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

Core I (aa1-149)

Nco I Bam HI Not I Eco RI Xho ISac I Sal 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 proteinFlexible linker

VP

4 VP2 VP3 VP1

HAV P1

Target PathogensHepatitis 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 I Core IINco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I

HAV P1 (aa1-791)

Flexible linker

Antigen insert site I

VP

4 VP2 VP3 VP1

125 KDa

Core I Core IINco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I

HBsAg (108-155)Flexible linker

Antigen insert site I

Antigen insert site II42 KDa

C2PR8HA_F2C2PR8HA_R2

C2PR8HA_F1C2PR8HA_R1

C2PR8HA_F2C2PR8HA_R2

C2PR8HA_F1C2PR8HA_R1

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)

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

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.

ptac

Substituting-in tac promoter (2)

3. Excise upstream elements from pET vector constructs between BglII and NcoI sites

1. Amplify tac region with primers to give BglII and NcoI flanking sites

2. Digest and purify PCR product

4. Clone digested PCR amplified tac promoter into tandem core constructs

Core I (aa1-149)

Nco I Bam HI Not I Eco RI Xho ISac I Sal I

Flexible linker

Antigen insert site I

Antigen insert site II

Nhe I

Core II (aa1-149)

pTAC 28b-CoHo7e

His

BglII

NcoI

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA

BglII

NcoI

TTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATA

BglIIBglII

NcoINcoI

+

tac constructs sent to Eden

• coHo7e

• coHo7sAg, e

• coHo7e, HAVP1

• coHo7sAg, HAVP1

50

37

150250

25

75

15/10

100

IPTG – – ––+ + ++ – – ––+ + ++

T7 T7tac tacPromoter T7 T7tac tac

Empty EmptysAg/HAV sAg/HAV

Preliminary Expression Trial (1)

50

37

150250

25

75

15/10

100

IPTG – – ––+ + ++ – – ––+ + ++

T7 T7tac tacPromoter T7 T7tac tac

HBsAg HBsAge,HAV e/HAV

Preliminary Expression Trial (2)

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)

60

40

30

cores

Analyses:BradfordSDS-PAGEWestern blotELISATEM

IPTG

French Press

14000 psi

Old Method – HA tandem core prep

27oC

Sonicat

ion

Lysis in Tris pH8, 5% glycerol, 5mM DTT, Prot. Inhib, benzonase:

Clarification: 50k x g spin

Soluble

Insoluble Pellet

30%sucrose

2 passes

HA – Tandem core purificationDiscontinuous sucrose gradient

M

Tot

al L

ysat

e

Inso

lubl

e Ly

sate

Sol

uble

Lys

ate

Load

Pel

let

2 3 4 5 6 7 8 9 10 11 12 30M

75

50

HA tandem prep – Anti-core WB

• Next pooled fractions 5 and 6

• Buffer exchange and concentrate

• Sent to ArecorLo

ad

Pel

let

2 3 4 5 7 8 9 10 11 126

Bio

tin M

r

coHo7e, HA1s – Batch 7

M

50

37

150

250

25

75

20/15/10

100

Pooled # 5 + 6

3 51 2 4MM

50

37

150

250

25

75

20/15/10

100

Pooled # 5 + 6

3 51 2 4

1 = Pooled fraction #5-62 = Buffer exchange3 = Centriprep 10,000 MWCO concentrate (0.8mg/ml)4 = 4-fold dilution (0.2mg/ml)5 = 8-fold dilution (0.1mg/ml)

IPTG

French Press

14000 psi

Alternative Method – sAg tandem core prep

27oC

Sonicat

ion

DRY ICE

EdenBiodesign

Lysis in elevated pH, no salt :

Clarification: 26k x g spin

Soluble

Insoluble

Sediment contaminant assemblies

S/NDialysed to pH 7.5

+ NaCl

30%sucrose

2 passes

60

40

30

cores

Pellet30%sucrose Pellet30%sucrose

S/NDialysed to pH 7.5

+ NaCl

Solubility and ‘Sediment-ability’

M Suc

rose

S/N

pH

7.5

Suc

rose

Pel

let

pH7.

5

Suc

rose

S/N

pH

8.5

Suc

rose

Pel

let

pH8.

5

Suc

rose

S/N

pH

9.5

Suc

rose

Pel

let

pH9.

5

M T I S

pH 7.5 pH 8.5 pH 9.5

T I S T I S

coHo7sAg indicated by arrow

50

37

75

Sedimentation at varying pH without NaCl

M

Suc

rose

S/N

pH

7.5

Suc

rose

Pel

let p

H7.

5

Suc

rose

S/N

pH

8.5

Suc

rose

Pel

let p

H8.

5

Suc

rose

S/N

pH

9.5

Suc

rose

S/N

pH

9.5

coH

o7sA

g,e

Lysa

te (

+ve

con

trol

)

Anti-core10E11

(1/4000 dilution)

50

37

75

coHo7sAg coHo7e

Sedimentation after dialysis to pH 7.5 with NaCl

M

NaClS/N P S/N P

pH7.5+–

Original pH

S/N P S/N P

pH8.5+–

S/N P S/N P

pH9.5+–

M

coHo7sAg prepDiscontinuous gradient analysis

coHo7sAg - Discontinuous sucrose gradient

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30 35

Fractions

Ab

s(49

0nm

)Disco1 anti-core

Disco2 anti-core

Disco1 anti-sAg

Disco2 anti-sAg

Quick Slide – (Apologies) Discontinuous gradient gel and WB

#3 #3 #10#10

Engineering inserts with spacers

Nco I Bam HI Not I Eco RI Nhe I Xho ISac I Sal I

Space(r) Shuttle

In vitro Assays

------------

ELISASPR

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 (?)