Design and Characterization of a DNA Vaccine Based on Spike with Consensus Nucleotide Sequence against Infectious Bronchitis Virus

Abstract

Avian coronavirus infectious bronchitis virus (IBV) causes severe economic losses in the poultry industry, but its control is hampered by the continuous emergence of new genotypes and the lack of cross-protection among different IBV genotypes. We designed a new immunogen based on a spike with the consensus nucleotide sequence (S_con) that may overcome the extraordinary genetic diversity of IBV. S_con was cloned into a pVAX1 vector to form a new IBV DNA vaccine, pV-S_con. pV-S_con could be correctly expressed in HD11 cells with corresponding post-translational modification, and induced a neutralizing antibody response to the Vero-cell-adapted IBV strain Beaudette (p65) in mice. To further evaluate its immunogenicity, specific-pathogen-free (SPF) chickens were immunized with the pV-S_con plasmid and compared with the control pVAX1 vector and the H120 vaccine. Detection of IBV-specific antibodies and cell cytokines (IL-4 and IFN-γ) indicated that vaccination with pV-S_con efficiently induced both humoral and cellular immune responses. After challenge with the heterologous strain M41, virus shedding and virus loading in tissues was significantly reduced both by pV-S_con and its homologous vaccine H120. Thus, pV-S_con is a promising vaccine candidate for IBV, and the consensus approach is an appealing method for vaccine design in viruses with high variability.

Keywords: DNA vaccine; consensus nucleotide sequence; heterologous challenge; infectious bronchitis virus; spike ectodomain.

Figure 1

Schematic diagram of the S_con gene and infectious bronchitis virus (IBV) DNA vaccine (pV-S_con) plasmid. (a) The S_con gene. The Kozak sequence (GCCACCATGG) and 6 × His-Tag gene were added to ensure its correct expression in a eukaryotic expression vector and enable detection, respectively. (b) The pV-S_con plasmid. The S_con gene was inserted into the pVAX1 vector between the multiple cloning sites BamHI and EcoRV.

Figure 2

Phylogenetic analysis of the consensus nucleotide sequence of spike protein (S-CON). (a) Genetic relatedness of S-CON and indicator strains. A neighbor-joining (NJ) tree illustrated the genetic distances between the S-CON (red) nucleotide sequence and the indicator strains. (b) Genotypes of S-CON and strains used in this study. Bootstrap values (n = 1000 replicates) <70% were not shown.

Figure 3

Expression of the S_con protein in vitro. (a) Expression of the S_con protein in HD11 cells analyzed by IFA. (b) Expression of the S_con protein in HD11 cells analyzed by Western blot.

Figure 4

Neutralization titer of immunized mice against the IBV Beaudette (p65) strain. Serum of mice immunized with pV-S_con or pVAX1 were serially diluted twofold with PBS in microplates and mixed with 100 EID₅₀ (50% chicken embryo infectious dose) of Beaudette (p65). PBS was used as the negative control. Virus neutralization (VN) titers were defined as the highest dilution where cells showed no cytopathic effect after 7 days of infection. There were three samples in each group and the value is expressed as the mean ± SEM. Ordinary one-way ANOVA was used for statistical analysis; ** p < 0.01.

Figure 5

IBV-specific antibodies in the serum. The antibody titers are shown as mean sample vs. positive control (S/P) values + SEM of each group. The threshold cut-off values of the IBV ELISA were 0.2. S/P = (Sample A (650) − NC (negative control) A (650))/(PC (positive control) A (650) − NC A (650)). Tukey’s multiple comparison test was used for statistical analysis; * p < 0.05, ** p < 0.01.

Figure 6

Analysis of the IL-4 (a) and IFN-γ (b) concentrations in the serum on the 14th and 28th dpv. Tukey’s multiple comparison test was used for statistical analysis. Concentrations are shown as mean values + SEM of each group; * p < 0.05, ** p < 0.01.

Figure 7

Viral shedding (a) and viral load in tissues (b) after M41 challenge. The formula of the standard curve is y = 37.302 − 3.155x, E = 107.5%, R² = 0.988. A multiple t-test was used for statistical analysis. Viral loads are shown as mean values + SEM of each group; ** p < 0.01.