Effects of interferon-γ knockdown on vaccine-induced immunity against Marek's disease in chickens

Abstract

Interferon (IFN)-γ has been shown to be associated with immunity to Marek's disease virus (MDV). The overall objective of this study was to investigate the causal relationship between IFN-γ and vaccine-conferred immunity against MDV in chickens. To this end, 3 small interfering RNAs (siRNAs) targeting chicken IFN-γ, which had previously been shown to reduce IFN-γ expression in vitro, and a control siRNA were selected to generate recombinant avian adeno-associated virus (rAAAV) expressing short-hairpin small interfering RNAs (shRNAs). An MDV challenge trial was then conducted: chickens were vaccinated with herpesvirus of turkey (HVT), administered the rAAAV expressing shRNA, and then challenged with MDV. Tumors were observed in 4 out of 10 birds that were vaccinated with HVT and challenged but did not receive any rAAAV, 5 out of 9 birds that were administered the rAAAV containing IFN-γ shRNA, and 2 out of 10 birds that were administered a control enhanced green fluorescent protein siRNA. There was no significant difference in MDV genome load in the feather follicle epithelium of the birds that were cotreated with the vaccine and the rAAAV compared with the vaccinated MDV-infected birds. These results suggest that AAAV-based vectors can be used for the delivery of shRNA into chicken cells. However, administration of the rAAAV expressing shRNA targeting chicken IFN-γ did not seem to fully abrogate vaccine-induced protection.

Figure 1

Mean number of copies of the viral genome [and standard error (SE)] of recombinant avian adeno-associated virus (rAAAV) in various tissues of chicks (16 per group) euthanized at 48 h, 120 h, 14 d, or 26 d after administration at 1 d of age of a lower dose [1 × 10⁹ genomic copies (black bars)] or a higher dose [1 × 10¹⁰ genomic copies (hatched bars)] of an rAAAV expressing short-hairpin small interfering RNA targeting chicken interferon-γ (rAAAV:IFN-γ-shRNA).

Figure 2

Necropsy data (mean and SE) for 6 groups of chicks (9 to 10 per group) 21 d after injection of a very virulent strain (RB1B) of Marek’s disease virus (MDV) or sham injection with phosphate-buffered saline at 5 d of age, some groups having been vaccinated with herpesvirus of turkey (HVT) on the day of hatch. A — body weight; B — frequency of gross tumors; C — ratio of spleen weight to body weight; D — ratio of weight of bursa of Fabricius to body weight. Group 1 — MDV-infected only; group 2 — MDV-infected after administration of all 3 rAAAV:IFN-γ-shRNAs (1 × 10¹⁰ genomic copies of each); group 3 — MDV-infected after HVT vaccination; group 4 — MDV-infected after HVT vaccination and administration of a control shRNA targeting enhanced green fluorescent protein (EGFP-S1 DS); group 5 — MDV-infected after HVT vaccination and administration of all 3 rAAAV:IFN-γ-shRNAs; group 6 — uninfected and unvaccinated controls. Asterisks indicate a significant difference (P ≤ 0.05) between this mean and the means for all the vaccinated groups and control group 6.

Figure 3

Mean MDV genome load (and SE) per 100 ng of DNA in feather follicle epithelium (FFE) at 4, 7, 10, 14, and 21 d after infection in groups 1 to 5. Asterisks indicate a significant difference (P ≤ 0.05) between this mean and the means for the unvaccinated groups.

Figure 4

Cytokine mRNA expression in spleen tissues of the 6 groups of chickens. IFN-γ (target) and β-actin (reference) gene expression in spleen was quantified by real time RT-PCR using SYBR. Target gene expression is presented relative to β-actin expression and normalized to a calibrator. Data is presented as mean expression (and SE) of each group. NS — not a significant difference.