TSB Q6 Meeting 03-Mar-2009 Hepatacore iQur Leeds Progress

Overview Introduction Transfer of key constructs to tac promoter; Hep B (HBsAg), Hep A (HAVP1), dual Hep A/B Tandem core purification HA-tandem core HBsAg-tandem core The “Space(r) shuttle” cloning: sAg, HA1s, HAVP1 Initial linkers Future work

The tandem core platform Homotandem core construct Core I (aa1-149) Nco I Bam HI Not I Eco RI Xho I Sac I Sal I Flexible linker Antigen insert site I Antigen insert site II Nhe I Core II pET 28b-CoHo7e His Tandem core protein Flexible linker 37 KDa Monomeric HBcAg (1-149) VLPs Heterotandem HBcAg 60nM Cryo-EM reconstructions of monomeric and tandem core particles. Performed by Dr R. Gilbert (University of Oxford)

Target Pathogens Hepatitis B virus Hepatitis A virus Enveloped virus 108 155 Enveloped virus Neutralising antigen surface antigen (HBsAg, aa124-137) Current vaccine – yeast expressed HBsAg VLPs 5 KDa insert Core I Core II Nco I Bam HI Not I Eco RI Nhe I Xho I Sac I Sal I HBsAg (108-155) Flexible linker Antigen insert site I Antigen insert site II 42 KDa VP4 VP2 VP3 VP1 HAV P1 Hepatitis A virus Non-enveloped virus Neutralising antigen – cluster of epitopes in VP1 and VP3 Current vaccines – live attenuated or inactivated whole virus 90 KDa insert Core I Core II Nco I Bam HI Not I Eco RI Nhe I Xho I Sac I Sal I HAV P1 (aa1-791) Flexible linker Antigen insert site I VP4 VP2 VP3 VP1 125 KDa

Influenza Antigens: Haemagglutinin and M2 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 M2 highly conserved between all strains – potential universal ‘flu antigen M2e (aa 1-24)

Sizes of the tandems discussed: Bacteria expression Sizes of the tandems discussed: CoHo7e (37kDa) CoHo7sAg,e (42kDa) CoHo7e,HAVP1 (125 kDa)

Substituting-in tac promoter (1) Eden vector containing the tac promoter pET vector containing the T7 promoter ptac With the exception of the T7 and tac promoters the remaining upstream region is conserved, therefore it is possible to interchange this entire region from EET2000 to the pET tandem core constructs.

Substituting-in tac promoter (2) Amplify tac region with primers to give BglII and NcoI flanking sites Digest and purify PCR product Clone digested PCR amplified tac promoter into tandem core constructs Excise upstream elements from pET vector constructs between BglII and NcoI sites + Core I (aa1-149) Nco I Bam HI Not I Eco RI Xho I Sac I Sal I Flexible linker Antigen insert site I Antigen insert site II Nhe I Core II pTAC 28b-CoHo7e His BglII NcoI

tac constructs sent to Eden coHo7e coHo7sAg, e coHo7e, HAVP1 coHo7sAg, HAVP1

Preliminary Expression Trial (1) 50 37 150 250 25 75 15/10 100 IPTG – + T7 tac Promoter Empty sAg/HAV

Preliminary Expression Trial (2) 50 37 150 250 25 75 15/10 100 IPTG – + T7 tac Promoter HBsAg e,HAV e/HAV

tac constructs expressed BL21(DE3) transformed with both pET and pTAC versions of coHo7e Other constructs also done Needs repeating Differing conditions (temp, media, etc)

Old Method – HA tandem core prep 2 passes Sonication 14000 psi IPTG French Press 27oC Pellet 30% sucrose 60 40 30 cores Analyses: Bradford SDS-PAGE Western blot ELISA TEM Lysis in Tris pH8, 5% glycerol, 5mM DTT, Prot. Inhib, benzonase: Clarification: 50k x g spin Soluble Insoluble

HA – Tandem core purification Discontinuous sucrose gradient Load Pellet 2 3 4 5 6 7 8 9 10 11 12 30 M M Total Lysate Insoluble Lysate Soluble Lysate 75 50

HA tandem prep – Anti-core WB Load Pellet 2 3 4 5 7 8 9 10 11 12 6 Biotin Mr Next pooled fractions 5 and 6 Buffer exchange and concentrate Sent to Arecor

coHo7e, HA1s – Batch 7 1 = Pooled fraction #5-6 2 = Buffer exchange M 50 37 150 250 25 75 20/15/10 100 Pooled # 5 + 6 3 5 1 2 4 1 = Pooled fraction #5-6 2 = Buffer exchange 3 = Centriprep 10,000 MWCO concentrate (0.8mg/ml) 4 = 4-fold dilution (0.2mg/ml) 5 = 8-fold dilution (0.1mg/ml)

Alternative Method – sAg tandem core prep 2 passes Sonication 14000 psi DRY ICE iQur LDS Delivery: Eden Biodesign IPTG French Press 27oC S/N Dialysed to pH 7.5 + NaCl 30% sucrose 60 40 30 cores Analyses: Bradford SDS-PAGE Western blot ELISA TEM Pellet 30% sucrose S/N Dialysed to pH 7.5 + NaCl Lysis in elevated pH, no salt : Clarification: 26k x g spin Soluble Insoluble Sediment contaminant assemblies

Solubility and ‘Sediment-ability’ Sucrose S/N pH7.5 Sucrose Pellet pH7.5 Sucrose S/N pH8.5 Sucrose Pellet pH8.5 Sucrose S/N pH9.5 Sucrose Pellet pH9.5 M T I S pH 7.5 pH 8.5 pH 9.5 75 50 37 coHo7sAg indicated by arrow

Sedimentation at varying pH without NaCl Sucrose S/N pH7.5 Sucrose Pellet pH7.5 Sucrose S/N pH8.5 Sucrose Pellet pH8.5 Sucrose S/N pH9.5 coHo7sAg,e Lysate (+ve control) Anti-core 10E11 (1/4000 dilution) 75 50 coHo7sAg coHo7e 37

Sedimentation after dialysis to pH 7.5 with NaCl S/N P pH7.5 + – Original pH pH8.5 pH9.5

coHo7sAg prep Discontinuous gradient analysis

Quick Slide – (Apologies) Discontinuous gradient gel and WB #3 #10

Engineering inserts with spacers Space(r) Shuttle Nco I Bam HI Not I Eco RI Nhe I Xho I Sac I Sal I In vitro Assays ------------ ELISA SPR

Shuttle first steps Cloned into shuttle Sequence verified and expression trials used to assess antigens

Designing spacers Theory Practical obstacles Compromise (with a small ‘C’)

Future work Repeat tac Tandem Core expression 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 (?)