Strategic Planning for Swine Disease Research Meeting Report

Transcription

1 Strategic Planning for Swine Disease Research Meeting Report December 4, 2012 Washington, DC

2 Strategic Planning for Swine Disease Research The and the Department of Homeland Security (DHS) National Center for Foreign Animal and Zoonotic Disease Defense (FAZD Center) convened a strategic planning meeting to discuss swine disease research efforts supported by federal or funds on December 4, 2012 in Washington, DC. The overall goal of the meeting was to discuss current research efforts focused on three key diseases of agricultural consequence affecting swine, African Swine Fever (ASF), Classical Swine Fever (CSF), and Foot and Mouth Disease (FMD), and to identify gaps in research and funding. Meeting participants addressed research and gaps in the following areas: Diagnostics o Sampling procedures o Utility of pen-side detection o Reagent and technology development Vaccines o Virus-specific research and development o Routes and methods of delivery o Antigenic coverage and serotype protection o Onset and duration of immunity o Use of vaccines developed for/validated in cattle for use in swine o Domestic manufacture and formulation of vaccines o Vaccine stockpiles and deployments o Live attenuated and recombinant vaccines o Adjuvant development Biotherapeutics o terferon-enhanced protection against FMD o Transmission studies o Current models of experimental challenge versus natural infection in swine o Mechanisms of protection in swine Page 1 of 32

3 Executive Summary Meeting Objectives This report summarizes discussion points and research gaps identified during a strategic planning for swine disease research meeting convened by the and the FAZD Center on December 4, Participants included 20 personnel representing the FAZD Center, the, the DHS Science and Technology Directorate (S&T), the United States Department of Agriculture (USDA) Agriculture Research Service (ARS), USDA Animal and Plant Health spection Service (USDA APHIS), the Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), the American Association of Swine Veterinarians (AASV), and Texas Veterinary Medical Diagnostic Laboratory (TVMDL). A full list of attendees may be found in Appendix A. The objectives of this meeting were to: Review the current state of vaccine, diagnostics, and biotherapeutics research involving three key foreign animal diseases (FADs) of consequence to the swine industry: ASF, CSF, and FMD Identify gaps in funding and research involving these three key diseases of consequence to the swine industry Prevent duplication of efforts in order to best utilize available research funding and resources Identify other areas in need of attention from research, government and industry representatives Facilitate communication between academia, government, and industry involved in research of diseases of consequence to the swine industry The meeting was structured as a round-table discussion organized by disease. Each disease was discussed in context of current and past Page 2 of 32

4 vaccine projects, as well as diagnostic and screening tools and biotherapeutic projects. Following a summary of each project, gaps in research and funding for each disease were identified. A list of current and past research projects discussed may be found in Appendix B. Page 3 of 32

5 Highlights of the Strategic Planning for Swine Disease Research Meeting Meeting Overview Meeting discussions were structured by disease (ASF, CSF, FMD, or multiple FADs), with each focus area further broken down to review vaccine, diagnostics and screening tools, or biotherapeutics research by pathogen of interest. Representatives or principal investigators from each research entity provided an overview of current and past research by project, followed by a discussion of gaps in research and funding. Discussion Summaries Discussion Topic: African Swine Fever ASF Vaccine Projects Table 1. ASF Vaccine Projects Round table discussion of research involving ASF began with USDA and DHS summarizing current and past research projects involving vaccine development targeted against ASF (Table 1). This has been identified as a high priority research area for the swine industry, as there are currently no effective vaccines available to combat this pathogen. The recent spread of outbreaks through the Caucuses and near the Eastern European border makes the need for an efficacious vaccine even more critical for protecting US industry. Several unique and ongoing recent research efforts funded by are helping address this need. Year 2011 Project Title Development of Multi- component Vaccines for African Swine Fever * vestigator stitution Mwangi TAMU 2011 Identification of African Swine Fever Candidates by Reverse Vaccinology Bounpheng TMVDL Page 4 of 32

6 Development of a Proof of Concept Rationally Designed Live Attenuated African Swine Fever Virus Vaccines A Comprehensive Research Program in African Swine Fever Towards the Development of Novel Countermeasures Borca Borca/Arzt USDA ARS USDA ARS *Project includes monoclonal antibody development. Gaps The following gaps were identified in the areas of ASF vaccine research and funding: Significant, sustained funding commitment o It is estimated that $2-3 million per year over the course of several years is the minimum amount of necessary funding to seamlessly support research o Though has made significant investment in vaccine research and development, sustained funding is needed to take initially successful proof of concept studies for lead candidate(s) through to final product Immunogenicity/challenge studies o Use attenuated and recombinant strains o Focus on promising candidates o Understanding host protective humoral and cellular immune mechanism o Understanding viral/host interactions at the cellular level crease workforce development o Need to train the next generation of ASF experts o is currently the only place in the U.S. that trains people in ASF research and animal and lab capacity is limited o Dichotomy between post-doctoral starting salaries versus student loan burden o Lack of sustained training/development programs ASF Diagnostics/Immunology Projects Round table discussions continued with a review of ASF diagnostics projects (Table 2). There was an active discussion about sampling for Page 5 of 32

7 virus detection from swine oral fluids, which is currently done for routine endemic disease testing as part of normal swine production practices. Meat juice collected in slaughter plants is also used as a routine sample for other pathogens, and could potentially be validated for FADs as well, depending on whether detection of antibodies versus isolation of virus is desired. Table 2. ASF Diagnostics/Immunology Projects Year Project Title vestigator stitution 2011 Comparison of different sample source and sample pooling for the detection and surveillance of ASF Dixon Pirbright DHS Development of fluorescent recombinant antibodies to detect African swine fever virus in tissue samples and infected cells Development of Monoclonal Antibodies Specific to ASFV Proteins A Comprehensive Research Program in African Swine Fever Towards the Development of Novel Countermeasures Identification of genetic signatures for African swine fever virus serologic group specificity Escribano Wu/Sayed Borca/Arzt Rock stituto Nacl. vestigacion y Tecnologia Agraria y Alimentaria (INIA) USDA APHIS USDA ARS University of Illinois Gaps The following gaps were identified in ASF diagnostics research and funding: Page 6 of 32

