The immunogenicity of plant-based COE-GCN4pII protein in pigs against the highly virulent porcine epidemic diarrhea virus strain from genotype 2

Abstract

Porcine epidemic diarrhea virus (PEDV) is a serious infectious causative agent in swine, especially in neonatal piglets. PEDV genotype 2 (G2) strains, particularly G2a, were the primary causes of porcine epidemic diarrhea (PED) outbreaks in Vietnam. Here, we produced a plant-based CO-26K-equivalent epitope (COE) variant from a Vietnamese highly virulent PEDV strain belonging to genotype 2a (COE/G2a) and evaluated the protective efficacy of COE/G2a-GCN4pII protein (COE/G2a-pII) in piglets against the highly virulent PEDV G2a strain following passive immunity. The 5-day-old piglets had high levels of PEDV-specific IgG antibodies, COE-IgA specific antibodies, neutralizing antibodies, and IFN-γ responses. After virulent challenge experiments, all of these piglets survived and had normal clinical symptoms, no watery diarrhea in feces, and an increase in their body weight, while all of the negative control piglets died. These results suggest that the COE/G2a-pII protein produced in plants can be developed as a promising vaccine candidate to protect piglets against PEDV G2a infection in Vietnam.

Keywords: COE; GCN4-pII motif; PEDV genotype 2; passive immunity; piglet protection; plant-based vaccine.

Figure 1

Production and characterization of COE/G2a-pII protein from plants. (A) Plant expression cassette containing a DNA sequence encoding the highly virulent NAVET/PEDV/PS6/2010 strain (NAVETCO, Vietnam) fused pII motif. CaMV35S-P, cauliflower mosaic virus (CaMV) 35 S promoter; pII, GCN4pII motif; KDEL, endoplasmic reticulum retention; CaMV35S-T, CaMV 35 S terminator. (B) Detection of COE/G2a protein expressed in N. benthamiana leaves by Western blotting assay. Anti-c-myc monoclonal antibody and HRP conjugated goat anti-mouse IgG were used as primary antibodies and secondary antibodies, respectively. Various amounts of H5N1-specific ScFv antibodies (23) were used to build the standard curve to calculate the accumulation of COE/G2a-pII protein in leaves by ImageQuant TL (Cytiva) after capturing by Amersham™ Imager 680 (Cytiva). (C) Detection of SEC-fractions containing COE/G2a-pII protein by Western blotting assay. (D) Analysis of IMAC-purified COE/G2a-pII protein by Coomassie Blue staining of SDS-PAGE. (E) Characterization of COE/G2a-pII protein by SEC. A standard kit including high molecular weight protein (75–2,000 kDa, GE) was used to estimate the molecular weight of COE/G2a-pII protein.

Figure 2

Humoral immune responses and cytokine responses in pregnant sows. Data are presented as mean ± standard deviation (SD). * indicates a statistically significant difference (p < 0.05). (A) Immunization and challenge experiment scheme in pregnant sows (~80 days of gestation, n = 1 per group) and piglets (n = 8 for COE/G2a-pII group and n = 2 for PBS group). The green arrow and red arrow indicate immunization and bleeding time, respectively. (B) PEDV-specific IgG antibodies in sow sera vaccinated with COE/G2a-pII or PBS were measured by a commercial ELISA kit. An S/P ratio > 0.35 was defined as positive with PEDV-specific IgG antibody. (C) COE-specific IgA antibodies in sow sera were evaluated by an ELISA using SEC-purified COE/G2a-pII as antigen. (D) Neutralizing antibodies in sow sera were analyzed by the virus-neutralizing assay. The highly virulent NAVET/PEDV/PS6/2010 strain (200 TCID50/0.1 ml) was used for the assay. A VN titer ≥ 8 was defined as positive with neutralizing antibody against PEDV. (E) IFN-γ levels in sow sera were measured based on the standard curve of recombinant porcine IFN- γ standard in a commercial ELISA kit.

Figure 3

Passive transfer of antibodies and cytokine from sows to piglets at the days 0 and 10 pc. Data are presented as mean ± standard deviation (SD). * indicates a statistically significant difference (p < 0.05). (A) PEDV-specific IgG antibodies in piglet sera from sows (n = 1 per group) vaccinated with COE/G2a-pII, or PBS were measured by a commercial ELISA kit. An S/P ratio > 0.35 was defined as positive with a PEDV-specific IgG antibody. (B) COE-specific IgA antibodies in piglet sera (n = 8 for COE/G2a-pII group and n = 2 for PBS group) were evaluated by an ELISA using SEC-purified COE/G2a-pII as antigen. (C) Neutralizing antibodies in piglet sera were analyzed by the virus-neutralizing assay. The highly virulent NAVET/PEDV/PS6/2010 strain (200 TCID50/0.1 ml) was used for the assay. A VN titer ≥ 8 was defined as positive with neutralizing antibody against PEDV. (D) IFN-γ levels in piglet sera were measured based on the standard curve of recombinant porcine IFN-γ standard in a commercial ELISA kit.

Figure 4

Clinical observation and the survival of piglets obtained passive immunity from sow vaccination. The value is presented as the mean ± standard deviation (SD). * means a statistically significant difference (p < 0.05). (A) Fecal score of piglets (n = 8 for COE/G2a-pII group and n = 2 for PBS group) was recorded daily after challenge with highly virulent NAVET/PEDV/PS6/2010 strain. (B) The body weight of piglets was calculated on the days 0 and 14 pc. (C) The clinical scores of piglets were analyzed daily after the challenge. (D) After the challenge, the survival rate of piglets was recorded daily.