Inactivated Pseudomonas PE(ΔIII) exotoxin fused to neutralizing epitopes of PEDV S proteins produces a specific immune response in mice

Abstract

Porcine epidemic diarrhea (PED) caused by the porcine epidemic diarrhea virus (PEDV), is a severe infectious and devastating swine disease that leads to serious economic losses in the swine industry worldwide. An increased number of PED cases caused by variant PEDV have been reported in many countries since 2010. S protein is the main immunogenic protein containing some B-cell epitopes that can induce neutralizing antibodies of PEDV. In this study, the construction, expression and purification of Pseudomonas aeruginosa exotoxin A (PE) without domain III (PEΔIII) as a vector was performed for the delivery of PEDV S-A or S-B. PE(ΔIII) PEDV S-A and PE(ΔIII) PEDV S-B recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The immunogenicity of PEDV S-A and PEDV S-B subunit vaccines were evaluated in mice. The results showed that PEDV-S-B vaccine could not only induce specific humoral and Th1 type-dominant cellular immune responses, but also stimulate PEDV-specific mucosal immune responses in mice. PEDV-S-B subunit vaccine is a novel candidate mucosal vaccine against PEDV infection.

Keywords: Neutralizing antibody; Porcine epidemic diarrhea virus; S protein; Th1-type immune response.

Fig. 1

Schematic and expression of the recombinant protein. a PE, full-length sequence of PE toxin contains four domains (Ia, II, Ib, III); PE (ΔIII) represents full-length sequence of a PE toxin deletant that deleted domain III. S-A, PEDV S1 aa 25-229; S-B, PEDV S aa 499-789+aa 1370-1378. b SDS-PAGE analysis of PE(ΔIII)-PEDV-S-A and PE(ΔIII)-PEDV-S-B proteins purified through affinity chromatography. Lane 1: PE(ΔIII)-PEDV-S-A (71 kDa). Lane 2: PE(ΔIII)-PEDV-S-B (80.5 kDa). c Western blot analysis of PE(III)-PEDV-S-A and PE(III)-PEDV-S-B proteins. Lane 1: PE(ΔIII)-PEDV-S-A. Lane 2: PE(ΔIII)-PEDV-S-B. Mouse anti-HisMAb and goat anti-mouse IgG-HRP were used as primary and secondary antibodies, respectively. PE, Pseudomonas aeruginosa exotoxin A; PEDV, porcine epidemic diarrhea virus

Fig. 2

PEDV-specific IgG and IgA in the sera of the immunized mice by indirect ELISA. a PEDV-specific IgG detected by indirect ELISA. b PEDV-specific IgA detected by indirect ELISA. Data were represented as the mean ± SEM. Different letters (a, b) indicate a statistically significant difference between the different experimental groups (P < 0.05). PEDV, porcine epidemic diarrhea virus

Fig. 3

Detection of PEDV-specific IgA in the small intestines of mice and PEDV-specific neutralizing antibodies. a Mucosal IgA detection. b Serum neutralizing antibody titers against PEDV. Data are represented as the mean ± SEM. Different letters (a, b) indicate a statistically significant difference between the different experimental groups (P < 0.05). PEDV, porcine epidemic diarrhea virus

Fig. 4

Lymphocyte proliferation responses measured by MTS assay in mice. Data were represented as the mean ± SEM. Different letters (a, b) indicate a statistically significant difference between different experimental groups (P < 0.05). PEDV, porcine epidemic diarrhea virusPEDV, porcine epidemic diarrhea virus

Fig. 5

Cytokine levels in the culture supernatants. IFN-γ (a), IL-2 (b), IL-4 (c), and IL-10 (d) levels were measured by double-antibody sandwich ELISA. Data were represented as the mean ± SEM. Different letters (a, b) indicate a statistically significant difference between different experimental groups (P < 0.05). PEDV, porcine epidemic diarrhea virusPEDV, porcine epidemic diarrhea virus