Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in E.coli as multiple epitope vaccine induces a protective immune response

Abstract

Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV). Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865) and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716), were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine) than that of mono-epitope peptide(rE2-a or rE2-b). Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals) vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.

Figure 1

Vaccination schedule and viral challenge period of in vivo experiment. Five-weeks-old pigs were acclimatized for 1 weeks and were given a first followed by a second immunization. Pigs immunized were inoculated intranasally with a lethal dose of CSFV on day 21 after the second immunization. Blood samples were collected once weekly from week 7. The health of each pig was assessed twice daily.

Figure 2

SDS-PAGE of recombinant proteins and Western blotting. A: SDS-PAGE of rE2-a, rE2-b and rE2-ba. Lane 1, lysates of the induced E. coli BL21(DE3) pET-32a(+); lane 2-4, lysates of the induced BL21(DE3) pET-rE2-a, BL21(DE3) pET-rE2-b and BL21 (DE3) pET-rE2-ba; lane 5, protein molecular weight markers; lane 6, lysates of the non-induced BL21(DE3) pET-32a(+). B: Western blotting of rE2-a, rE2-b and rE2-ba. Lane 1, protein molecular weight markers; lane 2-4, rE2-a, rE2-b; and rE2-ba.

Figure 3

Cytokine response to vaccination. (A) IL-4 (pg/ml); (B) IL-10 (pg/ml); (C) IFN-γ (pg/ml) expressed as the mean ± standard error. Data shown are representative of three separate experiments. Statistically significant differences (p < 0.05) are indicated with different letters.

Figure 4

Body temperature change (average body temperature curves of each group) of pigs after post-challenge of CSFV Shimen strain. Body temperature was measured every day. Symbol (◆) represents the average body temperature of Group A (immunized with rE2-a). Symbol (■) represents the average body temperature of Group B (immunized with rE2-b). Symbol (▲) represents the average body temperature of Group C (immunized with rE2-ba). Symbol (×) represents the average body temperature of Group D (immunized with HCLV). Symbol (*) represents the average body temperature of Group E (immunized with PBS). Obviously, the average body temperature of Groups A, B and C waves in the normal body temperature range (39.0 ± 0.5°C) after post-challenge (dpi with CSFV Shimen strain). In contrast, the average body temperature of Group E in the same period is 1~2°C higher. All pigs immunized with PBS died during the period from 14 to 21 dpi.