ELISAs TO ASSESS ANTIFMDV ANTIBODIES INDUCED BY VACCINATION IN BUFFALO AND SWINE SERUM SAMPLES F Mansilla J Sala M Trotta Piscitelli F Bessone M PérezFilgueira ID: 1046936
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1. APPLICATION OF INDIRECT AND AVIDITY ELISAs TO ASSESS ANTI-FMDV ANTIBODIES INDUCED BY VACCINATION IN BUFFALO AND SWINE SERUM SAMPLESF. Mansilla,; J. Sala, M. Trotta; . Piscitelli; F. Bessone, M. Pérez-Filgueira, S. Caspe, A. Capozzo Dr. Alejandra V. Capozzo, PhDHEAD OF THE APPLIED VETERINARY IMMUNOLOGY LAB. INSTITUTE OF VIROLOGY. INTA. BUENOS AIRES
2. 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. We cared more about cattle…. Vaccines are applied to cattle and buffaloes - Pigs are not vaccinated 51,429,848 5,011,446 101,076ARGENTINA 2015Consumption (per inhabitant) 60 Kg10 Kg3rd producer in South America2nd producer in South America3rd producer in South America
4. Animals < 2 years old -twice a year Animals > 2 years old once a year FMDV-free without vaccinationFMDV-free with vaccinationLatest outbreak: 2011O1 serotypeBorder beltAll susceptible species are vaccinatedWater buffaloesBubalus bubalisMediterranean breed
5. Serological surveillance of vaccine efficacyVNTLPBEGold StandardCan use any field strainRequires live virus manipulationRequires cell cultureTime consumingDifficult to harmonizeNot useful for high-throughputDedicated high-containment facilitiesConcordance with VNT (bovine serum samples)High throughputEasy to harmonizeUses inactivated virusRequires two antibody preparations: capture and detection antibodiesNot useful for rapid set up assay for new filed variants75-EPP% Cut-off valueFor each vaccine strainProsConsVNT and LPBE titers of virus-specific antibodies have been related to protection against infection in cattle (PPG)Virus neutralization testLiquid phase blocking ELISA
6. Do ELISAs match VNT when using buffalo serum samples?
7. LPBE: two different detector AbCut OFF - EPP 75%: LPBE titer 2.11 (Log10Titer , O1 Campos strain)LPBEYYYYYCapture Ab (rabbit)Detector AbMonoclonal Ab MIXGuinea-Pig PolyclonalLPBE-MabLPBE-HISDoes LPBE match VNT?N=91 samples with different VNT titersN=27 Log10 VNT titers < 1.66N=64 Log10 VNT titers >1.66Cut OFF - EPP 75%: VNT titer=1,6 Log10TCID50
8. Do both LPBE-Mab and LPBE-HIS perform equally when using buffalo samples?r2=0.87Pearson´s r= 0.88YES
9. Pearson´s r=0,4432 (0.2576 - 0.5972)Concordance between VNT and LPB-ELISAEPP 75%=2.1 (Log10 titer)EPP 75%=1,65 (Log10 titer)
10. Concordance between VNT and LPB-ELISALow specificity
11. Problem to solve: low specificity Mainly due to cross-reactive antibodies Natural antibodies Avidity ELISABinding strength Ag- AbLow-avidity binders are removed using UREASingle dilutionPurified antigenAvidity 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. 2012Accuracy 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. Indirect and avidity ELISAsIEYYIndirect ELISA, purified virus, single dilutionAEYYAvidity indirect ELISA: low avidity binders are washed way with Urea, purified virus, single dilutionYYYN=91 samples with different VNT titersCut OFF - EPP 70%: VNT titer=1,6 Log10TCID50Single dilution testPurified inactivated antigenNeither detector nor capture Ab neededThree steps
13. Concordance between VNT* and ELISAs* VNT is the accepted assay for vaccine efficacy testing in the European Pharmacopoeia.
14. LOW VNT titers<1.4HIGH VNT titers> 3Classified correctly with tests that measure total antibodiesVNT titers1.4 – 3.0Avidity ELISAImproving concordance with VNTVNT titers
15. Accuracy of combined indirect assessmentThe best agreement values (κ=80%) were obtained when combining results of both single dilution ELISAsAdditional benefit: high-throughput reproducible assessment that only takes a few hours of work to the result. Log 10Titer≤ 1.6 classify NEGATIVELog 10Titer≥ 3.5 classify POSITIVE1.6 < Log 10Titer < 3.5 RETESTIEAEOD ≤ 0.3 classify NEGATIVEOD ≥ 1.1 classify POSITIVE0.3 < OD < 1.1 RETEST88.892.6
16. 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. PIGS are not vaccinated in Argentina101,000-500,00040,000-100,00010,000-39,000<10,000Swine population (updated Nov. 2015)FMDV-free without vaccinationFMDV-free with vaccination(cattle and buffaloes)Latest outbreak: 2011O1 serotypeBorder beltAll susceptible species are vaccinated
18. Immunized with comercial vaccineDose: 2 ml; SCThe same applied in cattlePiglets (12 weeks old)Control group : immunized with oil adjuvant + PBS.2ml/dose; SCSerology against O1 Campos and A 24 Cruzeiro strains0dpv10dpv21dpv60dpvImmune responses to vaccination: experimental designLiquid phase blocking ELISA (LBPE)Indirect ELISA Avidity ELISAIsotype ELISAsn= 12n= 3
19. Immune responses to vaccination
20. Total abs: IndirectELISAAvidity ELISANeutralizing AbsTotal abs: LPBE
21. Do avidity and LPBE match VNT?Avidity correlates with VNT
22. ConclusionsAvidity ELISA emerges as a promising low-labor high throughput alternative to VNTAvidity matches better VNT titers than LPBE and does not require standardized sera, facilitating the assessment of antibodies against new field strainsIndirect ELISA and LPBE provide similar informationEveryone is invited to get the protocol/training and run their samples
23. JUAN SALA, DVMFLORENCIA MANSILLA, PhDMyrian TrottaMs BiotecAPPLIED VETERINARY IMMUNOLOGY LAB. INSTITUTE OF VIROLOGY. INTA. Capozzo´s LabMUCHAS GRACIAS