Anti-E1 Anti-E2

rE1E

2p7 p

rotei

n

VEE/SIN

-E1E

2P7

VEE/SIN

-E1E

2

33-35

rE1E

2P7 p

rotei

n

VEE/SIN

-E1E

2P7

VEE/SIN

-E1E

2

EndoH

35

25

15

75

50

35

25

r E1E

2p7 p

rotei

n)

VEE/SIN

-E1E

2P7

r E1E

2P7 p

rotei

n

VEE/SIN

-E1E

2P7

VEE/SIN

-E1E

2VEE/S

IN-E

1E2

EndoH

68-74

VEE/SIN

-NS34

5

VEE/SIN

-NS34

5

* **

VEE/SIN

-NS3

45

70

Anti-NS3

rSO

D-C33

C (NS3

)

NS4A/B: 35

VEE/SIN

-NS3

45

rSO

D-C10

0 (N

S4)

Anti-NS4

57

VEE/SIN

-NS3

45

rSO

D-NS5

(NS5

A/B)

Anti-NS5A Anti-NS5B

VEE/SIN

-NS3

45

69NS4B: 27

NS4A: 8

*

*

**

*

* *

* **

A

B

rE1E

2p7 p

rote

in

rE1E

2p7 p

rote

in

rE1E

2p7 p

rote

in

rE1E

2p7 p

rote

inrS

OD-N

S5 (N

S5A/B

)

Supplemental Fig. 1

Legend of supplemental Fig. 1

Detection of HCV protein expression from VEE/SIN replicon particles by Western blotting.A. Detection of E1 and E2 expression from the BHK cell lysates with VEE/SIN-E1E2 and VEE/SIN-E1E2p7 infection. VEE/SIN-NS345 is a negative control. rE1E2p7 protein is a positive control. The size was indicated corresponding to E1 (33-35kDa) and E2 (68-74kDa). EndoH: protein was treated with EndoH (Biolab) at 37ºC for 1 hour.B. Detection of NS3, NS4, NS5A and NS5B expression from the cell lysates with VEE/SIN-NS345 infection. rE1E2p7 is a negative control. SOD-C33C (a.a. 1192-1457), SOD-C100 (a.a. 1569-1931), and SOD-NS5 (a.a. 2054-2995) are recombinant proteins used as positive controls for the detection of NS3, NS4, NS5A and NS5B respectively. The size was indicated corresponding to NS3, NS4A, NS4B, NS4A/B, NS5A and NS5B. * Indicated the specific detection of the proteins.

CD4

IFN-

CD8

PBS (1,2,3)

IFN-

E1E2/MF59 (1,2,3)

E1E2/MF59/CpG(1,2,3)

VEE/SIN-E1E2 (1,2,3)

E1E2/MF59 (1,2)VEE/SIN-E1E2 (3)

E1E2/MF59/CpG (1,2)VEE/SIN-E1E2 (3)

PBS (1,2,3) E1E2/MF59 (1,2,3)

E1E2/MF59/CpG(1,2,3)

VEE/SIN-E1E2 (1,2,3)

E1E2/MF59 (1,2)VEE/SIN-E1E2 (3)

E1E2/MF59/CpG (1,2)VEE/SIN-E1E2 (3)

A BSupplemental Fig. 2

Legend of supplemental Fig. 2

Detection of CD4+ and CD8+ T cells responses by ICS/FACS.BALB/c mice (n = 10) received three injections i.m. at 3-week intervals by the immunogens as indicated. The spleen cells were harvested 2 weeks after the last immunization and stimulated with 10 g/ml of HCV specific peptides for ICS and FACS analysis. The data are presented as dot plots of ICS for CD4+ and IFN- after E1 peptide pool stimulation (A) and for CD8+ and IFN- after CD8 E2 peptide stimulation (B). Significant responses are indicated by circles in the upper-right quadrant.

Supplemental Fig. 3

E1E2/MF59 (1

,2,3)

E1E2/MF59/C

pG (1,2,3)

VEE/SIN

-E1E2 (1

,2,3)

VEE/SIN

-E1E

2 (1

,2);

E1E2/

MF59

(3)

VEE/SIN

-E1E

2 (1

,2);

E1E2/

MF59

/CpG (3

)

E1E2/

MF59

(1,2

);

VEE/SIN

-E1E

2 (3

)

E1E2/

MF59

/CpG (1

,2);

VEE/SIN

-E1E

2 (3

)

PBS (1,2,3)

*

0.0

0.2

0.4

0.6

0.8 MediumE1 poolE2 poolCD4 E2 pepCD8 E2 pep

% o

f CD8

+ T

cells

Legend of supplemental Fig. 3

VEE/SIN-E1E2 immunization and Prime/boost regimen with E1E2/MF59 and VEE/SIN-E1E2 could stimulate good CD8+ T cells responses.BALB/c mice (n = 10) were received three injections i.m. at 3-week intervals by the immunogens as indicated. The spleen cells were harvested at 2 weeks after the last immunization and stimulated with 10 g/ml of HCV specific peptides for ICS and FACS analysis. 20-mer over-lapping peptide pools for HCV-1a E1 region (E1 pool) and E2 region (E2 pool), and single peptide to E2 for CD4+ T cells (CD4 E2 pep) and CD8+ T cells (CD8 E2 pep) were used for stimulation. The data are presented as mean total percentages of CD8+IFN-+ cells from two pools (five mice per pool). * P<0.05 as compared with PBS control group.