Construction and immunogenicity of a recombinant fowlpox vaccine coexpressing S1 glycoprotein of infectious bronchitis virus and chicken IL-18

Abstract

Infectious bronchitis virus (IBV) poses a major threat to the chicken industry worldwide. In this study, we developed a recombinant fowlpox virus (rFPV) vaccine expressing the IBV S1 gene and chicken interleukin-18 gene (IL-18), rFPV-S1/IL18. Recombinant plasmid pSY-S1/IL18 was constructed by cloning chicken IL-18 into fowlpox virus transfer plasmid containing S1 gene and transfected into the chicken embryo fibroblasts cell pre-infected with S-FPV-017 to generate rFPV-S1/IL18. Expression of the recombinant proteins was confirmed by RT-PCR and IFA. We also constructed the recombinant fowlpox virus rFPV-S1 without IL-18. One-day-old chickens were vaccinated by wing-web puncture with the two rFPVs, and the induced humoral and cellular responses were evaluated. There was a significant difference in ELISA antibody levels (P<0.05) elicited by either rFPV-S1 or rFPV-S1/IL18. The ratios of CD4(+) to CD8(+) in chickens immunized with rFPV-S1/IL18 were significantly higher (P<0.05) than in those immunized with rFPV-S1. All chickens immunized with rFPV-S1/IL18 were completely protected (20/20) after challenge with the virulent IBV HN99 strain 43 days after immunization, while only 15 out of 20 of the chickens immunized with the rFPV-S1 were protected. Our results show that the protective efficacy of the rFPV-S1 vaccine could be enhanced significantly by simultaneous expression of IL-18.

Fig. 1

Schematic representations of fowlpox virus expression plasmids (pSY538, P11 and pSY681) and recombinant plasmids pSY-S1 and pSY-S1/IL18.

Fig. 2

PCR confirmation of presence of S1 and chicken IL-18 genes in rFPVs. 1, DNA molecular Marker (DL2000); 2, S1 gene in rFPV-S1; 3, S1 gene in rFPV-S1/IL18; 4, chicken IL-18 in rFPV-S1/IL18. Also, the results were double checked by DNA sequencing done on the PCR products. DNA sequencing results showed that S1 and chicken IL-18 genes are 1 675 bp and 597 bp, respectively, and consistent with predicted results.

Fig. 3

Detection of S1 expression by IFA. CEF cells were infected with (A) rFPV-S1 and (B) rFPV-S1/IL18 at a m.o.i. of 2.0. (C) Negative control cells were infected with S-FPV-017.

Fig. 4

Detection of antibodies in different vaccine inoculated groups by ELISA (n = 5, i.e. number of times the test was repeated). Values are expressed as mean optical density ± standard error. Statistically significant differences (P < 0.05) are indicated by * (compared with S-FPV-017 or PBS) or ** (compared with rFPV-S1 alone).

Fig. 5

The percentage of CD3⁺, CD4⁺CD3⁺ and CD8⁺CD3⁺ T-lymphocytes of different vaccine inoculated groups (n = 4, i.e. number of times the test was repeated). Values are expressed as mean counts ± standard error. Statistically significant differences (P < 0.05) are indicated by * (compared with S-FPV-017 or PBS) or ** (compared with rFPV-S1 alone).

Fig. 6

Diseased and dead chickens of different groups challenged with IBV HN99 strain. (A) Diseased chickens of different groups. Presence of one of the following can be used to judge morbidity: (1) coughing; (2) nasal discharge; (3) wheezing or dyspnea; (4) swinging of the head; (5) feathers erected, dullness. (B) The cumulative mortalities of different groups. Statistically significant differences (P < 0.05) are indicated by * (compared with rFPV-S1 or rFPV-S1/IL18).