N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Sae-Hae Kim; Byeol-Hee Cho; Kyung-Yeol Lee; Yong-Suk Jang

doi:10.4110/in.2018.18.e21

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Kim, Cho, Lee, and Jang: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Original Article

Immune Netw 2018;18(3):e21.

Published online: 4 June 2018

DOI: https://doi.org/10.4110/in.2018.18.e21

N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Sae-Hae Kim¹, Byeol-Hee Cho², Kyung-Yeol Lee³, Yong-Suk Jang^1,^2,^*

¹Department of Molecular Biology and the Institute for Molecular Biology and Genetics, Chonbuk National University, Jeonju 54896, Korea

²Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Chonbuk National University, Jeonju 54896, Korea

³Department of Oral Microbiology and Institute of Oral Bioscience, Chonbuk National University, Jeonju 54896, Korea

^*Correspondence to Yong-Suk Jang Department of Molecular Biology and the Institute for Molecular Biology and Genetics, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju 54896, Korea. E-mail: yongsuk@jbnu.ac.kr

Conflict of Interest The authors declare no potential conflict of interest.

Received 9 April 2017 Revised 11 June 2018 Accepted 12 June 2018

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Porcine epidemic diarrhea virus (PEDV) is a contagious coronavirus infecting pigs that leads to significant economic losses in the swine industry. Given that PEDV infection occurs in gut epithelial cells mainly via the fecal-oral route, induction of PEDV-specific immune responses in the mucosal compartment is required for protective immunity against viral infection. However, an effective mucosal vaccine against the currently prevalent PEDV strain is not available. In this study, we demonstrated that the N-terminal domain (NTD) of the spike (S) protein of PEDV represents a new vaccine candidate molecule to be applied via the mucosal route. We first established an Escherichia coli expression system producing the partial NTD (NTD_231–501) of the PEDV S protein. Orally administered NTD_231–501 protein specifically interacted with the apical area of M cells in the follicle-associated epithelium of Peyer's patch. Additionally, the NTD protein induced antigen-specific immune responses in both the systemic and mucosal immune compartments when administered orally. Collectively, we propose the NTD of the PEDV S protein to be a candidate mucosal vaccine molecule.

Keywords: Mucosal immunity, N-terminal domain, Porcine epidemic diarrhea virus, Vaccine

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Figure 1.

The NTD_231–501 of the PEDV S1 protein contains a sugar-binding domain. Schematic diagram shows the CTD and the NTD of the S protein encompassing amino acids 231–501.

Figure 2.

Expression of the NTD_231–501 of the PEDV S1 protein in E. coli. (A) The gene region encoding NTD_231–501 was cloned into the pGEX 4T–1 vector containing a GST tag. (B) Recombinant NTD_231–501 of the PEDV S1 protein was expressed by isopropyl β-D-1-thiogalactopyranoside induction and confirmed by Western blot analysis using an anti-GST Ab. (C) Recombinant NTD_231–501 of the PEDV S1 protein was purified by glutathione Sepharose chromatography.

Figure 3.

Recombinant NTD_231–501 of the PEDV S1 protein exhibited mucin-binding activity in a mucin-binding assay. (A) Recombinant NTD_231–501 of the PEDV S1 protein purified by GST affinity chromatography was incubated on an ELISA plate coated with mucin. Binding activity was determined using an HRP-conjugated anti-GST Ab followed by the addition of tetramethyl benzidine substrate. Numbers on the X-axis indicate the amounts of NTD or control proteins. (B) Whole PP incubated with recombinant NTD_231–501 of the PEDV S1 protein purified by GST affinity chromatography was stained with NKM 16-2–4 anti-M cell Ab (Alexa 488, green), anti-GST Ab (Alexa 594, red), or WGA (Alexa 405, blue). Samples were examined by confocal laser scanning microscopy. Dotted lines depict the M cells, and scale bars represent 20 μm. * p<0.05 indicates a significant difference between the NTD_231–501 protein and control recombinant protein.

Figure 4.

Oral mucosal immunization of recombinant NTD_231–501 of the PEDV S1 protein into mice induces Ag-specific immune responses in the systemic and mucosal compartments. Each group of mice was orally administered PBS, NTD_231–501, or NTD_231–501 with CT once a week for 3 wk. (A) Sera, (B) feces, and (C) LP cells were prepared at (A and B) 3 days and (C) 4 weeks after the last immunization. (A) The NTD_231–501-specific serum IgG isotype and NTD_231–501-specific fecal IgA were determined by ELISA. (C) The number of NTD_231–501-specific IgA Ab-secreting cells in the LP was determined by ELISPOT assay. Data are presented as means±standard error (n=3), and a representative result from three independent experiments is shown. * p<0.05 indicates a significant difference among the groups compared.

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