Priming with a DNA vaccine and boosting with an inactivated vaccine enhance the immune response against infectious bronchitis virus

Abstract

The methods of repeated immunization with inactivated vaccines have been used widely to increase antibody protection against infectious bronchitis virus (IBV). However, compared with DNA vaccines, these methods usually induce poor cellular responses. In the present study, specific pathogen-free (SPF) chickens were immunized intramuscularly with a DNA vaccine carrying the main IBV structural genes (pVAX1-S1, pVAX1-M, and pVAX1-N, respectively) and boosted with the IBV M41 strain inactivated vaccine to assess whether such a new strategy could enhance the immune responses against IBV. The protection efficacy of the DNA vaccine carrying different structural genes for priming was evaluated further. The chickens were immunized primely on day 7 and boosted 2 weeks later. After that, distribution of the DNA vaccine in vivo, the percentage of CD4+CD3+ and CD8+CD3+ subgroups of peripheral blood T-lymphocytes, and the specific IgG and virus neutralizing antibodies were measured. Chickens were then challenged by the nasal-ocular route with the IBV M41 strain 4 weeks after booster immunization. The results demonstrated that priming with a DNA vaccine encoding nucleocapsid protein (pVAX1-N) and boosting with the inactivated IBV vaccine led to the dramatic augmentation of humoral and cellular responses, and provided up to 86.7% rate of immune protection, providing an effective approach to protect chickens from IBV.

Fig. 1

Strategy for construction of the structural gene based DNA vaccine. The viral RNA was extracted, RT-PCR was carried out, and each of the structure genes was cloned into pVAX1 to construct the DNA vaccine.

Fig. 2

Identification of constructed DNA vaccine. Lane M1, Marker III; lane 1, pVAX1-M incised with HindIII and EcoRI was 678 bp; lane 2, pVAX1-S1 incised with HindIII and BamHI was 1610 bp; lane 3, pVAX1-N incised with restriction enzyme HindIII and EcoRI was 1231 bp; Lane M2, Marker D2000.

Fig. 3

Indirect immunofluorescence detection of the expressed structural protein in COS-7 cells. Transient expressed proteins were detected by immunofluorescent antibody assay at 36 h after transfection. Cells transfected with pVAX1-S1 (A), pVAX1-M (B), and pVAX1-N (C) showed green fluorescence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4

The percentage of CD4+CD3+ and CD8+CD3+ T-lymphocytes of different vaccination groups. The result was obtained from average of five sera in each group. The data were analyzed by software of Statistics Package for Social Science (SPSS). This test was conducted at 7 days after boosting.

Fig. 5

Antibody titers of different vaccination groups measured by ELISA. Sera from all the immunized animals were sampled weekly. The result was obtained from average of five sera in each group. The data of antibody titers were analyzed by software of Statistics Package for Social Science (SPSS). The results show the antibody titers of every immunized group on days 0, 7, 14, 21, 28, 35, 42, 49 after incubation.

Fig. 6

Level of IBV-specific neutralizing antibody. The sera were collected 28 days after boosting. Neutralizing titers indicate the highest serum dilution which protects half of the embryos from death (PD50). The higher serum dilution denotes the higher neutralizing titers.