Recipient Organization
BIOSTONE ANIMAL HEALTH LLC
2815 EXCHANGE BLVD STE 400
SOUTHLAKE,TX 760927515
Performing Department
(N/A)
Non Technical Summary
African swine fever (ASF) is a foreign animal disease caused by ASF virus (ASFV) and remains one of the largest threats to pork production worldwide. The U.S. has not been hit by ASF outbreaks, but in 2021, ASF was reported in Dominican Republic and Haiti, placing the U.S. on high alert to increase ASF surveillance and preparedness. ASF is routinely detected by enzyme linked immuno-sorbent assay (ELISA), which is a serological test for ASFV antibodies. There are currently no ASF ELISA approved by the USDA. However, ELISA can be used to screen for ASF, whereby positive results are verfied with a secondary confirmation test. The only approved confirmation tests face technical challenges, as they are time-consuming, employ ASF viruses, and require a high-security Biological Safety Level 3 (BSL-3) laboratory. Thus, ASF surveillance is limited by the number of BSL-3 facilities and their personnel. Our project goal is to develop an ELISA confirmation test, which uses ASFV proteins, rather than viruses, and will be accessible to a broad network of low-security BSL-2 laboratories. An accessible ELISA confirmation test has the potential to significantly improve ASF surveillance, containment, and prevention for the U.S. and global pork industry, resulting in better food security and biosecurity during emerging outbreaks.To develop an ELISA that has comparable performance to standard confirmation tests, we will identify multiple ASFV antigens, which are ASFV proteins capable of detecting host ASF antibodies. We will use a multiple antigen ELISA strategy that can significantly improve the chances of ASF confirmation by testing for multiple host antibodies. We previously identified three of these antigens, and we will evaluate 12 additional antigens in this project. The final ELISA design will use up to 11 ASF antigens capable of detecting 11 different ASF antibodies from the host. ELISA research and development will be conducted in our BSL-2 facility, and we will collaborate with a BSL-3 ASFV Reference Laboratory to validate prototypes with samples from ASFV-infected animals and compare our ELISA to standard confirmation tests.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
African Swine Fever (ASF) is a rapidly emerging disease of domesticated pigs caused by a highly virulent strain of ASFV. In 2021, ASFV was detected in close proximity to the U.S., in the Dominican Republic and Haiti, placing the U.S. on high alert for ASFV and ramping up surveillance and disease management efforts. ASF serological diagnostic tests and reagents have significant market demand to support domestic biosecurity to prevent ASF entry to the U.S. and for international disease management where ASF is endemic, emerging, or in close proximity. Currently, the immunoperoxidase test (IPT) or indirect fluorescent antibody test (IFAT) using ASFV infected cells, and immunoblotting are the only confirmatory serological methods available and recommended for ASF. However, enzyme linked immune-sorbent assay (ELISA) is an ideal format for surveillance due to its speed, low-cost, and technical accessibility (training, equipment, and biological safety). The goal of this project is to deliver new diagnostic reagents and assays for a confirmatory ASF ELISA. Currently available commercial ASF ELISA have sensitivity issues to detect certain ASFV strains, such as Estonia and Georgia. Many diagnostic targets have their strengths and weaknesses, but none can provide comparable performance as tests that use ASF-infect cells or cell lysates (IPT, IFAT, immunoblotting). Thus, to improve ELISA performance, a mixture of ASFV antigens must be used. We previously developed ASF ELISAs, and confirmed the strengths of p30, p54, and p72 antigens. In this project, we aim to develop a confirmatory ELISA using additional antigens to detect multiple ASF antibodies. Following reagent and ELISA development, we intend to validate the ELISA and pursue USDA-licensure. The ASF ELISA will be useful to maintain and prove ASF free status. In the event of an outbreak, this will support rapid recovery to regain market access.Technical ObjectivesObjective 1. Production of twelve different immunogenic ASFV proteinsWe will use baculovirus expression systems to express recombinant ASF antigens. We will employ 6x his-tag on each recombinant antigen to facilitate purification by immobilized metal affinity chromatography (IMAC). Each purified antigen will be screened for ASF antibody detection.Objective 2. Screening antigens by iELISA: benchmark >95% sensitivity and specificity2.1. Screen antigens by iELISA (a) antigen concentrations, microtiter plates and blocking buffers, (b) sample and reagent diluents, and (c) plates and reagent