1. Construction of Plasmids & Purification of Core-Haemagglutinin VLPs

2. Overview Introduction Cloning influenza haemagglutinin Transfer to Pichia vectors Expression of haemagglutinin construct in E.coli 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. The tandem core platform Tandem HBV core - eBFPAssembled CoHe7e,eBFP VLPs Core I Core II Linker eBFP Antigen insertion sites Single insertion into tandem core Core ICore II Nco I Bam HINot IEco RINhe IXho ISac ISal I eBFP Flexible linker Antigen insert site I Antigen insert site II Tandem core - eBFP,eGFP dimer Assembled CoHe7L3eBFP,eGFP VLPs Core I Linker Antigen insertion sites Core II eBFP eGFP Dual insertion into tandem core Core ICore II Nco I Bam HINot IEco RINhe IXho ISac ISal I eBFP Flexible linker Antigen insert site I Antigen insert site II eGFP

5. Target Pathogens Hepatitis B virus Enveloped virus Neutralising antigen surface antigen (HBsAg, aa124-137) Current vaccine – yeast expressed HBsAg VLPs 10 KDa insert 108 155 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

6. Target Pathogens Hepatitis A virus VP4 VP2 VP3 VP1 HAV P1 Non-enveloped virus Neutralising antigen – cluster of epitopes in VP1 and VP3 Current vaccines – live attenuated or inactivated whole virus 100 KDa insert 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 135 KDa

7. Cloning HA1 (PR8) PR8 strain of influenza obtained from NIBSC The virus was bulked in MDCK cells Mouse infection studies have been completed and the system is ready for protection studies HA1 globular domain encoding RNA was reverse transcribed and amplified by PCR Several different strategies to clone the HA1 globular domain into the CoHo7e vector failed

8. Cloning pPICZ C constructs Cloning CoHo7e and wt HBc149 was also attempted. pPICZ vectors were obtained from Invitrogen and large scale plasmid preps performed. After several attempts, the cloning was not successful.

9. Cloning Troubleshooting A list of possible reasons for unsuccessful cloning was drawn up. 1.Water supply – Building works adjacent to the lab had resulted in disruptions to the water supply that may have led to contamination. Cloning into commercial vectors may have been successful due to the inclusion of pure water in the cloning kits. 2.Suboptimal purification of vector and insert after digestion leading to contamination with impurities that inhibit ligation. 3.Problem with buffers used in electrophoresis of digested products for purification 4.Contaminated enzymes lead to improper overhangs for sticky end ligation 5.Incomplete digestion of vector

10. Getting the cloning back on track Firstly the CoHo7e,HA1s cloning was tackled. All plasmids involved in the cloning were prepared using an alternative large scale plasmid purification kit. Biorad Quantum Prep kit was used in place of Qiagen Midi/Maxi prep kits Three different approaches to cloning were employed. A wide range of insert:vector molar ratios and DNA concentrations were assessed.

11. Getting the cloning back on track Cloning strategies Core I (aa1-149) Nco I Bam HINot I Eco RI Xho ISac ISal I Flexible linker Antigen insert site I Nhe I Core II (aa1-149) pET 28b-CoHo7e Core I (aa1-149) Nco I Bam HINot IEco RI Xho I Sac ISal I Flexible linker Antigen insert site I Nhe I Core II (aa1-185) pET 28b-CoHe7e,HA1s Hi s HA1s HAVP1 Exchange HA1s only Exchange C-terminal region Exchange HA1s + core- coding sequence Successful cloning strategies

12. Current cloning status Three individuals gained CoHo7e,HA1s clones in one week. Unusual molecular ratios proved successful eg 30:1 (insert:vector). Lower total DNA concentrations gave more positive colonies. A new SOP has been written to ensure that these conditions are followed in the future.

13. Transfer to pPICZ-C vector All constructs to be used in this study must be transferred to the Pichia pastoris expression vector pPICZ-C For initial studies of tandem core expression in yeast, the following vectors are also required: 1.Wt HBc149 (to compare wt sequence with E.coli optimised sequences). 2.CoHo7e (empty E.coli optimised tandem core). 3.CoHo7e,eGFP (eGFP provides a simple assay for expression levels during optimisations). Each of these constructs have now been made and sent to Mologic for transformation and expression.

14. Transfer to pPICZ-C vector The tandem core-haemagglutinin construct CoHo7e,HA1s transfer to yeast for expression and use in stability and protection studies. The HA1s insert has been successfully transferred into pPICZ-C coHo7e and sent to Mologic. The following constructs are now being prepared: 1.CoHo7e,HAVP1 (HAVP1 sequence in core II) 2.CoHo7,sAg (HBV surface antigen in core I) 3.CoHo7sAg,HAVP1 (dual inserts- sAg in core I and HAVP1 in core II)

15. E.Coli expression and purification of CoHo7e,HA1s Expression and purification in E.coli was attempted to provide VLPs for protection and stability studies. Initial expression studies showed that the CoHo7e,HA1s is expressed in E.coli and is partially soluble. UninducedInducedMTotal lysate Insoluble lysateSoluble lysateCushion S/NCushion pellet 75 50 37 25 Expression cell pellet lysed by French press, sonicated and treated with 0.05% Tween-20 for 1 hr before clarification by centrifugation at 50,000 x ‘g’. Supernatant placed over 30% sucrose cushion and VLPs concentrated by centrifugation at 150,000 x ‘g’.

