1. London, 23 rd 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 Health Nastasja Hauck

2. 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

3. Set-up ΔHA stalk LAHVLPMOCK ΔHA 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 ): SAH

4. Workflow RNA extraction cDNA preparation Second strand synthesis Library amplification Sequencing Data analysis

5. Sequencing

6. 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…

7. UID method Adapted and modified from Vollmers et al, PNAS, 2013

8. 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

9. 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

10. UID method for virus project HA monomer

11. Nucleotide heterogeneity of LAH of pH1N1 virus Preliminary data Including all sequencesExcluding the ones that don’t meet our criteria

12. Alignment of LAH sequence on nucleotide and amino acid level Preliminary data On amino acid level On nucleotide level

13. 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

14. 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