8 Understand the application of research results from strains with differing virulence o Studies with moderate strains are needed for comparison to those with highly virulent strains (linear relationship?) Develop US-based reagents Develop and evaluate tools using moderate strains to improve anti-mortem detection o Matrices to best identify early infection Meat juice Oral fluids Tonsils o Comparison of sample selection methods and time course for window of detection Standard diagnostic scoring tools Active surveillance program in ASF endemic countries o Identify circulating strains Develop and use of a pen-side test (post-outbreak) o Established policy on pen-side testing o Outreach to producers Additional deep sequencing to support targeted research efforts (functional genomics) Discussion Topic: Classical Swine Fever CSF Vaccine Projects Discussion of CSF vaccine projects included discussion of research projects being performed by the FAZD Center and USDA ARS, including several recent efforts involving novel development technologies. The round table discussion emphasized the need for vaccination against highly virulent strains of CSF. Table 3 summarizes CSF vaccine projects. Page 7 of 32

9 Table 3. CSF Vaccine/Biotherapeutics Projects Year 2009 Project Title Identification of host factors interacting with classical swine fever virus proteins: development of novel anti- viral therapeutics. vestigator Borca stitution USDA ARS NAA National Pork 2009 FAD Countermeasure Development Roth/Riemser TABI, c Classical Swine Fever (CSF) Vaccine and Diagnostic Countermeasure Development Evaluation of Envelope Proteins for Rapid duction of Protective Immune Responses Against Classical Swine Fever Plant- based expression vectors for rapid, high- throughput development of animal vaccines Borca Borca Holtz USDA ARS USDA ARS NAA Caliber Biotherapeutics National Pork Gaps The following gaps were identified in CSF vaccine research and funding: formation on the EU live, attenuated marker vaccine candidate CP7_E2 alf (EU FP6 programme CSF project SSPE-CT ) o Onset of protection o Plans for pursuit of U.S. license Need for companion diagnostic assay to differentiate infected from vaccinated animals (DIVA) for the ARS CSF marker vaccine candidate Deployment strategy/policy for the conventional, live attenuated C- strain vaccine (non-diva) is not well defined Evaluate onset of protection and duration of immunity of subunit vaccines o Protective dose(s) Page 8 of 32

10 o Time-course of protection o Combined efficacy with biotherapeutics Potential to use early onset live attenuated vaccine at ground zero and a subunit vaccine in surveillance areas? Regulatory gaps Better understanding of host/virus relationships in vaccine production cells Identification of virulence factors/mechanism of protection for antivirals Evaluate use of amantadine to inhibit viral replication CSF Diagnostics Participants agreed that CSF endemic countries need diagnostic tools and procedures to detect the virus. Virulence varies between outbreaks and countries; however, establishing stronger international collaborations will help in both capacity building and validation of tests for domestic use. Table 4 summarizes CSF diagnostics projects. Table 4. CSF Diagnostics Projects Year Project Title vestigator stitution 2009 Foreign Animal Disease (FAD) Diagnostic Assay Development - Task 2: Diagnostic Technologies for Classical Swine Fever Mayr/Batonick USDA APHIS 2011 Development of classical swine fever virus diagnostic assays for porcine oral fluid samples Thanawongnuwech Chulalongkorn University Gaps The following gaps were identified in CSF diagnostics research and funding: Establish collaborations overseas for diagnostic field testing Develop US-produced reagents o Reagents needed for virus isolation and tissue staining Page 9 of 32

11 Commercial development and licensure of an improved CSF serology test Discussion Topic: Foot and Mouth Disease FMD Vaccine Projects Table 5. FMD Vaccine Projects Discussion of FMD vaccine projects began with an overview of the projects currently underway at Plum Island Animal Disease Center. On May 31 st, 2012, the first US-produced licensed vaccine for FMD was approved. The focus is primarily on bovine vaccination; however, DHS commercial partner has indicated they hope to work towards commercial development of the Adenovector-based FMD vaccine platform for swine. A summary of other past and present FMD vaccine research efforts conducted at and elsewhere may be found in Table 5. Year Project Title Development and Testing of a Subunit Vaccine for Foot- and- Mouth Disease Development of an Antiviral and Vaccine Approach to Control Foot- and- Mouth Disease Production of FMD Virus Pseudovirion Vaccine Candidates Using a Plant Transient System Research and Development of Molecular FMD Vaccines (historically cattle focused) vestigator Grubman Grubman Hiatt Brough and Butman stitution USDA ARS NAA USDA ARS NAA Kentucky BioProcessing GenVec National Pork National Pork Page 10 of 32

12 2011 Improved Challenge Systems for FMD Vaccine and Biotherapeutics Testing in Cattle and Pigs Arzt USDA ARS 2011 Development of VLP Vaccine as a Countermeasure for FMD Massare Novavax 2011 FMD DNA Vaccine Kern ovio Rational design of attenuated foot- and- mouth disease virus strains for development of improved disease countermeasures Testing and Evaluation of Human Adenovirus Replication Deficient Vectored FMD Vaccines (swine focus) Acquisition of Master Seed Virus (MSV) and Working Seed Virus (WSV) Lots for FMD Molecular Vaccine Countermeasures Bovine Adenovirus Vector- Based Vaccine for FMD Construction and Evaluation of Recombinant MVA- BN FMDV Candidates de los Santos David USDA ARS NAA Merial (CRADA/No Direct ) National Pork (CRADA/No Direct ) Miller Benchmark Biolabs Mittal Purdue University Weinberger Bavarian Nordic Page 11 of 32

