1. FMD Reference Laboratory THE USE OF NON-STRUCTURAL PROTEIN TESTS TO DIFFERENTIATE BETWEEN VACCINATED AND INFECTED ANIMALS Donald King Anna Ludi, Ginette Wilsden, Satya Parida and David Paton donald.king@pirbright.ac.uk donald.king@pirbright.ac.uk WRLFMD Team: Valerie Mioulet, Bryony Armson, Pip Hamblin, Nick Knowles, Kasia Bachanek-Bankowska, Lissie Hendry, Jemma Wadsworth, Britta Wood, Barsha Thapa, Bob Statham, Abid Bin-Tarif, Ashley Gray, Emma Fishbourne, Beth Johns, Mark Henstock, Alison Morris, Nick Lyons, Dexter Wiseman, Antonello Di Nardo, Trish Ryder, Sarah Belgrave

2. BBSRC National Virology Centre: The Plowright Building 2015: Occupied new high containment laboratory Houses all our work with “live” FMD and International Reference Laboratories for FMD, BT, PPR, ASF, AHS, Capripox

3. Submissions to WRLFMD Pirbright October 2013 – September 2015 Trinidad PAT Israel Hong Kong SAR Chinese Taipei South Korea Sequence exchange with China, Russia, India and BVI Reports for these samples can be found at: www.wrlfmd.org

4. Coordinating Global Networks OIE/FAO FMD Laboratory Network OIE and FAO Reference Centres (+ affiliates) Annual meeting and report Global surveillance and changing patterns in risk pathways Harmonised and improved lab capacity Brescia, Italy – November 2014

5. O/ME-SA/Ind2001 from the Indian sub-continent Since 2013 FMD Outbreaks in Saudi Arabia, UAE, Bahrain, Libya, Tunisia, Algeria and Morocco Increasing threats to Europe? Recent serotype O cases Knowles et al., (2015) TED

6. Samples collected in September 2015 Dairy cattle in Saudi Arabia Originating from the Indian sub- continent A further example of new “unexpected” FMDV movement between endemic pools New related outbreaks in Turkey (November 2015) Impact upon vaccination? (http://www.promedmail.org: Archive Number: 20151019.3727949)]. Recent serotype A cases

7. FMD serology: summary of different tests and approaches Principles of using NSP tests for differentiating infected from vaccinated animals ( DIVA) purposes Test performance and validation Use of NSP tests for surveillance Draft recommendations Overview of presentation

8. DAYS MEASUREMENT 1465432187 Diagnostic windows for FMD ● ● FMD virus in blood Clinical lesions FMDV-specific antibody responses Representative “in contact” cattle data from Alexandersen et al., 2003 and unpublished data from Pirbright FMD virus at mucosal sites

9. Evaluating population immunity (after vaccination) Investigating suspect cases and quantifying FMD prevalence Substantiating freedom from infection at population or individual animal level for trade FMD virus serology Purpose and use of tests:

10. Tests for antibodies to virus structural proteins Virus neutralisation Test (VNT)* Microtitre format Multiple cell lines described (IB-RS-2, BHK-21, lamb or pig kidney cells) Solid-phase competition ELISA (SPCE)* Liquid-phase blocking ELISA (LPBE)* Tests for antibodies to virus structural proteins Non-structural protein ELISA (NSPE) Indirect ELISA Enzyme-linked immunoelectrotransfer blot (EITB) * a prescribed test for international trade Many new commercial assays – versions of SPCE/NSPE FMD virus serology Methods described in OIE manual

11. ELISAAdvantagesDisadvantages NSPFast & easy to perform Detects infected animals regardless of vaccination One test for all serotypes Can be performed out of containment Commercially available Sensitivity less if low virus replication and for early infection LPBESerotype specific Can be performed out of containment Correlated to immunity from potency tests Like all tests for structural proteins, Se dependent upon good strain match Low Specificity (~96%) for infection surveys Limited availability from some reference labs Not always a homologous system Complicated to perform Polyclonal antibodies-use of rabbits & guinea pigs SPCEFast & easy to perform Serotype specific Can be performed out of containment Commercially available alternatives Not always a homologous system Polyclonal antibodies-use of rabbits & guinea pigs (commercial alternative tests use mAbs) VNTGold standard-OIE manual Serotype specific Easy to change virus strains Correlated to immunity from potency tests Containment facilities required Slow and laborious Cell-culture dependent Reproducibility issues CFTSerotype specific Can be used if ELISA reagents are not available Pro- or anti- complementary factors Less sensitive Lacks reproducibility Less economical FMDV serology Advantages & disadvantages of tests

