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

Outline Headless HA proteins incorporated into VLPs Immunological studies Challenge-protection assays

Outline Headless HA proteins incorporated into VLPs Immunological studies Challenge-protection assays

& Palese, Nat Struct Mol Biol 2009 Influenza virus hemagglutinin (HA)

Comparisons between the features of influenza HA globular head epitopes and HA stalk region epitopes Modified from Ellebedy et al, Front Immunol 2012 Receptor binding domain Viral envelope Globular head region Stalk region

Influenza virus life cycle highlighting the three distinct mechanism of actions of HA stalk-binding Abs Modified from Brandenburg et al, PlosOne 2013

Binding and neutralization mechanism of a mouse HA stalk-specific antibody, C179 Modified from Dreyfus et al, J Virology 2013 C179 inhibits the HA pH-induced conformational change that drives membrane fusion C179 binds multiple group 1 subtypes Exposure to low pH converts HA to a post-fusion state that is sensitive to trypsin digestion Pre-incubation with C179 prevents this conversion, retaining the HA in the protease-resistant

HA (monomer) Headless HA ΔHA stalk HA HA HA HA HA HA HA HA HA HA Schematic of headless HA constructs based on the structure of the HA protein of pH1N1 (A/Luxembourg/46/2009)

ΔHA stalk HA HA HA HA HA HA HA HA HA HA Schematic of headless HA constructs based on the structure of the HA protein of pH1N1 (A/Luxembourg/46/2009) Headless HA In vivo Abs to rHA No Yes (HA2.9) ongoing No ongoing Yes (HA2.3) ongoing No ongoing Good quantity Low quantity

Positions of the HA (HA2.3) epitopes on the structure of the trimeric HA protein of pH1N1 (A/Luxembourg/46/2009)

Positions of the HA (HA2.9) epitopes on the structure of the trimeric HA protein of pH1N1 (A/Luxembourg/46/2009)

Surfaces of the C179-binding, HA & HA epitopes on the structure of the trimeric HA protein C179 binding epitope HA (HA2.3) HA (HA2.9)

HA stalk-specific seroreactivity induced by sublethal dose infection with pH1N1 (A/Luxembourg/46/2009)

Outline Headless HA proteins incorporated into VLPs Immunological studies Challenge-protection assays

(rHA: H1; A/California/07/2009) Seroconversion - ELISA

HA (monomer) HA stalk … Peptide library spanning the HA stalk protein of pH1N1 (A/Luxembourg/46/2009) HA stalk peptide pool (15-mer peptides overlapping by 12 residues)

Positions of the CD4+ T cell epitopes on the structure of the HA protein of H1N1 (A/Luxembourg/46/2009) (N=6 per group)

Antiviral functions of CD4+ T cells Swain et al, Nat Rev Immunol 2012

Flow cytometry profiles of induced CD4+ T cells responses by the HA stalk peptides

* * * * * * * N=6 per group * P<0.5 Functional profile of the memory HA stalk-specific CD4+ T cells responses

Outline Headless HA proteins incorporated into VLPs Immunological studies Challenge-protection assays

Mouse-adapted influenza viruses Bouvier, Viruses 2010 A/Anhui/01/2013 (H7N9) BSL-2 BSL-3

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 Acknowledgment

HA