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FLUTCORE project meeting:

WP3- Immunogenicity studies

I-Na Lu PhD, CRP-SantéGroup Leader: Prof. Dr. Claude Muller

Institute of ImmunologyCentre de Recherche Public de la Santé (CRP-Santé)

Europa Royale Hotel, Riga

16th Sep 2014

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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

Wang & Palese, Nat Struct Mol Biol 2009

Influenza virus hemagglutinin (HA)

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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

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Influenza virus life cycle highlighting the three distinct mechanism of actions of HA stalk-binding Abs

Modified from Brandenburg et al, PlosOne 2013

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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

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HA(monomer)

HeadlessHA

ΔHA stalk

HA 292-351

HA 292-402

HA 292-474

HA 306-351

HA 306-402

HA 306-474

HA 351-402

HA 351-474

HA 403-474

HA 420-474

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

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ΔHA stalk

HA 292-351

HA 292-402

HA 292-474

HA 306-351

HA 306-402

HA 306-474

HA 351-402

HA 351-474

HA 403-474

HA 420-474

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

No

No

Yes (HA2.9)

ongoing

No

ongoing

Yes (HA2.3)

ongoing

No

ongoing

ongoing

Good quantity

Low quantity

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Positions of the HA403-474 (HA2.3) epitopes on the structure of the trimeric HA protein of pH1N1 (A/Luxembourg/46/2009)

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Positions of the HA292-474 (HA2.9) epitopes on the structure of the trimeric HA protein of pH1N1 (A/Luxembourg/46/2009)

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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)

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

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Outline

• Headless HA proteins incorporated into VLPs

• Immunological studies

• Challenge-protection assays

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

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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)

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

(N=6 per group)

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Antiviral functions of CD4+ T cells

Swain et al, Nat Rev Immunol 2012

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

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*

* **

*

*

* N=6 per group* P<0.5

Functional profile of the memory HA stalk-specific CD4+ T cells responses

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Outline

• Headless HA proteins incorporated into VLPs

• Immunological studies

• Challenge-protection assays

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Mouse-adapted influenza viruses

Bouvier, Viruses 2010

A/Anhui/01/2013 (H7N9)

BSL-2

BSL-3

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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

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HA403-474