere, it has been reported that a novel B-cell epitope, SE16, is the immunodominant region ( 722 SSTFNSTREL 731 ) of the PEDV S protein [20]. In our study, two aa mutations (F→L at 725; S→G at 727) were found in the strain CH/ XX/2015 (KU237231), and experimental studies are needed to determine whether these mutations affect vaccines and diagnostic reagents.…”

Porcine epidemic diarrhea virus: Molecular mechanisms of attenuation and vaccines

“…In addition, the S protein contains two intracellular sorting motifs, YxxΦ and/or KVHVQ at the cytoplasmic tail and deletion of both motifs reduced the virulence of PEDV in piglets [ 121 ]. S protein contains multiple B cell epitopes, such as 722 SSTFNSTREL 731 , 748 YSNIGVCK 755 , 764 SQYGQVKI 771 , and 1368 GPRLQPY 1374 [ [122] , [123] , [124] , [125] ]. The core neutralization epitope region (COE) of the S1 region has been widely applied to develop PEDV subunit vaccines.…”