Negligible effect of chicken cytokine IL-12 integration into recombinant fowlpox viruses expressing avian influenza virus neuraminidase N1 on host cellular immune responses

Abstract

In comparison to the extensive characterization of haemagglutinin antibodies of avian influenza virus (AIV), the role of neuraminidase (NA) as an immunogen is less well understood. This study describes the construction and cellular responses of recombinant fowlpox viruses (rFWPV) strain FP9, co-expressing NA N1 gene of AIV A/Chicken/Malaysia/5858/2004, and chicken IL-12 gene. Our data shows that the N1 and IL-12 proteins were successfully expressed from the recombinants with 48 kD and 70 kD molecular weights, respectively. Upon inoculation into specific-pathogen-free (SPF) chickens at 10⁵ p.f.u. ml^-1, levels of CD3+/CD4+ and CD3+/CD8+ populations were higher in the wild-type fowlpox virus FP9 strain, compared to those of rFWPV-N1 and rFWPV-N1-IL-12 at weeks 2 and 5 time points. Furthermore, rFWPV-N1-IL-12 showed a suppressive effect on chicken body weight within 4 weeks after inoculation. We suggest that co-expression of N1 with or without IL-12 offers undesirable quality as a potential AIV vaccine candidate.

Keywords: IL-12; avian influenza virus; cytokine; neuraminidase; recombinant fowlpox virus.

Fig. 1.

Overview of the cloning strategy. (a) Vector pEFL29 carrying N1 gene was recombined into FP9 genome. (b) Cytokine gene IL-12 expression cassette was recombined into rFWPV already carrying the N1 gene.

Fig. 2.

Recombination of IL-12 gene into rFWPV-N1 was verified using PCR after genomic DNA extraction using primer set pPC1.X-F and pPC1.X. Lanes 1, 3–8: intermediate viruses, which produced amplicons of 984 and 1934 bp; lane 2: rFWPV-N1, which has lost the IL-12 gene; lane 9: rFWPV-N1 carrying the IL-12 gene; lane 10: pPC1.X-IL-12 plasmid as positive control. M1 and M2 are 1 kb and 100 bp ladder markers, respectively (New England Biolabs).

Fig. 3.

Detection of recombinant proteins N1 from rFWPV-N1 at 48 kD (a) and IL-12 from rFWPV-N1-IL-12 at 70 kD (b). Lane 1: uninfected CEF as negative control; lane 2: CEF infected with WT FP9; lane 3: CEF infected with rFWPV-N1 (a) or rFWPV-N1-IL-12 (b). M is a protein ladder marker.