13 Exploiting the potential of leader proteinase coding sequence of foot- and- mouth disease virus to derive attenuated strains suitable for pathogenesis studies and development of improved countermeasures. Replicon- based Rapid Response Vaccines for FMD de los Santos USDA ARS NAA National Pork Kamrud Harrisvaccines Gaps The following gaps were identified in FMD vaccine research and funding: Test additional vaccine platforms in pigs that have shown promise or success in cattle o Host cross-species protection studies Conduct immunogenicity studies in pigs o Duration of immunity (key for eradication efforts) o Broad antigenic coverage o Cross-serotype protection (broadening immunity) o Protective dose(s) o Route of inoculation (e.g., subcutaneous or intradermal) o Method of delivery o Adjuvants o Antigen stability in different environments Define mechanisms of host protection o Utilize newly developed veterinary immunity research reagents o Utilize vaccinated animals from endemic countries as a resource to investigate vaccine efficacy and immunity Develop policies supporting vaccination and/or stamping out FMD globally via eradication efforts o Policies/schedules for available vaccines during different phases of an outbreak Stockpile and/or procure vaccine for emergencies Page 12 of 32

14 FMD Diagnostics o Number of vaccine doses o Number of serotypes/topotypes covered The discussion of FMD diagnostics projects began with a discussion of current research efforts funded by by performers outside the Federal government, including TVMDL, TABI, and national laboratories. This was followed by a review of past and ongoing efforts being performed at Plum Island. has a new monoclonal antibody development initiative that they hope will lead to U.S. production of reagents so they do not have to be purchased from abroad. This project feeds into R&D support for developing vaccines. FMD diagnostic projects at TVMDL,, and elsewhere are summarized in Table 6. Table 6. FMD Diagnostics Projects Year Project Title Diagnostic Evaluation of Multiplexed Reverse Transcription- PCR Microsphere Array Assay for Detection of Foot- and- Mouth and Look- Alike Disease Viruses Foot- and- Mouth Disease Multiplexed Nucleic Acid Assay Enhancements and Validation FAD Countermeasure Development vestigator Hindson Beckham (collaboration with LLNL) Roth (collaboration with Prionics) stitution University of California Lawrence Livermore National Laboratory USDA APHIS Terminated TABI, c. Page 13 of 32

15 2011 Development of 3ABC ELISA for Detection of FMD Antibody in fected Animals Regardless of Vaccination (Prototype kit and bench validation) Rieder/Sayed/ Bounpheng USDA ARS /FADDL/TVMDL Deployment of FMD Serology to the National Animal Health Laboratory Network (swine and cattle together) vestigating potential existence of chronic, persistent foot- and- mouth disease virus infection in domestic pigs; implications for disease control strategies Negative Cohort Study for Validation of the SVANODIP FMDV- Ag test Development and Characterization of Monoclonal Antibodies to Foot- and- Mouth Disease Virus Structural Proteins Establishment of a FMD Antisera Bank Molecular Epidemiology and Biosurveillance of FMD Endemic Regions of Middle East, Southeast Asia and Africa Mctosh (collaboration with NAHLN/TVMDL) Arzt Tomlinson/ Mctosh Bounpheng USDA APHIS USDA ARS NAA National Pork USDA/FADDL/ NAHLN TVMDL/ LLNL/ BIOO Sci Wu USDA APHIS Rodriguez USDA ARS Page 14 of 32

16 2012 Pen- side detection of Foot- and- Mouth Disease virus by a portable microfluidics PCR system Bounpheng/ Mctosh USDA/FADDL/ TVMDL Gaps The following gaps in FMD diagnostics research and funding were identified: Validate 3ABC ELISA in swine vaccinated with Ad5 vectored vaccine Develop US-based reagents o Most reagents developed with bovine model o Need sera to test diagnostics and detection methods o Issues with continuity of supply/cost of international reagents Validate different sample types across multiple serotypes Explore other modalities for FMD detection (aerosol, thermal imaging) Explore value of penside testing in outbreak situations with industry FMD Biotherapeutics Discussion of FMD biotherapeutics began with a review of research that had originally been done in cattle and is now being translated to the swine model. USDA presented research highlighting the use of interferon lambda as an adjuvant to FMD vaccines to generate a protective B-cell response. addition, USDA ARS has promising research involving adenovirus vectors that can offer protection as an adjuvant to the current Ad5 vaccine within one week. Challenges involved in modeling natural infection were also discussed. Table 7 summarizes past and present FMD biotherapeutics studies conducted by the USDA and others. Page 15 of 32

17 Table 7. FMD Biotherapeutics Studies Year Project Title vestigator stitution 2009 Foreign Animal Disease (FAD) Countermeasure Development - Identification of Biotherapeutic Candidates to Control FMDV Grubman USDA ARS 2011 Countermeasures for Foot- and- Mouth Disease Utilizing Diagnostics, Biotherapeutics, a Novel Vaccine Platform, and Improved Challenge Systems - Identification of Biotherapeutic Candidates to Control FMDV de los Santos/ Grubman USDA ARS Gaps The following gaps in research and funding were identified with regards to FMD biotherapeutics in swine: Establish biotherapeutic adjuvant utility with other vaccines Evaluate site of replication/timecourse of infection in swine o Oral challenge more closely models natural infection than intradermal or foot o Transmission studies need to be done in swine o Identify tissues to target for biotherapeutics Need to define persistence in swine versus cattle via carrier studies Discussion Topic: Multiple Foreign Animal Diseases (FADs) Multiple FAD Diagnostic Projects Several multiplex and cross-pathogen projects on FADs have been conducted at Plum Island and elsewhere. All of the projects are diagnostic in nature. The diagnostic projects conducted by, TVMDL, and others are summarized in Table 8. Page 16 of 32

18 Table 8. Multiple FAD Diagnostic Projects Year Project Title vestigator stitution 2008 Enhancements of High- Throughput Diagnosis for FADs Mctosh USDA APHIS & 2008 Foreign Animal Disease Diagnostic Assay Development: Vesicular Disease Reagent Production Development of a multiplex RT- qpcr assay for surveillance of FADs during routine testing of oral fluid samples The Matrix- Chaperone: Ambient Temperature Biospecimen Collection, Transport & Banking For Simplified Animal Disease Screening (swine and cattle). Assessment of Two Methods for Nucleic Acid Stability and Viral activation of FAD Viruses Collected from Oral Fluid Rope Samples Development of Pan- viral DNA Microarrays for the Detection of Emerging and Foreign Animal Diseases Jia Bounpheng/Mctosh USDA APHIS TMVDL/ FADDL Hogan tegenx Mayr Mctosh USDA APHIS USDA APHIS Gaps The following gaps were identified involving multiple FAD diagnostic research projects and funding: Develop universal protocols for sample collection and preparation Establish working relationships and collaborations with laboratories in countries where each disease is endemic Page 17 of 32

