Progress 07/01/21 to 06/28/22
Outputs
Target Audience:Our overall target audience is the swine industry. During this project, we primarily reach our audience in research laboratories at regulatory agencies, which are concerned with the development of swine health and diagnostic products. We have communicated significant details of our progress and ongoing efforts with the USDA - Plum Island Animal Disease Center and the Canadian Food Inspection Agency - National Center for Foreign Animal Disease. These government sector labs will provide significant support in the development and dissemination of our African Swine Fever diagnostic products in the event that our commercial product receives regulatory approval for sales in the US and Canada. Our target market and future audiences will also include swine farmers and distributors of diagnostic products. In the process of R&D, we have connected with international distributors who are aware of our products in development. These distributors serve the international markets where African Swine Fever is endemic, and they will have a significant role in marketing and distributing our final diagnostic products to end-users. Changes/Problems:We faced challenges to achieve sensitive detection of ASF I177L antibodies. This is an issue that is directly related to the concentration (titer) of antibodies an ASF-infected animal produces against the I177L antigen. To overcome this challenge, we have vigorously developed and evaluated ELISA reagents (buffers, diluents, etc.) for every step in ELISA production and ELISA test/sample processing. We established a proprietary internal catalogue of >40 buffer formulas, which have been deeply analyzed in check-board titrations. From this effort we managed to improve sensitivity of the test >40-fold. We continue evaluating new buffer, based on the identity of key components. In addition to our work in reagent development, we have initiated I177L monoclonal antibody development, which can lead us to helpful alternatives to iELISA, such as bELISA. Due to the challenges faced, we did not reach our validation benchmarks with the I177L iELISA. What opportunities for training and professional development has the project provided?The project has provided training opportunities for new hires to develop their skills at the bench. This has included conducting replicates during ELISA validation or learning how to produce an ELISA plate, ELISA reagents, and managing ELISA data. It is also a learning opportunity for ELISA technicians on the project to understand the global impact of ASF, the impact of their work on ASF diagnostics, and the potential role they could have in ASF management. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?This is the final report. While there are no further reporting periods, our effort to develop the ASF DIVA is ongoing, and we will continue to work with the USDA and CFIA to accomplish this goal. We are currently awaiting approval of a NACA cooperative research agreement with Douglass Gladue of USDA Plum Island Animal Disease Center, the laboratory where the ASF vaccine was invented. We also have an ongoing collaboration with Dr. Aruna Ambagala at the Canadian National Center for Foreign Animal Disease, which is one of 7 OIE-recognized ASF reference laboratories in the world. We intend to continue submitting grant proposals to help fund ongoing work on the project. The remaining primary goal is to improve diagnostic performance of the I177L antibody test. There are a few stages of R&D to finalize the ELISA format and reagent formulation, but thus far, our progress has been positive and performance of the I177L test continues to improve.
Impacts
What was accomplished under these goals?
Impact Statement African swine fever (ASF) is a rapidly emerging disease of domesticated pigs caused by a highly virulent strain of African swine fever virus (ASFV). In 2018, ASF spread to China, then to other countries including Mongolia, Vietnam, Cambodia, Laos, North Korea, South Korea, and Myanmar. In 2020, the spread continued to Papua New Guinea, India, and wild boars in Germany. In 2021, ASFV reached domestic pigs in Germany, Europe's largest pork producing country. Europe's most recent emergence was in Italy in 2022, where 6 cases were identified in wild boars. In 2021, ASFV was detected close to the U.S., in the Dominican Republic and Haiti, resulting in the U.S. being on high alert for potential introduction of ASFV. Although the U. S. has not suffered an ASFV outbreak, there is a substantial risk, which could cost the pork industry $15 billion (2-year recovery) and up to $50 billion (10-year recovery) after eradication. Many countries, including the US are developing ASF vaccines to prevent the introduction or spread of ASF. A vaccine will reduce ASF prevalence in international markets and reduce the risk of transmission to the US, improving our biosecurity. The ASF vaccine is currently being manufactured and widely tested in Vietnam. If successful, it will be the first ever commercially available and approved ASF vaccine. Diagnostic antibody tests are a critical part of ASF management. However, test results can be confounded by vaccinations, because a vaccine and a virus can cause host animals to produce similar antibodies. One way to overcome this challenge is to develop a test for Differentiating Infected from Vaccinated Animals, also known as a DIVA test. DIVA tests are critical for monitoring infections within a vaccinated population. An ASF DIVA test would prevent culling vaccinated animals during an ASF outbreak. However, the ASF vaccine does not have a companion diagnostic DIVA test. For our project, we developed an ASF DIVA test using a common testing platform called Enzyme Linked Immuno-Sorbent Assay (ELISA). We produced several proteins in our