FLUTCORE project meeting: WP3- Immunogenicity studies I-Na Lu PhD, CRP-Santé Group Leader: Prof. Dr. Claude Muller Institute of Immunology Centre de Recherche Public de la Santé (CRP-Santé) Europa Royale Hotel, Riga 16th Sep 2014 www.crp-sante.lu Outline • Headless HA proteins incorporated into VLPs • Immunological studies • Challenge-protection assays www.crp-sante.lu Outline • Headless HA proteins incorporated into VLPs • Immunological studies • Challenge-protection assays www.crp-sante.lu Influenza virus hemagglutinin (HA) Wang & Palese, Nat Struct Mol Biol 2009 www.crp-sante.lu Comparisons between the features of influenza HA globular head epitopes and HA stalk region epitopes Stalk region Globular head region Receptor binding domain Viral envelope Modified from Ellebedy et al, Front Immunol 2012 www.crp-sante.lu Influenza virus life cycle highlighting the three distinct mechanism of actions of HA stalk-binding Abs Modified from Brandenburg et al, PlosOne 2013 www.crp-sante.lu Binding and neutralization mechanism of a mouse HA stalk-specific antibody, C179 C179 binds multiple group 1 subtypes C179 inhibits the HA pH-induced conformational change that drives membrane fusion Exposure to lowwith pH C179 converts HA to this a Pre-incubation prevents post-fusion that is to conversion,state retaining thesensitive HA in the trypsin digestion protease-resistant Modified from Dreyfus et al, J Virology 2013 www.crp-sante.lu Schematic of headless HA constructs based on the structure of the HA protein of pH1N1 (A/Luxembourg/46/2009) ΔHA stalk HA 292-351 HA 292-402 HA 292-474 HA 306-351 HA 306-402 HA 306-474 HA (monomer) Headless HA HA 351-402 HA 351-474 HA 403-474 HA 420-474 www.crp-sante.lu Schematic of headless HA constructs based on the structure of the HA protein of pH1N1 (A/Luxembourg/46/2009) In vivo Abs to rHA Headless HA Good quantity Low quantity No ΔHA stalk No HA 292-351 No HA 292-402 No HA 292-474 Yes (HA2.9) HA 306-351 ongoing HA 306-402 No HA 306-474 ongoing HA 351-402 ongoing HA 351-474 ongoing HA 403-474 Yes (HA2.3) HA 420-474 ongoing www.crp-sante.lu Positions of the HA403-474 (HA2.3) epitopes on the structure of the trimeric HA protein of pH1N1 (A/Luxembourg/46/2009) www.crp-sante.lu Positions of the HA292-474 (HA2.9) epitopes on the structure of the trimeric HA protein of pH1N1 (A/Luxembourg/46/2009) www.crp-sante.lu Surfaces of the C179-binding, HA292-474 & HA403-474 epitopes on the structure of the trimeric HA protein C179 binding epitope HA403-474 (HA2.3) HA292-474 (HA2.9) www.crp-sante.lu HA stalk-specific seroreactivity induced by sublethal dose infection with pH1N1 (A/Luxembourg/46/2009) www.crp-sante.lu Outline • Headless HA proteins incorporated into VLPs • Immunological studies • Challenge-protection assays www.crp-sante.lu Seroconversion - ELISA (rHA: H1; A/California/07/2009) www.crp-sante.lu Peptide library spanning the HA stalk protein of pH1N1 (A/Luxembourg/46/2009) … HA (monomer) HA stalk HA stalk peptide pool (15-mer peptides overlapping by 12 residues) www.crp-sante.lu Positions of the CD4+ T cell epitopes on the structure of the HA protein of H1N1 (A/Luxembourg/46/2009) (N=6 per group) www.crp-sante.lu Antiviral functions of CD4+ T cells Swain et al, Nat Rev Immunol 2012 www.crp-sante.lu Flow cytometry profiles of induced CD4+ T cells responses by the HA stalk peptides www.crp-sante.lu Functional profile of the memory HA stalk-specific CD4+ T cells responses * * * * * * * N=6 per group * P<0.5 www.crp-sante.lu Outline • Headless HA proteins incorporated into VLPs • Immunological studies • Challenge-protection assays www.crp-sante.lu Mouse-adapted influenza viruses BSL-2 BSL-3 A/Anhui/01/2013 (H7N9) Bouvier, Viruses 2010 www.crp-sante.lu Acknowledgment Prof. Dr. Claude P Muller (project supervisor) Sophie Farinelle (animal exp., ELISA, ELISpot) Aliki Papakonstantinou (molecular cloning) Aurélie Sausy (expansion of viruses) Institute of Immunogy, CRP-Santé René Brons (flow cytometry) Core Facility of Flow Cytometry, CRP-Santé Dr. Catherine Leonard (ELISpot, ICS assay) Laboratory of Immunogenetics and Allergology, CRP-Santé Dr. Mike Whelan (project supervisor) Alex Ramirez (animal exp.) Sophie Maucourant (molecular cloning) iQur Ltd www.crp-sante.lu HA403-474 www.crp-sante.lu