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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.

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Related in: MedlinePlus

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.
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fig3: 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.

Mentions: A key question to address was how long after the last immunization could T cells remain functional in mice? To answer this question, we immunized BALB/c and/or C57BL/6J mice one to three times with wtNS3/4A-pVAX1 or coNS3/4A-pVAX1 DNA vaccines and thereafter waited 1, 2, 3, 4, 6, 12 and 16 months. At indicated time points, mice were challenged with NS3/4A-expressing tumor cells or splenocytes were monitored in vitro for detection of CTL responses (Figures 3a and b). We found that one immunization protected C57BL/6J mice from tumor development when challenged 1, 3 and 6 months after the last immunization (P<0.001; Figure 3a). Not surprisingly, mice immunized three or four times were protected against tumor growth when challenged 4 months after the last immunization (data not shown). In addition, three monthly immunizations protected BALB/c mice from tumor development when challenged 16 months after the last immunization irrespective of administration route (P<0.01; Figure 3a). Importantly, we obtained similar results regarding long-term protective immune responses in two mouse strains. 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 in groups of C57BL/6J mice immunized two times (Figure 3b). Additionally, in groups of mice immunized once, we detected lytic activity for up to 6 months (Figure 3b). However, the lytic activity was at a lower magnitude after one immunization compared with two immunizations. Next, we were interested in comparing NS3/4A-specific immune activation in wt and IFNγR2−/− mice as determined by IFN-γ and IL-2 production by Enzyme-Linked ImmunoSpot (ELISpot), lytic activity by a cytotoxicity assay and quantification of NS3/4A-specific T cells using pentamer staining. Groups of five mice were immunized once with gene gun (2 μg) or intramuscular immunization (50 μg) and 2 weeks later were killed and immune responses subsequently monitored. We noted that wt mice immunized intramuscularly primed stronger IFN-γ production compared with mice immunized transdermally using gene gun (Figure 4a). As expected, no IFN-γ production was detected in IFNγR2−/− mice (Figure 4a). However, we found detectable levels of IL-2-production in both wt and IFNγR2−/− mice immunized intramuscularly, whereas the IL-2 production were weak or absent in mice immunized transdermally using gene gun (data not shown). To determine if the primed NS3/4A-specific T cells were functional, a cytotoxicity assay was used. Both immunized wt and IFNγR2−/− mice had detectable CTLs that were able to lyse NS3/4A target cells (Figure 4a). However, it was noted that IFNγR2−/− mice immunized transdermally had lower levels of CTLs with lytic activity. Quantification of NS3-specific CD8+ T cells revealed that wt mice immunized intramuscularly or transdermally, and IFNγR2−/− mice immunized intramuscularly primed similar frequencies of T cells, whereas IFNγR2−/− mice immunized transdermally had low frequencies of positive T cells (P<0.05 (wt) and P<0.01 (IFNγR2−/−); Figure 4b). Also, representative dot plots of flow cytometry analysis are shown (Figure 4b).


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 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.
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Related In: Results  -  Collection

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

fig3: 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.
Mentions: A key question to address was how long after the last immunization could T cells remain functional in mice? To answer this question, we immunized BALB/c and/or C57BL/6J mice one to three times with wtNS3/4A-pVAX1 or coNS3/4A-pVAX1 DNA vaccines and thereafter waited 1, 2, 3, 4, 6, 12 and 16 months. At indicated time points, mice were challenged with NS3/4A-expressing tumor cells or splenocytes were monitored in vitro for detection of CTL responses (Figures 3a and b). We found that one immunization protected C57BL/6J mice from tumor development when challenged 1, 3 and 6 months after the last immunization (P<0.001; Figure 3a). Not surprisingly, mice immunized three or four times were protected against tumor growth when challenged 4 months after the last immunization (data not shown). In addition, three monthly immunizations protected BALB/c mice from tumor development when challenged 16 months after the last immunization irrespective of administration route (P<0.01; Figure 3a). Importantly, we obtained similar results regarding long-term protective immune responses in two mouse strains. 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 in groups of C57BL/6J mice immunized two times (Figure 3b). Additionally, in groups of mice immunized once, we detected lytic activity for up to 6 months (Figure 3b). However, the lytic activity was at a lower magnitude after one immunization compared with two immunizations. Next, we were interested in comparing NS3/4A-specific immune activation in wt and IFNγR2−/− mice as determined by IFN-γ and IL-2 production by Enzyme-Linked ImmunoSpot (ELISpot), lytic activity by a cytotoxicity assay and quantification of NS3/4A-specific T cells using pentamer staining. Groups of five mice were immunized once with gene gun (2 μg) or intramuscular immunization (50 μg) and 2 weeks later were killed and immune responses subsequently monitored. We noted that wt mice immunized intramuscularly primed stronger IFN-γ production compared with mice immunized transdermally using gene gun (Figure 4a). As expected, no IFN-γ production was detected in IFNγR2−/− mice (Figure 4a). However, we found detectable levels of IL-2-production in both wt and IFNγR2−/− mice immunized intramuscularly, whereas the IL-2 production were weak or absent in mice immunized transdermally using gene gun (data not shown). To determine if the primed NS3/4A-specific T cells were functional, a cytotoxicity assay was used. Both immunized wt and IFNγR2−/− mice had detectable CTLs that were able to lyse NS3/4A target cells (Figure 4a). However, it was noted that IFNγR2−/− mice immunized transdermally had lower levels of CTLs with lytic activity. Quantification of NS3-specific CD8+ T cells revealed that wt mice immunized intramuscularly or transdermally, and IFNγR2−/− mice immunized intramuscularly primed similar frequencies of T cells, whereas IFNγR2−/− mice immunized transdermally had low frequencies of positive T cells (P<0.05 (wt) and P<0.01 (IFNγR2−/−); Figure 4b). Also, representative dot plots of flow cytometry analysis are shown (Figure 4b).

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