12. PolyAVPg LVP4VP2VP3VP12A2B2C3A3B3C3D 5’3’ 5’ UTR3’ UTR Viral structural proteins (VP1, VP2, VP3 are on surface) Viral non-structural proteins that elicit antibodies 2B, 2C, 3ABC, 3D FMD Viral Proteins Structural proteins (SP) and non-structural proteins (NSP) are expressed during replication Following exposure to infection, vaccinated animals show an antibody response to NSP, particularly 3AB, 3ABC, (Mackay et al., 1998), 2B (Berger et al., 1990; Inoue et al., 2006) and/or 3C, 2C, and occasionally 3A (Mackay et al., 1998).

13. FMD Viral Proteins NSPs are responsible for replication of the viral RNA and processing of the viral proteins

14. FMD Serology Basis of differentiating infected from vaccinated animals ( DIVA): Vaccinated - infected NSPs Infection with live virus SP antibodies - Serotype-specific - Correlate to protection NSP antibodies - Pan-serotype reactive - Used for DIVA testing Viral non-structural proteins (NSP) Inactivated purified virus (structural proteins, SP) Live virus KEY TO FIGURE Vaccination with purified vaccine Vaccinated - infected NSPs Vaccine virus preparation Purification

15. Spectrum of host scenarios: Unvaccinated animals Unvaccinated animals infected with FMDV (recovered) Unvaccinated animals infected with FMDV (carrier state) SP and NSP tests can be used Vaccinated animals not infected with FMDV Vaccinated animals infected with FMDV (recovered) Vaccinated animals infected with FMDV (carrier state) This status impacts upon the performance of NSP tests

16. FMD Carriers Definition: an animal from which FMD virus can be isolated >28 days after infection Virus can be recovered from the pharynx and dorsal soft palate Ruminants, but not pigs can become carriers Evidence of virus transmission from carriers comes from field but not experimental studies

17. NSP Test performance Points to consider when adopting a DIVA approach: Sensitivity: FMDV NSP antibody responses normally reflect extent of FMD virus replication Ability to detect infected individuals in a vaccinated population is dependent upon degree of FMDV replication and may be more difficult than in non-vaccinated animals Specificity: Even after purification, most vaccines contain residual NSPs Multiple vaccination regimes can generate anti-NSPs responses that generate false positives in the tests

18. Development of SP and NSP antibodies NSP antibody induction is T cell dependent and they appear later than SP antibody Causes of NSP antibody detection – Infection – Vaccination – Other non-specific In case of infection, ruminants can be – Fully recovered – Carriers NSP SP

19. Comparative test performance Validation of NSP assays: Cedi-diagnostics kit (now PrioCHECK ELISA - ThermoFisher) SVANOVIR  FMDV 3ABC-Ab ELISA (Svanova, Uppsala, Sweden) CHEKIT-FMD-3ABC (Bommeli Diagnostics, Bern, Switzerland) UBI  FMDV NS ELISA (United Biomedical Inc., New York, USA) 3ABC Trapping ELISA (IZSLER, Italy) NCPanaftosa-screening ELISA (Panaftosa, Brazil)

20. Brescia workshop May 2004 Study used 3,551 sera representing different histories Sera from naïve, vaccinated (European vaccines/one dose), infected and vaccinated-and-infected animals Vaccinated and non-vaccinated carrier animals Sera collected at different times after vaccination and at different points after infection 6 tests evaluated (competitive/indirect) 2/3 (n=2579) from cattle, rest from sheep (n=703) and pigs (n=269)

21. Available NSP Kits evaluated by Brescia Workshop PrioCHECK® FMDV NS ELISA (formerly Cedi-diagnostics) – Competitive 3ABC ELISA kit with trapping mAb – Overnight protocol IDEXX FMD 3ABC Ab Test (formerly Bommeli Chekit) – Cattle and sheep – 3 hr protocol Boehringer Ingelheim; SVANOVIR® FMDV 3ABC-Ab ruminant (formerly Svanova) – Indirect 10 plate ELISA kit for cattle, sheep, goats UBI® FMDV NS ELISA (cattle) and UBI® FMDV NS ELISA (swine) – Commercial availability ?

