1. TSB Q5 Meeting 16-Dec-2008 Hepatacore iQur Leeds Progress

2. Overview Introduction Yeast expression – issues Non-adjuvanted coHo7e,HA1s Dual tandem core purification attempts Alternative ‘flu antigens in tandem core Transfer of new promoter; Transfer of dual Hep A/B The case for “Spacers”: sAg in Core I versus Core II Future work

3. The tandem core platform Core I (aa1-149) Nco IBam HINot IEco RIXho I Sac ISal I Flexible linker Antigen insert site I Antigen insert site II Nhe I Core II (aa1-149) pET 28b-CoHo7e His Homotandem core construct Monomeric HBcAg (1-149) VLPs Heterotandem HBcAg VLPs 60nM Cryo-EM reconstructions of monomeric and tandem core particles. Performed by Dr R. Gilbert (University of Oxford) 37 KDa Tandem core protein Flexible linker

4. VP4 VP2 VP3 VP1 HAV P1 Target Pathogens Hepatitis B virus Enveloped virus Neutralising antigen surface antigen (HBsAg, aa124-137) Current vaccine – yeast expressed HBsAg VLPs 5 KDa insert 108 155 Non-enveloped virus Neutralising antigen – cluster of epitopes in VP1 and VP3 Current vaccines – live attenuated or inactivated whole virus 90 KDa insert Hepatitis A virus Core ICore II Nco I Bam HINot IEco RINhe IXho ISac ISal I HAV P1 (aa1-791) Flexible linker Antigen insert site I VP4 VP2 VP3 VP1 125 KDa Core ICore II Nco I Bam HINot IEco RINhe IXho ISac ISal I HBsAg (108-155) Flexible linker Antigen insert site I Antigen insert site II 42 KDa

5. Pathogen and useful model: Influenza Haemagglutinin H1 serotype (PR8) HA1 globular domain cloned into homo-tandem core Functional assay to confirm conformation of the haemagglutinin Protection studies can be done in a mouse model Express and purify from E.coli for optimisation assays Optimise haemagglutination, biophysical (EM, CD) and other in vitro assays

6. Yeast – PICZ revisited

7. Yeast expression - Revisited Previous experiments may have been overly diluted compared to some values in the literature Cross-reactivity observed in previous western blots Using pPICZ-C as the expression vector, constructs were transformed and induced and larger pellets were received from Mologic for high cell density lysis by vortex + glass beads Non-transformed P. pastoris (X33 strain) Wt HBc149 CoHo7e,eGFP Total and soluble lysates subjected to SDS-PAGE, blotted on to PVDF membrane and probed with several monoclonal antibodies: Anti-core: 10E11, (13A9, H5F6 – data not presented) Anti-EGFP Anti-mouse secondary (-ve control)

8. More Yeast Western blots High Cell density Lysis Western blot revealed that only eGFP construct was detectable No evidence of cross reactivity of the secondary antibodies 50 37 75 100 150 250 25 20 15 / 10 TSTSTSTSTSTSTSTSTSTSTSTS X33 HBc 149 eGFP X33 HBc 149 eGFP X33 HBc 149 eGFP X33 HBc 149 eGFP Coomassie10E11 1/ 10,000 Dil. Anti-eGFP 1/ 4,000 Dil. 2 o Ab only 1/ 5,000 Dil.

9. IPTG French Press 15000 psi Pellet 30% sucrose Method – tandem core prep 27 o C 60 50 40 30 20 10 cores 20 60 Analyses: Bradford SDS-PAGE Western blot ELISA TEM Sonication DRY ICE M ologic Ltd. Detergent incubation: 1 hour, RT, rotating mixer Clarification: 50k x g spin  Soluble  Insoluble ARCHIVES 2007

10. Expression of cores in yeast AB Whole Lysate 37 50 64 98 22 A B Western Blot of samples from lysis and clarification yeast expressed material. A. CoHe7e B. CoHe7e,eGFP 37 50 64 98 22 A B AB Soluble fraction Sucrose soluble fraction ABABBA Concentrated protein 1.Sand and Liquid nitrogen lysis with benzonase and protease inhibitors 2.Clarify 1: Spun at 26,000 xg 3.Clarify 2: Added sucrose to 0.15M and spun at 150,000 xg 4.Sucrose cushion concentration: supernatant layered over 30% sucrose cushion. Spun at 150,000xg ARCHIVES 2007

11. It can be done

12. Non-Adjuvanted Immunogen Immunogen = coHo7e,HA1s Expressed and purified from E.coli lysates Material used in immunogenicity (iQur) and stability studies (Arecor) To date immunogen has been mixed with adjuvant = Imject® (aluminium hydroxide, Alum) Aggregation and sedimentation hindered previous studies without adjuvant

13. 0%, 10%, 20%, 40% sucrose tandem-HA stored at 2 – 8 o C over 8 days Samples were centrifuged (12,000 x g) for 10 minutes Pellet and supernatant samples were analysed by SDS-PAGE and Bradford Addition of sucrose helps resist aggregation and resultant sedimentation of HA-tandem coHo7e,HA1s minus Adjuvant Sucrose stabilisation 60 220 100 45 30 20

14. Purification of Dual sAg, HA tandem core Last time we discussed: Initial expression and solubility indicated workable amounts Soluble fraction processed by AmSO 4 ppt, sucrose gradients tightly at 40%:60% interface Attempted solvent extraction to remove potential lipid contaminants – no effect, meaning protein or CHO Plus/ minus detergent (TW20): soluble lysates were analysed for antigenicity (10E11, anti-sAg (mAbs), anti-PR8 (pAb)) demonstrated no preference for detergent Core response was strong sAg weak in comparison to control but titrated PR8 good, but evidence of strong cross-reactivity E.coli or core Strong reducing conditions (beta-mercaptoethanol)

