Induction of Genotype Cross-Reactive, Hepatitis C Virus-Specific, Cell-Mediated Immunity in DNA-Vaccinated Mice

Abstract

A universal hepatitis C virus (HCV) vaccine should elicit multiantigenic, multigenotypic responses, which are more likely to protect against challenge with the range of genotypes and subtypes circulating in the community. A vaccine cocktail and vaccines encoding consensus HCV sequences are attractive approaches to achieve this goal. Consequently, in a series of mouse vaccination studies, we compared the immunogenicity of a DNA vaccine encoding a consensus HCV nonstructural 5B (NS5B) protein to that of a cocktail of DNA plasmids encoding the genotype 1b (Gt1b) and Gt3a NS5B proteins. To complement this study, we assessed responses to a multiantigenic cocktail regimen by comparing a DNA vaccine cocktail encoding Gt1b and Gt3a NS3, NS4, and NS5B proteins to a single-genotype NS3/4/5B DNA vaccine. To thoroughly evaluate in vivo cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses against Gt1b and Gt3a HCV peptide-pulsed target cells, we exploited a novel fluorescent-target array (FTA). FTA and enzyme-linked immunosorbent spot (ELISpot) analyses collectively indicated that the cocktail regimens elicited higher responses to Gt1b and Gt3a NS5B proteins than those with the consensus vaccine, while the multiantigenic DNA cocktail significantly increased the responses to NS3 and NS5B compared to those elicited by the single-genotype vaccines. Thus, a DNA cocktail vaccination regimen is more effective than a consensus vaccine or a monovalent vaccine at increasing the breadth of multigenotypic T cell responses, which has implications for the development of vaccines for communities where multiple HCV genotypes circulate.IMPORTANCE Despite the development of highly effective direct-acting antivirals (DAA), infections with hepatitis C virus (HCV) continue, particularly in countries where the supply of DAA is limited. Furthermore, patients who eliminate the virus as a result of DAA therapy can still be reinfected. Thus, a vaccine for HCV is urgently required, but the heterogeneity of HCV strains makes the development of a universal vaccine difficult. To address this, we developed a novel cytolytic DNA vaccine which elicits robust cell-mediated immunity (CMI) to the nonstructural (NS) proteins in vaccinated animals. We compared the immune responses against genotypes 1 and 3 that were elicited by a consensus DNA vaccine or a DNA vaccine cocktail and showed that the cocktail induced higher levels of CMI to the NS proteins of both genotypes. This study suggests that a universal HCV vaccine can most readily be achieved by use of a DNA vaccine cocktail.

Keywords: DNA vaccines; ELISpot; HCV; NS5; consensus; hepatitis; multiantigenic; multigenotypic; vaccine.

FIG 1

DNA vaccines and detection of antigen expression. (A) Schematic diagram of the bicistronic DNA plasmid vaccines. pNS5B-PRF (7.3 kb) and pNS345B-PRF (9.8 kb) were based on the control plasmid pSV40-PRF (5.5 kb). Codon-optimized sequences in pNS5B-PRF that represent the HCV NS5B consensus sequence or Gt1b or Gt3a sequences were cloned into the multiple-cloning site with a Kozak sequence for enhanced mammalian expression under the control of the CMV promoter and flanked by the bovine growth hormone polyadenylation (BGH pA) site for termination of transcription. The expression of the cytolytic protein PRF was under the control of the weaker SV40 promoter and flanked by a downstream SV40 polyadenylation (pA) sequence. The pNS345B-PRF plasmid was modeled on pNS5B-PRF; the HCV genotype 1b or 3a NS3, NS4, and NS5B sequences were cloned into the multiple-cloning site as described previously (29). pSV40-PRF was used as a control plasmid, in which PRF was expressed from the SV40 promoter but no antigen was cloned under the control of the CMV promoter. (B to D) Photomicrographs of HEK 293T cells transfected with the different DNA vaccines (B and C) or vaccine controls (D) and stained with anti-HCV antibodies, anti-PRF, or a mixture of both (29, 30), showing colocalization of PRF and HCV antigens within transfected cells.

FIG 2

Cell-mediated responses to DNA vaccines as determined by ELISpot analysis. Groups of BALB/c mice (n = 7 for G1 to G4 and n = 3 for G5) were vaccinated with DNA encoding the NS5B consensus sequence or the NS5B consensus sequence (25 μg) plus an equal dose of SV40-PRF (25 μg), a cocktail comprised of 25 μg each of Gt1b and Gt3a sequences encoding NS5B, a similar cocktail comprised of 50 μg each of Gt1b and Gt3a sequences, or a control (G5). At 14 days postvaccination, the splenocytes were harvested and stimulated with peptides representing the NS5B protein (4 μg), and IFN-γ-secreting cells were detected as described in Materials and Methods. The data are presented as mean (n = 7) ± SEM SFU per 1 × 10⁶ splenocytes, and the unpaired nonparametric Mann-Whitney U test was used to analyze the statistical significance. Note that the scale of the y axis differs in different panels. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

