OS16 - 4.P2.a Application of Indirect and Avidity ELISA Tests to Assess Anti-FMDV Antibodies Induced by Vaccination in Buffalo and Swine Serum Samples - A. Capozzo

1. Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016 APPLICATION OF INDIRECT AND AVIDITY ELISAs TO ASSESS ANTI-FMDV ANTIBODIES INDUCED BY VACCINATION IN BUFFALO AND SWINE SERUM SAMPLES F. Mansilla,; J. Sala, M. Trotta; . Piscitelli; F. Bessone, M. Pérez-Filgueira, S. Caspe, A. Capozzo Dr. Alejandra V. Capozzo, PhD HEAD OF THE APPLIED VETERINARY IMMUNOLOGY LAB. INSTITUTE OF VIROLOGY. INTA. BUENOS AIRES

2. Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016 Current situation: FMD free, with or without vaccination. Vaccines: oil-adjuvanted, tetravalent (O1 Campos - A24 Cruzeiro – A/Arg/2001- C3 Indaial). Locally produced, purified, high quality. Vaccines are applied to cattle and buffaloes Young animals (< 2 years old) are vaccinated twice a year Animals > 2 years old are vaccinated once a year Goal: Study antibodies elicited by current vaccines in all susceptible species. ARGENTINA

3. Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016 We cared more about cattle…. Vaccines are applied to cattle and buffaloes - Pigs are not vaccinated 51,429,848 5,011,446 101,076 ARGENTINA 2015 Consumption (per inhabitant) 60 Kg 10 Kg 3rd producer in South America 2nd producer in South America 3rd producer in South America

4. OS16 Animals < 2 years old -twice a year Animals > 2 years old once a year FMDV-free without vaccination FMDV-free with vaccination Latest outbreak: 2011 O1 serotype Border belt All susceptible species are vaccinated Water buffaloes Bubalus bubalis Mediterranean breed

5. OS16 Serological surveillance of vaccine efficacy VNT LPBE Gold Standard Can use any field strain • Requires live virus manipulation • Requires cell culture • Time consuming • Difficult to harmonize • Not useful for high-throughput Dedicated high- containment facilities Concordance with VNT (bovine serum samples) High throughput Easy to harmonize Uses inactivated virus • Requires two antibody preparations: capture and detection antibodies • Not useful for rapid set up assay for new filed variants 75-EPP% Cut-off value For each vaccine strain Pros Cons VNT and LPBE titers of virus-specific antibodies have been related to protection against infection in cattle (PPG) Virus neutralization test Liquid phase blocking ELISA

6. OS16 Do ELISAs match VNT when using buffalo serum samples?

7. OS16 • LPBE: two different detector Ab Cut OFF - EPP 75%: LPBE titer 2.11 (Log10Titer , O1 Campos strain) LPBE Y Capture Ab (rabbit) Detector Ab Monoclonal Ab MIX Guinea-Pig Polyclonal LPBE-Mab LPBE-HIS Does LPBE match VNT? N=91 samples with different VNT titers N=27 Log10 VNT titers < 1.66 N=64 Log10 VNT titers >1.66 Cut OFF - EPP 75%: VNT titer=1,6 Log10TCID50

8. OS16 Do both LPBE-Mab and LPBE-HIS perform equally when using buffalo samples? 0 1 2 3 4 5 6 0 1 2 3 4 5 6 2.40 1.20 1.40 1.91 2.31 LPBE-HIS LPBE-Mabs r2=0.87 Pearson´s r= 0.88 YES

9. OS16 2 3 4 VNT titer 0 1 2 3 4 0 2 4 6 VNT titer LPBE-Mabstiter 2.5 B D Pearson´s r=0,4432 (0.2576 - 0.5972) Concordance between VNT and LPB-ELISA EPP 75%=2.1 (Log10 titer) EPP 75%=1,65 (Log10 titer)

10. OS16 1 Assay Specifications Readout Sensitivity Specificity Accura LPBE-HIS Serial-dilution liquid- phase blocking ELISA. Uses HIS to reveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 LPBE- Mab Serial-dilution liquid- phase blocking ELISA. Uses a mixture of Mab to reveal 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 IE Single dilution indirect ELISA OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 AE Single dilution indirect ELISA with an Urea washing step 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 Specifications Readout Sensitivity Specificity Accuracy κ value Le agr erial-dilution liquid- ase blocking ELISA. Uses HIS to reveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 0.34 Mi erial-dilution liquid- ase blocking ELISA. es a mixture of Mab to reveal 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 0.35 Mi ngle dilution indirect ELISA OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 0.51 W ngle dilution indirect LISA with an Urea washing step 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 0.62 W Readout Sensitivity Specificity Accuracy κ value Level of agreement uid- ISA. eal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 0.34 Minimal uid- ISA. Mab 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 0.35 Minimal irect OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 0.51 Weak irect rea 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 0.62 Weak Concordance between VNT and LPB-ELISA Low specificity

