Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection

Abstract

We have recently demonstrated that immunization with hepatitis C virus-like particles (HCV-LPs) generated in insect cells can elicit both humoral and cellular immune responses in BALB/c mice. Here, we evaluate the immunogenicity of HCV-LPs in HLA2.1 transgenic (AAD) mice in comparison to DNA immunization. HCV-LP immunization elicited a significantly stronger humoral immune response than DNA immunization. HCV-LP-immunized mice also developed stronger HCV-specific cellular immune responses than DNA-immunized mice as determined by using quantitative enzyme-linked immunospot (ELISpot) assay and intracellular cytokine staining. In BALB/c mice, immunization with HCV-LPs resulted in a >5 log10 reduction in vaccinia titer when challenged with a recombinant vaccinia expressing the HCV structural proteins (vvHCV.S), as compared to 1 log10 decrease in DNA immunization. In HLA2.1 transgenic mice, a 1-2 log10 reduction resulted from HCV-LP immunization, whereas no reduction was seen from DNA immunization. Adoptive transfer of lymphocytes from HCV-LP-immunized mice to naive mice provided protection against vvHCV.S challenge, and this transferred immunity can be abrogated by either CD4 or CD8 depletion. Our results suggest that HCV-LPs can induce humoral and cellular immune responses that are protective in a surrogate HCV challenge model and that a strong cellular immunity provided by both CD4 and CD8 effector lymphocytes may be important for protection from HCV infection.

Fig. 1.

Anti-E1/E2 titers in HCV-LP- or DNA-immunized AAD mice. The AAD mice were immunized, and the serum was harvested and assayed 2 wk after the last immunization for anti-E1/E2 as described in Materials and Methods. The anti-E1/E2 titer by endpoint dilution in ELISA of each mouse is shown. Mean values are expressed as geometric mean titers ± SEM and shown as bars on the left. Sera tested negative by ELISA were assigned an arbitrary titer of one-half of the initial dilution of 100. The P value is shown above. These data are representative of three similar experiments.

Fig. 2.

CD4 T helper responses in HCV-LP- or DNA-immunized AAD mice. AAD mice were immunized, and splenocytes were harvested 2 wk after the last immunization as described in Materials and Methods.(A) Proliferative response. Splenocytes were stimulated in vitro for 5 days with 5 μg/ml recombinant HCV core or E1/E2 proteins. Data from individual mice are shown, and the mean SI ± SEM is indicated as bar on the left. SI of >3 (dotted line) is considered positive. IFN-γ (B) and IL-4 (C) ELISpot assays of splenocytes after stimulation with recombinant HCV core or E1/E2 protein are shown. Spots from individual mice are shown, and the mean ± SEM is indicated as bar on the left. The P values are shown. These data are representative of three similar experiments.

Fig. 3.

CD8 CTL responses in HCV-LP- or DNA-immunized AAD mice. AAD mice were immunized and splenocytes were harvested 2 wk after the last immunization as described in Materials and Methods.(A) The cytotoxic activities (percentage specific lysis) of splenocytes against core or E2 peptide target are indicated at an effector-to-target cell ratio of 30:1. Data from individual mice are shown, and the mean percentage specific lysis ± SEM is indicated as the bar on the left. (B) Intracellular cytokine staining. Splenic lymphocytes were stimulated in vitro with HCV CTL peptide (core or E2) and then analyzed for intracellular IFN-γ staining. Representative fluorescence-activated cell sorter flowgrams (10,000 events were collected for counting) are shown in Upper (percentages of IFN-γ⁺CD8⁺ cells are shown in the right upper quadrants) and summary of percentages of IFN-γ-producing CD8⁺ cells with means ± SEM are shown in Lower. For background determination, lymphocytes cultured for 5 days with HCV CTL core or E2 peptide were stimulated without any peptide and assayed for ICS. Individual data are shown after subtraction of the background. For both CTL and ICS assays, splenocytes were stimulated with an irrelevant peptide (HBV core 18–27 peptide) as control, and the results showed background activities (<5% specific lysis for CTL and <0.2% for ICS). The P values are shown. These data are representative of three similar experiments.

Fig. 4.

Vaccinia growth curve in BALB/c mice. BALB/c mice were inoculated i.p. with 10⁶ or 10⁷ pfu of vvHCV.S or vvLacZ in triplicate. The ovaries were harvested at various times, and the mean vaccinia titers were determined and plotted.

Fig. 5.

Vaccinia titers in immunized BALB/c mice challenged with vvHCV.S or vvLacZ. Mice were killed 5 days after challenge. Vaccinia titers in ovaries were determined. Data are shown for each mouse, and the mean vaccinia titer ± SEM is indicated. (A) Mice (six or seven per group) were immunized with HCV-LP or control preparation and then challenged with vvHCV.S or vvLacZ 2 wk after the third immunization. (B) Mice (three to five per group) were immunized with p7020.S or p7020 (control vector) and then challenged with vvHCV.S or vvLacZ. (C) Vaccinia titers in immunized AAD mice challenged with vvHCV.S or vvLacZ. Six mice per group were immunized with HCV-LP, p7020.S, or control. Mice were killed, and vaccinia titers in ovaries were determined. Data are shown for each mouse, and the mean vaccinia titer ± SEM is indicated. The P values are shown. These data are representative of two similar experiments.

Fig. 6.

Vaccinia titer in adoptive transferred BALB/c mice. Ten BALB/c mice were immunized three times with HCV-LP. Two weeks after the third immunization, splenic lymphocytes were harvested and pooled. After washing with PBS extensively, CD4 or CD8 cells were depleted. Cells (3 × 10⁷) with or without depletion were injected into syngenic naive BALB/c mice through the tail vein. After adoptive transfer (24 h), the mice were challenged with 10⁶ pfu of vvHCV.S i.p. Mice were killed 4 days after challenge, and then vaccinia titers in ovaries were determined. Data are shown for each mouse, and the mean vaccinia titer ± SEM is indicated. The P values are shown. These data are representative of three similar experiments.