19 Address questions surrounding transport of samples from outbreak zones to laboratories for testing Research usable/optimal sample matrices for multiplex assays Develop capacity to work on FADs in other laboratories o Use ABSL-2 containment where possible to make efficient use of high containment space Emphasize multi-institution collaborations in calls for proposals Emphasize partnerships that facilitate transfer of technology earlier in the process for projects that are leading to usable diagnostic tools, vaccines or biotherapeutics Outcomes and Next Steps The meeting reported here was an unusual opportunity for scientists from research entities involved in vaccine, diagnostics, and biotherapeutics research for three diseases of consequence in swine, African Swine Fever, Classical Swine Fever, and Foot-and-Mouth Disease, to entertain open discussion with funding agencies and industry representatives to identify gaps in research and funding. Sharing information on technologies currently in development will help prevent duplication of efforts and ensure responsible discharge of research funding. Meeting participants agreed that the meeting was fruitful in that it allowed participants to openly discuss research projects conducted at multiple institutions and funded by multiple agencies. The outcomes of the discussion will be used by the swine industry to devise a national strategy for future ASF, CSF, and FMD research and funding. Page 18 of 32

20 Appendix A: Meeting Participants Name Organization Mr. Bobby Acord Council Dr. James Anthony Program Manager, Chemical and Biological Division, DHS S&T Dr. Tammy Beckham Director, FAZD Center and TVMDL Dr. Lisa Becton Director, Swine Health formation and Research, National Pork Dr. Melissa Berquist Associate Director, FAZD Center* Dr. Manuel Borca Head, Swine Diseases Research, Dr. Mangkey Bounpheng Molecular Diagnostics Section Head, TVMDL Dr. David Brake Scientific Consultant, Plum Island Animal Disease Center, DHS Dr. Matthew Coats Program Manager, Office of University Programs, Dr. Michelle Colby Agriculture Defense Branch Chief, Chemical and Biological Division, Dr. Bruce Harper Director of Science, Plum Island Animal Disease Center, Dr. Mike King Manager, Science Communications, Dr. Greg Mayr Microbiologist, Diagnostic Services Section, USDA APHIS FADDL Dr. Juergen Richt Director, CEEZAD, Kansas State University Dr. Luis Rodriguez Research Leader, Plum Island Animal Disease Center, USDA ARS Dr. Abu Sayed Microbiologist, USDA APHIS FADDL Dr. Harry Snelson Director of Communications, AASV Dr. Paul Sundberg Vice President, Science & Technology, Dr. Sabrina Swenson Virologist, USDA APHIS* Dr. Patrick Webb Director, Swine Health Programs, * Participated via teleconference/webinar. Page 19 of 32

21 Appendix B: Full List of Projects Sorted by Disease All meeting participants received a full list of African Swine Fever, Classical Swine Fever, and Foot-and-Mouth Disease projects, as well as multiple foreign animal disease (FAD) projects, compiled by the FAZD Center, the,, and USDA, sorted by disease. The collated list follows, beginning on the next page. Page 20 of 32

22 vestigator stitution Comments ASF Diagnostics 2011 Comparison of different sample source and sample pooling for the detection and surveillance of ASF Dixon Pirbright DHS This project will compare and validate pooled sample matricies, including oral fluids, nasal swabs, blood, and serum, for the molecular detection of ASFV. Project will include a methods comparison between USDA NAHLN PCR, Pirbright PCR, and serological assay for detection. ASF Diagnostics 2011 Development of fluorescent recombinant antibodies to detect African swine fever virus in tissue samples and infected cells Escribano stituto Nacl. vestigacion y Tecnologia Agraria y Alimentaria (INIA) The present project pretends to develop new reagents to cover a gap in the ASFV diagnosis. ASF Diagnostics 2012 A Comprehensive Research Program in African Swine Fever Towards the Development of Novel Countermeasures Borca/Arzt USDA ARS Vx/Dx end Objective 1: Development of challenge model for ASF to characterize pathogenesis and evaluate novel countermeasure products. Objective 2: Determining immune mechanisms of protection induced by attenuated strains. Objective 3: Functional genomics. ASF Diagnostics 2012 Development of Monoclonal Antibodies Specific to ASFV Proteins Wu/Sayed USDA APHIS Vx/Dx end The end product of this project is a battery of monoclonal antibodies against ASF proteins p30, p72, p54, and 8-DR. These will be produced and made available to ARS and DHS for their research needs. They can also be potentially used in the development or improvement of diagnostic assays in FADDL. It is expected that the knowledge acquired from ARS and DHS characterization will assist in this process. ASF Diagnostics 2012 Identification of genetic signatures for African swine fever virus serologic group specificity Rock University of Illinois Identify genetic signature (s) for ASFV serologic group specificity and determine serologic group specificity for currently untyped ASFV isolates in VNIIVViM strain collection ASF Vaccine 2011 Development of Multi-component Vaccines for African Swine Fever Mwangi Texas A&M Research Foundation end The objective of this proposal is to clone and express 5 candidate ASFv genes in eukaryotic (HEK) and adenovirus expression platforms. The eukaryotic platform will be used to produce recombinant proteins to serve as immunogens for MAb and rabbit PcAb reagent production (2 ASFV targets). The adenovirus platform will be used to produce (3) vaccine candidates for swine proof-ofconcept safety and immunogencity study at TAMU.

