Location: Produce Safety and Microbiology Research
2022 Annual Report
Objectives
Objective 1: Develop mass spectrometry, immunological, and in vitro prion amplification techniques to detect, structurally define, and distinguish among CWD strains in order to predict their ability to transmit to new animal species- Develop a laboratory test that can be certified as an official method for the USDA CWD Herd Certification Program that is sensitive, CWD-specific, repeatable, reproducible, cost-effective, and can detect CWD in easy to collect samples (e.g., oral fluids, feces, blood, skin) from cervids.
Sub-objective 1.A: Develop mass spectrometry-based methods to improve detection of CWD prions and distinguish among prion strains.
Sub-objective 1.B: Detect covalent modification of prions by Western blot.
Sub-objective 1.C: Improve detection of CWD prions using prion amplification methods and glycosylated recombinant PrP (grPrP).
Objective 2: Develop rapid immunoassays and molecular diagnostic methods for early detection of emerging pathogens-Develop diagnostic tests that can be registered with the USDA-APHIS Center for Veterinary Biologics that is sensitive, specific, reproducible, and cost-effective to detect emerging animal pathogens in easy to collect samples (e.g., oral fluids, feces, blood, skin).
Sub-objective 2.A: Generate monoclonal antibodies (mAbs) against SARS-CoV-2 and SVA antigens to develop immunoassays used for diagnostic detection of viral infection in farm animals.
Sub-objective 2.B: Develop lateral flow and colorimetric assays integrated with highly specific aptamers for rapid detection of SARS-CoV-2, senecavirus A (SVA), and influenza A virus (IAV-S, H1N1) in farm animals.
Approach
The approach will address the development of rapid antemortem tests for the early detection of transmissible spongiform encephalopathies and other animal diseases such as SARS-CoV-2, senecavirus A (SVA), and influenza A virus (IAV-S, H1N1). Objective 1 will develop mass spectroscopy, immunological, and in vitro prion amplification techniques to detect, structurally define, and distinguish CWD strains. Objective 2 will develop pen-side/point-of-care/pre-clinical diagnostic methods involving immunological and non-immunological-based tools targeting emerging and re-emerging viral pathogens, specifically SARS-CoV-2, SVA, and IAV-S (H1N1). Under Objective 1, mass spectrometry-based methods will be developed to improve the detection of CWD prions and distinguish among prion strains by conformation-dependent differences of amino acids. In addition, Western blot will be utilized to detect any covalent modifications present in specific amino groups of lysines present in CWD prions. Prion amplification methods by real-time quaking-induced conversion (RT-QuIC) and glycosylated recombinant prion proteins (grPrP) will also be used to improve detection of CWD prions. Under Objective 2, monoclonal antibodies will be generated against SARS-CoV-2 and SVA antigens, while highly-specific aptamers will be generated via systematic evolution of ligands by exponential enrichment (SELEX) to target SARS-CoV-2, SVA, and IAV-S (H1N1). These recognition elements will be integrated into pen-side diagnostic tools, mainly lateral flow assay (LFA) and gold nanoparticles detection platforms, and ultimately directly applied on animal and environmental samples.
Progress Report
This is a new project that started in March 2022 and continues work from expired project 2030-32000-010-000D.
In support of Sub-objective 1A, ARS scientists have optimized conditions to detect methionine oxidation in hamster, bank vole, cervid, and ovine recombinant prion proteins. In addition, the conditions have been optimized to facilitate lysine acylation of prions from sheep and cervids. Brain tissue from homozygous (ARQ) sheep naturally infected with classical scrapie and brain tissue from homozygous (ARR) and heterozygous (AHR/ARR) sheep experimentally infected with atypical scrapie have been acquired. Samples of brain tissue from white-tailed deer experimentally infected with the Wisc-1 and H95+ strains of CWD have been obtained. Well-characterized hamster-adapted scrapie strains have been oxidized with hydrogen peroxide and chloramine T to quantify the extent of methionine oxidation.
In support of Sub-objective 1B, ARS scientists have synthesized new acylating agents. The 8G8 and 6D11 monoclonal antibodies (mAbs) have been obtained from commercial vendors. mAb N2 was obtained through an incoming materials transfer agreement. These mAbs have been tested against sheep and deer prion protein (PrPC), derived from brain homogenates and acylated with synthetic reagents. These results indicate that when the lysine (PrPC confirmation) in the epitopes of these mAbs is acylated, the mAbs do not bind, and PrPC is not detected by Western blot.
In support of Sub-objective 1C, ARS scientists have synthesized genes to express the native bank vole prion protein (109M) and a polymorphic variant (109I). Both genes have had their codons optimized for expression in Escherichia coli (E. coli). The plasmid has been cloned into the BL21 strain of E. coli. The bank vole (109I) recombinant prion protein (rPrP) has been overexpressed in this strain of E. coli. The protein has been successfully purified and analyzed by mass spectrometry.
In support of Sub-objective 2A, immunization of mice with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigenic protein variants for the generation of monoclonal antibody-producing hybridoma cell lines is ongoing. Various adjuvant and immunization regimes have been deployed, and animal immune responses were confirmed by immunoassay. Hybridoma technology, cell cloning, and high-throughput enzyme-linked immunosorbent assay (ELISA) screening are progressing toward the selection of distinct hybridoma cell lines that produce novel monoclonal antibodies against SARS-CoV-2 variants. These anti-SARS-CoV-2 monoclonal antibodies will be used to develop improved animal immunodiagnostic assays. In collaboration with partners, existing anti-SARS-CoV-2 antibody cohorts generated against viral spike proteins have been evaluated. Antibody pairs that selectively bind recombinant spike protein variants from SARS-CoV-2 by lateral flow immunoassay have been identified. Efforts are underway to enhance lateral flow reporter sensitivities as a prerequisite for the detection of native viral particles when targeting spike protein antigens.