FTA analysis of in vivo NS5B-specific T cell responses following consensus NS5B or cocktail vaccination. The Gt1b and Gt3a NS5B-specific in vivo CTL responses were examined following vaccination of mice with DNA encoding the consensus NS5B protein or a cocktail encoding Gt1b NS5B and Gt3a NS5B. Female BALB/c mice (n = 6 or 7 per group for G1 to G4 and n = 3 for G5) were vaccinated as described in Materials and Methods, and 13 days after the final vaccination, 1.3 × 10⁶ peptide-pulsed cells (for each target cell cluster) or mock-pulsed target cells were transferred i.v. Fifteen hours after FTA challenge, splenocytes were harvested and analyzed by flow cytometry to determine the surviving number of peptide-pulsed target cells relative to the number of unpulsed target cells. (A) Flow diagram of the FTA analysis used in the experiments. Doubly discriminated lymphocytes were gated prior to analysis of the Gt1b and Gt3a targets based on CPD emission, as shown. For simplicity, the plots show an example in which Gt1b targets from a G4-vaccinated mouse were gated. All the peptide-pulsed cell targets were gated and analyzed for the percent recovery relative to that of mock-pulsed targets to determine the specific FTA cell loss, using the following equation: % cell loss = [(% mock-pulsed targets − % peptide-pulsed targets)/% mock-pulsed targets] × 100. For Th cell response analysis, B220⁺ cells from Gt1b or Gt3a targets were gated, and the geometric mean fluorescence intensity (GMFI) of CD69 was determined by flow cytometry. The values plotted in panel C reflect the GMFI of CD69 on peptide-pulsed B220⁺ targets minus the GMFI of CD69 on mock-pulsed B220⁺ targets. The representative histogram plot shows CD69 expression on B220⁺ cell targets pulsed with 10 μg/ml of Gt1b NS5B pool 2 (black line) or 0 μg/ml of Gt1b NS5B pool 2 (gray filled area). (B and C) Bar graphs showing the means and SEM of Gt1b- and Gt3a-specific CTL responses against target cells pulsed with 10 μg/ml of NS5B pools 1 to 3 (P1 to P3) (B) and Th cell responses for the same targets, based on the geometric mean fluorescence intensity of CD69 on FTA B220⁺ cells (C). The statistical significance of the data was determined as described in Materials and Methods. Note that only comparisons where at least one of the groups being compared had a statistically significant enhancement of the responses relative to those of the control group are shown. *, P < 0.05; **, P < 0.01.

FIG 4

In vivo multiantigenic and multigenotypic CTL responses resulting from monovalent and cocktail vaccinations. Female BALB/c mice (n = 6 or 7 per group for G6 to G9 and n = 4 for G10) were vaccinated as described in Materials and Methods. Thirteen days later, 6.5 × 10⁵ peptide-pulsed cells (for each target cell cluster) or mock-pulsed target cells were injected i.v., and RBC-depleted splenocytes were harvested 15 h later and analyzed by flow cytometry to determine the percentage of surviving peptide-pulsed target cells. (A) FTA design for the experiment. For simplicity, the target cell matrix for the Gt1b population (CPD^lo) is shown, but the same matrix was used to label the Gt3a population (CPD^hi). (B and C) Means and SEM of Gt1b-specific (B) and Gt3a-specific (C) CTL responses. The statistical significance of the data was determined as described in Materials and Methods. Note that only comparisons where at least one of the groups being compared had a statistically significant enhancement of the responses relative to those of the control group are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

In vivo multiantigenic and multigenotypic Th cell responses resulting from monovalent and cocktail vaccinations. The graphs show means and SEM representing the GMFI of CD69 (above the mock level) on FTA B220⁺ cells pulsed with 10 μg/ml peptides for the groups of mice described in the legend to Fig. 4. (A) Th cell responses to Gt1b NS3 (left) and NS5B (right) peptides. (B) Th cell responses to Gt3a NS3 (left) and NS5B (right) peptides. The statistical significance of the data was determined as described in Materials and Methods. Note that only comparisons where at least one of the groups being compared had a statistically significant enhancement of the responses relative to those of the control group are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Cumulative IFN-γ ELISpot response to the heterologous or cocktail vaccines. Groups of female BALB/c mice (n = 7) were vaccinated with DNA encoding HCV NS3/NS4/NS5B sequences of Gt1b (G6) or Gt3a (G7), a cocktail comprised of 25 μg each of Gt1b and Gt3a sequences (G8), a cocktail comprised of 50 μg each of Gt1b and Gt3a sequences (G9), or 50 μg of control SV40-PRF (G10). Fourteen days after the last dose of DNA vaccine, the splenocytes were harvested and stimulated with either Gt1b or Gt3a peptides representing the NS3, NS4A, or NS5B protein (4 μg), and IFN-γ-secreting cells were detected as described in Materials and Methods. The data are presented as mean (n = 7) and SEM SFU per 1 × 10⁶ splenocytes, and the unpaired nonparametric Mann-Whitney U test was used to analyze the statistical significance of the data.