23 ASF Vaccine 2011 ASF Vaccine 2011 Identification of African Swine Fever Candidates by Reverse Vaccinology Development of a Proof of Concept Rationally Designed Live Attenuated African Swine Fever Virus Vaccines vestigator stitution Comments Bounpheng TMVDL Vx/Dx Borca USDA ARS Vx/Dx end end The objective of this proposal is to evaluate a novel approach, reverse vaccionology (RV), for the identification and development of ASFV vaccine candidates. RV will be used to identify novel in silico candidates and rank the DHS BAA candidates (i.e., p30, p54, p72, CD2v). Two protein expression and delivery platforms will be evaluated. The in silico and top ranked DHS candidates will be expressed in mammalian HEK 293 cells and fused to an immunogenic inducing tag. addition these candidates will be expressed in the highly safe poxvirus modified vaccinia Ankara (MVA) vaccine vector to enhance cellular immunity. Recombinant vaccines will be tested for immunogenicity and safety in pigs. The PIs anticipate that these candidates (alone or synergistically combined) will be highly immunogenic and induce strong humoral and cellular responses. Protein vaccines are highly safe and can induce strong humoral responses. addition, MVA-based vaccines are an attractive vaccine delivery technology because when administered to the host they can induce in vivo expression of one or more specific antigens. These newly expressed antigens are processed and presented by professional antigen-presenting cells, resulting in the induction of antibody responses with high avidity, as well as major histocompatibility complex class I-restricted cytotoxic T-lymphocyte (CTL) responses. This pattern of responses is similar to that induced by live attenuated vaccines. addition, the induction of B and T cell immune responses eliminates the need for adjuvants. These features are major advantages of viral vectors when compared to other vaccine delivering platforms. Objective: 1. Develop recombinant African Swine Fever Virus (ASFV) strains by deletion of one or more viral genes already described as responsible for inducting attenuation of highly virulent ASF strains. 2. Test attenuated ASFV strains for their ability to induce protection against challenge with homologous, well-characterized, virulent ASFV isolates. 3. Evaluate patterns of heterologous protection among genetically heterogeneous ASFV strains. Approach: Develop recombinant African Swine Fever Virus (ASFV) strains by deletion of one or more viral genes already described as responsible for inducting attenuation of highly virulent ASF strains. Critical ASFV genes that are responsible for inducing attenuation of highly virulent ASF strains will be deleted individually or as a group. This deletion should confer virus replication but not disease production. 2. Test attenuated ASFV strains for their ability to induce protection against challenge with homologous, well-characterized, virulent ASFV isolates. This will be done through: Testing strains for in vivo attenuation, testing for efficacy against homologous challenge, determination of minim protective dose response, evaluation of protection profile for at least one attenuated ASFV vaccine candidate, and the evaluation of lead vaccine candidate to induce sterile immunity. 3. Evaluate and confirm cross-protection conferred by lead vaccine candidate (obj. 2) by using genetically diverse ASFV strains. Page 22 of 32

24 vestigator stitution Comments CSF Diagnostics 2009 Diagnostic Technologies for Classical Swine Fever Mayr/Batonick USDA APHIS Vx/Dx end The current CSF antibody ELISA has limited specificity in excluding antibody to ruminant pestiviruses. Identification and characterization of the E2 glycoprotein epitopes that discriminate between swine and ruminant pestivirus are essential in the design of a next generation ELISA. For epitope mapping, a panel of monoclonal antibodies was obtained in collaboration with the Central stitute for Animal Diseases Control, Lelystad, the Netherlands. The CSF target epitopes were defined, and the ELISA was designed and is being optimized and validated. Such an ELISA will have vital application in the current national surveillance program and during recovery of an outbreak. CSF Diagnostics 2011 Development of classical swine fever virus diagnostic assays for porcine oral fluid samples Thanawongnuwe ch Chulalongkorn University The primary objective of this project is to optimize and validate technology capable of rapidly identifying premises infected with classical swine fever virus (CSFV) following its introduction into North America or other CSFV-free areas using oral fluid samples. Achievement of this objective will also provide technology for improved surveillance in CSFV endemic areas, thereby enhancing elimination and control efforts. The specific objectives are focused on demonstrating the feasibility of detecting CSFV in oral fluids by modified PCR-based methods. CSF Vaccine 2009 Identification of host factors interacting with classical swine fever virus proteins: development of novel anti-viral therapeutics. Borca USDA ARS NAA CSF Vaccine 2009 FAD Countermeasure Development Roth /Riemser TABI, c. Vx/Dx Results obtained enable the identification of several host proteins interacting with CSFV structural protein Core. Core protein is the major contributor to the virus capsid. Several of these interactions have been studied in detail and the regions of the CSFV Core protein interacting with the host proteins were identified. Mutant CSFV viruses having altered these regions have been demonstrated that have severely altered their ability to produce disease in swine. Therefore, the manipulation of the identified host-virus interactions allowed the development of attenuated strains of virus which may constitute a tool for the further development of live attenuated vaccine against classical swine fever. Additionally, this knowledge may open the possibility of designing bio therapeutic compounds that could alter those critical interactions that may limit the spread of the disease. CVB Approval of Permittee License for Importation and Distribution of CSF Modified Live Vaccine, Chinese strain (C strain). Riemser Arzneitmittel AG (GDR) is currently producing a EU licensed vaccine.. The vaccine (C strain) was recently excluded from the USDA APHIS 2013 end date Select Agent List. Plans are in to import master seed virus (modification (MSV) and master seed cell bank (MSB) for safety testing in swine to 2014 end by FADDL,. If satisfactory, MSV and MSB will sent to USDA date planned) CVB for additional testing. Submission of various testing reports to USDA CVB are in. GDR manufacturing facility has been inspected by USDA CVB I&C. CSF Vaccine 2010 Classical Swine Fever (CSF) Vaccine and Diagnostic Countermeasure Development Borca ARS USDA Vx/Dx Objective 1: Develop a genetically stable version of FlagT4 virus (FlagT4c). Objective 2: Viral-vectored vaccine (backup candidate):. POC safety and efficacy studies using baculovirus-vectored 2013 end date experimental vaccine. Objective 3: Obtain select agent exclusion for FlagT4c vaccine candidate and transition to AH partner (CRADA in place). Page 23 of 32

