1. Cloning and development of constructs

2. Cloning overview Construct design Cloning into pPICZ plasmid (bacteria) Yeast cloning (P. pastoris KM71H) Determination of insert copy number (qRT-PCR) Construct confirmation (sequencing) Final clone Isolation of single cell clones Selection of high copy clones (2 mg/ml zeocin) Determination of insert copy number (qRT-PCR)

3. Current constructs Double insert (no empty core) Truncated product Improved by alteration of fermentation method Single insert (one empty core) Works really well

4. Constructs in development

5. K1, SP(M2e) 3 SP Colony morphology after freezing steps Sequencing of VLP coding sequence CFU stability after freezing steps Stability of Insert copy number after freezing steps

6. LAH HA2.3, SP(M2e)3SP Co1-2 C61S The presence of a linker in tandem core should allow both cores to move independently A disulphide bridge may form between both cores (cys 61) The flexibility of the linker may be fundamentally impaired due to the structural limitations imposed by a disulphide bridge Mutation of the cys 61 residue to a ser in both cores The linker is now the only connection between the two cores. This should allow more movement of the two cores

7. LAH HA2.3, SP(M2e)3SP Co1-2 C61S Design of Co1a-C61S and Co2a-C61S First cloning step Substitution of Co1a with Co1a-C61S Second cloning step Substitution of Co2a with Co2a-C61S Yeast cloning and selection of high copy clones P. pastoris KM71H transformation and selection of high copy clones using 2 mg/ml zeocin Selection of the highest copy clone with qRT-PCR

8. LAH HA2.3, SP(M2e)3SP Co1-2 C61S Anti-M2 (14C2) HA2.3,(M2e) 3 1:2 HA2.3,(M2e) 3 1:4 HA2.3,(M2e) 3 1:8 rHBc (50ng) Marker HA2.3,(M2e) 3 Anti-core (10E11) HA2.3,(M2e) 3 1:2 HA2.3,(M2e) 3 1:4 HA2.3,(M2e) 3 1:8 rHBc (50ng) Marker HA2.3,(M2e) 3 HA2.3,(M2e) 3 1:2 HA2.3,(M2e) 3 1:4 HA2.3,(M2e) 3 1:8 rHBc (50ng) Marker HA2.3,(M2e) 3 SDS-PAGE LAH HA2.3, SP(M2e) 3 SP Co1-2 C61S LAH HA2.3, SP(M2e) 3 SP TEM

9. LAH HA2.3, K1 Construct generation Substitution of SP(M2e)3SP with K1 insert Yeast cloning and selection of high copy clones P. pastoris KM71H transformation and selection using 1 mg/ml zeocin Selection of low, medium and high copy clones with qRT-PCR

10. LAH HA2.3, K1-K1, SP(M2e)3SP A Yeast cloning and selection of high copy clones P. pastoris KM71H LAH HA2.3K1 # 20 transformation and selection of high copy clones using 2 mg/ml zeocin Selection of the highest copy clone with qRT-PCR

11. LAH HA2.3, K1-K1, SP(M2e)3SP A Marker Control CLControl S Control IN #33 CL #33 S#33 IN 10- 15- 20- 25- 37- 50- 75- 100- 150- 250- KDa 10- 15- 20- 25- 37- 50- 75- 100- 150- 250- KDa 10- 15- 20- 25- 37- 50- 75- 100- 150- 250- KDa #32 CL #32 S #32 IN Marker#51 CL #51 S#51 IN #49 CL #49 S #49 IN Marker#56 CL #56 S #56 IN #44 CL #44 S #44 IN Anti-M2 (14C2) Control: LAHHA2.3,K1 Anti-core (10E11)

12. LAH HA2.3, K1-K1, SP(M2e)3SP B BamHI BamHI and BglII

13. BamHI LAH HA2.3, K1-K1, SP(M2e)3SP B Ligation BamHI Ligation BglII BamHI G CCTAG BglII GATCT A

14. LAH HA2.3, K1-K1, SP(M2e)3SP B Bacterial cloning transformation into E. Coli DH5α cells Selection of positive clones Low salt LB agar containing zeocin PCR amplification of Core 1 Marker#1#2#3#4#5#6#7#8#10#9K1M2 HA2.3K1 negative 500- 1000- bp Marker #11#12#13#14 #15 #16#17#18 #20 #19 K1M2 HA2.3K1 negative 500- 1000- bp 1500- 2000- 3000- Marker#2#3#5#8#10#9 #12#13#18 #20 Cut back ( BamHI+BglII )

15. LAH HA2.3, K1- SP(M2e)3SP, K1 (quad core) Two tandem cores linked together Two antigens (LAH HA2.3 and M2e 3 ) One empty core (K1) in each tandem core

16. Cloning status Construct design Bacterial cloning Yeast cloning Yeast high copy clones RCB WCB MCB K1, SP(M2e) 3 SP LAH HA2.3, K1-K1, SP(M2e) 3 SP A LAH HA2.3, K1 LAH HA2.3, SP(M2e) 3 SP Co1-2 C61S LAH HA2.3, K1-SP(M2e) 3 SP, K1 (VLP2) LAH H3, K1 (VLP1) LAH HA2.3, SP(M2e) 3 SP LAH HA2.3, K1-K1, SP(M2e) 3 SP B

17. Summary VLP1 and VLP2 RCBs transferred to 3P VLP3 (K1,M2 3 ) RCB also prepared and ready for transfer Concerns about truncation of VLP1 Five alternatives in development Cys61 mutant Single inserts Double transformations Quad core Need to make alternative RCB if new VLP is used