Long-term functional duration of immune responses to HCV NS3/4A induced by DNA vaccination

Abstract

We have investigated the ability of hepatitis C virus non-structural (NS) 3/4A-DNA-based vaccines to activate long-term cell-mediated immune responses in mice. Wild-type and synthetic codon optimized (co) NS3/4A DNA vaccines have previously been shown to be immunogenic in mice, rabbits and humans, although we have very poor knowledge about the longevity of the immune responses primed. We therefore analyzed the functionality of primed NS3/4A-specific immune responses in BALB/c (H-2(d)) and/or C57BL/6J (H-2(b)) mice 1, 2, 3, 4, 6, 12 and 16 months after the last immunization. Mice were immunized one, two, three or four times using gene gun delivery to the skin or by intramuscular administration. Immunological responses after immunization were monitored by protection against in vivo challenge of NS3/4A-expressing syngeneic tumor cells. In addition, functionality of the NS3/4A-specific T cells was analyzed by a standard cytotoxicity assay. First, we identified a new unique murine H-2(d)-restricted NS3/4A cytotoxic T lymphocyte (CTL) epitope, which enabled us to study the epitope-specific immune responses. Our results show that the coNS3/4A vaccine was highly immunogenic by determination of interferon-γ/tumor necrosis factor-α production and lytic cytotoxic T cells, which could efficiently inhibit in vivo tumor growth. Importantly, we showed that one to four monthly immunizations protected mice from tumor development when challenged up to 16 months after the last immunization. When determining the functionality of NS3/4A-specific T cells in vitro, we showed detectable lytic activity up to 12 months after the last immunization. Thus, NS3/4A-based DNA vaccines activate potent cellular immune responses that are present and function in both BALB/c and C57BL/6J mice up to 12-16 months after the last immunization. The induction of long-term immunity after NS3/4A DNA immunization has not been shown previously and supports the use of NS3/4A in hepatitis C virus vaccine compositions.

Figure 1

Priming of in vitro detectable CTLs against K^d- and D^b-binding peptides in H-2^d (BALB/c) and H-2^b (C57BL/6J) mice. Groups of five mice were immunized two times with 2 μg coNS3/4A plasmid DNA using transdermal gene gun (gg) delivery (a, c, e and g) or left untreated (non-immunized) (b, d, f and h). Two weeks after the last immunization, the specific lytic activity was determined using peptide-loaded RMA-S cells at E:T ratios of 60:1, 20:1 and 7:1 in a standard ⁵¹Cr-release assay. Specific lysis above 10% was considered positive. Each line indicates an individual mouse.

Figure 2

Evaluation of the ability of different immunogens to prime HCV NS3/4A-specific tumor-inhibiting responses after DNA immunization. Groups of 8–10 C57BL/6J or BALB/c mice were either left untreated or were given one (a), two or four monthly (b) gene gun (gg) immunizations of 2 μg plasmid DNA of the indicated immunogens. At 2 weeks after the last immunization, mice were subcutaneously inoculated with 1 × 10⁶ NS3/4A-expressing EL-4 (a) or NS3/4A-expressing SP2/0-Ag14 (b) cells. Tumor sizes were measured through the skin at days 6–19 after tumor inoculation. Values are given as the mean tumor size±s.e.m. Also given is the P-value obtained from the statistical comparison of immunized and control groups using the area under the curve (AUC) and analysis of variance. In (c), the NS3-specific lytic activity after two immunizations with indicated immunogens in groups of five C57BL/6 and IFNγR2−/− mice are shown. In (d), the percentage of IFN-γ- or TNFα-producing CD8⁺ T cells in groups of five immunized C57BL/6 mice (five mice per pool) with indicated immunogens. In (e and f), representative dot plots from each group of mice showing the IFN-γ- and TNFα-positive CD8⁺ T cells.