Recombinant proteins have been generated against the external capsid proteins of the Senecavirus-A. These proteins have been purified, biochemically characterized, and used for immunization of mice and the generation of hybridoma cell lines producing specific monoclonal antibodies. A predominant Senecavirus-A strain has been secured from our partners for propagation in cell culture as a source of native virus for laboratory evaluation of immunoassay performance. To date, a cohort of monoclonal antibodies generated against the capsid VP2 protein has been partially characterized, and a sandwich enzyme-linked immunosorbent assay has been constructed. Current efforts are focused on the applicability of these VP2 antibodies and immunoassay formats for the detection of native viruses.
In support of Sub-objective 2B, research has continued on the development of aptamer-based lateral flow assay (LFA) for the early detection of emerging pathogens [influenza virus A – H1N1 in swine, SARS-CoV-2 variants of concerns (VOCs: Alpha, Delta, and Omicron variants), and Senecavirus A or SVA] that cause animal diseases. Target viral proteins were used as baits to generate highly-specific in-house aptamers through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). As an iterative process, each round of SELEX narrows down the aptamer sequences from a random pool of DNA libraries which can eventually generate aptamer sequences that are highly specific with excellent binding affinity to the target viral proteins. For SVA, aptamers have been developed (SELEX – Round 8) and sequenced using VP1 recombinant protein with further optimizations needed. For H1N1, HA protein (Round 5) was used, while both spike and nucleocapsid proteins (Round 3) were used for SARS-CoV-2 VOCs during SELEX. The generated aptamer sequences would then be incorporated onto the LFA system as capture and detection elements for the rapid and on-site detection of viral agents. In parallel to the in-house aptamer generation, available published aptamer sequences targeting VOCs were also successfully incorporated into the LFA system. These efforts have shown the sensitivity of aptamers and compatibility with LFA platforms.
Accomplishments
1. Detecting prion strains by measuring methionine oxidation. Prions cause chronic wasting disease (CWD), a rapidly spreading disease of wild deer and elk. CWD negatively impacts the $30 billion U.S. hunting industry. Prions impart disease through their distinct shapes. Unfortunately, differences in shape are difficult to characterize by conventional means. ARS researchers in Albany, California, used mass spectrometry to detect shape differences of hamster-adapted prion strains by measuring the surface exposure of the amino acid methionine. This approach allowed the researchers to define a prion strain by its shape instead of relying on more empirical and cumbersome conventional methods. This will provide regulators with the tools to manage the emergence of new prion strains.
2. Normal cellular prion protein enables spread of misfolded proteins. Protein misfolding or prion-like diseases, such as Parkinson’s disease, cost Americans untold misery and at least $50 billion annually. These diseases spread from cell-to-cell by binding to the natively expressed prion protein (PrPC). As part of a large international collaboration, ARS researchers in Albany, California, showed that binding of the misfolded protein that causes Parkinson’s disease to PrPC allows the protein to enter a cell, thereby spreading it from one cell to another. The part of PrPC that the misfolded proteins attaches to was also identified. This information can be used to develop drugs to prevent the binding and consequent intercellular spread of misfolded proteins. In principle, such drugs may be a general means of treating prion and prion-like diseases, such as Parkinson’s disease.
3. Novel VP2 capsid antibodies against Senecavirus-A. No reliable rapid tests are available for detecting Senecavirus A (SVA) infection in swine. ARS researchers in Albany, California, have designed, engineered, and expressed three viral capsid proteins (VP1-3) from Senecavirus-A (SVA) in bacteria. These recombinant viral VP capsid proteins have been affinity purified and biochemically characterized. Mice were immunized with the VP2 protein and hybridoma technology used to generate and select unique hybridoma cell lines expressing anti-VP2 monoclonal antibodies. Novel monoclonal antibodies to viral capsid proteins will be used by researchers to develop pen-side SVA diagnostic immunoassays.
4. Aptamers are an alternative option for lateral flow assay detection of SARS-CoV-2 in animals. In the United States, 17 mink farms have been affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all of which have had associated human cases. Currently, there are no federally authorized rapid and handheld detection tools for SARS-CoV-2 testing in animals. Researchers in Albany, California, have collaborated with USDA Animal and Plant Health Inspection Service (APHIS), ARS National Animal Disease Center, and Centers for Disease Control and Prevention One Health to develop aptamer-based lateral flow assays (LFA) for pen-side rapid detection. Aptamers have been shown to be highly compatible and stable in the LFA system as its main detection component to screen for SARS-CoV-2. This inexpensive technology is an excellent option in addition to immune-based detection tools to effectively address current diagnostic needs related to animal diseases by USDA APHIS, veterinarians, farmers, and other regulatory agencies.
Review Publications
Silva, C.J. 2022. Chronic wasting disease (CWD) in cervids and the consequences of a mutable protein conformation. ACS Omega. 7(15):12474–12492. https://doi.org/10.1021/acsomega.2c00155.
Thom, T., Schmitz, M., Fischer, A., Correia, A., Correia, S., Llorens, F., Pique, A., Mobius, W., Domingues, R., Zafar, S., Stoops, E., Silva, C.J., Fischer, A., Outeiro, T.F., Zerr, I. 2021. Cellular prion protein mediates a-synuclein uptake, localization, and toxicity in vitro and in vivo. Movement Disorders. 37(1):39-51. https://doi.org/10.1002/mds.28774.
