EuFMD 2024 - S.Berryman FMD vaccine quality_a universal test for antigen based on detection of VP4.pdf

EuFMD 2024 - S.Berryman FMD vaccine quality_a universal test for antigen based on detection of VP4.pdf

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  Alcalá de Henares(Madrid, Spain), 29-31 October 2024 EuFMD Open Session | OS24 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 S Berryman1, A Asfor1, A Yasmin1, C Grant1, A Ludi1, E Perez1, E Benham1, N Howe1, A Burman1, E Brocchi2, S Grazioli2, D King1, T Tuthill1 1 Pirbright Institute, Ash Road, Pirbright, UK 2Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Brescia, Italy
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 2 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 VP4 + VP4 - VP4 Heat Acid +VP2 +VP2 ELISAs to Assess Vaccine Integrity – Principle: VP4 is present in intact antigen but absent from dissociated subunits. Problem – Sufficient 146S content crucial to FMDV vaccine effectiveness. Current gold standard to measure 146S (sucrose density gradient) is slow, low throughput and requires expensive equipment Solution – Universal pan serotypic ELISA tests for 146S/Total Antigen – Faster, higher throughput, no requirement for ultracentrifuge etc. Intact antigen (146S) Protective Response Unstable Dissociated subunits (12S) Reduced Immunogenicity
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 3 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 VP4 + VP4 - VP4 Both VP4 and VP2 Mab are pan serotypic Intact antigen (146S) Protective Response Unstable Dissociated subunits (12S) Reduced Immunogenicity Heat Acid +VP2 +VP2 ELISAs to Assess Vaccine Integrity – Principle: VP4 is present in intact antigen but absent from dissociated subunits. VP4 lost on particle dissociation – ELISA to detect VP4 detects intact antigen VP2 is present in intact and dissociated antigen – VP2 ELISA detects total antigen
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 Vaccine Integrity ELISA - Using VP4 Mab to Quantify Intact Antigen Universal Capture (Integrin) Intact Antigen (146S) VP4 Mab HRP Secondary VP4 Mab Doesn’t Bind Dissociated Antigen (12S) Universal Capture (Integrin) No Signal ELISA Signal ELISA Plate Coat plate with Integrin Universal Capture Reagent Block wells using non-fat dry milk Add Vaccine Sample/ Positive control Treat with crosslinker – Required to make VP4 epitope available for antibody binding Add anti-VP4 Mab HRP Secondary Add anti-Ms HRP Secondary antibody Add ELISA Substrate (OPD/TMB)
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 Vaccine Integrity ELISA - Using VP2 Mab to Quantify Total Antigen Universal Capture (Integrin) Intact Antigen (146S) VP2 Mab HRP Secondary VP2 Mab Dissociated Antigen (12S) Universal Capture (Integrin) ELISA Signal ELISA Plate Coat plate with Integrin Universal Capture Reagent Block wells using non-fat dry milk Add Vaccine Sample/ Positive control Add anti-VP2 Mab HRP Secondary Add anti-Ms HRP Secondary antibody Add ELISA Substrate (OPD/TMB) Higher ELISA signal due to greater epitope availability
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 Purified 146S Particles require treatment with crosslinker to be detected by VP4 Mab Treat with doubling quantities of crosslinker post integrin capture Purified Virus separated on sucrose gradient – Fractions Assayed by Capture ELISA – Fractions -/+ Crosslinker Treatment – VP4 Mab Pentamers Virions Integrin Capture ELISA – 20ng Purified 146S Virus Per Well – Post Capture Crosslinker Treatment – VP4 Mab
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 VP4 Mab shows specificity for 146S virions over heat/acid dissociated material (12S pentamers) Purified Virus (+- Acid dissociation) separated on sucrose gradient – Fractions Assayed by Capture ELISA – Fractions Detected by VP2 Mab Pentamers Virions Pentamers Virions VP4 Mab VP2 Mab Purified virus – Vast majority is intact virions Acid treatment – 146S virions dissociated in to pentamers Purified Virus (+- Acid dissociation) separated on sucrose gradient – Fractions Assayed by Capture ELISA – Fractions Detected by VP4 Mab VP4 Mab – Detects 146S virions in intact sample but not 12S pentamers in dissociated sample
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 Application of Integrity ELISA to vaccine samples: ELISA vs Density Gradients Data from ELISA matches that from sucrose density gradient analysis. Vaccine A 0.0 0.5 1.0 1.5 2.0 2.5 Total antigen Intact antigen V P 2 V P 4 VP4 (dissociated) Vaccine B Fractions OD 492 0.0 0.5 1.0 1.5 2.0 2.5 0 0.5 1 1.5 2 2.5 3 0 10 20 0 0.5 1 1.5 2 2.5 3 0 10 20 Total antigen Intact antigen V P 2 V P 4 VP4 (dissociated) Fractions OD 492 Vaccine C 0.0 0.5 1.0 1.5 2.0 2.5 Total antigen Intact antigen V P 2 V P 4 VP4 (dissociated) 0 0.5 1 1.5 2 2.5 3 0 10 20 O D 4 9 2 Fractions OD 492 OD 492 OD 492 OD 492 VP4/VP2 ELISA Gradient Analysis Intact antigen Dissociated antigen Intact antigen Dissociated antigen
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 Application of Integrity ELISA to vaccine samples: ELISA vs Serology Data from ELISA matches that from vaccine serology Vaccine A 0.0 0.5 1.0 1.5 2.0 2.5 Total antigen Intact antigen V P 2 V P 4 VP4 (dissociated) Vaccine B OD 492 0.0 0.5 1.0 1.5 2.0 2.5 Total antigen Intact antigen V P 2 V P 4 VP4 (dissociated) OD 492 Vaccine C 0.0 0.5 1.0 1.5 2.0 2.5 Total antigen Intact antigen V P 2 V P 4 VP4 (dissociated) OD 492 VP4/VP2 ELISA Vaccine Serology 10 100 1000 No titre 10 100 1000 No titre 10 100 1000 No titre VNT Titre VNT titre VNT titre Serotype 1 Serotype 2 Serotype 3 Serotype 1 Serotype 2 Serotype 3 Serotype 1 Serotype 2 Serotype 3
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  OS24 Alcalá de Henares(Madrid), 29-31 October 2024 ● Developed universal pan-serotypic ELISAs to assess the 146S content (VP4 ELISA) and total antigen content (VP2 ELISAs) of vaccine antigens both before and after formulation. Quicker and higher throughput than current gold standard. ● Ongoing B&M GATES Foundation funded project to further develop and validate the tests, working with a number of vaccine companies ● Prototype Kit version of ELISA developed with IZSLER and recently transferred to vaccine companies for testing/evaluation ■ Contains reagents required for tests – plates, antibodies, buffers etc ■ Simplified workflow: --Plates precoated with integrin and preblocked --HRP-conjugated VP4/VP2 Mabs (eliminates need for secondary antibody) ● Serotype specific versions of ELISAs under development – Replace integrin as capture with serotype specific capture reagent – would allow components of multivalent samples to be assessed separately 10 Foot-and-mouth disease vaccine quality: A universal test for intact antigen based on detection of VP4 Summary and Further Developments
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  Thank you !. Stephen Berryman [email protected] #os24 #movefast