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

Ahlén G, Holmström F, Gibbs A, Alheim M, Frelin L - Gene Ther. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Division of Clinical Microbiology, F68, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4126484&req=5

fig2: 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.

Mentions: Next, we wanted to evaluate the efficiency of the in vivo primed NS3/4A-specific CTL responses. We therefore used a tumor inhibition model, in which we can characterize the in vivo functionality of the NS3/4A-specific cellular immune response by determining the inhibition of tumor growth in vivo. We have previously established two cell lines stably expressing NS3/4A,17,22 which enable us to evaluate tumor-inhibiting immune responses after NS3/4A immunization. Protection against NS3/4A-expressing EL-4 lymphoma cells were evaluated in C57BL/6J mice and protection against NS3/4A-expressing Sp2/0 myeloma cells were evaluated in BALB/c mice. First, we were interested in the in vivo growth characteristics of these cell lines in naive C57BL/6J mice and BALB/c mice. To determine if the stably transfected cell lines have similar growth properties compared with the parental cell lines, we inoculated equal numbers of cells into groups of 5–7 C57BL/6J or BALB/c mice. We noted that the four cell lines had similar in vivo growth curves with palpable tumors from around day 5 after inoculation. Without any treatment, the tumors continued to grow for more than 2 weeks after inoculation. No difference in growth properties between the NS3/4A-Sp2/0 and the parental Sp2/0 cell lines were seen (P=NS; Supplementary Figure 2A). Moreover, we did not see any differences between the growth properties of the NS3/4A-EL-4 and the parental EL-4 cell lines (P=NS; Supplementary Figure 2B). Next, we were interested in whether DNA immunization using NS3/4A-based vaccines could protect mice from tumor growth. Specifically, we immunized groups of 8–10 C57BL/6J mice once by gene gun with 2 μg of wild-type (wt)NS3-pVAX1, wtNS3/4A-pVAX1 or coNS3/4A-pVAX1 DNA vaccines. Control mice were left non-immunized. Two weeks after immunization, mice were challenged with a subcutaneous inoculation of 1 × 106 NS3/4A-EL-4 cells, and tumor growth was monitored for up to 3 weeks (Figure 2a). Our results show that NS3/4A-based vaccination mediated protection against tumor growth (P<0.001), whereas NS3-based vaccines protected less efficiently against tumor growth, although statistically significantly (P<0.05; Figure 2a; Frelin et al.17,22). Thus, inclusion of the NS3 cofactor NS4A is essential for optimal NS3 immunogenicity. Similarly, groups of 9–10 BALB/c mice were immunized two or four times at monthly intervals with 2 μg of wtNS3/4A-pVAX1 or coNS3/4A-pVAX1 using gene gun. Two weeks after the last immunization, mice were challenged with a subcutaneous inoculation of 1 × 106 NS3/4A-Sp2/0 cells and thereafter tumor growth was monitored for up to 3 weeks. All groups of immunized mice were protected against tumor growth, whereas non-immunized mice developed tumors (P<0.01 and <0.001). There was no significant differences between mice immunized with wtNS3/4A-DNA or coNS3/4A-DNA (Figure 2b). Moreover, no statistical difference was found between groups of mice immunized two or four times (Figure 2b). In addition to monitoring the in vivo functionality of the vaccine-primed T-cell responses, we were also interested in determining the in vitro lytic activity of NS3/4A-based DNA vaccines in wt and IFNγR2−/− mice. We therefore immunized groups of five mice two times with 2 μg of wtNS3-pVAX1, wtNS3/4A-pVAX1 or wtNS3/4AΔRGT-pVAX1 (construct with deficient NS3 proteolytic activity), or coNS3/4A-pVAX1. Again, only NS3 constructs including NS4A primed potent NS3-specific lytic activity. There was no significant difference in the in vitro lytic activity between plasmids with a functional or a deficient NS3 protease (Figure 2c), although the trend was toward less lytic activity with a deficient protease. Moreover, C57BL/6J and IFNγR2−/− mice immunized with coNS3/4A-pVAX1 primed equally strong lytic activities. Mice were also immunized three times with similar results (data not shown). We were also interested in the cytokine profile of mice immunized with NS3/4A-based vaccines. We found that vaccines containing NS3 and NS4A were superior with regard to the amount of cytokines produced upon in vitro stimulation (Figures 2d–f). The coNS3/4A-pVAX1 vaccine produced the highest levels of IFN-γ and TNFα followed by wtNS3/4A-pVAX1. Mice immunized with wtNS3-pVAX1 rarely had any production of cytokines with results similar to non-immunized controls. Immunized and non-immunized mice stimulated without peptide had similar levels of IFN-γ and TNFα-production as non-immunized mice stimulated with the NS3-CTL peptide. Representative dot plots of flow cytometry analysis are shown in Figures 2e and f.


