2013 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.
Amendment 5: Objective 1 will be expanded to include obtaining the regulatory approval process necessary to complete duration of protective immunity against FMDV infection following vaccination with the newly licensed Ad5FMDV reombinant vaccine outside BSL-3 conditions.
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.
Determination of MHC class I and class II Restricted FMDV-Specific T Lymphocyte Responses Following AdFMD Vaccination in Cattle. Six BoLA class I encoded proteins identified as being dominantly expressed in the University of Vermont (UVM) research cattle were synthesized and expressed in bacteria. The proteins have now been tested to determine preferred peptide binding motifs of each BoLA class I protein. These data are now being incorporated into the predicting T cell epitopes developed by the University of Copenhagen, and once these new data are calculated, the algorithm will be applied to the structural protein amino acid sequence of FMDV, strain A24 Cruizero. 8 BoLA class II proteins that are also dominantly expressed in the UVM research cattle have been expressed as recombinant proteins by collaborators at the University of Copenhagen. These proteins were used to develop a new peptide binding assay for BoLA class II proteins. This technology will now be used to analyze the binding of peptides from a library corresponding to structural protein amino acid sequence of FMDV, strain A24 Cruizero.
Antigenic profiling of FMDV 3D polymerase and evaluation as a candidate for DIVA diagnostic test development. This project has been completed in 2013. Main achievements are as following: 1. Using a pannel of monoclonal antibodies raised against the FMDV 3D protein and western analysis, we have uncovered differential reactivities against the 3D proteins of Aphtoviruses. Three mAbs were shown to exclusively react with the FMDV 3D, while three others cross-reacted with FMDV, ERV and BRV 3D proteins making them less suitable for assay design. This information has been transferred to APHIS for their used in the optimization of ELISA protocols. 2. We have expressed the FMDV 3D protein and chimeras with the BRV-2 3D in E.coli using a His-tag system for the improvement (more specific test) of 3D-based DIVA diagnostic tests. These reagents have also aid in mapping antigenic epitopes in these proteins. 3. We have assessed the feasibility of recombinant 3D protein of aphthoviruses to be used in VIAA , indirect and competitive ELISA test. The FMDV 3D portein as well as plasmid and SOPs were transferred to APHIS for their use in VIAA test performed routinely. 4. Based on the results of bench validation performed in collaboration between ARS and APHIS, a competitive ELISA was selected as the method of choice to be used in discrimination between FMDV infected versus vaccinated animals. Assessment of the attributes and limitations of the assay were reported. Activities concluded in FY 2013.
Identification of bovine type I interferons with enhanced biotherapeutic potency to control FMDV. In addition to the best members IFNs alpha and beta genes, the best IFN w was identified. The IFN alpha gene was mutated to create four glycosylation sites in order to extend its half life in animals, but the mutations reduced the antiviral activity by several folds. In an animal trial, steers inoculated with infectious Ad5 recombinant virus particles containing the best IFN alpha gene had anti-FMDV activity in the sear. Sera collected at 24 hours post injection completely protected LFBK cells against FMDV infection in cell culture. The activity gradually decreased but remained detectable at day 5. In contrast, injection with the same doses of the recombinant virus containing the mutated IFNA alpha gene barely produced detectable antiviral activity. These results suggest that higher in vitro activity can be translated into higher in vivo activity. The activities for this task were completed in FY 2013.
Identification of biotherapeutic candidates to control FMDV.
Sub-objective 1. Determine PAMP candidates for subsequent in vivo pilot potency studies based on results from cell culture dose responses studies using Venezuelan equine encephalitis. In FY 2013 studies were initiated to evaluate the in vitro activity of PAMPS in mice. Sub-objective 2. Evaluation of one or more PAMP candidates with/without Ad5-IFN for further development and evaluation. We cloned one member of the porcine INF – gamma in a bacterial expression system and in the Ad5 vector and we constructed the Ad5 vector containing porcine IFN-gamma 3 and demonstration expression and biological activity in cell culture. Swine potency/efficacy studies were conducted using polyICLC and Ad-pIFN alpha and gamma. Studies using Ad5-poIFNgamma 3 indicate 100% protection. Sub-objective 3. Testing of a novel candidate with antiviral activity against FMDV. Studies were initiated to construct Ad5 vectors containing other cytokines. Porcine MIP3 alpha and porcine IP 10 have been cloned in the Ad5 vectors.