Development of Serological Tests to Differentiate Between Animals Infected with and Vaccinated Against Avian Influenza

Avian influenza virus (AIV) poses a global threat to the poultry industry, with the ongoing resurgence of highly pathogenic AIV (HPAI) H5Nx (where Nx is neuraminidase (NA) subtypes N1 to N9) causing substantial economic losses across Europe, the Americas, Asia, and Africa. The currently dominant H5Nx clade 2.3.4.4b has disseminated globally among domestic poultry, wild birds, and various other mammalian species, including dairy cattle and humans. Current inefficient methods to control virus transmission and subsequent mass culling strategies highlight the need for new approaches, particularly in distinguishing between infected and vaccinated birds in AIV surveillance programs. Widely used targeted killed virus vaccines often mitigate disease impact, however, these vaccines lack tests that could differentiate infected from vaccinated animals (DIVA). This limitation impedes surveillance efforts and leads to widespread trade restrictions, causing significant losses in export markets and restricting AIV vaccine use in regions enforcing strict stamp-out policies, such as Europe and North America. This project aims to develop and validate enzyme-linked immunosorbent assays (ELISAs) capable of measuring and distinguishing antibody titers in animals infected with or vaccinated against AIV. These assays will detect antibodies specific to genetically improved vaccines that include antigenically matched haemagglutinin (HA) antigens to field viruses, along with distinct neuraminidase (NA) and nucleoprotein (NP) genes incorporated via the reverse genetics (RG) method. The focus is to produce widely adoptable, inexpensive ELISA tests that enable differentiation between infected and vaccinated animals using inactivated HPAI H5 vaccines produced through the RG method, with an emphasis on efficacy, safety, and cost-effectiveness. The ELISA platform developed in this project will detect antibodies specific to NA, HA, and NP induced by vaccination or infection. NA ELISAs will identify antibodies against all NA subtypes found in birds (N1-N9), distinguishing between those from vaccination (e.g., H5N2) and natural exposure (e.g., H5N1). Similarly, HA ELISAs will measure antibodies specific to economically significant AIV subtypes infecting poultry, such as H5, H7, or H9, indicating whether animals were exposed to the same HA subtype in the vaccine or a different one due to field exposure. These assays will also assess vaccine potency and immunity duration, ensuring successful vaccination and seroconversion. Additionally, the NP ELISA, in a competitive format, will differentiate antibodies specific to the NP included in the vaccine, derived from laboratory-adapted strains via the reverse genetics approach, from those targeting the NP of field strains. In summary, the lack of optimal DIVA assays has been a major barrier to implementing effective vaccination programs for HPAI, particularly in regions enforcing strict culling policies. This project aims to improve global acceptance of cost-effective killed virus vaccines by (a) reducing the prevalence and economic impact of H5Nx HPAI in vaccinated poultry flocks; (b) enabling farmers to identify AIV-infected animals accurately, thereby minimising unnecessary culling and facilitating the export of healthy, vaccinated animals, thereby promotes sustainable and affordable animal protein production; (c) reducing financial burden on taxpayers; (d) helping regions achieve and declare freedom from disease status; (e) decreasing virus prevalence in the environment, thereby reducing zoonotic transmission risks; and (f) providing a distinguishable vaccine to support AIV control strategies in regions with strict culling policies. These outcomes directly translate into the long-term strategic goals of BBSRC, contributing to sustainable agriculture, benefiting farming communities, improving animals and public health, boosting economies, and reducing the environmental impact of the virus.