23 rd September 2015 B cell response and escape mutants Identification of protective hemagglutinin stalkspecific B cell receptor sequences Identification of antigenic sites on the HA stalk ID: 410640
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Slide1
London, 23rd September 2015
B cell response and escape mutants
Identification of protective hemagglutinin stalk-specific B cell receptor sequencesIdentification of antigenic sites on the HA stalk of pH1N1 and phenotypic variation of escape mutants
Luxembourg Institute of Health
Nastasja HauckSlide2
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
protectionSlide3
Set-up
Δ
HA stalk
LAH
VLP
MOCK
Δ
HA includes LAH and VLP
LAH
and VLP
VLP
ΔHA stalk (HA
292-553
):
HA
306-474
:
HA2.3 (HA
403-474
):
LAH (HA
420-474
):
SAHSlide4
WorkflowSlide5
SequencingSlide6
Advantages of using the IonTorrent
Deep coverage High throughput
Problems the IonTorrent is creatingEspecially when there are homopolymers the change in pH is often not detected properlyIndelsHow we handle the challenges…Slide7
UID method
Adapted and modified from Vollmers et al, PNAS, 2013 Slide8
Ongoing work
First we will continue analysing the data on DNA levelNext we will continue with the sequencing and then analysing on RNA levelComparing DNA and RNA levelSlide9
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 stressSlide10
UID method for virus project
HA monomerSlide11
Nucleotide heterogeneity of LAH of pH1N1 virus
Preliminary data
Including all sequencesExcluding the ones that don’t meet our criteriaSlide12
Alignment of LAH sequence on nucleotide and amino acid level
Preliminary data
On amino acid levelOn nucleotide levelSlide13
Ongoing work
The next step will be to process the lungs from three mice that were immunized but still died after the challengeWe 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 dieThat’s where B cell project and virus project come togetherSlide14
Thank you for your attention!
Thank you to:Prof. Dr. Claude P. MullerDr. I-Na LuSophie FarinelleAurélie Sausy
Regina SinnerJosiane KirpachJean-Philippe BürckertWilliam Faison