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

2. 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

3. 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)

4. Target Pathogens Hepatitis B virus Hepatitis A virus Enveloped virus
108
155
Enveloped virus
Neutralising antigen surface antigen (HBsAg, aa )
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 ( )
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

5. 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)

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

7. 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.

8. 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

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

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

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

12. 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)

13. 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

14. 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

15. 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

16. coHo7e, HA1s – Batch 7 1 = Pooled fraction #5-6 2 = Buffer exchangeM 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)

17. 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

18. 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

19. 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

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

21. coHo7sAg prep Discontinuous gradient analysis

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

23. 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

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

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

26. 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 (?)