22. Infection in a unvaccinated population Infection in a vaccinated population Vaccinated and infected [detection of CARRIERS] Performance of NSP tests specificity 97.2 - 98.5% sensitivity 100% Similar performance between tests and for sheep and pigs specificity 96.9 - 99.5% sensitivity 38*-74% *Some groups of sera yield test se as low as 25% specificity >99% sensitivity 86.4* - 93.9% *for the 3 best performing tests At 28-100 dpi improved sensitivity compared to entire vaccinated population Diagnostic sensitivity (se) and specificity (sp) in cattle Used as screening tests

23. Findings from Brescia workshop (1) All kits detect infection with high Se in unvaccinated animals For vaccinated cattle, only 1 kit comparable to NCPanaftosa- screening index test in OIE Manual: Cedi-diagnostics Sp and Se for detection of carriers in vaccinated cattle around 99% and 90% respectively Single dose vaccination with European manufactured vaccines had no impact on Sp Other commercial kits had similar Sp but less Se than Cedi- diagnostics kit for detecting infection in vaccinated cattle Most false-positive samples recorded in non-infected cattle had borderline values in the tests

24. Findings from Brescia workshop (2) Retesting in the same test improved the Sp Sera reacting as false-positive in one test were mostly negative in the other, but serial testing reduces the test system Se – Se and Sp test covariance For vaccinated and infected animals: – Se greater for carriers than non-carriers – Se greater in diseased than subclinically infected animals – Se wanes over time (but can last at least 3 years)

25. Elnekave et al., 2015 Longevity of NSP responses in cattle PrioCHECK 3ABC NS Repeatedly vaccinated NSP specific antibodies detected up to 3 years after infection

26. FMD Serology Use of NSP Tests Useful to confirm past infection or to substantiate its absence: – In FMD-free zones without vaccination – In FMD-free zones without vaccination after use of emergency vaccination – In FMD-free zones with regular vaccination – In FMD endemic countries and zones where vaccination is not practised According to PCP status, NSP tests can be used: – as part of a prophylactic vaccination campaign to assess the underlying burden of disease in a region, – or to provide robust evidence that a region (or zone) is free from FMD).

27. Use of NSP tests in endemic settings To support sero-surveillance exercises that assess the prevalence of infection in livestock and in wildlife Especially where the results for SP tests might be complicated by the presence of vaccine-induced antibodies Following infection, NSP sero-conversion usually takes 7-14 days after which these antibodies can be detected in serum for months, or even years, depending upon the amount of virus replication Important that only high-quality vaccines (that have been purified to remove contaminating NSPs) are deployed Study designs usually focus on younger animals (< 18 months of age), since repeated vaccination, even with high quality vaccines, can generate positive signals in the NSP ELISAs that may provide a false indication of FMDV infection

28. Use of NSP tests in endemic settings As countries move towards OIE FMD-freedom (with vaccination), NSP surveys play an important role to confirm the absence of FMDV circulation in livestock populations These exercises involve the testing of large numbers of sera, Important to adopt a layered testing approach to accommodate the expected number of false positives (Brocchi et al., 2006) Tests with high diagnostic sensitivity (such as a 3ABC ELISA) used to screen the sera Positive results are confirmed using a second NSP antibody assay at least equivalent sensitivity and specificity To rule out the false-positives, epidemiological investigations and analysis of probang samples by real-time RT-PCR (or SP tests) may be recommended.

29. Use of NSP tests in endemic settings It is important to define the design prevalence of the study (such as to detect 5% prevalence with 95% confidence) since this will impact upon the interpretation of data. If prevalence is low - may face Sp problems due to repetitive vaccination Rather than employing random sampling, the adoption of a risk-based sampling strategy may be more effective to recognize rare events Retests for continuing seropositivity and seroconversion widely used in South America

30. Why Check Specificity of NSP tests? To understand impact of different amounts of vaccination on NSP test Sp Values specific for a given vaccine, vaccination regime and NSP test system However, numbers of samples needed to distinguish the difference between Sp in subpopulations depends on how much difference there is Therefore local pilot studies are required to evaluate test Sp to support DIVA testing with use of vaccination in different settings

31. Interpreting NSP seroreactors Understand NSP responses to vaccine in use Target surveillance to reduce non-specific results – Risk based to reduce overall scale of testing – Focus on 6-12 month old animals where possible Number, strength and clustering of seroreactors Revisits and repeat and additional testing Complementary investigations and evidence – Disease – Epidemiological links

