National Center for Foreign Animal and Zoonotic Disease Defense: Agricultural Screening Tools Portfolio

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1 National Center for Foreign Animal and Zoonotic Disease Defense: Agricultural Screening Tools Portfolio FAZD Center Annual Meeting Dr. Melissa Berquist Associate Director June 27 th, 2013

2 BIO Overview Transition/delivery focused portfolio Projects selected address national, high priority gaps Strong partnerships with USDA and DHS ensure project relevance and transition potential Currently, 15 research projects involving industry, academic, and government partners Agricultural screening tools program supports business continuity planning and integrates with IAS and EOS portfolios Current agricultural screening tools portfolio investment: $4M 2

3 BIO Mission To perform cutting edge research that enhances knowledge and provides state-of-the-art capacity for the identification, control, response, and eradication of foreign animal, emerging and/or zoonotic diseases of agricultural and public health significance 3

4 Programmatic Areas of BIO Portfolio Basic Applied Future Reagent Development Ag Screening Tools Validated Test Kits EVOLVING RESEARCH PORTFOLIO 4

5 2004 Blue Ribbon Panel Sponsored by the White House Office of Science and Technology Policy Prioritized likelihood and potential consequences of biological terrorism directed against US livestock Focus on vaccination/protection and detection/diagnostic technologies 5

6 Foreign Animal Disease R&D Plan ( ) R&D efforts most likely to detect, control and eradicate FADs Top priority diseases: FMD, RVF, HPAI, END Recent discussions with USDA and DHS have added CSF and ASF as high priority Four key areas for R&D Epidemiological and economic modeling Veterinary countermeasures Decontamination and disposal Basic research 6

7 2011 GAO Report: Food/Ag Defense Response Coordination Lack of defined coordination among agencies makes response challenging FAZD plays a critical role in bringing together diverse stakeholders DHS needs to lead inter-agency implementation of HSPD-9 FAZD supports DHS efforts to develop countermeasures and other response technologies 7

8 Workshops to Coordinate and Enhance the Agro-Security Enterprise Defining the Needs and Requirements for Agricultural Screening Tools (Nov 2010) Agricultural Industry Perspective of Utilizing Agricultural Screening Tools (April 2011) Recommendations for Establishing Policies and Concepts of Operations for Use of Agricultural Screening Tools (Oct 2011) Policies and Concept of Operations Recommendations for the Use of Agricultural Screening Tools (May 2012) 8

9 BIO Transition Pathway Needs Assessment Gap identification Stakeholder meetings ID of emerging disease Risk of disease spread Gap assessment and prioritization Need/feasibility assessment Implementation goals Surveillance plan Funding Training PT and reference materials NAHLN Methods Technical Working Group Processes Review proposals addressing specific gaps Validation Methods comparison Consistent with OIE guidelines Analytical and diagnostic sensitivity/specificity Repeatability Dossier Review Available samples Fitness for purpose Fitness for use Recommendations provided to VS FAD investigation Surveillance/early detection Response Recovery Implementation Develop testing algorithm Finalize surveillance plan Response and notification plans Distribute SOPs Provide training Provide reference materials Provide proficiency tests Determine funding mechanism Determine data collection Finalize guidance documents Communication across all involved stakeholder groups Monitor performance of assay Monitor performance of assay Review effectiveness of surveillance program 9

10 Program Area: Agricultural Screening Tools 2010 to Present Program Principal Investigators Dr. Tammy Beckham, FAZD Center Dr. Mangkey Bounpheng, TVMDL Dr. Alfonso Clavijo, PAHO Dr. Scott Adams, VMRD, Inc Dr. Linda Dixon, Pirbright Institute Dr. Donald King, Pirbright Institute Dr. Michael McIntosh, USDA APHIS Dr. Brian Bird, CDC Dr. Ute Stroeher, CDC Dr. Bob Rowland, KSU 10

