Immune Netw.  2018 Jun;18(3):e21. 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

• Kim SH
• Cho BH
• Lee KY
• Jang YS

• KMID: 2414556
• DOI: http://doi.org/10.4110/in.2018.18.e21

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 (NTD231-501) of the PEDV S protein. Orally administered NTD231-501 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.

Keyword

Mucosal immunity; N-terminal domain; Porcine epidemic diarrhea virus; Vaccine

MeSH Terms

Coronavirus
Epithelial Cells
Epithelium
Escherichia coli
Immunity, Mucosal
Porcine epidemic diarrhea virus*
Swine

Figure

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  Figure 1. The NTD231–501 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.

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  Figure 2. Expression of the NTD231–501 of the PEDV S1 protein in E. coli. (A) The gene region encoding NTD231–501 was cloned into the pGEX 4T–1 vector containing a GST tag. (B) Recombinant NTD231–501 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 NTD231–501 of the PEDV S1 protein was purified by glutathione Sepharose chromatography.

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  Figure 3. Recombinant NTD231–501 of the PEDV S1 protein exhibited mucin-binding activity in a mucin-binding assay. (A) Recombinant NTD231–501 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 NTD231–501 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 NTD231–501 protein and control recombinant protein.

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  Figure 4. Oral mucosal immunization of recombinant NTD231–501 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, NTD231–501, or NTD231–501 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 NTD231–501-specific serum IgG isotype and NTD231–501-specific fecal IgA were determined by ELISA. (C) The number of NTD231–501-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.

Reference

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