London, 23rd September 2015

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 HealthNastasja Hauck

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

Set-up

ΔHA stalk LAH VLP MOCK

ΔHA includes LAH and VLP

LAH and VLP

VLP

ΔHA stalk (HA292-553):

HA306-474 :

HA2.3 (HA403-474):

LAH (HA420-474):

SAH

Workflow

RNA extraction

cDNA preparation

Second strand

synthesis

Library amplification Sequencing Data analysis

Sequencing

Advantages of using the IonTorrentDeep coverage High throughput

Problems the IonTorrent is creatingEspecially when there are homopolymers the change in pH is often not detected properlyIndels

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

9678

683108

8844

3833

32

22

21

1306 N°1

N°2

N°3

N°4

N°5

N°6

N°7

N°8

N°9

N°10

others 9678

683

10888 44 3833 32 22 21

N°1 N°2

N°3 N°4

N°5 N°6

N°7 N°8

N°9 N°10

Preliminary data

Including all sequences Excluding the ones that don’t meet our criteria

Alignment of LAH sequence on nucleotide and amino acid level

Preliminary data

On amino acid level

On nucleotide level

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