Protective Efficacy of H9N2 Avian Influenza Vaccines Inactivated by Ionizing Radiation Methods Administered by the Parenteral or Mucosal Routes

Abstract

H9N2 viruses have become, over the last 20 years, one of the most diffused poultry pathogens and have reached a level of endemicity in several countries. Attempts to control the spread and reduce the circulation of H9N2 have relied mainly on vaccination in endemic countries. However, the high level of adaptation to poultry, testified by low minimum infectious doses, replication to high titers, and high transmissibility, has severely hampered the results of vaccination campaigns. Commercially available vaccines have demonstrated high efficacy in protecting against clinical disease, but variable results have also been observed in reducing the level of replication and viral shedding in domestic poultry species. Antigenic drift and increased chances of zoonotic infections are the results of incomplete protection offered by the currently available vaccines, of which the vast majority are based on formalin-inactivated whole virus antigens. In our work, we evaluated experimental vaccines based on an H9N2 virus, inactivated by irradiation treatment, in reducing viral shedding upon different challenge doses and compared their efficacy with formalin-inactivated vaccines. Moreover, we evaluated mucosal delivery of inactivated antigens as an alternative route to subcutaneous and intramuscular vaccination. The results showed complete protection and prevention of replication in subcutaneously vaccinated Specific Pathogen Free White Leghorn chickens at low-to-intermediate challenge doses but a limited reduction of shedding at a high challenge dose. Mucosally vaccinated chickens showed a more variable response to experimental infection at all tested challenge doses and the main effect of vaccination attained the reduction of infected birds in the early phase of infection. Concerning mucosal vaccination, the irradiated vaccine was the only one affording complete protection from infection at the lowest challenge dose. Vaccine formulations based on H9N2 inactivated by irradiation demonstrated a potential for better performances than vaccines based on the formalin-inactivated antigen in terms of reduction of shedding and prevention of infection.

Keywords: H9N2; formalin-inactivated; irradiated; mucosal; subcutaneous; vaccines.

Figure 1

Negative stain TEM images of virus (× 180,000). (A) Live untreated H9N2. (B) formalin-inactivated H9N2; and (C) irradiated H9N2. Red arrows indicate viral glycoprotein spikes.

Figure 2

Effects of subcutaneous (SC) vaccination upon infection at different H9N2 challenge doses. (A–C) qRRT-PCR results of tracheal swabs collected from all the challenged birds. (D–F) Serological test results of blood samplings performed before and after challenge. The numbers above bars indicate a number of NP-ELISA positive birds out of tested birds.

Figure 3

Models of the shedding levels of irradiated and formalin-inactivated vaccines and a non-vaccinated control for 12 days post infection (DPI) for three challenge doses (10³, 10⁴, and 10⁶ EID₅₀). For each group, the dots represent the measured observations, the solid line represents the fitted model, and the shaded contour shape represents the 95% confidence intervals of the fitted model. For each plot, the inset compares the overall effect of the group on the shedding, with the solid circle representing the effect value and the vertical bars the 95% confidence interval. Panels (A–C) show models of shedding in ON vaccinated birds upon challenge with 10³, 10⁴, and 10⁶ EID₅₀ of H9N2, respectively; panels (D–F) show models of shedding in SC vaccinated birds upon challenge with 10³, 10⁴, and 10⁶ EID₅₀ of H9N2, respectively.

Figure 4

Effects of mucosal (oculo-nasal, ON) vaccination upon infection at different H9N2 challenge doses. (A–C) qRRT-PCR results of tracheal swabs collected from all the challenged birds. (D–F) Serological test results before and after challenge. The numbers above bars indicates the number of NP-ELISA positive birds out of tested birds.