25 vestigator stitution Comments CSF Vaccine 2011 Evaluation of Envelope Proteins for Rapid duction of Protective Immune Responses Against Classical Swine Fever Borca USDA ARS NAA progess The main objective of the research project is to evaluate native and modified forms of CSF envelope proteins for their capacity to induce rapid protective immune response against the CSFV. CSFV envelope proteins are expressed as fusion proteins along with different immunological adjuvants. The improved efficacy of the novel constructs will evaluated compared with the native version of the proteins in terms of induced immune response and protection. CSF Vaccine 2012 Plant-based expression vectors for rapid, high-throughput development of animal vaccines Holtz Caliber Biotherapeutics OUP The main objective of this research project is to demonstrate proof of principle for rapid expression, testing, and commercial-scale production capacity using a novel plant-based approach. The CSF E2 protein is targeted for a potential recombinant subunit vaccine for use in later stages of a disease outbreak as a possible compliment to a live attenuated vaccination strategy. Page 24 of 32

26 vestigator stitution Comments FMD Biotherapeutics 2009 Foreign Animal Disease (FAD) Countermeasure Development - Identification of Biotherapeutic Candidates to Control FMDV Grubman USDA ARS Treatment of swine with poly IC alone or in combination with Ad5-pIFNα confers early protection against FMD (Dias, C. C. A, Moraes, M. P., Diaz- San Segundo, F., de los Santos, T., and Grubman, M. J. Porcine type I interferon rapidly protects swine against challenge with multiple serotypes of foot-and-mouth disease virus. J. t. Cyt. Res. 31: , 2011). FMD Biotherapeutics 2011 Countermeasures for Foot-and-Mouth Disease Utilizing Diagnostics, Biotherapeutics, a Novel Vaccine Platform, and Improved Challenge Systems - Identification of Biotherapeutic Candidates to Control FMDV de los Santos/ Grubman USDA ARS end Overarching Goal: Identification of one or more biotherapeutic candidates alone or with Ad-IFNs for transition to Targeted Advanced Development program for further evaluation and development. FMD Diagnostics 2008 Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and- Mouth and Look-Alike Disease Viruses Hindson University of California Lawrence Livermore National Laboratory A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-pcr (mrt- PCR) amplification coupled with a microsphere hybridization array and flowcytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rrt-pcr) assays for FMDV. The diagnostic accuracy of the mrt-pcr assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mrt-pcr assay results were compared to those of two singleplex rrt-pcr assays, using virus isolation with antigen enzymelinked immunosorbent assays as the reference method. The diagnostic sensitivity of the mrt-pcr assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rrt-pcr assays used in combination. addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. terestingly, the mrt-pcr detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mrt-pcr assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rrt-pcr assays. FMD Diagnostics 2010 FAD Countermeasure Development Roth (collaboration with Prionics) TABI, c end date CVB Approval of License for Importation and Distribution of Prionics PrioCHECK FMD-NS 3ABC ELISA Page 25 of 32

27 FMD Diagnostics 2011 Evaluation and Validation of 3ABC ELISA for Detection of FMD Antibody in fected Animals Regardless of Vaccination vestigator Sayed (collaborative with Clavijo/Bounpheng and Rieder projects above and below) stitution USDA APHIS end Comments Project is directly linked to Diagnostic 2011 Bounpheng and Rieder projects. The 3ABC ELISA kit (PrioCHECK FMD-NS (3ABC ELISA) in used in routine diagnosis. This test is costly, is imported from Europe, and requires an overnight incubation to perform. Recent evaluations of this commercial test done at FADDL on a conclusive panel of sera showed that the sensitivity was 0.93 for pigs, 0.9 for cattle and sheep, and specificity was 0.94 for pigs, 0.88 for cattle and 0.73 for sheep. order to develop an improved assay, USDA ARS at produced the recombinant 3ABC protein. Test optimization was conducted at by ARS using the Mab from FAZD (Clavijo). The optimized assay along with standard reagents (3ABC clone and hybridomas), a detailed protocol describing optimization conditions and preparation of reagents, and evaluation data specifying the analytical performance of the assays (specificity and sensitivity), were transferred to FADDL for further analytical evaluation and full validation for the test s intended use. >100 positive and 500 negative samples were evaluated in version 1.0 and showed improved performance over PrioCHECK FMD-NS (3ABC ELISA). Field evaluation is being planned for version 2.0. The ideal test will be a differential ELISA to rapidly demonstrate freedom from infection with greater performance characteristics, less cost, and the capacity to be manufactured in the US. FMD Diagnostics 2011 Development of 3ABC ELISA for Detection of FMD Antibody in fected Animals Regardless of Vaccination Rieder (collaborative with Clavijo/Bounpheng and Sayed projects above) USDA ARS Project is directly linked to Diagnostic 2011 Sayed and Bounpheng projects. Developed a celisa that uses a FMDV 3ABC recombinant protein and a monoclonal antibody (FAZD-Clavijo) specific for an immunodominant B-cell epitope on the 3B protein that will be compatible with either next generation FMD molecular vaccines (e.g., AdFMD platform) or current, high quality inactivated vaccines in which NSPs have been removed. After bench evaluation and assay optimization, the assay and associated reagents were transitioned to APHIS FADDL and TVMDL for assay validation (positive and negative samples). FMD Diagnostics 2011 Deployment of FMD Serology to the National Animal Health Laboratory Network (swine and cattle together) Mctosh (collaboration with NAHLN/TVMDL) USDA APHIS end This effort aims to deploy FMD DIVA Serology (Prionics PrioCHECK FMD- NS (3ABC ELISA) to a subset of the NAHLN within the first year of funding with subsequent years to expand the capabilities to all appropriate NAHLN State Veterinary Diagnostic Laboratories. By deploying the 3ABC ELISA to the NAHLN, this effort will augment the national capacity to not only detect antibodies to FMDV during an outbreak but to discriminate between vaccinated and unvaccinated animals (DIVA) during recovery. FMD Diagnostics 2011 Negative Cohort Study for Validation of the SVANODIP FMDV-Ag test Tomlinson/Mctosh/ Beckham USDA/FADDL/ NAHLN/FAZD The objective is to perform a negative cohort study for the SVANODIP FMDV-Ag FMD assay, utilizing samples from the US National herds. Page 26 of 32