Figure 3

Evaluation of the longevity of HCV NS3/4A-specific tumor-inhibiting responses. Protection against tumor growth was evaluated 1, 3, 6 or 16 months after one or three DNA immunizations (a). Groups of 4–12 C57BL/6J mice were either left untreated or were given one (gene gun (gg) delivery) or three (gg or intramuscular delivery) immunizations of 2 μg wtNS3/4A-pVAX1 or coNS3/4A-pVAX1 plasmid DNA. A dose of 100 μg wtNS3/4A-pVAX1 was used for the intramuscular immunizations. Tumor sizes were measured through the skin at days 6–20 after tumor inoculation. Values are given as the mean tumor size±s.e.m. Also given is the P-value obtained from the statistical comparison of immunized and control groups using the AUC and analysis of variance. In (b), the presence of lytic T-cell responses was determined at 1, 3 and 6 months after a single transdermal gg immunization and at 1, 2, 3, 4, 6 and 12 months after two monthly transdermal gg immunizations with 2 μg coNS3/4A-pVAX1. Each group consisted of five mice. Two weeks after the last immunization, specific lytic activity was determined using peptide-loaded RMA-S cells at E:T ratios of 60:1, 20:1 and 7:1 in a standard ⁵¹Cr-release assay. Specific lysis above 10% was considered positive. Each line indicates an individual mouse.

Figure 4

Priming of NS3-specific immune responses after intramuscular and transdermal DNA immunization. Groups of five C57BL/6J or IFNγR2−/− mice were immunized using intramuscular needle injection, transdermal gene gun (gg) injection or left untreated. At 2 weeks after the last immunization, mice were killed and splenocytes harvested for determination of T-cell responses. In (a, rows 1 and 3), the number of IFN-γ spot-forming cells (SFCs) by ELISpot assay was determined 36 h after in vitro stimulation of splenocytes with CTL and T-helper peptides or recombinant NS3 protein at the indicated concentrations. Results are given as mean SFCs per 10⁶ (±s.d.), the cutoff was set to 50 SFCs per 10⁶ splenocytes. In (a, rows 2 and 4), the lytic activity determined by a ⁵¹Cr-release assay using peptide-loaded (GAVQNEVTL) RMA-S cells from individual mice at E:T ratios of 60:1, 20:1 and 7:1 is shown. Specific lysis above 10% was considered positive. Each line indicates an individual mouse. In (b), expansion of NS3-specific CD8⁺ T cells was determined using direct ex vivo pentamer staining. GAVQNEVTL epitope-specific CD8⁺ T-cell frequencies are shown as the percentage of GAVQNEVTL pentamer-positive CD8⁺ T cells (±s.d.). Also, representative dot plots from each group are shown. The presence of a statistical difference (Mann–Whitney U-test) was indicated as follows: P<0.05 and P<0.01. NS, not significant.

Figure 5

Evaluation of in vitro detectable CTLs and tumor-inhibiting responses in C57BL/6J mice immunized with wtNS3/4A-pVAX1 or wtNS3/4AΔ5,9-pVAX1 plasmid DNA. (a) Groups of five C57BL/6J mice were immunized once with 2 μg plasmid DNA using transdermal gene gun (gg) delivery or were left untreated (non-immunized). Two weeks after the last immunization, the NS3-specific lytic activity was determined using peptide-loaded (GAVQNEVTL) RMA-S cells at E:T ratios of 60:1, 20:1 and 7:1 in a standard ⁵¹Cr-release assay. Specific lysis above 10% was considered positive. Each line indicates an individual mouse. In (b), the in vitro peptide stabilization of MHC–peptide complexes on transporter associated with antigen processing 2-deficient RMA-S cells transfected with H-2D^b is shown. Binding affinities were determined by flow cytometry measuring the mean fluorescence intensity (MFI) for each peptide in descending concentrations. (c) Groups of 10 C57BL/6J mice were either left untreated or were given one gg immunizations of 2 μg wtNS3/4A or wtNS3/4AΔ5,9 plasmid DNA. At 2 weeks after immunization, mice were subcutaneously inoculated with 1 × 10⁶ NS3/4A-expressing EL-4 cells. Tumor sizes were measured through the skin at days 6–18 after tumor inoculation. Values are given as the mean tumor size±s.e.m. Also given is the P-value obtained from the statistical comparison of wtNS3/4A and non-immunized or wtNS3/4AΔ5,9 and non-immunized or wtNS3/4A and wtNS3/4AΔ5,9 using the AUC and analysis of variance. NS, not significant.