AN ENZYME-LINKED IMMUNOSORBENT ASSAY TO DETECT ANTIBODIES AGAINST AVIAN INFLUENZA VIRUS (AIV) SUBTYPE H5 AND H7 IN COMMERCIAL POULTRY

The United States is suffering from an ongoing Avian Influenza Virus (AIV) outbreak. Historically, the AIV subtypes H5 and H7 pose the greatest risk of mutating into highly pathogenic avian influenza (HPAI) strains that cause deadly outbreaks. However, current diagnostic methods to detect AIV H5 and H7 subtypes have limitations (time, cost, process volume). Our project goal is to commercialize an H5 and H7 test using enzyme linked immune-sorbent assay (ELISA) formats. ELISA would reduce the cost, time, and efforts of AIV management by providing a high-throughput screening method to diagnose high-risk AIV subtypes at an earlier stage of surveillance. ELISA is frequently used for disease surveillance, but only as a pre-screening tool; it is typically followed by a gold-standard test, such as the Hemagglutination Inhibition assay to determine the H5 or H7 subtypes. There are no USDA-approved AIV subtype ELISAs, which provides us with a great opportunity to develop and commercialize these diagnostic tools for commercial poultry. New ELISA tools will be easily integrated into the current infrastructure and could have an immediate impact for AIV surveillance in the poultry industry. Our team has ongoing research and development efforts for competitive ELISA (cELISA) prototypes to detect chicken and turkey antibodies against AIV subtypes H5 and H7. However, each ELISA prototype is at a different stage of development. Our project efforts will focus on continuing optimization, validation, and eventually commercialization of these two products. We will collaborate with Canada's National Center for Foreign Animal Disease, which provides us with an excellent opportunity to validate each ELISA with extensive sample collections including antiserum from birds infected with North American,Eurasian, and African AIV strains, historic samples collected from past H5 and H7 outbreaks, and samples collected from the current H5 and H7 outbreaks in North American (Canada, U.S., and Mexico).Objectives1. Develop cELISA to detect AIV H5 antibodies1.1. Screening monoclonal antibodies against AIV H5 antigens The success of a cELISA is dependent upon producing monoclonal antibodies (mAb) that have binding characteristics similar to the host antibody that is being detected. We will screen previously generated hybridoma cell lines for competitive binding against a panel of antisera collected from birds infected with different AIV H5 strains. We will also evaluate H5 mAb competitive binding against H5 antiserum collected from Mexico and Ghana.1.2. Develop and optimize AIV H5 cELISA We will use mAb screening and validation data to identify gaps in diagnostic sensitivity, and we will strategically optimize mAb and antigen combinations to eliminate gaps to achieve full diagnostic coverage. ELISA optimization will include: (a) Optimize antigen concentrations, microtiter plates, and blocking buffers. (b) Optimize sample and reagent diluents and enzyme linked reporters. (c) Optimize plates and reagent preservatives.1.3. Validation benchmark: We aim to reach specificity and sensitivity of >98% for the AIV subtype H5 cELISABioStone will conduct preliminary validation of specificity with AIV negative chicken sera (n=500) and an H5-positive sera sample (n=1) from chicken. Validation experiments will be continued in Berhane lab using sera from chicken and turkey, which are AIV-negative (n=300) or AIV H5-positive (n=100). To evaluate the impact of antigenic drift in groups of H5 subtypes that have been endemic to and evolving in Mexico since the1990s, they will also evaluate the ELISA with LPAI H5-positive (n=50) and AIV-negative (n=50) chicken serum samples collected from Mexico in 2022. They will evaluate additional samples from the 2021 Ghana H5N1 outbreak (n=25). They will evaluate cross-reactivity with antisera for AIV subtype H1, H2, H3, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16 infected poultry (n=1 per subtype), and they will evaluate cross-reactivity with antisera from poultry infected with six non-AIV avian viruses: avian paramyxovirus type 1 (n=1), type 2 (n=1), and type 3 (n=1), and avian metapneumovirus subtype A (n=1), subtype B (n=1), and subtype C (n=1).2. Develop cELISA to detect AIV H7 antibodies2.1. Screening monoclonal antibodies against AIV H7 antigens We will screen previously generated hybridoma cell lines for competitive binding against a panel of antisera collected from birds infected with different AIV H7 strains. We will also evaluate H7 mAb competitive binding against H7 antiserum collected from Mexico.2.2. Develop and optimize AIV H7 cELISA We will use mAb screening and validation data to identify gaps in diagnostic sensitivity, and we will strategically optimize mAb and antigen combinations to eliminate gaps to achieve full diagnostic coverage. ELISA optimization will include: (a) Optimize antigen concentrations, microtiter plates, and blocking buffers. (b) Optimize sample and reagent diluents and enzyme linked reporters. (c) Optimize plates and reagent preservatives.2.3. Validation benchmark: We aim to reach specificity and sensitivity of >98% for the AIV subtype H7 cELISABioStone will conduct preliminary validation of specificity with AIV negative chicken sera (n=500) and an H7-positive sera control (n=1) from chicken. Validation experiments will be continued in Berhane lab using sera from chicken and turkey, which are AIV-negative (n=300) or AIV H7-positive (n=100). AIV positive samples have been collected from different global regions and different years (from routine surveillance and major HPAI outbreaks), which includes birds infected with North American and Eurasian strains. The panel will also include the HPAI H7-positive (n=50) and AIV-negative (n=50) chicken serum samples collected from Mexico in 2022. They will evaluate cross-reactivity with antisera for AIV subtype H1, H2, H3, H4, H5, H6, H8, H9, H10, H11, H12, H13, H14, H15, and H16 infected poultry (n=1 per subtype), and they will evaluate cross-reactivity with antisera from poultry infected with six non-AIV avian viruses: avian paramyxovirus type 1 (n=1), type 2 (n=1), and type 3 (n=1), and avian metapneumovirus subtype A (n=1), subtype B (n=1), and subtype C (n=1).