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
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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
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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
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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