11 Develop a 3B-FMDV celisa Diagnostic Kit Leverages initial OUP investment to develop 3B monoclonal antibody Supports FMD vaccinate-to-live policy to differentiate vaccinated from infected animals, based on the detection of antibodies to FMDV non structural proteins (NSP) Strong industry/government/academic/international collaboration Kit utilizes USDA ARS developed antigen with FAZD developed antibody Testing of FMDV serum samples from infected and vaccinated cattle at the Pirbright Institute and USDA APHIS FADDL Testing of FMDV positive wildlife and SAT+ cattle samples at USDA ARS Small scale negative cohort testing at TVMDL 11

12 Develop a 3B-FMDV celisa Diagnostic Kit FAZD DHS/S&T DHS OUP TVMDL, BIOO USDA ARS/APHIS VMRD, Inc End user Investment from OUP in FMDV monoclonal antibody development Evaluation and selection of best monoclonal and antigen Kit development, bench validation, and performance improvement Delivery of v2.0 FMDV ELISA pilot kits to DHS June 2010 December 2013 Validation in swine Transition to NAHLN September2014 September

13 Develop a 3B-FMDV celisa Diagnostic Kit Good separation of positive and negative results Enables diagnostic serological testing of FMDV susceptible species Enables U.S. manufacturing and production of critical reagent PrioCHECK FMDV NS test (Prionics) is not licensed by USDA CVB for importation, distribution, and use Additional funding to support transition to NAHLN (large scale negative cohort) and validation in swine scheduled for FY

14 Develop a 3B-FMDV celisa Diagnostic Kit: Outcomes No import permit required More rapid response with US-based manufacturing Lower cost and faster results celisa results obtained in hours rather than days DIVA compatible with newly licensed DHS-Ad5 vaccine Unequivocally distinguish vaccinated from infected animals Improved performance over gold standard celisa detects all seven FMDV serotypes 99.8% diagnostic specificity; 100% diagnostic sensitivity 14

15 Optimization and Validation of an FMDV RT-PCR Assay for Bulk Tank Milk Samples FMD Bulk Milk Screening Tool Support Secure Milk Supply plan and business continuity Restore normal milk movement during an FMD outbreak Need identified during Ag Screening Tools workshops and by the US dairy industry Partners: USDA APHIS Plum Island, Pirbirght (IAH), Wisconsin, California, Dairy industry Public/private/international collaboration Strong management/subject matter expertise from FAZD Links to dairy industry, NAHLN MTWG, network laboratories Animal work at Pirbright Field collection (bulk tanks) performed internationally Bangladesh, Tanzania Negative cohort performed in NAHLN laboratories Assay development coordinated with NAHLN MTWG 15

16 Days 0-7 Acclimatize animals for background/negative sample collection Closed room for virus challenge Linked rooms Day 7 Inoculate non-milking cattle ( ) with FMDV Milking room Day 9 Direct inoculates mixed with milking cattle ( ) for 5 days Space available (if needed) Milking room Days 9-37 Direct inoculates mixed with milking cattle Space available (if needed) Space available (if needed) Milking room 16

17 Optimization and Validation of an FMDV RT-PCR Assay for Bulk Tank Milk Samples Incorporates internal positive control Similar results for fresh vs frozen and infected vs spiked milk Strong reproducibility with infected animal samples tested at FADDL and Pirbright Milk fat content does not appear to inhibit detection Strain amplification efficiency of % (most 99% or better) TaqMan Assay Run at FADDL TaqMan Assay Run at Pirbright Strong interlaboratory reproducibility Animal ID 108 Animal ID 825 Animal ID 867 Animal ID

18 Optimization and Validation of an FMDV RT-PCR Assay for Bulk Tank Milk Samples: Outcomes Deploy a standardized high-throughput protocol for detection of FMDV in milk to the NAHLN Meets need identified by dairy industry to support business continuity during an outbreak Supports product movement from uninfected premises Includes validation of a sensitive molecular screening tool using the existing dairy industry milk transportation and quality control infrastructure End users: dairy industry; federal, state, and local government emergency planners; and veterinary diagnostics labs 18