preservatives buffers. We will screen twelve ASFV proteins that were previously characterized to be antigenic or immunogenic but have not yet been used to develop diagnostic tests. Antigens will be evaluated by their ability to detect ASF antibodies from ASF-infected animals.2.2. Validate antigens by iELISA and select top four candidates. We will select four antigens based on their sensitivity to detect ASFV antibodies, from multiple ASFV strains, early stages of infection, and ASFV vaccinated pigs. Then, we will use four new antigens, three well-characterized antigens (p30, p54, and p72), and a negative control antigen to develop an eight-antigen confirmation test. Following initial screening, we will work with the Canadian Food Inspection Agency, National Center for Foreign Animal Disease (NCFAD), collaborating with the OIE-recognized ASF Reference laboratory, Headed by Dr. Aruna Ambagala. Dr. Ambagala has an extensive sample collection for validating ASF diagnostics. His lab will perform validation experiments to evaluate the performance of each antigen identified in the ELISA screen.2.3. Initiate mAb development for top four antigen candidates. Four validated antigens, based on their sensitivity to detect ASFV antibodies, will be used to initiate mAb development. The mAb reagents can be used in research and diagnostics, but as a long-term process, mAb development and validation will not be conducted in the scope of our Phase I project. However, new mAb may provide valuable tools and positive controls for ELISA improvement and commercialization during a Phase II project.Objective 3. Develop confirmation test: benchmark >99.9% sensitivity and >99.75% specificity?3.1. Optimize compatibility of eight iELISA conditions. During validation we will develop several ELISA reagents (buffers, diluents, etc.) for each of the antigens individually. In this objective, they will be evaluated for their compatibility with the other antigens to determine if reformulation will be necessary. Objective 2 will be conducted in such a way that we will have reference data to identify compatible formulas and avoid incompatible conditions. 3.2. Validate iELISA confirmation test. Following development, we will work with Dr. Ambagala (NCFAD) to validate performance of the iELISA confirmation test, using the same strategies, sample sets, and comparison as performed for single-antigen iELISA screening.
Project Methods
EffortsAlthough the staff are formally trained in ELISA development and production, there are opportunities to receive a greater depth of training and education. The research and development process will involve staff scientists directing and training technicians as needed. During development and/or troubleshooting, it is common to review primary literature to generate new ideas or protocols. Designing, developing, and training technicians to perform new protocols is expected. There are 12 antigens and ELISAs to evaluate in this project, each of which potentially have different biochemical properties and have the potential to present different challenges. Therefore, we will make considerable efforts in educating team members and experiential learning.EvaluationAntigen ProductionThe codon-optimized gene sequences of EP402R (CD2V), EP153R (C type lectin), A151R, B438L (p49), B602L (p72 chaperone), CP530R, D117L, E199L, F317L, I329L, K78R, and H108R will be synthesized and cloned into a baculovirus transfer vector with c-terminal histidine tags (6xHis-tags). Baculoviruses encoding recombinant His-tagged ASF proteins will be generated, plaque purified, amplified, and tittered in Spodoptera frugiperda (Sf9) insect cells. For optimum antigen expression, Trichopulsia ni (Tni) cells will be infected with optimalmultiplicity of infection (MOI) and collection time post-infection to maximize protein yields. Following infection, Tni cells and supernatant (media) will be separated, and Tni cells will be lysed and separated into soluble and insoluble protein fractions. Expressed protein will be evaluated for expression level, solubility, and secretion into culture media. His-tagged ASFV proteins will be purified by immobilized metal affinity chromatography (IMAC). Expression and purification will be evaluated by Western blot and Coomassie gel staining.Screening antigens by iELISAScreening will require optimization of each iELISA, which includes (a) antigen concentrations, microtiter plates and blocking buffers, (b) sample and reagent diluents, and (c) plates and reagent preservatives buffers.