lab, which are derived from the ASF virus and vaccine, using the genetic sequences of each protein and a protein expression system that employs insect cells. Each protein is a key component to develop the ELISA antibody tests. The DIVA ELISA uses two tests to differentiate infections from vaccinations, one that detects ASF virus antibodies (p54 and p72) and one that discriminates the ASF vaccine antibodies (I177L). Our laboratory developed prototypes for both ELISAs and conducted preliminary validation studies. We collaborated with academic and government labs to confirm the diagnostic performance using laboratory animals and samples collected from the field. We worked with service labs, USDA Plum Island Animal Disease Center, Vietnam National University of Agriculture, and CFIA Center for Foreign Animal Disease, who collectively tested > 600 swine sera samples. Through our collaborative work, we validated one ELISA (ASF p54/p72 combo iELISA) with >98% sensitivity and specificity. The second ELISA (ASF I177L iELISA) requires more development to improve prototype sensitivity. Notably, we have improved sensitivity >40-fold thus far, and we expect to reach benchmark performance in the coming year. We are moving forward with additional validation work with both ELISAs. We aim to apply for a USDA product license for both tests, which comprise the DIVA test kit, and we will market it as a companion diagnostic product for the ASF vaccine. 1. Develop an I177L iELISA to reach a benchmark of specificity >98-99% and sensitivity to detect antibodies as early as 7-9 days post infection (dpi). 1.1. Express and purify I177L antigens 1) We expressed and purified I177L antigens. 2) We collected data for cell culture conditions, protein purity, and yield. 3) We optimized protocols that result in high I177L yield >30mg/L culture and purity >90%. 4) We have a reliable source of I177L antigens. 1.2. Optimize iELISA (a) antigen concentrations, (b) microtiter plates and blocking buffers, (c) sample and reagent diluents, and (d) plates and reagent preservatives buffers 1) We developed several prototypes, which had increasing sensitivity at each stage of development. 2) We analyzed positive and negative controls, and we analyzed ASF-positive and ASF-negative pig sera to optimize the ELISA specificity and sensitivity. 3) We obtained >90% between negative and positive samples at 1:40 dilution with early prototypes. Our most recent prototype achieved 95% separation at 1:1600. 4) We identified formulation strategies to improve I177L diagnostics. 1.3. Validate iELISA with naturally infected and experimentally infected pigs 1) We collected preliminary validation data. 2) We evaluated the performance of the I177L iELISA in two ASF-vaccinated animals, 20 ASF-positive animals, and >200 ASF-negative animals. 3) We acquired small data sets with minimal statistical power. 4) The I177L iELISA can detect ASF for the DIVA test, but it has not been validated at a large scale. 1.4. Initiate monoclonal antibody production for I177L 1) We initiated monoclonal antibody production for I177L. 2) We use the I177L iELISA to evaluate the presence of I177L antibodies at each stage of production, including test bleeds from injected mice and all stages of hybridoma culture development. 3) Monoclonal antibody production is ongoing. 4) The I177L protein is sufficiently antigenic to induce antibody production in mice. 2. Develop combo p54/p72 iELISA to reach a benchmark of specificity >98-99% and sensitivity to detect antibodies as early as 7-9 dpi and maintain detectability up to 35 dpi. 2.1. Express and purify p72 antigens 1) We expressed and purified p72 antigens. 2) We collected data for cell culture conditions, protein purity, and yield. 3) We established optimized protocols that result in high yield >30mg/L culture and purity >90%. 4) We have a reliable source of p72 antigens. 2.2. Optimize p72 stand-alone iELISA (a) antigen concentrations, (b) microtiter plates and blocking buffers, (c) sample and reagent diluents, and (d) plates and reagent preservatives buffers 1) We initiated development of both p72 iELISA and p72/p54 combo iELISA. 2) We analyzed positive and negative controls, and we analyzed ASF-positive and ASF-negative pig sera to optimize the iELISA specificity and sensitivity. 3) We collected minimally sufficient data to determine that a combination p54/p72 format performed better than a single p72 iELISA. 4) The p72 ELISA is inferior to the p54/p72 iELISA. 2.3. Develop a combined p54/p72 iELISA based on stand-alone optimization data 1) We developed the p54/p72 iELISA. 2) We analyzed positive and negative controls, and we analyzed ASF-positive and ASF-negative pig sera to optimize the iELISA specificity and sensitivity. 3) We achieved >98% specificity in preliminary validation. 4) The p54/p72 combo iELISA was successfully developed. 2.4. Validate iELISA with naturally infected and experimentally infected pigs 1) We validated the p54/p72 iELISA with service labs and collaborators. 2) We evaluated diagnostic performance with >600 pig sera samples, which includes a variety of ASFV-vaccinated, ASFnegative, ASF-positive, and experimentally ASF-infected samples from North America, Vietnam, and China. 3) We achieved >98% specificity and sensitivity, and the iELISA could detect infections as early as 10 dpi. We have ongoing validation work to evaluate performance beyond 22dpi. 4) The p54/p72 combo iELISA performed well under all conditions. It will be a critical component for a DIVA test, but it is also an exceptional test for ASF infection antibodies regardless of vaccination status. 2.5. Initiate monoclonal antibody production for p72 The p72 monoclonal antibodies were intended for alternative bELISA format. Since the iELISA format was successful, the antibody was not produced.
Publications
|