Transcript Nastasja - Flutcore

B cell response and escape mutants
• Identification of protective hemagglutinin stalk-specific B cell receptor sequences
• Identification of antigenic sites on the HA stalk of pH1N1 and phenotypic variation of
escape mutants
Luxembourg Institute of Health
Nastasja Hauck
London, 23rd September 2015
Identification of protective hemagglutinin stalk-specific B cell receptor sequences
What does the repertoire look like in vaccinated compared to unvaccinated mice?
Antibody heavy chain (V, D, J gene usage; lengths and features of the complementarity determining
region 3 (CDR3))
Can we identify a protective clone?
The mouse study is a proof of concept for the future human research
identification of novel correlates of immunity and protection
SAH
ΔHA stalk (HA292-553):
HA306-474 :
HA2.3 (HA403-474):
LAH (HA420-474):
Set-up
ΔHA stalk
LAH
VLP
ΔHA includes LAH and VLP
LAH and VLP
VLP
MOCK
Workflow
RNA
extraction
cDNA
preparation
Second
strand
synthesis
Library
amplification
Sequencing
Data analysis
Sequencing
Advantages of using the IonTorrent
Deep coverage
High throughput
Problems the IonTorrent is creating
Especially when there are homopolymers the change in pH is often not detected properly
Indels
How we handle the challenges…
UID method
Adapted and modified from Vollmers et al, PNAS, 2013
Ongoing work
First we will continue analysing the data on DNA level
Next we will continue with the sequencing and then analysing on RNA level
Comparing DNA and RNA level
Identification of antigenic sites on the HA stalk protein of pH1N1 virus and phenotypic
variation of escape mutants
If a mouse is immunized and then checked for good antibody titres and still dies after the challenge
what has happened? Has the virus “managed” to escape?
The influenza virus exists naturally as a quasi species
Antibody induced stress
UID method for virus project
HA monomer
Nucleotide heterogeneity of LAH of pH1N1 virus
Including all sequences
Excluding the ones that don’t meet our criteria
22
88
32
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N°1
1306
N°1
683
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108
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683
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9678
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others
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Preliminary data
Alignment of LAH sequence on nucleotide and amino acid level
On nucleotide level
On amino acid level
Preliminary data
Ongoing work
The next step will be to process the lungs from three mice that were immunized but still died after
the challenge
We want to compare the distribution/ proportion of the different sequences between the original
stock and those three lungs
Comparing this to lungs from mice that didn’t die
That’s where B cell project and virus project come together
Thank you for your attention!
Thank you to:
Prof. Dr. Claude P. Muller
Dr. I-Na Lu
Sophie Farinelle
Aurélie Sausy
Regina Sinner
Josiane Kirpach
Jean-Philippe Bürckert
William Faison