1. Confirmation of positive clones Screening of positive clones Selection of high copy number clones Selection of positive clones FLUTCORE vaccine yeast constructs Construct design Bacterial cloning Molecular and morphological Characterization Yeast cloning Cell bank Molecular cloning

2. Constructs design Synthesis of inserts (GeneArt ) Standard cloning manipulation (restriction digestion and ligation) Heterotandem construct PHe7K1K1

3. Bacterial cloning Cloning of inserts into pPICZ C plasmid using specific restriction sites Plasmid transfection into E. Coli DH5α using the heat-shock method Growth of positive clones using selective medium (low salt LB agar containing 0.1 mg/ml of zeocin) pPICZ C plasmid

4. Screening and confirmation of positive clones Screening of positive clones via colony PCR using insert specific primers M1 2 3456 M: DNA ladder 1: Clone 1 2: Clone 2 3: Clone 3 4: Clone 4 5: Clone 5 6: Negative control Sequencing of inserted DNA fragments of two positive clones

5. Yeast cloning Plasmid linearization using pmeI restriction site (present on the plasmid sequence) P. Pastoris KM71H transformation (electroporation) using linearized plasmids (5 µl) and electro competent yeast cells (80 µl) Incubation of transformed cells in: 1 ml of 1 M sorbitol at 30° C for 90 minutes and 10 ml of YPD at 30° C and 250 rpm for 90 minutes Note: no antibiotics are used at tis stage

6. Yeast cloning More inserts = More antibiotic resistance gene Integration of zeocin resistance gene together with target inserts

7. Selection of high copy clones: zeocin Selection of 24 clones (pool of cells) showing the highest OD 600 gDNA extraction of the selected clones Growth of cloned yeast cells using selective medium: YPDS with increasing concentrations of zeocin (0.2 and 2 mg/ml)

8. Selection of high copy clones: qRT-PCR Selection of the highest copy clone (pool of cells) via qPCR Single-cell colonies Isolation of 8 single-cell colonies on YPD agar Growth of selected single-cell colonies and gDNA extraction Selection of the highest copy clone via qPCR

9. Yeast Clones CloneCore 1Core 2 Product MW (KDa) 1K1 42.5 2LAH HA2.33sp-(M2) 3 -3sp58.8 3LAH H3K148.2 43sp-LAH H1/H3/HB-3sp3sp-(M2) 3 -3sp71.6 5LAH H1/H33sp-(M2) 3 -3sp63.7 6LAH H33sp-(M2) 3 -3sp56.8 7K1LAH H348.2 8M2a-LAH H1-M2bLAH H358.9 9LAH H13sp-(M2) 3 -3sp56.8 10LAH H1LAH H353.7 11M2a-LAH H1-M2bM2a-LAH H3-M2c64.1 12K13sp-(M2) 3 -3sp51.2 VLP1 VLP2

10. Research Cell Bank (RCB) Master Cell Bank (MCB) and Working Cell Bank (WCB) generated and analysed following the same criteria used for the RCB RCB Morphology Cell viability Insert copy number Genetic identity Stocks of P. pastoris KM71H transformed with VLP coding sequences

11. RCB generation and characterization

12. Single-cell colony stocks VLP1VLP2 Samples inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours

13. VLP1 morphology Liquid culture1 day at -80° C 6 days at -80° C30 days at -80° C Samples inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours

14. VLP2 morphology Liquid culture1 day at -80° C 6 days at -80° C30 days at -80° C Samples inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours

15. Morphology ColourFormMarginElevation Colony size (mm) Liquid cultureWhiteRoundedEntireRaised2.87±0.13 Stocks after 1 day at - 80°C WhiteRoundedEntireRaised2.08±0.10 Stocks after 6 days at - 80°C WhiteRoundedEntireRaised2.23±0.08 Stocks after 30 days at - 80°C WhiteRoundedEntireRaised2.6±0.06 ColourFormMarginElevation Colony size (mm) Liquid cultureWhiteRoundedEntireRaised2.64±0.11 Stocks after 1 day at - 80° C WhiteRoundedEntireRaised2.85±0.08 Stocks after 6 days at - 80° C WhiteRoundedEntireRaised2.71±0.14 Stocks after 30 days at - 80° C WhiteRoundedEntireRaised2.82±0.04 VLP2

16. Cell viability: CFU determination VLP1VLP2 Serial dilution (1:10) of stock samples were prepared, inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours

17. Insert copy number: qRT-PCR VLP1VLP2 gDNA extracted from each sample was used to perform qRT-PCR

18. Genetic identity: PCR Amplification of the entire VLP coding fragments M: DNA ladder 1: VLP1 single-cell stock 2: VLP1 RCB stock before storage 3: VLP1 RCB stock after storage 4: VLP1 negative control 5: VLP2 single-cell stock 6: VLP2 RCB stock before storage 7: VLP2 RCB stock after storage 8: VLP2 negative control

19. Genetic identity: sequencing Full VLP sequence Pic upstream F Mid core1 F SacI F SalI R NheI R Pic downstream R Correspondence between expected and cloned nucleotide sequences

20. Conclusions 1.Cloning of VLP coding sequences into P. pastoris KM71H 2.Isolation of single-cell clones 3.Selection of the highest copy number clones 4.Generation and characterization of cell banks a.Same cell morphology before and after stock storage at -80° C b.No significant differences in cell viability before and after storage at -80° C and at different time points c.Stability of insert copy number before and after storage at -80° C d.Proof of genetic identity

22. P. Pastoris KM71H