Recipient Organization
BIOSTONE ANIMAL HEALTH LLC
2815 EXCHANGE BLVD STE 400
SOUTHLAKE,TX 760927515
Performing Department
(N/A)
Non Technical Summary
Avian Influenza Virus (AIV) can be transmitted by migrating birds over long distances, and AIV outbreaks can pose a major threat to commercial poultry worldwide. The United States is suffering from an ongoing AIV outbreak, which has resulted in a loss of >58 million birds. The last AIV outbreak in the United States (2014) resulted in a loss of 50 million poultry, a direct loss of $1.6 billion, and additional recovery cost estimated at $1.7 billion. The cost of the current outbreak is still forthcoming. While some strains of AIV pose a relatively low risk, the greatest risk comes from AIV subtypes H5 and H7, which can become highly pathogenic avian influenza (HPAI) and cause deadly outbreaks. One favorable method to diagnose AIV is the enzyme linked immuno-sorbent assay (ELISA), which detects antibodies from blood samples. ELISA is preferrable because it is relatively faster and less expensive than alternative methods. However, AIV ELISAs that are used to screen commercial poultry require a second lab test to identify AIV subtypes H5 and H7. There is no commercially available ELISA that can diagnose AIV subtypes H5 and H7. Therefore, our project goal is to develop an ELISA to diagnose AIV subtypes H5 and H7, which can improve the time and cost of responding to a deadly AIV outbreak. New ELISA tools will be easily integrated into the current infrastructure and could have an immediate impact for AIV surveillance and prevention in the poultry industry.Our team previously conducted research and development for competitive ELISA (cELISA) to detect chicken and turkey antibodies against AIV subtypes H5 and H7. We have developed an H5 cELISA and an H7 cELISA prototype, which are at different stages of development. This project will focus on continuing to develop the H5 and H7 cELISA by using laboratory approaches that will improve their diagnostic performance. We will collaborate with Canada's National Center for Foreign Animal Disease (CFAD), which will provide us with exceptional support to evaluate each cELISA. Samples from different countries will be evaluated to address the issue of AIV originating from migratory birds over long distances. The CFAD maintains a collection of samples from poultry infected with American, Eurasian, and African AIV subtypes, historic samples collected from past outbreaks, and samples collected from the current outbreaks in North American (Canada, U.S., and Mexico). Our project will result in bringing the H5 and H7 cELISA prototypes to an advanced stage of development, which will allow us to pursue a Phase II project for large-scale global validation and commercialization.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
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).
Project Methods
EffortsEfforts will be achieved through ongoing evaluation of ELISA through our collaboration with Canadian Center for Foreign Animal Disease (CFAD). We will maintain communications and potentially work with multiple prototypes during the optimization process. We will provide CFAD with reagents, data, and instruction. We will work collaboratively to troubleshoot and identify sources of error when needed. From this experience, we will deliver new knowledge.EvaluationMonoclonal antibody evaluation. We will screen hybridoma cell lines for the production (and secretion) of H5-specific antibodies in the culture supernatants using iELISA and Western blot (WB). ELISA plates will be coated with H5 antigens from North American and Eurasian strains to evaluate H5 specificity. Supernatants will be used directly to examine H5 mAb reactivity with both antigens. To evaluate H5 specificity by WB, H5 antigens will be resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose for WB. WB will be probed using hybridoma supernatants as the primary antibody. A commercial anti-mouse-HRP will be used as the secondary antibody for both assays, iELISA and WB. We will evaluate H5 mAb candidates based on specificity to North American and Eurasian H5 antigens. The selected antibodies will also be screened for competitive binding against a panel of antisera collected from birds infected with different AIV H5 strains (from U.S., Canada, Mexico, and Ghana). The AIV H7 monoclonal antibodies will be screened and evaluated by the same approach, but the H7 competitive assay screen will not include the H5 antisera from Ghana.cELISA evaluation. During optimization, BioStone will conduct preliminary validation of specificity with AIV negative chicken sera (n=500) and an H5-positive sera sample (n=1) or an H7-positive sera samples (n=1) from chicken. Validation experiments will be continued in Berhane lab at CFAD using sera from chicken and turkey, which are AIV-negative (n=300), AIV H5-positive (n=100), 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. 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). For H7 cELISA, they will use a panel that includes 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 subtypes H1, H2, H3, H4, H5*, 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). Cross-reactivity of AIV H5* will only be evaluated on the H7 cELISA and cross-reactivity of AIV H7* will only be evaluated on the H5 cELISA.The Berhane lab will use samples that were previously subtyped and confirmed AIV negative by Np-based ELISA and confirmed AIV H5-positive or H7-positive by standard HI test methods. We will use these data to determine the accuracy of the ELISA. Data from all samples will be used to evaluate performance of the ELISA sensitivity and specificity to determine if additional optimization is necessary, and we will prepare data and analysis according to CVB requirements, using the statistical test package for R, DiagTestKit.ELISA data will be collected on 96-well plate readers, as optical density (OD) at 450 nm, and saved in Excel file formats. These files will be shared between labs to evaluate and discuss results. Analysis of cELISA data is based on Inhibition Percent (IP) relative to the negative control, and evaluation of correct diagnosis is determined by the IP cut-off values calculated by the receiver operating characteristic (ROC) curve analysis.