28 vestigator stitution Comments FMD Diagnostics 2011 vestigating potential existence of chronic, persistent foot-and-mouth disease virus infection in domestic pigs; implications for disease control strategies Arzt USDA ARS NAA (a) Determine optimal route of direct inoculation of donor pigs for contact experiments; compare efficacy of intra-oral (IO) and heel bulb intradermal (HBI) FMDV inoculation as administration route using FMDV, serotype O. (b) Characterize FMDV acute pathogenesis parameters (shedding, viremia, tissue-specific distribution) of infection in contact transmission studies using FMDV, serotype O.(c) Characterize FMDV post-acute (i.e. suspect persistent) pathogenesis parameters (shedding, tissue-specific distribution) of infection in contact transmission studies using FMDV, serotype O.(d) Characterize FMDV post-acute (i.e. suspect persistent) pathogenesis parameters (shedding, tissue-specific distribution) of infection in contact transmission studies using FMDV, serotypes A, and Asia1. FMD Diagnostics 2012 Development of a Prototype and Bench Validation of a 3BFMDV Competitive ELISA Kit Clavijo/ Bounpheng (collaborative with Sayed and Rieder projects below) TMVDL/Pirbright DHS 2013 end The goal of this effort is to develop a competitive ELISA against the FMDV 3B non-structural protein for use in a diagnostic kit for early detection of FMDV. Project is directly linked to Diagnostic 2011 Sayed and Rieder projects. FMD Diagnostics 2012 Development of 3D ELISA for Detection of FMDV Antibody in fected Swine Immunized with Ad5-FMD(-3D) Vaccine Sayed USDA APHIS end Develop an optimized and validated ELISA to discriminate Ad5-FMD vaccinated from FMD infected swine with objectives: (A) Identify the dominant antigenic region(s) of 3D protein in swine; (B) Develop Mab(s) against the identified antigenic fragment/peptide of 3D; (C) Develop an ELISA for detection of FMDV antibodies in infected swine; (D) Evaluate the suitability of the 3D ELISA as a DIVA test for Ad5-FMDV vaccinated/challenged swine. It was discovered that different epitopes are recognized in swine and in cattle. FMD Diagnostics 2012 Pen-side detection of Foot-and-Mouth Disease virus by a portable microfluidics PCR system Bounpheng/ Mctosh USDA/FADDL/ TVMDL The overall objective of this effort is to perform a comprehensive feasibility study of a novel pen-side PCR system (PanNAT ) for sensitive detection of FMD virus in the field from oral swabs of infected cattle. This proof of principle study will be performed using samples collected from experimentally inoculated cattle and pigs. FMD Diagnostics 2012 Development and Characterization of Monoclonal Antibodies to Foot-and-Mouth Disease Virus Structural Proteins Bounpheng TVMDL/ LLNL/ BIOO Sci end The objective of this project is to develop a set of Mabs to FMDV using two protein expression systems and to evaluate a bioinformatics approach to develop FMDV cross reactive Mabs to facilitate research and development of FMDV vaccine and diagnostics. Cross reactive Mabs will be developed using in silico ranked linear epitopes from VP1, VP2, and VP3 FMDV sequences. Diverse specificity Mabs will be developed using recombinant proteins from the structural proteins VP1, VP2 and VP3 of selected serotypes of FMDV; these recombinant proteins will be produced in E. coli and baculovirus expression systems. The specificity of these Mabs will be evaluated and the epitope identified. These Mabs will have the following specificities: 1. Linear epitopes of the structural proteins VP1, VP2 and VP3. 2. Conformational epitopes from baculovirus expressed structural proteins VP1 of FMDV serotypes O, A and Asia1. 3. Cross reactive Mabs between all serotypes of FMDV. 4. Serotype (s) FMDV specific. Mabs to linear and conformational epitopes of FMDV serotype A and O will be produced. FMD Diagnostics 2008 Foot-and-Mouth Disease Multiplexed Nucleic Acid Assay Enhancements and Validation Beckham (collaboration with LLNL) USDA APHIS Terminated Bench validation was completed; no further work was authorized. Page 27 of 32