15. Discontinuous gradient of coHo7sAg, HA1s soluble lysate with B-ME Strong reducing conditions disrupt tight banding at the 60:40% sucrose interface M T I S 1 3 5 7 8 9 11 13 15 16 17 19 21 23 24 25 27 29 31 33 Discontinuous sucrose gradient fractions 60 220 100 45 30 20 60%40%20%Sucrose %

16. Alternative treatments Heat treatment (+/- urea) to clarify away from E.coli proteins - Urea TISHIHSM + Urea TISHIHSM 72 kDa

17. Modified Purification I coHo7e run alongside sAg,HA dual tandem Lysed, clarify, heat + clarify, dialysis, sucrose cushion Gels, WB, ELISA, Bradford Lysis and clarify TISMIST EmptysAg,HA Heat treatment and clarify HIHSSHIHSMS EmptysAg,HA

18. Modified Purification II Start: (Sol lysate) coHo7e = 40 mgcoHo7sAg,HA1s = 42.4 mg End: (Cushion) coHo7e = 10.8 mgcoHo7sAg,HA1s = 4.8 mg (Total Protein) Proportion of tandem core in these preps is low at the end of first stage These results suggests that it is not feasible to purify away from contaminants by these methods Dialysed material over sucrose cushion Cushion pellet EmptysAg,HA AmSO 4 ppt 20%30%40%20%30%40% EmptysAg,HA

19. Sizes of the tandems discussed: HBc149 (wild type sequence) (17kDa) CoHo7e (37kDa) CoHo7e,eGFP (65kDa) CoHo7e,HA1s (67kDa) CoHo7sAg,e(42kDa) CoHo7sAg,HA1s(72kDa) CoHo7e,HAVP1(125 kDa) Expression shifted from yeast to bacteria More Bacteria expression Sizes of the tandems discussed: HBc149 (wild type sequence) (17kDa) CoHo7e (37kDa) CoHo7e,eGFP (65kDa) CoHo7e,HA1s (67kDa) CoHo7sAg,e(42kDa) CoHo7sAg,HA1s(72kDa) CoHo7e,HAVP1(125 kDa) Expression shifted from yeast to bacteria

20. Expression of HAV P1 (it expressed…!!) 32.5 25 47.5 62 83 175 16.5 6.5 ARCHIVES 2007

21. E.Coli expression of HAVP1 and sAg coHo7e coHo7sAg,e coHo7e,HAVP1 10E11 1/ 2,000 Dil. Anti-HBsAg 1/ 1,000 Dil. 50 37 75 100 150 250 25 20 15 / 10 IPTG -+-+-+-+-+-+ Empty HAVP1 sAg Empty HAVP1 sAg -+-+-+ Empty HAVP1 sAg Coomassie

22. Restriction sites within antigen insert Size issues FINALLY AFTER MULTIPLE APPROACHES Dual Hep A/ B candidate coHo7sAg,HAVP1 Ligations at 1:10 ratioLigations at 1:20 ratio PIC3.5K coHo7sAg,HAVP1 HBsAg (108-155) Nco I Bam HINot IEco RINhe IXho ISac ISal I HAV P1 (aa 1-791) VP4 VP2 VP3 VP1 pET coHo7sAg,HAVP1

23. Alternative ‘Flu inserts

24. Neuraminidase (NA) clone verification Viral RNA isolation  RT  PCR with RT template  pJET shuttle  Ligate Colony screen verified by BamHI digest: Negative = linearise with BamHI in core I Positive = Band release cutting BamHI in core I and BamHI internal to NA sAgEmpty ~1400bp ~1600bp -ve CTRL -ve CTRL # 1 - 5

25. PCR from template containing HA1 long (pJET::HA1l ) Double digest of PCR product (Eco RI – Nhe I) yielding insert Ligate into coHo7e,HA1s (Eco RI – Nhe I digested) Release of HA1s indicates successful digest Gel purify vector backbone Colonies screened by PCR # 1 - 60 -ve CTRL HA1-long (HA1l) clone verification

26. 1kb Ladder 100bp Ladder Tac promoter PCR Bgl II – Nco I Core I (aa1-149) Nco IBam HINot IEco RIXho I Sac ISal I Flexible linker Antigen insert site I Antigen insert site II Nhe I Core II (aa1-149) pET 28b-CoHo7e His Substitution of T7 promoter in the pET coHo7e vector

27. Promoter‘FluHep A/B tac-coHo7ecoHo7e,NAcoHo7sAg,HAV tac-coHo7sAg,ecoHo7e,M2ecoHo7HAV,e All the others…coHo7e,HA1l Promoter‘FluHep A/B tac-coHo7ecoHo7e,NAcoHo7sAg,HAV tac-coHo7sAg,ecoHo7e,M2ecoHo7HAV,e All the others…coHo7e,HA1l Cloning summary

28. sAg – Core I vs Core II Second look Core I has extra residues around the e1 loop due to Not I restriction site being 8 nt in length »Extra SERINES at the front and at the back of the sAg insert Core II has less non-core derived sequence M U I U I sAg in Core I sAg in Core II M sAg in Core I sAg in Core II MU I U I

29. Engineering inserts with spacers Nco I Bam HINot IEco RINhe IXho ISac ISal I Space(r) Shuttle In vitro Assays ------------ ELISA SPR

30. Future work Cloning remaining constructs into Tac Tandem Core vector Improving antigen and core independent folding –Orientation of inserts (core I or core II) –Engineering spacers for antigen sequences –Variations of insert sequences Development of in vitro screen –SPR –ELISA Yeast (?)