FIG. 6.

A combination prime/boost immunization regimen elicits broad cellular responses to HCV structural and nonstructural proteins, as well as cross-neutralizing antibodies. Groups of 10 BALB/c mice were immunized as indicated. For the combination immunization regimen, priming with E1E2/MF59/CpG and NS345Poly/IMX vaccines and boosting with VEE/SIN-E1E2 and VEE/SIN-NS345 were done individually to separate muscles for two different antigens at weeks 0, 3, and 6. At week 8, two pools of spleen cells were prepared (five spleens per pool) and stimulated with HCV-specific peptides prior to staining for intracellular IFN-γ and fluorescence-activated cell sorter analysis. The mean values obtained from the two pools are shown. Panels: A, CD4⁺ cells stimulated by E1 and E2 peptides; B, CD8⁺ cells stimulated by E1 and E2 peptides; C, CD4⁺ cells stimulated by NS3, -4, and -5 peptides; D, CD8⁺ cells stimulated by NS3, -4, and -5 peptides; E, cross-neutralizing antibody activity elicited against JFH-1 2a HCVcc. Serum obtained at week 8 was diluted 1:100 and preincubated with virus for 1 h at 37°C prior to infection of Huh-7 cells. Three days later, luciferase activity in cell lysates was determined. Data are expressed as percent inhibition (based on means from triplicate assays) relative to control mice immunized with PBS. *, P < 0.05 compared with the medium control and the PBS-immunized group. IMX, Iscomatrix.