Induction of immune response in chickens primed in ovo with an inactivated H9N2 avian influenza virus vaccine

Abstract

Objective: Infection of chickens with low pathogenic avian influenza virus, such as H9N2 virus, culminates in decreased egg production and increased mortality and morbidity if co-infection with other respiratory pathogens occurs. We have previously observed the induction of antibody- and cell-mediated immune responses after intramuscular administration of an H9N2 beta-propiolactone inactivated virus vaccine to chickens. Given the fact that in ovo vaccination represents a practical option for vaccination against H9N2 AIV in chickens, in the current study, we set out to characterize immune responses in chickens against a beta-propiolactone inactivated H9N2 virus vaccine after primary vaccination in ovo on embryonic day 18, and secondary intramuscular vaccination on day 14 post-hatch. We also included the Toll-like receptor 21 ligand, CpG ODN 2007, and an oil emulsion adjuvant, AddaVax™, as adjuvants for the vaccines.

Results: Antibody-mediated immune responses were observed after administering the secondary intramuscular vaccine. Cell-mediated immune responses were observed in chickens that received the beta-propiolactone inactivated H9N2 virus combined with AddaVax™. Our results demonstrate that adaptive immune responses can be induced in chickens after a primary in ovo vaccination and secondary intramuscular vaccination.

Keywords: Antibody; Beta-propiolactone; Cell-mediated; CpG ODN; H9N2 avian influenza virus; In ovo; Intramuscular; Toll-like receptor 21; Vaccine.

Fig. 1

HI titers in serum against H9N2 AIV. Average serum HI titers 21 and 28 days ph, from 10 chickens per group. Chickens were vaccinated in ovo on ED18 and received a second vaccination 14 days ph. Vaccines consisted of 15 µg of BPL inactivated H9N2 virus administered alone (BPL), with AddaVax™ (BPL + Add), or with 2 µg CpG ODN 2007 (BPL + CpG). One group received just PBS as a negative control (PBS). Serum was collected weekly following hatch. HI titers were first observed in serum 7 days post secondary vaccination. Group means that share the same letter did not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using Duncan’s Multiple Range Test and differences in means were considered significant if p < 0.05

Fig. 2

IgY and IgM titers in serum against H9N2 AIV. Average serum IgY titers a 21 days and b 28 days ph and average serum IgM titers c 21 days and d 28 days ph, from 10 chickens per group. Chickens were vaccinated in ovo on ED18 and received a second vaccination 14 days ph. Vaccines consisted of 15 µg of BPL inactivated H9N2 virus administered alone (BPL), with AddaVax™ (BPL + Add), or with 2 µg CpG ODN 2007 (BPL + CpG). One group received just PBS as a negative control (PBS). Serum was collected weekly following hatch. Antibody titers were first observed in serum 7 days post secondary vaccination. Group means that share the same letter did not differ significantly. Standard error of the mean is indicated with error bars. Data were analyzed using Duncan’s Multiple Range Test and differences in means were considered significant if p < 0.05

Fig. 3

IFN-γ production in stimulated splenocyte supernatants and IFN-γ and IL-2 expression in stimulated splenocytes. Chickens were vaccinated in ovo on ED18 and received a second vaccination 14 days ph. Vaccines consisted of 15 µg of BPL inactivated H9N2 virus administered alone (BPL), with AddaVax™ (BPL + Add), or with 2 µg CpG ODN 2007 (BPL + CpG). One group received just PBS as a negative control (PBS). Five spleens from 5 birds per group were collected 10 days post secondary vaccination. Splenocytes were isolated and seeded in 48 well plates and stimulated with 1 μg/ml of BPL inactivated H9N2 virus. Supernatants were collected a 48 and 72 h after stimulation and supernatant IFN-γ concentrations were quantified. IFN-γ expression in splenocytes was quantified at b 12 h and c 24 h post-stimulation and IL-2 expression was quantified at d 12 h and e 24 h post-stimulation. Gene expression was quantified relative to β-actin expression. Group means that share the same letter did not differ significantly. Standard error of the mean is indicated with error bars. Data was analyzed using Duncan’s Multiple Range Test and differences in means were considered significant if p < 0.05