The impact of innate and humoral immune mechanisms on vaccine induced protection against avian influenza H9N2 in broilers

Avian influenza virus (AIV) (H9N2) is a highly contagious avian respiratory disease that causes significant economic losses in the poultry industry sector. Although immunological responses to AIV (H9N2) vaccinations have been extensively studied, little is known about how inactivated vaccines interact with the host’s innate and adaptive immune responses. Five commercially available inactivated avian influenza virus (AIV) H9N2 vaccines, each containing different virus strains and adjuvant compositions, were administered via subcutaneous injection to 4-day-old commercial broiler chicks. The effects of vaccination on the kinetics of chicken toll-like receptor (TLR) 5 and 7 pathways, along with their downstream cytokines and the expression profiles of major histocompatibility complex class I and 2 (MHC-1 and 2) expression profiles were investigated. Quantitative real-time PCR analysis demonstrated that immunization with inactivated H9N2 AIV vaccines activated pattern recognition receptors (PRRs), specifically TLR-5 and TLR-7, along with their downstream cytokines. These included the proinflammatory interleukin-6 (IL-6), antiviral interferon-alpha, and myxoma resistance protein 1 (Mx1). This activation was observed as early as 48 h post-vaccination and persisted for up to 7 days in the spleens of the vaccinated birds. Subcutaneous administration of either inactivated vaccine A or B elicited the highest expression levels of IL-6, MHC class I, and MHC-1 and MHC-2 molecules. Hemagglutination-inhibition (HI) antibody titers against avian influenza were > 5 log2 from two to five weeks post-vaccination, with vaccine A inducing significantly higher responses at weeks three, four, and five. Following intraocular challenge with 106 EID50/ml of a locally isolated avian influenza virus strain, vaccines A, B, and E conferred complete (100%) protection against mortality. Notably, vaccine A demonstrated superior clinical protection and resulted in minimal histopathological lesions in the tracheal and pulmonary tissues.