Location: Foreign Animal Disease Research2011 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. Amendment 3, Objective 1b Enhanced: To enhance the fidelity of data generated, a robust system to check and confirm the predicting algorithm is warranted to generate a peptide library for the viral protein, P1 region of the FMDV. This library will allow for systematic analysis of the bovine immune responses to confirm the predictions fo the computer and identify peptides that the algorithm might miss. These data will be used to improve the functionality of the predictor program, identifying T cell epitopes which induce immune response. 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. Amend 3: Obj 2a: include comprehnesive bench standardization of competiive ELISA 3D test.
3. Progress Report
Several in vivo experiments were performed involving inoculation of cattle with Ad5-luciferase followed by euthanasia, necropsy and tissue collection. Tissues were screened by RT-PCR and luminometry. One manuscript was drafted describing novel techniques developed. Identified the BoLA class I and class II genes that are the predominant genes expressed in the experimental herd of Holstein cattle at the University of Vermont. These first molecules are being synthesized and expressed in analysis in vitro. Completed and coordinated efforts with USDA, APHIS to conduct bench validation of FMD DIVA test. A large quantity of antigens was produced and transferred to USDA, APHIS for use in conducting validation of ELISA test. Cloned all bovine type I interferon genes and developed several new bioassays needed to screen for those IFN molecules with the highest anti-FMDV specific activity in cell culture. The top members of interferon alpha and beta subtypes were identified and have estimated anti-FMDV activities ranging from 70-560 times higher than the IFN previously developed by ARS. Successfully identified and tested in vitro and in vivo bovine IFN lambda 3. Demonstrated that treatment of cattle with bovine IFN lambda 3 delays disease for 7-9 days suggesting that this IFN may cover the window of time required for vaccine-induced adaptive immunity. We have also cloned porcine IFN lambda 3 and characterized it in vitro. Performed dose response potency study in swine using polyICLC. A manuscript is currently under preparation. Performed efficacy study in swine with poly ICLC, Ad5pIFNalpha combination. Demonstrated that in combination, induced enhanced protection. Studies were initiated to examine gene induction by real-time RT-PCR and will continue in FY 2012.