16. E.Coli expression and purification of CoHo7e,HA1s Resuspended sucrose cushion pellet is separated on a discontinuous sucrose density gradient (20-60%). A contaminating E.coli band (~ 37KDa) sediments with the CoHo7e,HA1s protein. 182 116 82 64 49 37 26 19 15 3 6 912 15 18212427303336 M Fractions collected from the bottom of the gradient

17. E.Coli expression and purification of CoHo7e,HA1s Fractions containing CoHo7e,HA1s are combined and concentrated by centrifugation The protein pellet is resuspended and centrifuged over a continuous sucrose density gradient (20-60%). The contaminating E.coli band sediments with the CoHo7e,HA1s protein. 182 116 82 64 49 37 26 369121518212427303336 M Fractions collected from the bottom of the gradient

18. Removing the contaminant HBV core VLPs are resistant to up to 3M urea. If the contaminant is not as urea-stable it may be possible to denature the contaminant while retaining the VLPs The sucrose density gradient purified material was treated with 0,1,2 or 4M urea prior to separation on a 5ml continuous sucrose density gradient. Urea treatment

19. Removing the contaminant Urea treatment

20. Removing the contaminant Urea treatment 0M 2M 4M 1M 12345678101112131415169 12345678101112131415169 12345678101112131415169 12345678101112131415169 Fractions collected from the top of the gradient 182 116 82 64 49 37 26 182 116 82 64 49 37 26 182 116 82 64 49 37 26 182 116 82 64 49 37 26

21. Removing the contaminant HBV core VLPs may be heated to 60°C without loss of integrity. Heating of the E.coli cell lysate causes precipitation of contaminating proteins. Heat treatment pH 7.5pH 8.5pH 9.5 TotSolTotSol Tot Sol M12345 1.Total lysate 2.Insoluble fraction 3.Soluble fraction 4.Sucrose cushion pellet 5.Sucrose cushion supernatant 182 116 82 64 49 37 26 182 116 82 64 49 37 26

22. Removing the contaminant Fetuin is a serum protein that binds to haemagglutinin. Fetuin was coupled to CnBr activated sepharose and used to pull down the CoHo7e,VLPs (batch purification) Affinity purification (Fetuin) M1234567M M1234567M 1.Input 2.Flowthrough 3.Wash 1 4.1.5M NaCl 1 5.1.5M NaCl 2 6.3M NaCl 7.Sepharose beads 182 116 82 64 49 37 26 182 116 82 64 49 37 26

23. Removing the contaminant Using surface charge characteristics of the VLPs, it may be possible to separate them from the E.coli contaminant. Anion exchange chromatography CIM monolith QA disk

24. Removing the contaminant Clarified cell lysate was bound to the column low salt (0.1M NaCl) conditions and eluted in 1M followed by 2M NaCl (stepwise elution). Anion exchange chromatography Input FT Wash M67811121314 1516 2223 182 116 82 64 49 37 26 1M NaCl2M NaCl

25. Removing the contaminant Clarified cell lysate was bound to the column low salt (0.1M NaCl) conditions and eluted in 0.1- 1M NaCl gradient. Anion exchange chromatography Fractions viewed by EM

26. Removing the contaminant Anion exchange chromatography 182 116 82 64 49 37 26 19 15 M Input 4349515355 M 576163656771 2M Wash FT1 3741 FT2 FT3 Wash 394547596983

27. Removing the contaminant At least 50% of the CoHo7e,HA1s protein is insoluble. An inclusion body preparation showed that insoluble core protein is present in inclusion bodies. Denaturing and renaturing experiments using urea, high pH and detergent are being done in an attempt to prepare VLPs from the inclusion body material Purification of inclusion bodies

28. Future work Cloning 1.Transfer CoHo7e,HA1, CoHo7e,sAg and CoHo7e,HAVP1 to pPICZc vector (iQur/UoL) Expression & purification of VLPs 1.Further investigate the use of anion exchange purification and inclusion body protein refolding to produce CoHo7e,HA1s VLPs (iQur/UoL) 2.Express CoHo7e,HA1s in P. pastoris (Mologic) and purify the resulting protein (iQur/UoL) 3.Optimise expression of CoHo7e,eGFP in P. pastoris and compare localisation in yeast cells with CoHe7e,eGFP 4.Express CoHo7e and wt monomeric core in Pichia to compare expression (Mologic) 5.Purify CoHo7e and HBc149 core VLPs compare solubility 6.Express CoHo7e,HA1s in P. pastoris (Mologic) and purify the resulting protein (iQur/UoL)