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

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

3. Single gene expression in E.coli
Improved purification protocol developed
AmS precipitation
tº treatment
DEAE AEC
S500 SEC
HBc176_K HBc176_M2e

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

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

6. 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

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

8. 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

9. 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

10. 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???

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

12. 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

13. 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

14. 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…

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

16. 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