11. OS16 Problem to solve: low specificity • Mainly due to cross-reactive antibodies • Natural antibodies Avidity ELISA Binding strength Ag- Ab Low-avidity binders are removed using UREA Single dilution Purified antigen Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle. Lavoria MÁ, Di-Giacomo S, Bucafusco D, Franco-Mahecha OL, Pérez- Filgueira DM, Capozzo AV. Vaccine. 2012 Accuracy of traditional and novel serology tests for predicting cross-protection in foot-and-mouth disease vaccinated cattle. Brito BP, Perez AM, Capozzo AV. Vaccine. 2014

12. OS16 Indirect and avidity ELISAs IE Indirect ELISA, purified virus, single dilution AE Avidity indirect ELISA: low avidity binders are washed way with Urea, purified virus, single dilution • N=91 samples with different VNT titers Cut OFF - EPP 70%: VNT titer=1,6 Log10TCID50  Single dilution test  Purified inactivated antigen  Neither detector nor capture Ab needed  Three steps

13. OS16 Concordance between VNT* and ELISAs * VNT is the accepted assay for vaccine efficacy testing in the European Pharmacopoeia. 1 Assay Specifications Readout Sensitivity Specificity Accuracy LPBE-HIS Serial-dilution liquid- phase blocking ELISA. Uses HIS to reveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 LPBE- Mab Serial-dilution liquid- phase blocking ELISA. Uses a mixture of Mab to reveal 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 IE Single dilution indirect ELISA OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 AE Single dilution indirect ELISA with an Urea washing step 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 Specifications Readout Sensitivity Specificity Accuracy κ value Level agreem Serial-dilution liquid- phase blocking ELISA. Uses HIS to reveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 0.34 Minim Serial-dilution liquid- phase blocking ELISA. Uses a mixture of Mab to reveal 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 0.35 Minim Single dilution indirect ELISA OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 0.51 Wea Single dilution indirect ELISA with an Urea washing step 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 0.62 Wea ons Readout Sensitivity Specificity Accuracy κ value Level of agreement liquid- ELISA. reveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 0.34 Minimal liquid- ELISA. of Mab l 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 0.35 Minimal indirect OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 0.51 Weak indirect n Urea tep 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 0.62 Weak 1 Assay Specifications Readout Sensitivity Specificity Accuracy LPBE-HIS Serial-dilution liquid- phase blocking ELISA. Uses HIS to reveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 LPBE- Mab Serial-dilution liquid- phase blocking ELISA. Uses a mixture of Mab to reveal 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 IE Single dilution indirect ELISA OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 AE Single dilution indirect ELISA with an Urea washing step 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 Specifications Readout Sensitivity Specificity Accuracy κ value Level agreem Serial-dilution liquid- phase blocking ELISA. Uses HIS to reveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 0.34 Minim Serial-dilution liquid- phase blocking ELISA. Uses a mixture of Mab to reveal 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 0.35 Minim Single dilution indirect ELISA OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 0.51 Weak Single dilution indirect ELISA with an Urea washing step 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 0.62 Weak ons Readout Sensitivity Specificity Accuracy κ value Level of agreement liquid- ELISA. eveal Titer 84.2 (75.2 – 91.0) 48.4 (37.7 – 59.1) 0.72 0.34 Minimal liquid- ELISA. of Mab 84.6 (75.5 – 91.3) 48.6 (97.8 – 59.2) 0.73 0.35 Minimal indirect OD value 71.2 (68.0 – 80.4) 84.4 (73.0 – 89.6) 0.75 0.51 Weak indirect n Urea ep 74.6 (64.5 – 83.3) 93.83 (85.0 – 98.8) 0.81 0.62 Weak

14. OS16 LOW VNT titers <1.4 HIGH VNT titers > 3 Classified correctly with tests that measure total antibodies VNT titers 1.4 – 3.0 Avidity ELISA Improving concordance with VNT VNT titers

