Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model

Abstract

DNA vaccines express antigens intracellularly and effectively induce cellular immune responses. Because only chimpanzees can be used to model human hepatitis C virus (HCV) infections, we developed a small-animal model using HLA-A2.1-transgenic mice to test induction of HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) and protection against recombinant vaccinia expressing HCV-core. A plasmid encoding the HCV-core antigen induced CD8(+) CTLs specific for three conserved endogenously expressed core peptides presented by human HLA-A2.1. When challenged, DNA-immunized mice showed a substantial (5-12 log(10)) reduction in vaccinia virus titer compared with mock-immunized controls. This protection, lasting at least 14 mo, was shown to be mediated by CD8(+) cells. Thus, a DNA vaccine expressing HCV-core is a potential candidate for a prophylactic vaccine for HLA-A2.1(+) humans.

Figure 1

Specificity of CTL lines raised against three HLA-A2-binding peptides by immunization with the HCV-core DNA vaccine. Spleen cells from mice immunized 6 mo previously with the HCV-core DNA vaccine were stimulated in vitro with the indicated peptides at 10 μM. After three to four weekly in vitro stimulations, the effector CTL were tested for lysis of 3,000 C1R-AAD target cells/well in the presence of the indicated concentrations of each peptide, at an effector-to-target cell ratio of 100:1. Lysis on targets in the absence of peptide was <5%, as shown on the left axis. Furthermore, each line killed targets only with the homologous peptide, and lysis on targets with each of the other two respective peptides was ≤5% (data not shown). Similar results were obtained 2 mo and 14 mo after immunization. At all three time points, no specific CTL were detected after similar peptide stimulation of spleen cells from mice immunized with a control DNA vaccine plasmid (<5% lysis).

Figure 2

Blocking of the CTL activity against three HCV-specific peptides by anti-CD8, anti-CD4, or anti-HLA-A2 antibodies. CTL were expanded by in vitro peptide stimulation of spleen cells from mice immunized 6 mo earlier with the HCV-core DNA vaccine. The CTL were tested for killing of C1R-AAD target cells in the presence of the indicated peptides at 10 μM. The effector-to-target ratio was 50:1. The blocking antibodies indicated were present throughout the 6-hr ⁵¹Cr-release assay as a 1:5 dilution of hybridoma culture supernatant. Background lysis on targets without peptide was ≤5%.

Figure 3

Cytotoxicity of AAD CTL lines against endogenously expressed protein after stimulation with the core-specific peptides 1073.07, C7A2, and 939.20.1 6 mo after immunization with AC7. C1R-AAD target cells were infected for 2 hr with rVV expressing HCV-core or measles HA as a control, at a multiplicity of infection of 10, washed, incubated for an additional 14 hr, and then ⁵¹Cr labeled. Lysis by the CTL lines stimulated with each of the three peptides is shown as a function of effector-to-target ratio, with 3,000 target cells per well.

Figure 4

Vaccinia virus titers in ovaries of mice challenged with 10⁷ pfu of vaccinia/HCV-core or vaccinia/measles HA i.p. 2 mo after immunization. Two months after immunization with either the AC7 HCV-core DNA vaccine or mock DNA vaccine (plasmid only), the mice were challenged i.p. with 1 × 10⁷ pfu of rVV expressing HCV-core or measles HA as a control. In the groups indicated, the mice were treated with 0.5 mg anti-CD8 antibody daily for 2 d before challenge, on the day of challenge, and on the day after challenge. Five days after challenge, the ovaries were harvested and the vaccinia titer measured by plaquing on BSC-1 cells. Two independent experiments are shown in a and b.

Figure 5

Titer of vaccinia/HCV-core in ovaries of mice 6 mo after immunization. Six months after immunization with the indicated DNA vaccines, mice were challenged as described in Fig. 4. There was a statistically significant protection with a 7-log₁₀ reduction in virus titer in the group immunized with AC7 and not treated with anti-CD8 antibody, compared with mice immunized with AC7 and treated with anti-CD8 antibody (P = 0.000057 by Student's t test). Thus, protection persisted 6 mo after immunization and was CD8 dependent.

Figure 6

Titer of vaccinia/HCV-core in ovaries of mice 14 mo after immunization. Fourteen months after immunization with AC7 or mock DNA vaccines, mice were challenged with HCV-core rVV, as indicated in Fig. 4. There was a statistically significant protection with a 5-log₁₀ reduction in virus titer in the group immunized with AC7 and not treated with anti-CD8 antibody, compared with mice immunized with AC7 and treated with anti-CD8 antibody (P = 0.0000015 by Student's t test). Thus, protection persisted at least 14 mo after immunization and was CD8 dependent.