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Anti-E1Anti-E2 rE1E2p7 protein VEE/SIN-E1E2P7 VEE/SIN-E1E2 33-35 rE1E2P7 protein VEE/SIN-E1E2P7 VEE/SIN-E1E2 EndoH 35 25 15 75 50 35 25 r E1E2p7 protein) VEE/SIN-E1E2P7 r E1E2P7 protein VEE/SIN-E1E2P7 VEE/SIN-E1E2 EndoH 68-74 VEE/SIN-NS345 * * * 70 Anti-NS3 rSOD-C33C (NS3) NS4A/B: 35 VEE/SIN-NS345 rSOD-C100 (NS4) Anti-NS4 57 VEE/SIN-NS345 rSOD-NS5 (NS5A/B) Anti-NS5AAnti-NS5B VEE/SIN-NS345 69 NS4B: 27 NS4A: 8 * * * * * * * * ** A B rE1E2p7 protein rSOD-NS5 (NS5A/B) Supplemental Fig. 1

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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.

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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) AB Supplemental Fig. 2

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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.

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Supplemental Fig. 3 E1E2/MF59 (1,2,3) E1E2/MF59/CpG (1,2,3) VEE/SIN-E1E2 (1,2,3) VEE/SIN-E1E2 (1,2); E1E2/MF59 (3) VEE/SIN-E1E2 (1,2); E1E2/MF59/CpG (3) E1E2/MF59 (1,2); VEE/SIN-E1E2 (3) E1E2/MF59/CpG (1,2); VEE/SIN-E1E2 (3) PBS (1,2,3) *

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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.

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