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

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

WP1 – evaluation of back-up strategy I Single HBc gene expression II Development of long homodimer constructs III Chemical coupling of HA2.3

Single gene expression in E.coli Improved purification protocol developed AmS precipitation tº treatment DEAE AEC S500 SEC HBc176_K1 HBc176_M2e

Single gene expression in E.coli Expression and purification of HA2.3 (403-474) antigen TEV 6His MBP HA2.3 - Cys Advantage: Trimer according to SEC Disadvantages: Low outcome Hard to scale-up Expensive due to TEV cleavage

Single gene expression in E.coli Structure determination of HA2.3 (403-474) antigen Cys Cys Trimeric state confirmed (A) Resembles HA post-fusion structure (B)

Single gene expression in E.coli Expression optimization of HA2.3 antigen HA2.3-C HA2.3-N Construct with C-Cys gives better production Purification method developed -C -N Trimer structure retained Suitable for chemical coupling

Single gene expression in E.coli All the components ready HA2.3-C C176_M2e + c176_K1-HA2.3 = vaccine candidate

Development of long homodimer constructs Optimization of connector linker ? Band of ~21-22 kDa detected instead of ~44 kDa Strong signal in Ouchterlony assay The highest titers with GGSx6 linker

Development of long homodimer constructs Characterization of HBcDi6 construct Thermally stable even after 65 ºC VLPs detected by EM Two bands detected by MS: ~21.5 and 22.5 kDa

Development of long homodimer constructs Construction and characterization of HBcDi6_K1_M2e Band of ~23-24 kDa instead of ~47 kDa VLPs detected by EM Both monomers present in VLPs???

Chemical coupling of HA2.3 to VLPs + + HS- -NH2 Sulfo-KMUS S- -NH

Chemical coupling of HA2.3 to VLPs Coupling to Qb VLPs Conclusion: Coupling of HA2.3 to VLPs is possible Qb+HA2.3 HA2.3 non-bind Qb VLPs Qb VLPs+linker Qb VLPs+linker+HA2.3

Chemical coupling of HA2.3 to VLPs Coupling to HBc176_K1 VLPs K1+HA2.3 VLPs after coupling HA2.3 K1 VLPs+linker K1 VLPs+linker+HA2.3

Chemical coupling of HA2.3 to VLPs Coupling to HBc176_K1_M2e VLPs K1_M2 dimer Optimization done: linkers concentrations K1_M2+HA2.3? K1_M2 monomer HA2.3 No promising results up to now…

Coming back to “component” vaccine HA + All the components obtained in E.coli system Purification fast and scalable High-quality VLPs Immunogenicity tests need to be performed

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