32. Follow-up of seroreactors Most serosurvey designs target part of the population to achieve a certain confidence – e.g. to detect infection with 95% confidence of detecting infection if >2% of herds infected, containing >10% infected animals Need to consider Se and Sp of whole tests system Any positive finding are evidence of infection UNLESS RULED OUT ON JUSTIFIABLE GROUNDS OIE requires follow-up of all seroreactors Problem that if tests have relatively low individual animal Se, need to test large numbers to compensate Then get Sp problems Also difficult to prove a negative (S American experience)

33. Serosurveys for infection in FMD-free zones without vaccination Choice of using NSP or SP tests SP tests – need to know serotype or use multiple tests, so use for confirmation NSP tests – do not need to know serotype, so use for screening Animals testing positive in both NSP and SP tests should be followed up: – Consider number and strength of reactors on same epi unit – Check for clustering – Check for clinical signs – Check for epidemiological risks – Additional damples to check for continuing seropositivity and seroconversion – Probang sampling of ruminants for testing by RT-PCR (Se ~50%)

34. Serosurveys for infection in FMD-free zones without vaccination after use of emergency vaccination Only NSP tests applicable Delay sampling until 1 month after last case or vaccination, whichever later Use of different NSP tests for screening and confirmation improves Sp but may reduce Se, so single optimal test with retesting of positives in same assay may be best – Need to factor overall Se/Sp into survey design Animals testing positive should be followed up (as per previous slide)

35. Test Follow-up Algorithm From: The use of serosurveys following emergency vaccination, to recover the status of "foot-and-mouth disease free where vaccination is not practised". Paton DJ, Füssel AE, Vosloo W, Dekker A, De Clercq K. (2014) Vaccine 32(52):7050-6. Review.The use of serosurveys following emergency vaccination, to recover the status of "foot-and-mouth disease free where vaccination is not practised".

36. More Recently Available NSP Tests Innovative Diagnostics, IDVet; ID SCREEN® FMD NSP COMPETITION – Competitive 3ABC ELISA kit (5 or 10 plate formats) Excelsior Bio-System Incorporation; Sentinel FMDV NSP Ab ELISA kit – Competitive 3ABC ELISA kit with trapping mAb – Overnight protocol Ubio Biotechnology Systems Ltd; Bovine FMD NSP Antibody Test and Swine FMD NSP Antibody Test

37. Available NSP Penside Tests Bionote; Anigen Rapid FMD NSP Ab Test Kit – Immunochromatographic detection in whole blood, plasma and serum from cattle, sheep and pigs Qbiotech; FMD NSP Ab Test – Sandwich lateral flow immunochromatographic assay. Claimed Se and Sp of 94.2% and 98.5% respectively Excelsior Bio-System Incorporation; Sentinel® Foot and Mouth Disease Virus NSP Antibody Rapid Test – Validated in Taiwan on 169 swine sera. Relative to PrioCHECK: Se = 90.2%, Sp = 96.9%. Chen et al. J Vet Med Sci. 2009 Jun;71(6):703-8. Span Biotech Ltd; One-Step FMD NSP Ab Test – sandwich lateral flow immunochromatographic assay for detection NSP Ab in animal serum. Ease of use for small numbers of samples Reliability issues: extent of validation and QC

38. 36 cattle sera from different experimental studies conducted at Pirbright 4 groups of cattle were vaccinated and then infected by contact or inoculation: – developed FMD and became carriers (n=3) – no clinical signs and became carriers (n=17) – developed FMD and did not become a carrier (n=1) – no clinical signs and did not become carriers (n=3) 2 groups of cattle were unvaccinated and then infected by contact or inoculation (all developed FMD): – became carriers (n=7) – did not become carriers (n=5) NSP Pirbright Panel

39. Draft recommendations: [1] According to PCP status, member states should define the purpose of sero-surveys (i.e., as part of a prophylactic vaccination campaign to assess the underlying burden of disease in a region, or to provide robust evidence that a region (or zone) is free from FMD). [2] Member states should compile a list of all vaccines (including details of manufacturer, specific FMDV strains and formulations) that are currently deployed in the region. [3] Data should be collated from experimental and field trials (corresponding to the vaccines used) to determine the performance of NSP tests after application of different doses of the particular vaccines that are used in the region, in a range of livestock host species. [4] Field data on extent of NSP sero-prevalence in vaccinated herds that become infected should be collected to inform design prevalence of future serological surveys.

40. Acknowledgements Support for the WRLFMD and research projects Collaborating FMD Reference Laboratories and field teams Partners within the OIE/FAO FMD Lab Network