2012 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.
TASK 1A – Vector-Host Interaction of AdFMDV in Cattle. The goal of the current project is to determine biodistribution of human adenovirus 5 (HuAd5) vector and expression of inserted transgenes (FMDV-A24 or other) in bovine tissues (injection site and lymphoid tissues) subsequent to inoculation. Molecular techniques and immunomicroscopy will be used to characterize biodistribution.
During FY 2012 the optimization of Adenovirus-FMD (AdFMD) detection by multichannel immunofluorescence (IFA) took place. Adeno hexon has been localized in combination with various cell markers, including FMDV antigens and with Luciferase at injection sties and minimally in regional lymph nodes.
The milestones for this task are complete. A manuscript entitled “Early detection and visualization of human adenovirus serotype 5-viral vectors carrying foot-and-mouth disease virus or luciferase transgenes in cell lines and bovine tissue” has been published in the Journal Vaccine. This manuscript includes the biodistribution/immunopathogenesis data for in vitro and in vivo experiments utilizing Ad5-FMDV-A24 and Ad5-Luc. This task has now been completed.
TASK 1B – Determination of MHC Class 1 and Class 2 Restricted FMDV-Specific T Lymphocyte Responses Following AdFMD Vaccination in Cattle. Several in vivo experiments were performed in FY 2012 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. Six BoLA class I encoded proteins and 8 BoLA class II proteins have been shown to be expressed in some combination by 90% of the UVM research herd. All of these genes are now expressed as recombinant proteins by collaborators at the University of Copenhagen. These proteins are now available for analysis of binding peptides derived from the proteome of FMDV towards the identification of T cell epitopes of FMDV. Completed and coordinated efforts with USDA and 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.
TASK 2A – Antigenic profiling of FMDV 3D polymerase and evaluation as a candidate for DIVA diagnostic test development. A competitive ELISA test based on the FMDV 3D polymerase and selected monoclonal antibodies specific for FMDV was developed. The assay was bench tested for feasibility with good results by collaborative efforts between ARS and APHIS. ARS has transferred the FMDV 3D-based cELISA protocols and reagents to APHIS for further validation. The information provided also included the results of additional parallel 3D-based assays (3ABC, VIAA, indirect ELISAs for FMDV and BRV 3D) performed during bench validation by ARS. In addition, ARS supplied APHIS with a large batch of purified 3D protein to be used on their diagnostic assays for suspicious FMDV-infected biological specimen. We also mapped relevant antigenic epitopes in the FMDV 3D pol protein using recombinant proteins constructed with the FMDV and the closely related bovine rhinitis 3D proteins.
TASK 2B – Identification of Bovine Type 1 Interferons with Enhanced Biotherapeutic Potency to Control FMDV. The activities of these clones were higher than those of previously identified type I IFN. The highest IFN alpha and IFN beta clones are being tested in animals. In addition the most potent interferon alpha clone has been mutated to create four glycosylation sites in order to extend its half life in animals. Animal experiments will be conducted to test the protective effect against FMDV infection in animals.
Tasks 3a and 3b – Identification of Biotherapeutic Candidates to Control FMDV.
Viruses including FMDV induce host innate immune response through host pattern recognition receptors that detect non-host pathogen associated molecular patterns (PAMPs) and activate host transcription factors that upregulate IFN and other anti-viral genes. It is hypothesized that co-treatment of livestock with both Ad5-IFN and various PAMPs will result in a broader, enhanced and prolonged antiviral and protective response as compared to Ad5-IFN treatment alone. We demonstrated that polyICLC can induce a very rapid innate response in swine and protect animals challenged one day later with FMDV. This project was completed prior to Q4 FY11. Based on our results, in FY13 we hope to continue with our polylCLC PAMP studies in swine and cattle and initiate studies in susceptible animals with VEE replicons. We also demonstrated that cattle inoculated with Ad5-bovine IFN lambda when challenged one day later with FMDV are either protected or have significantly delayed and reduced disease.