A DNA prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge

Abstract

The present study was undertaken to determine immune response and protection efficacy of a spike (S) protein fragment containing neutralizing epitopes (4F/4R) of turkey coronavirus (TCoV) by priming with DNA vaccine and boosting with the recombinant protein from the corresponding DNA vaccine gene segment. Turkeys were vaccinated by priming with either one dose (G1-750DP) or two doses (G3-750DDP) of 750μg DNA vaccine expressing 4F/4R S fragment and boosting with one dose of 200μg 4F/4R S fragment. One dose of 100μg DNA vaccine mixed with polyethyleneimine (PEI) and sodium hyaluronate (HA) followed by one dose of 750μg DNA vaccine and one dose of 200μg 4F/4R S fragment were given to the turkeys in group G2-100DPH. After infectious challenge by TCoV, clinical signs and TCoV detected by immunofluorescence antibody (IFA) assay were observed in less number of turkeys in vaccination groups than that in challenge control groups. TCoV viral RNA loads measured by quantitative real-time reverse transcription-PCR were lower in vaccinated turkeys than those in challenge control turkeys. The turkeys in G3-750DDP produced the highest level of TCoV S protein-specific antibody and virus neutralization (VN) titer. Comparing to the turkeys in G1-750DP, significantly less TCoV were detected by IFA in the turkeys in G2-100DPH receiving an extra dose of 100μg DNA mixed with PEI and HA. The results indicated that DNA-prime protein-boost DNA vaccination regimen targeting TCoV S fragment encompassing neutralizing epitopes induced humoral immune response and partially protected turkeys against infectious challenge by TCoV.

Fig. 1

Expression of encoded TCoV 4F/4R spike protein fragment (482–678) from plasmid pTriEx3-4F/4R in COS-7 was detected by immunofluorescent antibody assay (IFA) 72 h after transfection. (A) COS-7 cells transfected with pTriEx3-4F/4R using FuGEGE^® HD transfection reagent (Roche) show positive signals to turkey anti-TCoV serum, 400×; (B) COS-7 cells transfected with pTriEx3-4F/4R in DNA-CLIPEI-HA (DPH) complex show positive signals to turkey anti-TCoV serum, 400×; (C) COS-7 cells transfected with vector pTriEx3 using FuGEGE^® HD transfection reagent have negative immunofluorescence to turkey anti-TCoV serum, 400×; and (D) COS-7 cells without transfection have negative immunofluorescence to turkey anti-TCoV serum, 400×.

Fig. 2

Western blotting of TCoV 4F/4R spike protein fragment (482–678) reacted to the serum collected from TCoV-infected turkeys. Lane “NC” is TCoV 4F/4R S protein reacted to the serum from non-infected turkey. Lane “M” is protein marker in kDa. Lane “anti-TCoV” is TCoV 4F/4R S protein reacted to the serum from TCoV-infected turkey. The darker band at 26 kDa is monomer of TCoV 4F/4R S protein and the lighter band below 50 kDa is dimer of TCoV 4F/4R S protein.

Fig. 3

Serum antibody levels determined by TCoV spike protein-based ELISA (O.D.450 nm) in trial 1 (A) and trial 2 (B). The gray bars are for turkeys in the negative control groups (NC-2V and NC-3V). The white empty bars are for turkeys in the vector control groups (VC-2V and VC-3V). The black bars are from turkeys primed with one dose of 750 μg pTriEx3-4F/4R (S-DNA) and boosted with one dose of 200 μg TCoV 4F/4R S fragment (482–678) (S-Pro) (G1-750DP). The bars with black and white strips are from turkeys primed with one dose of 100 μg pTriEx-4F/4R in DPH-complex, one dose of 750 μg S-DNA, and boosted with S-Pro (G2-100DPH). The bars with checker broad are from turkeys primed with 2 doses of 750 μg S-DNA and boosted with S-Pro (G3-750DDP). The different letters above each bar at each time point indicate significant differences (p < 0.05).

Fig. 4

Serum virus neutralization (VN) titers against TCoV 540 determined by VN assay using 22-day-old turkey embryonated eggs in trial 1 (A) and trial 2 (B). In trial 1, turkeys received two doses of vaccines: NC-2V, negative control turkeys; VC-2V, turkeys received one dose of 750 μg plasmid vector pTriEx3 and PBS; G1-750DP, turkeys primed with one dose of 750 μg pTriEx3-4F/4R (S-DNA) and boosted with one dose of 200 μg TCoV 4F/4R S fragment (482–678) (S-Pro). In trial 2, three doses of vaccines were given: NC-3V, negative control turkeys; VC-3V, turkeys received two doses of 750 μg plasmid vector pTriEx3 and PBS; G2-100DPH, turkeys primed with one dose of 100 μg pTriEx-4F/4R in DPH-complex and one dose of 750 μg S-DNA and boosted with S-Pro; G3-750DDP, turkeys primed with 2 doses of 750 μg S-DNA and boosted with S-Pro. The different letters above each bar at each time point indicate significant differences (p < 0.05).

Fig. 5

Fold changes of turkey interferon gamma (IFNγ) mRNA levels in the spleens of turkeys in the vector control and vaccinated groups before challenge and at 3 and 10 days after challenge (dpi) in 2 trials. In trial 1 (A), turkeys in group VC-2V received one dose of 750 μg plasmid vector pTriEx3 and PBS and turkeys in G1-750DP primed with one dose of 750 μg pTriEx3-4F/4R (S-DNA) and boosted with one dose of 200 μg TCoV 4F/4R spike fragment (482–678) (S-Pro). In trial 2 (B), turkeys in group VC-3V received two doses of 750 μg plasmid vector pTriEx3 and PBS, turkeys in G2-100DPH primed with one dose of 100 μg pTriEx-4F/4R in DPH-complex and one dose of 750 μg S-DNA and boosted with S-Pro, and turkeys in G3-750DDP primed with 2 doses of 750 μg S-DNA and boosted with S-Pro. The different letters above each bar at each time point indicate significant differences (p < 0.05).