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

Ahlén G, Holmström F, Gibbs A, Alheim M, Frelin L - Gene Ther. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126484&req=5

fig2: 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.
Mentions: Next, we wanted to evaluate the efficiency of the in vivo primed NS3/4A-specific CTL responses. We therefore used a tumor inhibition model, in which we can characterize the in vivo functionality of the NS3/4A-specific cellular immune response by determining the inhibition of tumor growth in vivo. We have previously established two cell lines stably expressing NS3/4A,17,22 which enable us to evaluate tumor-inhibiting immune responses after NS3/4A immunization. Protection against NS3/4A-expressing EL-4 lymphoma cells were evaluated in C57BL/6J mice and protection against NS3/4A-expressing Sp2/0 myeloma cells were evaluated in BALB/c mice. First, we were interested in the in vivo growth characteristics of these cell lines in naive C57BL/6J mice and BALB/c mice. To determine if the stably transfected cell lines have similar growth properties compared with the parental cell lines, we inoculated equal numbers of cells into groups of 5–7 C57BL/6J or BALB/c mice. We noted that the four cell lines had similar in vivo growth curves with palpable tumors from around day 5 after inoculation. Without any treatment, the tumors continued to grow for more than 2 weeks after inoculation. No difference in growth properties between the NS3/4A-Sp2/0 and the parental Sp2/0 cell lines were seen (P=NS; Supplementary Figure 2A). Moreover, we did not see any differences between the growth properties of the NS3/4A-EL-4 and the parental EL-4 cell lines (P=NS; Supplementary Figure 2B). Next, we were interested in whether DNA immunization using NS3/4A-based vaccines could protect mice from tumor growth. Specifically, we immunized groups of 8–10 C57BL/6J mice once by gene gun with 2 μg of wild-type (wt)NS3-pVAX1, wtNS3/4A-pVAX1 or coNS3/4A-pVAX1 DNA vaccines. Control mice were left non-immunized. Two weeks after immunization, mice were challenged with a subcutaneous inoculation of 1 × 106 NS3/4A-EL-4 cells, and tumor growth was monitored for up to 3 weeks (Figure 2a). Our results show that NS3/4A-based vaccination mediated protection against tumor growth (P<0.001), whereas NS3-based vaccines protected less efficiently against tumor growth, although statistically significantly (P<0.05; Figure 2a; Frelin et al.17,22). Thus, inclusion of the NS3 cofactor NS4A is essential for optimal NS3 immunogenicity. Similarly, groups of 9–10 BALB/c mice were immunized two or four times at monthly intervals with 2 μg of wtNS3/4A-pVAX1 or coNS3/4A-pVAX1 using gene gun. Two weeks after the last immunization, mice were challenged with a subcutaneous inoculation of 1 × 106 NS3/4A-Sp2/0 cells and thereafter tumor growth was monitored for up to 3 weeks. All groups of immunized mice were protected against tumor growth, whereas non-immunized mice developed tumors (P<0.01 and <0.001). There was no significant differences between mice immunized with wtNS3/4A-DNA or coNS3/4A-DNA (Figure 2b). Moreover, no statistical difference was found between groups of mice immunized two or four times (Figure 2b). In addition to monitoring the in vivo functionality of the vaccine-primed T-cell responses, we were also interested in determining the in vitro lytic activity of NS3/4A-based DNA vaccines in wt and IFNγR2−/− mice. We therefore immunized groups of five mice two times with 2 μg of wtNS3-pVAX1, wtNS3/4A-pVAX1 or wtNS3/4AΔRGT-pVAX1 (construct with deficient NS3 proteolytic activity), or coNS3/4A-pVAX1. Again, only NS3 constructs including NS4A primed potent NS3-specific lytic activity. There was no significant difference in the in vitro lytic activity between plasmids with a functional or a deficient NS3 protease (Figure 2c), although the trend was toward less lytic activity with a deficient protease. Moreover, C57BL/6J and IFNγR2−/− mice immunized with coNS3/4A-pVAX1 primed equally strong lytic activities. Mice were also immunized three times with similar results (data not shown). We were also interested in the cytokine profile of mice immunized with NS3/4A-based vaccines. We found that vaccines containing NS3 and NS4A were superior with regard to the amount of cytokines produced upon in vitro stimulation (Figures 2d–f). The coNS3/4A-pVAX1 vaccine produced the highest levels of IFN-γ and TNFα followed by wtNS3/4A-pVAX1. Mice immunized with wtNS3-pVAX1 rarely had any production of cytokines with results similar to non-immunized controls. Immunized and non-immunized mice stimulated without peptide had similar levels of IFN-γ and TNFα-production as non-immunized mice stimulated with the NS3-CTL peptide. Representative dot plots of flow cytometry analysis are shown in Figures 2e and f.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Division of Clinical Microbiology, F68, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

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.

Show MeSH
Related in: MedlinePlus