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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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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-2Db 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.
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fig5: 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-2Db 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.

Mentions: Next, we were interested in testing the specificity of the murine H-2b CTL epitope. Hence, we compared mice immunized transdermally two times using wtNS3/4A DNA vaccines containing the wt sequence (GAVQNEVTL) or a mutated sequence where positions 5 and 9 had been changed to alanine instead of an asparagine and leucine, respectively (GAVQAEVTA). Two weeks after the last immunization, mice were killed and the cytolyic activity was monitored using target cells presenting the wt sequence. We showed that only mice immunized with a DNA vaccine containing the wt sequence demonstrated T cells with lytic activity (Figure 5a). By mutating positions 5 and 9, we markedly affected the ability of the peptide to bind the MHC H-2Db molecule (Figure 5b). Also, the single mutations markedly affected the MHC-peptide-binding affinity and it was only the wt peptide that had a strong binding affinity for H-2Db (Figure 5b). Polymorphism within the GAVQNEVTL epitope was rare with only one mutation at position 5 and none at position 9 found in 40 published GenBank isolates (Supplementary Figure 3). Next, we were interested in whether DNA immunization using the wtNS3/4A or mutated DNA vaccine could protect mice from tumor growth. Specifically, we immunized groups of 10 C57BL/6J mice once by gene gun with 2 μg of indicated DNA vaccine. 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 5c). Our results show that wtNS3/4A-based vaccination mediated protection against tumor growth (P<0.01), whereas wtNS3/4AΔ5,9-based vaccine only mediated a partial protection (P=NS; Figure 5c). There was no statistically significant difference between groups of mice immunized with wtNS3/4A or wtNS3/4AΔ5,9 vaccines (P=NS; Figure 5c).


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 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-2Db 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.
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Related In: Results  -  Collection

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fig5: 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-2Db 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.
Mentions: Next, we were interested in testing the specificity of the murine H-2b CTL epitope. Hence, we compared mice immunized transdermally two times using wtNS3/4A DNA vaccines containing the wt sequence (GAVQNEVTL) or a mutated sequence where positions 5 and 9 had been changed to alanine instead of an asparagine and leucine, respectively (GAVQAEVTA). Two weeks after the last immunization, mice were killed and the cytolyic activity was monitored using target cells presenting the wt sequence. We showed that only mice immunized with a DNA vaccine containing the wt sequence demonstrated T cells with lytic activity (Figure 5a). By mutating positions 5 and 9, we markedly affected the ability of the peptide to bind the MHC H-2Db molecule (Figure 5b). Also, the single mutations markedly affected the MHC-peptide-binding affinity and it was only the wt peptide that had a strong binding affinity for H-2Db (Figure 5b). Polymorphism within the GAVQNEVTL epitope was rare with only one mutation at position 5 and none at position 9 found in 40 published GenBank isolates (Supplementary Figure 3). Next, we were interested in whether DNA immunization using the wtNS3/4A or mutated DNA vaccine could protect mice from tumor growth. Specifically, we immunized groups of 10 C57BL/6J mice once by gene gun with 2 μg of indicated DNA vaccine. 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 5c). Our results show that wtNS3/4A-based vaccination mediated protection against tumor growth (P<0.01), whereas wtNS3/4AΔ5,9-based vaccine only mediated a partial protection (P=NS; Figure 5c). There was no statistically significant difference between groups of mice immunized with wtNS3/4A or wtNS3/4AΔ5,9 vaccines (P=NS; Figure 5c).

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