A Multiantigenic DNA Vaccine That Induces Broad Hepatitis C Virus-Specific T-Cell Responses in Mice

Abstract

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI.

Importance: Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.

FIG 1

Schematic diagram of the bicistronic DNA vaccines used in the study. The DNA vaccine backbone, pVAX, was used to develop the following DNA vaccines: (i) HCV (poly)protein under the control of the CMV promoter and (ii) VSVG or PRF under the control of the SV40 promoter. An additional construct, which has a pLenti backbone, encodes mCherry, an NLS, and MAVS fused as a single protein under the control of the CMV promoter.

FIG 2

Gating strategy used to detect CD8 and CD4 T cells producing multiple cytokines in the splenocyte populations of vaccinated mice. Mice were vaccinated with three 10-μg doses or three 25-μg doses of DNA intradermally, and splenocytes were harvested on day 14 after the final vaccination for ICS. Cytokine profiles were determined by using flow cytometry. Splenocytes were gated on the lymphocyte population, followed by doublet discrimination, and then gated on CD3⁺ CD44⁺ cells and finally CD4⁺ or CD8⁺ cells to assess the frequency of IFN-γ, TNF-α, and IL-2. Representative blots for IFN-γ-, TNF-α-, and IL-2-positive cells are shown.

FIG 3

HCV protein expression and protease activity. (A) To detect HCV protease cleavage of MAVS, Huh-7 cells were cotransfected with a plasmid (pRep) encoding a fluorescent reporter protein, i.e., mCherry, fused to an NLS and the C-terminal region of MAVS, as well as different constructs encoding various HCV proteins. At 24 h posttransfection, fluorescence was observed by fluorescence microscopy. The panels (left to right) show the intracellular localization of mCherry in cells cotransfected with pRep and either pNS3, pNS3/4A, pNS34B5B, and pNS345B, respectively. In general, cells which lack NS3/4A protease show punctate cytoplasmic fluorescence, whereas cells that express a functional NS3/4A protease show nuclear fluorescence. (B and C) Detection of HCV proteins in the lysates of transfected cells. HEK293T cells were transfected with DNA encoding NS3 with or without VSVG (B) and HCV proteins with or without PRF (C). At 48 h posttransfection, cell lysates were examined by Western blotting and probed with pooled HCV gt3 patient sera. Bicinchoninic acid and β-actin were used as loading controls. The predicted sizes of the HCV proteins are shown. Blots are representative of three independent experiments. (D) To detect perforin expression, HEK293T cells were transfected with plasmids in which perforin expression was controlled by the SV40 promoter. At 48 h posttransfection, the cells were fixed and permeabilized, and immunofluorescence was performed as described in Materials and Methods. The immunofluorescence images are representative of all perforin-expressing constructs.

FIG 4

NS3 is more immunogenic than NS3/4A in C57BL/6 mice. Mice were vaccinated three times at 2-week intervals with the respective constructs, and splenocytes were harvested 14 days after the final vaccination. (A) Splenocytes were restimulated in duplicate, with overlapping peptides representing the complete HCV NS3 protein (gt3a), and IFN-γ secretion was measured by ELISPOT assay. The PHA control from the pVAX group is representative of all PHA-stimulated groups. The number of spots in unstimulated splenocytes was subtracted from the number in peptide-stimulated cells to generate the net number of NS3-specific SFU. The data are shown as the mean SFU/10⁶ splenocytes ± the SEM. Cytokine profiles were determined using flow cytometry. Splenocytes were gated on CD3⁺, CD44⁺ cells and then CD4⁺ or CD8⁺ cells to assess the frequency of IFN-γ-producing CD4⁺ T_EM cells (B), IFN-γ-producing CD8⁺ T_EM cells (C), TNF-α-producing CD4⁺ T_EM cells (D), and TNF-α-producing CD8⁺ T_EM cells (E). Each symbol represents an individual mouse, and the data show the means (n = 7) ± the SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney test).

FIG 5

NS3 coexpressed with PRF is more immunogenic than NS3 alone. Splenocytes from vaccinated mice were stimulated with NS3 peptides, and cytokine production was analyzed using ICS and flow cytometry. Memory CD4⁺ and CD8⁺ T cells were gated to assess the frequency of IFN-γ-producing CD8⁺ T_EM cells (A), TNF-α-producing CD8⁺ T_EM cells (B), IL-2-producing CD8⁺ T_EM cells (C), IFN-γ/TNF-α-double-producing CD8⁺ T_EM cells (D), TNF-α/IL-2-double-producing CD8⁺ T_EM cells (E), IFN-γ/IL-2-double-producing CD8⁺ T_EM cells (F), IFN-γ/TNF-α/IL-2-triple-producing CD8⁺ T_EM cells (G), and the proliferation of CFSE-labeled CD3⁺ CD8⁺ T cells (H). Splenocytes were labeled with CFSE and restimulated for 5 days with peptides representing the C-terminal third of NS3; the proliferation of CD3⁺ CD8⁺ T cells is shown as a percentage of total CD3⁺ CD8⁺ T cells. (I) Frequency of IFN-γ-producing CD4⁺ T_EM cells after NS3 peptide stimulation. Each symbol represents an individual mouse, and the data show the means (n = 8 to 9) ± the SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney test).

FIG 6

The adjuvant activity of PRF is retained in DNA vaccines encoding multiple HCV proteins. Mice were vaccinated twice with equimolar amounts of the plasmid DNA at 2-week intervals, and splenocytes from vaccinated animals were restimulated with overlapping peptides representing NS3 peptides (A), NS4 peptides (B), and NS5B peptides (C). (D) NS3, NS4, and NS5B combined analysis. (E and F) IFN-γ response in splenocytes from mice vaccinated with pNS345B-PRF or pNS3-PRF and restimulated with NS3 peptides (E) or with pNS345B-PRF or pNS4B5B and restimulated with NS4 and NS5B peptides (F). The PHA control from the pVAX group is representative of all PHA-stimulated groups. The number of spots in unstimulated splenocytes was subtracted from the number in peptide-stimulated cells to generate the net number of specific SFU. The data are shown as the mean SFU/10⁶ splenocytes ± the SEM. Each symbol represents an individual mouse, and the data show the mean (n = 7) ± the SEM. *, P ≤ 0.05; ***, P ≤ 0.001 (Mann-Whitney test).