(a) Optimize antigen concentrations, microtiter plates and blocking buffers for iELISA. We will analyze high binding, medium binding, and low binding plates, as well as different blocking buffers to coat the antigens to optimize assay sensitivity and specificity. Denatured antigens will be refolded by dialysis prior to coating. Blocking buffers are applied to coated wells to inhibit non-specific antigen-antibody interactions. We will determine optimal blocking buffer components, which typically consist of a pH buffer, nonionic detergents, and protein as blocking agent. Antigen coating concentration and buffer formulation will undergo optimization by checkerboard titration. (b) Optimize sample and reagent diluents for iELISA. We will optimize sample diluents, and antibody conjugate diluents to reduce the background and increase assay sensitivity. We will evaluate different formulas, which commonly include a pH buffer and non-specific protein sources such as BSA, horse serum, goat serum, rabbit serum, milk, or fish proteins to reduce assay background. (c) Optimize plates and reagent preservatives buffers for iELISA. We will consider proper application of preservatives to both the assay plates and immunoassay reagents. The stability of each reagent with preservatives will be evaluated at different temperatures (-20°C, 4°C, 25°C, and 37°C) to ensure at least 1-year shelf life.Validating iELISASensitivity will be evaluated as a percentage, false negative rate, of ASFV-positive samples. Specificity will be evaluated as a percentage, false positive rate, of ASFV-negative samples. iELISA 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 and personnel to evaluate results. Analysis and interpretation of iELISA data is based on Percent Positivity (PP) relative to the positive control. PP cut-offs to determine negative/positive results will be calculated based on a receiver operating characteristic (ROC) curve analysis.At BioStone, we will conduct iELISA optimization and preliminary validation of specificity using ASFV negative serum samples (n=500) and ASFV positive serum (n=1). If we achieve specificity >95%, we will produce prototypes for validation, for each antigen and different coating concentrations. Dr. Ambagala will continue validation in his lab (NCFAD). First, he will determine optimum antigen concentrations by evaluating sensitivity for each iELISA with an ASF reference serum panel of negative, weak positive and strong positive ASF serum (n=20). Once the optimal antigen coating concentration is identified, BioStone will produce a larger batch of iELISA prototypes to complete validation. Dr. Ambagala will evaluate optimized ELISA with field samples, confirmed ASF-negative (n=100) and ASF-positive (n=100), ASF-vaccinated (n=50), and experimental ASF-infected animals. For experimental. infections, serum samples will be collected from pigs inoculated with highly, moderately, and low virulent ASFV strains, 7-14 dpi (n=30) and 15-35 dpi (n=30). We will also evaluate validation samples sets with the commercial PPA and ID Screen ASF diagnostic kits. We will review data from all twelve ELISAs and conclude which four (or more) antigens are the best candidates for an ELISA confirmation test.Validating Confirmation Test FormatWe will compare all eight iELISA antigen conditions: seven ASF antigens plus one negative control antigen. We will determine which antigens appear incompatible with each other based on their plate type, coating buffers, and blocking buffers selected during the iELISA antigen screening. We will select confirmation test conditions base on each antigen and empirically determine which conditions are tolerated by which antigens. If specific antigens lose sensitivity or specificity, we will re-formulate buffers and diluents.Validation will be conducted using the samples and analyses described above. BioStone will conduct preliminary validation prior to sending prototype to Dr. Ambagala, who will verify performance with a reference serum panel. If the ELISA can detect all samples on the panel, then they will be further evaluated using field samples and samples from experimental ASF-infections. The final prototype will be evaluated with three controls on each plate: a negative, a weak positive, and a strong positive ASF sample. By employing seven ASFV antigens, the confirmation test will detect multiple antibodies against different strains, of different virulence, and vaccines, which could otherwise be missed by a single antigen. A positive confirmation test for different conditions will exhibit a profile of at least two positive wells across the panel and is not dependent on detecting all seven ASFV antibodies However, a single positive well will be interpreted as a suspected/ambiguous result. If our analyses determine that seven antibody targets are limiting diagnostic performance (sensitivity and specificity), we will increase the number of antibody targets, by reformating the 96-well plates (8x12) to coat 11 ASFV antigens (and one negative control) along the 12-well axis. This would reduce the total tests that can be run per plate but will increase sensitivity by detecting 11 different ASFV antibodies per sample.