15. OS16 Accuracy of combined indirect assessment The best agreement values (κ=80%) were obtained when combining results of both single dilution ELISAs Additional benefit: high-throughput reproducible assessment that only takes a few hours of work to the result. TEST 1 Conditions (test 1) TEST 2 Selection (test 2) Sensitivity Specificity Accuracy K value (VNT) a LPBE OD ≤ 1.6 - classify NEGATIVE OD > 1.6 RETEST AE Cut off= 0.7 87.9 (0.80 – 0.93) 90.6 (0.84 – 0.96) 87.8 0.764 AE OD ≤ 0.35-classify NEGATIVE OD ≥ 1.1 Classify POSITIVE 0.35 < OD < 1.1: RETEST IE Cut off= 1.1 94.8 (0.87 – 0.99) 84.4 (0. 87 – 0.91) 91.1 0.803 1 Log 10Titer≤ 1.6 classify NEGATIVE Log 10Titer≥ 3.5 classify POSITIVE 1.6 < Log 10Titer < 3.5 RETEST IE AE OD ≤ 0.3 classify NEGATIVE OD ≥ 1.1 classify POSITIVE 0.3 < OD < 1.1 RETEST 88.8 92.6

16. OS16 • The combination of two single-dilution high-throughput ELISA may afford a simpler and faster alternative to the VNT. • Provided a thorough validation is performed, these assays may be useful for monitoring the effectiveness of vaccination campaigns in buffalo populations. Further analysis ( > 600 samples). Consider changing cut-off values? Conclusions

17. OS16 PIGS are not vaccinated in Argentina101,000-500,000 40,000-100,000 10,000-39,000 <10,000 Swine population (updated Nov. 2015)FMDV-free without vaccination FMDV-free with vaccination (cattle and buffaloes) Latest outbreak: 2011 O1 serotype Border belt All susceptible species are vaccinated

18. OS16 Immunized with comercial vaccine Dose: 2 ml; SC The same applied in cattle Piglets (12 weeks old) Control group : immunized with oil adjuvant + PBS. 2ml/dose; SC Serology against O1 Campos and A 24 Cruzeiro strains 0dpv 10dpv 21dpv 60dpv Immune responses to vaccination: experimental design •Liquid phase blocking ELISA (LBPE) •Indirect ELISA •Avidity ELISA •Isotype ELISAs n= 12 n= 3

19. OS16 0 15 30 45 60 0 1 2 3 4 IgG2 IgG1 IgM DPV Ig1titerO1Campos (Log10) Immune responses to vaccination

20. OS16 Total abs: Indirect ELISA Avidity ELISA Neutralizing Abs Total abs: LPBE 30 45 60 Placebo Vaccinated DPV 0 15 30 45 60 0.5 1.0 1.5 2.0 2.5 3.0 Placebo Vaccinated DPV VNTtiterO1Campos (Log10) 30 45 60 DPV 0 15 30 45 60 0 10 20 30 Vaccinated DPV Avidityindex(%) 0 15 30 45 60 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Placebo Vaccinated DPV LPBEtiterO1Campos (Log10) 0 15 0.5 1.0 1.5 2.0 2.5 3.0 VNTtiterO1Campos (Log10) 0 15 30 45 60 0.0 0.5 1.0 1.5 2.0 2.5 DPV IEO1Campos (OD450nm) 0 15 0 10 20 30 Avidityindex(%)

21. OS16 Do avidity and LPBE match VNT? Correlation 0.5 1.0 1.5 2.0 2.5 3.0 0 10 20 30 40 r2 =0.70 Pearson r 95% confidence interval 0.8191 0.6422 to 0.9131 VNT titer Avidityindex(%) Correlation 0.5 1.0 1.5 2.0 2.5 3.0 0 1 2 3 4 5 r2 =0.45 Pearson r 95% confidence interval 0.6750 0.4861 to 0.8037 VNT titer LPBE-HIStiter Avidity correlates with VNT

22. OS16 Conclusions Avidity ELISA emerges as a promising low-labor high throughput alternative to VNT • Avidity matches better VNT titers than LPBE and does not require standardized sera, facilitating the assessment of antibodies against new field strains • Indirect ELISA and LPBE provide similar information Everyone is invited to get the protocol/training and run their samples

23. OS16 JUAN SALA, DVM FLORENCIA MANSILLA, PhD Myrian Trotta Ms Biotec APPLIED VETERINARYIMMUNOLOGYLAB. INSTITUTEOF VIROLOGY.INTA. Capozzo´s Lab MUCHAS GRACIAS

OS16 - 4.P2.a Application of Indirect and Avidity ELISA Tests to Assess Anti-FMDV Antibodies Induced by Vaccination in Buffalo and Swine Serum Samples - A. Capozzo

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