2010 Annual Report
1a.Objectives (from AD-416)
The Foreign Animal Disease Research Unit (FADRU) at Plum Island Animal Disease Center (PIADC) is the primary USDA laboratory carrying out research on foreign animal diseases (FAD) of livestock, such as foot-and-mouth disease (FMD) that could be accidentally or deliberately introduced into the United States in acts of agro-terrorism. The Department of Homeland Security (DHS) PIADC is tasked with developing bio-defense control mechanisms, inclusive of veterinary countermeasures. This collaborative research agreement addresses the threat of an introduction and subsequent outbreak of FMD or other high-consequence FAD and the development of improved countermeasure technologies. This IAA has three objectives all of which address Foot-and-Mouth Disease Countermeasures research gaps: 1- improvement of Ad5-FMDV empty capsid vaccine in cattle; 2- improvement of biotherapeutics against FMDV in cattle and 3- improvement of FMD countermeasures through bioinformatics.
1. Host-Pathogen Interaction will include a) the vector-host interaction of adenoviurus FMD (AdFMD) vaccine in cattle and b) determination of major histocompatibilty complex (MHC) class I and class II restricted FMDV-specific lymphocyte responses following AdFMD vaccination in cattle.
Amendment 3: Objective 1 Enhanced: A peptide library and associated equipment to conduct tetramer studies based on predictor FMV peptide binding to dominant Class I and II recombinant molecules will be procured and utilized. These results will be used to better predict the capability of peptide epitopes which can be used to induce an immune response against FMDV.
2. Bioinformatics objective will address knowledge gaps for countermeasures and diagnostic technologies for high consequence FAD, specifically, a) antigenic profiling of FMDV 3D polymerase and evaluation as a candidate for DIVA diagnostic test development and b) identification of bovine Type I interferon with enhanced biotherapeutic potency to control FMDV.
3. Biotherapeutics objective will focus on a) evaluation of Type III interferons as potential biotherapeutics to control FMDV and b) identification of biotherapeutic candidates to control FMDV.
1b.Approach (from AD-416)
Objective 1a, host-pathogen interaction of the huAd5 vector will be studied by inoculating cattle with huAd5 vectors containing reporter genes or FMDV capsid and tracking its distribution and transgene expression in target tissues such as muscle and regional lymph nodes. The identification and tracking tissue distribution of AdFMD vaccines will be useful for future design and assessment of AdFMD vaccine with increased potency. Objective 1b. ARS, PIADC will utilize the experimental closed Holstein herd from the University of Vermont, to conduct tissue typing analysis of Class I major histocompatibility complex (MHCI) among herd members using PCR developed in collaboration with the University of Copenhagen, Denmark. Relevant Class I genes corresponding to animals in the experimental herd will be expressed in E.coli for analysis of peptide binding in vitro. These data will be used to predict peptides of FMDV capsids from strains A24, A22, O1 Manisa and O1 Campos.
Objective 2a, will be accomplished by sequence analysis and determination of antigenic profiles of FMDV and bovine rhinovirus polymerase proteins and the determination of cross-reactivity between them. Identification and expression of FMDV-specific 3D epitopes by recombinant technologies will be conducted. Proof of concept for specific and sensitivity of ELISA tests will be developed. Objective 2b will be accomplished by cloning and expressing bovine IFNA. The expressed IFNA will be tested for antiviral activity. The bIFNA with the highest activity will be identified.
Objective 3a, will be accomplished through identification and cloning of all members of the type III IFN gene family of cattle using Ad5-vectors. Examination of antiviral properties against FMDV will be conducted. Dose-response experiments will be conducted in-vitro. A study of IFN stimulated gene induction and specific type III receptor gene expression by real time RT-PCR and/or microarray analysis, will be conducted to examine gene induction after treatment with type III IFN alone or in combination with other Biotherapeutics. 3.b. will be accomplished by conducting vaccine challenge studies on swine and cattle utilizing different doses of polyICLC in combination with the optimal dose of Ad5-IFNa, and a suboptimal dose of AD5-IFNa. The cellular components of the innate immune response including natural killer cells, dendritic cells will be examined. Based on these studies, the optimal amounts of polyICLC and Ad5-IFNa will be administered to swine and cattle and challenged with FMDV.
Optimization of systems and tools for tracking tissue and microscopic (cellular) distribution of Ad5-FMD vaccine. These tools to track the distributionof Ad5-vectors in cattle will be used to identify the interaction of these vectors with immune cells. To identify the dominant Class I and II alleles in genetically-identifiable cattle, we obtained sequence data. This information will allow the study of T cell responses of cattle against FMDV infection or vaccination. Using bioinformatics, a recombinant modified 3Dpol antigen was produced to aid in the development of an improved FMD DIVA test with Ad5-FMD molecular vaccines. The results indicate that a competitive ELISA is the best method to use. Bioinformatic analysis was used to identify 51 Type I interferon (IFN) proteins in cattle. Testing of the in-vitro expressed IFN proteins demonstrated biological activity on several of these IFN molecules. Novel bovine and swine IFN were tested as biotherapeutic candidates in-vitro and in-vivo. Results indicate a delay and reduction in severity of infection treated with these biotherapeutic candidates. Demonstrated that bovine IFN alpha and polyIC in combination, synergistically inhibited FMDV replication. Potency and efficancy studies were conducted in swine.
This project was monitored through email and telephone exchange. Meetings were held between ARS and DHS staff at PIADC.