Application of Indirect and Avidity ELISAs for Anti-FMDV Antibody Assessment in Buffalo and Swine Serum Samples

In this study, researchers utilized indirect and avidity ELISAs to evaluate anti-foot-and-mouth disease virus (FMDV) antibodies induced by vaccination in serum samples from buffalo and swine in Argentina. The country is FMD-free, with vaccines applied to cattle and buffaloes but not to pigs. The research aimed to study antibodies elicited by current vaccines in all susceptible species, emphasizing serological surveillance for vaccine efficacy through various methods like virus neutralization tests and liquid phase blocking ELISAs.

• ELISA
• Foot and Mouth Disease
• Antibody Assessment
• Vaccination
• Argentina

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1. 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. P rez-Filgueira, S. Caspe, A. Capozzo Dr. Alejandra V. Capozzo, PhD HEAD OF THE APPLIED VETERINARY IMMUNOLOGY LAB. INSTITUTE OF VIROLOGY. INTA. BUENOS AIRES Open Session of the EuFMD - Cascais Portugal 26-28 October 2016

2. ARGENTINA 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. Open Session of the EuFMD - Cascais Portugal 26-28 October 2016

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

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

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

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

7. Does LPBE match VNT? LPBE: two different detector Ab Cut OFF - EPP 75%: LPBE titer 2.11 (Log10Titer , O1 Campos strain) LPBE-Mab Monoclonal Ab MIX LPBE Detector Ab Y LPBE-HIS Guinea-Pig Polyclonal Capture Ab (rabbit) 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 OS16

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

9. Concordance between VNT and LPB-ELISA Pearson s r=0,4432 (0.2576 - 0.5972) A B 6 6 LPBE-Mabs titer LPBE-HIS titer 4 4 EPP 75%=2.1 (Log10 titer) 2 2 0 0 0 1 2 3 4 0 1 2 3 4 VNT titer VNT titer C D EPP 75%=1,65 (Log10 titer) 2.0 2.5 OS16 2.0 1.5 AIE- OD 1.5 IE- OD 1.0 1.0 0.5 0.5 0.0 0.0 0 1 2 3 4 0 1 2 3 4 VNT titer VNT titer

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

11. Problem to solve: low specificity Avidity and subtyping of specific antibodies applied to the indirect assessment of heterologous protection against Foot-and-Mouth Disease Virus in cattle. Lavoria M , Di-Giacomo S, Bucafusco D, Franco-Mahecha OL, P rez- Filgueira DM, Capozzo AV. Vaccine. 2012 Mainly due to cross-reactive antibodies Natural antibodies 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 Avidity ELISA Binding strength Ag- Ab Low-avidity binders are removed using UREA Purified antigen Single dilution OS16

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

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

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

15. Accuracy of combined indirect assessment Level of agreement K value (VNT) TEST 1 Conditions (test 1) TEST 2 Selection (test 2) Sensitivity Specificity Accuracy Log 10Titer 1.6 classify NEGATIVE LPBE 87.9 OD 1.6 - classify NEGATIVE OD > 1.6 RETEST Log 10Titer 3.5 classify POSITIVE 1.6 < Log 10Titer < 3.5 RETEST 90.6 AE Cut off= 0.7 87.8 0.764 Moderate (0.80 0.93) (0.84 0.96) OD 0.3 classify NEGATIVE OD 1.1 classify POSITIVE OD 0.35-classify NEGATIVE OD 1.1 Classify POSITIVE 0.35 < OD < 1.1: RETEST 0.3 < OD < 1.1 RETEST AE IE Cut off= 1.1 88.8 92.6 94.8 84.4 IE AE 91.1 0.803 Strong (0.87 0.99) (0. 87 0.91) The best agreement values ( =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. OS16

16. Conclusions 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? OS16

17. Border belt Latest outbreak: 2011 O1 serotype All susceptible species are vaccinated FMDV-free with vaccination (cattle and buffaloes) Swine population (updatedNov. 2015) FMDV-free without vaccination PIGS are not vaccinated in Argentina <10,000 10,000-39,000 40,000-100,000 101,000-500,000 OS16

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

19. Immune responses to vaccination IgG2 IgG1 IgM 4 Ig1 titer O1 Campos 3 (Log10) 2 1 0 0 15 30 DPV 45 60 OS16

20. Placebo Vaccinated Placebo Vaccinated 4.0 3.0 4.0 3.0 Placebo Vaccinated Placebo Vaccinated LPBE titer O1 Campos LPBE titer O1 Campos VNT titer O1 Campos VNT titer O1 Campos 3.5 3.5 2.5 2.5 3.0 3.0 (Log10) (Log10) (Log10) (Log10) 2.0 2.0 Neutralizing Abs Total abs: LPBE 1.0 2.5 2.5 2.0 2.0 1.5 1.5 1.5 1.5 1.0 1.0 1.0 0.5 0.5 0.5 0.5 0 15 30 DPV 45 60 0 15 30 DPV 45 60 0 15 30 DPV 45 60 0 15 30 DPV 45 60 30 30 2.5 2.5 Vaccinated Vaccinated 2.0 2.0 Avidity index (%) Avidity index (%) Total abs: Indirect ELISA IE O1 Campos IE O1 Campos (OD 450nm) (OD 450nm) 20 20 Avidity ELISA 1.5 1.5 1.0 1.0 10 10 0.5 0.5 0.0 0 0.0 0 0 15 30 DPV 45 60 0 15 30 DPV 45 60 0 15 30 DPV 45 60 0 15 30 DPV 45 60 OS16

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

22. 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 OS16

23. MUCHAS GRACIAS Capozzo s Lab JUAN SALA, DVM Myrian Trotta Ms Biotec FLORENCIA MANSILLA, PhD APPLIED VETERINARY IMMUNOLOGY LAB. INSTITUTE OF VIROLOGY. INTA. OS16