Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes

Abstract

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

Keywords: DNA vaccine; Newcastle disease virus; antibody response; inactivated vaccine.

Fig. 1. Map of the DNA vaccines. The DNA plasmids were constructed by cloning the fusion (F) gene into the NheI and MluI\I sites and the hemagglutinin-neuraminidase (HN) gene into SalI and NotI of the expression vector pIRES.

Fig. 2. Western blot analysis. Vero cells were transfected with the constructs; 48 h post-transfection, gene expression was evaluated by Western blot analysis. (A) M, protein ladder; Lines 1–2, immunoblotting for the cells transfected with pIRES-F/HN, co-transfected with the DNA plasmids pIRES-HN and pIRES-F; Line 3, cells transfected with pIRES-F; Line 4, control cells transfected with the parental plasmid. (B) M, protein ladder; 1, immunoblotting of the cells transfected with virus as a positive control; Lines 2–3, cells transfected with pIRES-HN; Line 4, control cells transfected with the parental plasmid.

Fig. 3. Indirect immunofluorescence test. At 48 h post-transfection, vero cells transfected with pIRES-HN (B), pIRES-F (C), pIRES-F/HN (D) and/or the empty plasmid (A) as a negative control. Cells transfected with pIRES/HN and pIRES/F were treated with chicken anti-NDV polyclonal antibody, while cells transfected with pIRES-F/HN were treated with anti-HN and anti-F monoclonal antibody as the primary antibodies. Acll cells were then treated with FITC-conjugated goat anti chicken IgY as a secondary antibody. The cells were observed under the 20× objective of an inverted fluorescence microscope.

Fig. 4. S/P ratio column chart with SD bars per week as determined by ELISA. The error bars show the standard deviation of the means. Group 1, pIRES-F; Group 2, pIRES-HN; Group 3, pIRES-HN/F; Group 4, pIRES-HN+pIRES-F; Group 5, pIRES-F+inactivated vaccine; Group 6, pIRES-HN+inactivated vaccine; Group 7, pIRES-F/HN+inactivated vaccine; Group 8, pIRES-HN+pIRES-F+inactivated vaccine; Group 9, pIRES+ inactivated vaccine; Group 10, inactivated vaccine; Group 11, pIRES.