TSB Q5 Meeting

16-Dec-2008

Hepatacore

iQur Leeds Progress

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

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

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

Yeast – PICZ revisited

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)

More Yeast Western blotsHigh Cell density Lysis

• Western blot revealed that only eGFP construct was detectable• No evidence of cross reactivity of the secondary antibodies

50

37

75100150250

2520

15 / 10

T S T S T S T S T S T S T S T S T S T S T S T S

X33HBc149 eGFP X33

HBc149 eGFP X33

HBc149 eGFPX33

HBc149 eGFP

Coomassie 10E111/ 10,000 Dil.

Anti-eGFP1/ 4,000 Dil.

2oAb only1/ 5,000 Dil.

IPTG

French Press

15000 psi

Pellet30%sucrose

Method – tandem core prep

27oC

605040302010

cores

20

60

Analyses:BradfordSDS-PAGEWestern blotELISATEM

Sonicat

ion

DRY ICE

MologicLtd.

ARCHIVES 2

007

Expression of cores in yeast

A B

WholeLysate

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

A B

Solublefraction

Sucrosesolublefraction

A B AB B A

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 2

007

It can be done

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

coHo7e,HA1s sucrose stability

0

0.1

0.2

0.3

0.4

0.5

0.6

Neat S/N Pellet

Sample

Co

nc

(mg

/ml) 0%

10%

20%

40%

• 0%, 10%, 20%, 40% sucrose tandem-HA stored at 2 – 8oC 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 AdjuvantSucrose stabilisation

M M P S/N P S/N P S/N P S/N

0% 10% 20% 40%

M M P S/N P S/N P S/N P S/N

0% 10% 20% 40%

60

220

100

45

3020

Purification of Dual sAg, HA tandem core

Last time we discussed:• Initial expression and solubility indicated workable amounts• Soluble fraction processed by AmSO4 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)

Discontinuous gradient ofcoHo7sAg, 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

3020

60% 40% 20%Sucrose %

Alternative treatments

• Heat treatment (+/- urea) to clarify away from E.coli proteins

- Urea

T I S HI HSM

+ Urea

T I S HI HSM

72 kDa

Modified Purification I

• coHo7e run alongside sAg,HA dual tandem• Lysed, clarify, heat + clarify, dialysis, sucrose cushion

• Gels, WB, ELISA, Bradford

Lysis and clarify

T I SM I ST

Empty sAg,HA

Heat treatment and clarify

HI HSS HI HSM S

Empty sAg,HA

Modified Purification II

• Start: (Sol lysate) coHo7e = 40 mg coHo7sAg,HA1s = 42.4 mgEnd: (Cushion) coHo7e = 10.8 mg coHo7sAg,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

Cushio

n pe

llet

Cushio

n pe

llet

Empty sAg,HA

AmSO4 ppt

20% 30% 40% 20% 30% 40%

Empty sAg,HA

• 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

Expression of HAV P1 (it expressed…!!)

32.5

25

47.5

62

83

175

16.56.5

ARCHIVES 2

007

E.Coli expression of HAVP1 and sAg

coHo7e

coHo7sAg,e

coHo7e,HAVP1

10E111/ 2,000 Dil.

Anti-HBsAg1/ 1,000 Dil.

50

37

75

100

150

250

252015 / 10

IPTG - + - + - + - + - + - +

Em

pty

HA

VP

1

sAg

Em

pty

HA

VP

1

sAg

- + - + - +

Em

pty

HA

VP

1

sAg

Coomassie

Restriction sites within antigen insertSize issuesFINALLY AFTER MULTIPLE APPROACHES

Dual Hep A/ B candidatecoHo7sAg,HAVP1

Ligations at 1:10 ratio Ligations at 1:20 ratio

PIC

3.5

K c

oH

o7

sA

g,H

AV

P1

HBsAg (108-155)

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

HAV P1 (aa 1-791)

VP

4 VP2 VP3 VP1

pET coHo7sAg,HAVP1

Alternative ‘Flu inserts

Neuraminidase (NA) clone verificationViral 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 # 1 - 5

• 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

1kb

La

dd

er

10

0b

p L

ad

de

r

Ta

c p

rom

ote

r

PCR

Bgl II – Nco ICore 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

Substitution of T7 promoterin the pET coHo7e vector

Promoter ‘Flu Hep A/B

tac-coHo7e coHo7e,NA coHo7sAg,HAV

tac-coHo7sAg,e coHo7e,M2e coHo7HAV,e

All the others… coHo7e,HA1l

Promoter ‘Flu Hep A/B

tac-coHo7e coHo7e,NA coHo7sAg,HAV

tac-coHo7sAg,e coHo7e,M2e coHo7HAV,e

All the others… coHo7e,HA1l

Cloning summary

sAg – Core I vs Core IISecond 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

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

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 (?)