29 vestigator stitution FMD Diagnostics 2012 Establishment of a FMD Antisera Bank Wu USDA APHIS FMD Diagnostics 2012 Molecular Epidemiology and Biosurveillance of FMD Endemic Regions of Middle East, Southeast Asia and Africa Rodriguez USDA ARS end Comments A steady supply of antisera is needed for at least two of DSS s routine diagnostic assays, ELISA s, and virus neutralization tests. addition to the diagnostic applications, the availability of a wide range of well characterized antisera has been identified by Reagent and Vaccine Services Section (RVSS) as a gap in the development of new diagnostic assays and the improvement of existing ones. This was recently experienced by the RVSS in the development of the FMD liquid phase blocking ELISA (LPBE), and it is expected to be even more critical during the validation phase. The validation of serologic assays, a mission critical element for Proficiency Validation Service Section (PVSS), requires significant amounts and varieties of FMD antisera. Goals: 1. Gain a better understanding of the epidemiology of FMDV strains circulating in selected endemic regions of Central and Southeast Asia and Africa. 2. Determine the phylogenetic relationships of FMDV strains in endemic regions. 3. Determine the antigenic relationship (vaccine matching) of FMDV strains circulating in selected regions with currently available vaccine strains. FMD Vacccine 2012 Development of novel foot and mouth disease virus with multiple mutations for evaluation as live attenuated DIVA vaccine candidates de los Santos, Rieder APHIS USDA Progress, 1 yr timeline The objective of this project is to improve current strategies to generate FMDV containing specific attenuating factors and antigenic markers and evaluate their potential as vaccine candidates. This proposal builds upon previous and ongoing research where mutations are being introduced into the leader protein of FMDV in order to attenuate the virus and derive virus strains that could be used as vaccine candidates FMD Vaccine 2000 Development and Testing of a Subunit Vaccine for Foot-and-Mouth Disease Grubman USDA ARS NAA We have tested the potency and efficacy of replication-defective recombinant human adenoviruses containing the capsid and 3C proteinase coding regions of foot-and-mouth disease virus (FMDV) as vaccine candidates in swine. This vaccine, which lacks the coding region of several FMDV nonstructural proteins, has a number of advantages over the current whole virus inactivated vaccine including the ability to readily distinguish vaccinated from infected animals using current technology. oculation of swine with this vaccine resulted in either complete protection from disease after virulent homologous virus challenge or significant reduction in clinical signs as compared to co-housed un-inoculated control animals. The optimal vaccine regimen tested was a low dose initial inoculation followed by a high dose boost. All animals inoculated by this regimen were completely free of disease after challenge. creased efficacy of the two-dose regimen was associated with heightened levels of FMDV specific IgG1 and IgG2 antibodies. Expression and processing of the FMDV capsid precursor protein, requiring a biologically active 3C proteinase, was essential for induction of FMDV-specific neutralizing antibodies and protection from challenge. This vaccine is safe since there is no evidence of sero-conversion of co-housed unvaccinated control animals. These experimental results suggest that the Ad5-FMDV virus vector is a promising vaccine candidate against FMD and additional efforts to improve its potency and efficacy are underway. FMD Vaccine 2004 Development of an Antiviral and Vaccine Approach to Control Foot-and-Mouth Disease Grubman USDA ARS NAA this study we have demonstrated that delivery of the combination of our FMD subunit vaccine and interferon alpha can enhance the long-term protection afforded swine with the vaccine alone. Furthermore, we are developing additional antiviral reagents, i.e.., interferon beta, that may allow us to supplement our current approach as well as develop a more comprehensive understanding of the interactions between FMDV and its host. Page 28 of 32

30 FMD Vaccine 2010 Research and Development of Molecular FMD Vaccines (historically cattle focused) vestigator stitution Brough and Butman GenVec end Comments Research milestones are associated with the construction, in vitro and in vivo (cattle focus). Several AdFMD vaccine candidates have been made and tested in cattle. Development milestones are associated with manufacturing process improvements and stability and potency assessment of AdFMD vaccine candidates producted under improved manufacturing processes. Development milestones are aligned with DHS S&T Vx and Dx CRADA with Merial. FMD Vaccine 2011 Development of VLP Vaccine as a Countermeasure for FMD Massare Novavax Goals is construction and testing (cattle focus) of virus-like particle. recombinant, subunit FMDV vaccine produced in insect cells. POC cattle 2014 end study planned for Spring FMD Vaccine 2011 FMD DNA Vaccine Kern ovio (CRADA/No Direct ). End date TBD. Goal is construction and testing (swine focus) of FMD DNA vaccines based on ovio's SynCon technology and Cellectra delivery (electroporation) device. POC swine study planned for Spring FMD Vaccine 2011 Testing and Evaluation of Human Adenovirus Replication Deficient Vectored FMD Vaccines (swine focus) David Merial (CRADA/No Direct ). End date TBD. Goal is testing and evaluation of (3) AdFMD vaccine candidates in swine, production of master seed vacine viruses, and improvement of vaccine manufacturing process. One POC efficacy study in swine has been completed and target efficacy level was not achieved. Efforts are ongoing to improve vaccine potency. FMD Vaccine 2012 Acquisition of Master Seed Virus (MSV) and Working Seed Virus (WSV) Lots for FMD Molecular Vaccine Countermeasures Miller Benchmark Biolabs end date (if all option years awarded) Goal is construction of MVA-BN-FMDV vaccine candidate for POC testing (cattle focus). Major tasks include i) evaluation and identification of optimal vector parameters, ii) construction and evaluaton of two recombinatn MVA-BN-FMDV constructs expressing 3C in trans, and iii) production and formulation of optimal MVA-BN FMDV candidate (research grade). FMD Vaccine 2012 Bovine Adenovirus Vector-Based Vaccine for FMD Mittal Purdue University Goal is construction and testing (cattle focus) of replication competent. and replication deficient bovine adenovirus vectored FMD vaccine 2014 end candidates. FMD Vaccine 2012 Construction and Evaluation of Recombinant MVA-BN FMDV Candidates Weinberger Bavarian Nordic end Goal is construction of MVA-BN-FMDV vaccine candidate for POC testing (cattle focus). Major tasks include i) evaluation and identification of optimal vector parameters, ii) construction and evaluaton of two recombinatn MVA-BN-FMDV constructs expressing 3C in trans, and iii) production and formulation of optimal MVA-BN FMDV candidate (research grade). FMD Vaccine 2012 Replicon-based Rapid Response Vaccines for FMD Kamrud Harrisvaccines. Goals is rapid, large-scale (50-100K doses) of VEE replicon particle FMDV 2014 end vaccine candidate (cattle focus). FMD Vaccine 2010 Production of FMD Virus Pseudovirion Vaccine Candidates Using a Plant Transient System Hiatt Kentucky BioProcessing Vx/Dx. The key goal of the project is to produce FMDV pseudovirion structures in plants and develop scaleable manners for purification and vaccine production. To date, efforts to produce pseudovirion structures in tobacco plants have failed. Modified SOW is in to use an alernative strategy to improve capsid production and solubility. FMD Vaccine 2011 Countermeasures for Foot-and-Mouth Disease Utilizing Diagnostics, Biotherapeutics, a Novel Vaccine Platform, and Improved Challenge Systems - Improved Challenge Systems for FMD Vaccine and Biotherapeutics Testing in Cattle and Pigs Arzt USDA ARS end The primary goal of this proposed research is the optimization of new, simulated natural systems for challenging cattle and pigs with foot-andmouth disease virus (FMDV). These novel systems will be optimized in studies using naïve animals and subsequently validated in vaccinatechallenge experiments. Additionally, the novel systems will be used to assess pigs and steers ability to transmit FMDV in the pre-clinical phase of disease. Page 29 of 32