19 Multiplex RT-qPCR Assay for Endemic and FAD Swine Surveillance Molecular (RT-qPCR) approach to multiplex testing for endemic and FADs ASFV, CSFV, FMDV, PRRS and SIV in oral fluid samples Optimize nucleic acid (NA) purification from oral fluid samples Simultaneous detection of pathogens using oral fluid diagnostic samples and experimentally infected animals (proof of concept) Oral fluid samples Easily collected during normal business practices Represent a pooled sample from which to detect a single positive infection Cost effective for non-invasive group disease surveillance Enables rapid sampling during an outbreak to monitor disease progression Partners: TVMDL, USDA APHIS Plum Island, National Pork Board, Smithfield, Chulalongkorn University (Thailand) 19

20 Multiplex RT-qPCR Assay for Endemic and FAD Swine Surveillance: Outcomes Optimized multiplex assay for endemic and FADs Pooled oral fluid sample is non-invasive and reduces burden of sample collection Potential commercialization in to a kit format for wide scale use 20

21 Comparison of Different Sample Sources and Sample Pooling for ASF Molecular-based approach to ASF testing Oral fluid, nasal swab, blood and serum Optimized processing yields more accurate results Methods comparison of two different assays Compliments other molecular approaches in development Options are needed to accommodate different sample types Oral fluids are processed similarly to standard serum samples Compare potential inhibition in pooled samples for PCR-based and immunoassay approaches Animal experiments completed May 2013 Partners: Pirbright Institute, USDA APHIS Plum Island Image courtesy FLI 21

22 Comparison of Different Sample Sources and Sample Pooling for ASF: Outcomes Diagnostics for ASF identified as a high priority need Use of pooled samples satisfies user needs for a noninvasive test that can be collected as needed by animal care personnel during normal business practices Current spread of disease in Eastern Europe/Russia represents high risk for US swine population Provides an optimized, bench-validated assay that can be applied to a variety of scenarios Includes methods comparison with currently NAHLNapproved qpcr assay for ASF 22

23 RVF DIVA ELISA Re-start in 2013 Leverages CDC investment in DIVA-compatible vaccine candidate Economically advantageous for restoring trade Large library of samples from RVF-endemic African countries for bench validation Partners: CDC National Center for Emerging and Zoonotic Infectious Diseases 23

24 Development of a Fluorescent Microsphere Immunoassay for Rift Valley Fever New start in 2013 Adapts Bio-Plex and MAGPIX (magnetic beads) to new immunoassay Initial tests for IgM and IgG Abs to RVFV Bovine and ovine serum Complimentary approach to DIVA ELISA Rule out infections that mimic RVF Image courtesy Luminex Corp 24

25 Program Area: Sample Preparation 2011 to Present Program Principal Investigators Dr. Mike Hogan, IntegenX Dr. Blanca Lupiani, TAMU CVM Dr. Pam Ferro, TVMDL Dr. Benjamin Lepene, Ceres Nanosciences Dr. Kylene Kehn-Hall, George Mason Univ 25

26 Matrix-Chaperone: Ambient Temperature Biospecimen Collection Ambient temperature, dry state collection, transport and banking of biospecimens Reduces need for cold chain during disease outbreak sampling Leverages DOD investment in human health sampling technology Demonstrated dry state preservation of immunoglobulin, nucleic acid, and live virus stored at room temperature for at least 5 days Phase II (FY2013) includes additional live animal testing for further validation of the technology Partners: IntegenX, TVMDL, and TAMU CVM 26

27 Universal Sample Preparation Tools for Rift Valley Fever Proteins Nanoparticle bait chemistry for RVFV antigens Concentrates and enriches diagnostic analytes for sample preparation (pre-test step) Removes interfering high-abundance analytes Nanoparticles: active after heat and detergent treatment Capture results in inactivation of RVFV Potential to adapt to multiple different antigens Phase II funded via Year 4 (FY2013) OUP Workplan and Agricultural Screening Tools project Incorporate nanoparticle capture in to ELISA and Luminex workflow Leverages previous DHS investment at CDC and KSU Partners: Ceres Nanosciences, George Mason University 27

28 Program Area: Reagent Development 2011 to Present Program Principal Investigators Dr. Mangkey Bounpheng, TVMDL Dr. Alfonso Clavijo, TVMDL Dr. Lance Ford, BIOO Scientific Dr. Adam Zelma, LLNL 28

