1. Latvian Biomedical Research and Study Centre (BMC), Riga, Latvia

2. Testing of HA stalk constructs Report summary Testing of M2e constructs Evaluation of long homodimer constructs Evaluation of new M2e constructs Testing of K1K1 construct Evaluation of new HA stalk constructs

3. Comparison of CoHe constructs carrying different HA stalk fragments WP1 - part I - tandem core NoConstruct Mw, kDa 1pET coHe7HA1(306-351), empty46.8 2pET coHe7HA2(306-402), empty52.3 3pET coHe7HA3(403-474), empty50.2 4pET coHe7LAH(420-474), empty49.4 5pET coHe7∆HAStalk,empty77.9 6pET coHe7empty, Headless HA78.0 7pET coHe7empty, HA2(306-402)52.4 8pET coHe7empty, HA4(296-402)~55.0 9pET coHe7empty, LAH(420-474)49.5 All except No5 and No6 produces product of corresponding Mw

4. Solubility of recombinant HBc-HA proteins At least part of protein is in soluble fraction No5 is insoluble. No6 is degraded No detectable immunodiffusion bands, except No6 WP1 - part I - tandem core

5. Synthesis and solubility of recombinant HBc-M2e proteins Minor part of protein is in soluble fraction No detectable immunodiffusion bands, except No5; confirmed VLP formation NoConstruct 1pETcoHe7e,M2eSP 2pETcoHe7e,M2e C19S 3pETcoHe7e,M2e C17,19S 4pETcoHe7e,M2e C17,19S SP 5pETcoHe7(M2e C17,19S ) 3 SP WP1 - part I - tandem core

6. Purification of KM71H pHe7 K1,K1 (codon-optimized) from P.pastoris Immunodiffusion (+) Survives AmS precipitation Confirmed VLP formation WP1 - part I - tandem core

7. Testing of HA stalk constructs Testing of M2e constructs Evaluation of long homodimer constructs Evaluation of new M2e constructs Testing of K1K1 construct Evaluation of new HA stalk constructs WP1 - part II – single gene expression

8. Evaluation of new M2e constructs WP1 - part II – single gene expression 1 176 G 4 SG 4 SLLTEVETPIRNEWGCRCNDSSD TEVETPIRNEWGSRSNDSSD

9. Evaluation of new M2e constructs WP1 - part II – single gene expression TEVETPIRNEWGSRSNDSSD SLLTEVETPIRNEWGSRSNDSSD tº treatmentAmS precipitationS500 chromatography VLPs for immunization?

10. Evaluation of new HA stalk constructs WP1 - part II – single gene expression HA2.3 (403-474) as the most prospective immunologically No VLPs detected by expression in tandem core Chemical coupling as a backup strategy linker M2e HA3 HA2.3 needed for coupling as a single protein K

11. Expression and purification of HA2.3 as a single protein WP1 - part II – single gene expression HA3 - Cys MBP TEV 6His 1. Expression 3-4. TEV cleavage 2. Ni-affinity 5.-7. AEC HA2.3 trimer? SEC profile aggregates and VLPs HA3 monomer

12. Expression and purification of HBc176_K1 VLPs WP1 - part II – single gene expression 1 176 G 4 SG 4 SGGS K GGSG 4 SG 4 S tº treatmentAmS precipitationS500 chromatography

13. Chemical coupling of HA2.3 to HBc_K1 VLPs WP1 - part II – single gene expression -NH 2 + HS- -NH S- Sulfo-KMUS +

14. Design of long homodimer construct WP1 - part II 1 176 GGSx1→GGSx3→GGSx5 No detectable expression in E. coli GGSx6→GGSx9 Constructs in progress linker M2e HA2.3 K

15. Our group Dr.biol. Tatjana Voronkova Inara Akopjana Dr.biol. Kaspars Tars Dr.biol. Andris Kazaks Svetlana Kotelovica Special thanks for electron microscopy pictures: Dr. Velta Ose