29 Production and Characterization of Novel Reagents for Rift Valley Fever Develop monoclonal antibodies with different specificities to RVFV for diagnostic/research use All 12 MAbs produced against Baculovirus N protein specifically reactive to the Bac N protein MAbs Bac-1, Bac-8, Bac-9, Bac-11, and Bac-12 were also reactive to E.coli N protein, indicating shared epitopes Isotyping of MAbs (produced against E.coli, Bacculovirus, and inactivated whole RVFV) were successfully performed 30 Mabs delivered to USDA ABADRU in Fall 2012 Partners: TVMDL, UTMB, USDA ABADRU 29

30 Development and Characterization of Monoclonal Antibodies to FMDV Structural Proteins Bioinformatics approach Cross reactive MAbs developed using in silico ranked linear epitopes from VP1, VP2, and VP3 FMDV sequences Two protein expression system Linear epitopes of E. coli-expressed VP1, VP2, VP3 Conformational epitopes of baculovirus-expressed FMDv A VP1, FMDV O VP1, and FMDV Asia 1 VP1 Linear epitopes of peptides of all FMDv serotypes Recombinant proteins produced in E. coli and baculovirus expression systems Partners: TVMDL, BIOO Scientific, LLNL 30

31 Program Area: Penside Diagnostics 2013-present Program Principal Investigators Dr. Mangkey Bounpheng, TVMDL Dr. John Gerdes, Micronics Dr. Don King, Pirbright Institute 31

32 Penside Detection of Foot-and-Mouth Disease Virus by a Portable Microfluidics PCR System New start in 2013 PanNAT Results in 30 minutes High sensitivity field deployable PCR Single-use disposable cartridge FMDV FMDV FMDV Oral swab suspension deposit into sample well Nucleic acid purification Purified nucleic acid FMDV PCR in 3 wells Hands free, closed system (sample in, results out) Chemistry produces positive FMDV amplification with good separation of two independent targets biplexed in the same reaction well Partners: TVMDL, Micronics 32

33 Development of a Multiplex RT-LAMP Assay for FMDV New start in 2013 Develop an improved potential penside diagnostic tool for FMD Multiplex RT-LAMP assay for FMDV and VSV Optimize sample preparation approaches for field use Establish an improved decision algorithm that can be applied to suspect cases of FMD and VSV in the US Partners: Pirbright Institute, USDA ARS, and Forsite Diagnostics 33

34 Development of a Multiplex RT-LAMP for FMDV Improved differential diagnostics Companion to portable PCR field deployable equipment Highly sensitive and less susceptible to interference from sample matrices such as OP fluids and fecal samples 34

35 Comparison of FAZD Penside Diagnostics Projects SVANOVA Validation Current gold standard Not yet validated for use in US Produced internationally Requires extraction of sample from tissue Results in 10 minutes (excluding extraction) Portable microfluidics PCR system Closed cartridge system (sample in, results out) Sample to results in 30 minutes Multiplex RT-LAMP Assay Simple readout Could be used penside Potential differential diagnostic 35

36 Program Area: Proposals New Starts in 2013 Program Principal Investigators Dr. Sandy Rogers, TVMDL Dr. Garry Adams, TAMU CVM Dr. Scott Adams, VMRD, Inc 36

37 Multi-species competitive ELISA for Rift Valley Fever Virus Proposed for FY2013 Agricultural Screening Tools project funding Validation of specific mab and RVFV nucleoprotein antigen for use in a multi-species celisa Leverages previous DHS investment in RVFV reagent development and vaccine immunogenicity studies Output: two pilot lots of kits for use with multispecies samples and transition to NAHLN as a research use kit via MTWG Partners: TVMDL, TAMU CVM and VMRD 37

38 BIO Portfolio Impacts FAZD Center is making critical contributions supporting agricultural, animal, and public health preparedness Development of agricultural screening tools and novel vaccine technologies supports agricultural industries and promotes business continuity in the face of a disease event Improving detection and countermeasure technologies will allow for earlier detection and disease control and more rapid outbreak recovery 38

39 The Agricultural Enterprise QUESTIONS? 39