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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

Priming of in vitro detectable CTLs against Kd- and Db-binding peptides in H-2d (BALB/c) and H-2b (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.
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fig1: Priming of in vitro detectable CTLs against Kd- and Db-binding peptides in H-2d (BALB/c) and H-2b (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.

Mentions: We have previously identified an NS3 major histocompatibility complex (MHC) class I CTL epitope in C57BL/6J (H-2b) mice that bound H-2Db molecules with high affinity,22 which has been used for monitoring NS3/4A-specific CTL responses. In the present study, we wanted to expand the immunological analysis of NS3/4A-specific immune responses by evaluating immune responses to NS3/4A in another mouse haplotype. To evaluate NS3/4A-based DNA vaccines in BALB/c mice, we needed to identify an H-2d-restricted NS3/4A CTL epitope. To identify new MHC class I epitopes within the NS3/4A protein, we synthesized 68 20-mer peptides (10-amino-acid (aa) overlap) spanning the entire NS3/4A protein. All 68 peptides were screened for binding to the H-2d molecule as determined by stabilization of MHC–peptide complexes on transporter associated with antigen processing 2-deficient RMA-S cells23,24 transfected with H-2Kd, H-2Dd or H-2Ld. This extensive analysis revealed one 20-mer peptide (e.g. amino-acid sequence: TVRLRAYMNTPGLPVCQDHL) that could stabilize the H-2Kd molecule. This same region has been suggested to contain a CTL epitope previously, but it has not been fine mapped.25 Hence, we decided to fine map this NS3/4 A-specific H-2d CTL epitope. To fine map the H-2d epitope, a panel of ten 10-mer peptides with 9 aa overlap was synthesized (Supplementary Figure 1A). To determine IFN-γ production in response to the 10-mer peptides, BALB/c mice were immunized once with 100 μg coNS3/4A-pVAX1 intramuscularly, and 2 weeks later the animals were killed. Spleen cells were restimulated for 4 h in vitro in the presence of the 10-mer peptides and the frequency of IFN-γ-producing CD8+ T cells was determined by flow cytometry (Supplementary Figure 1B). We could identify two peptides (numbers 3 and 5) that were able to induce high IFN-γ production by CD8+ T cells (Supplementary Figure 1B). At the same time, we performed an in vitro peptide stabilization of MHC–peptide complexes on transporter associated with antigen processing 2-deficient RMA-S cells transfected with H-2Kd, H-2Dd or H-2Ld. Our results revealed that only peptides 3, 4, 5 and 6 could bind to the H-2Kd molecule and not the other two H-2 alleles (Supplementary Figure 1B). Moreover, we analyzed the cytotoxic potential of spleen cells from BALB/c coNS3/4A-pVAX1 or peptide-immunized mice by performing a 5-day restimulation using peptides 3, 4 and 5. The cytolytic activity of the NS3/4A-specific CTLs was determined by a conventional 51Cr-release assay on peptide-loaded RMA-S target cells. Functional testing revealed that all three peptides bound to MHC class I and were recognized and eradicated by NS3/4A-specific CTLs (data not shown). Based on all the above data, peptide number 5 (amino-acid sequence: AYMNTPGLPV, positions 1541–1550 in the HCV polyprotein) was selected for all further experimental procedures as it had the highest binding affinity for the H-2Kd molecule and it also induced high levels of IFN-γ production by CD8+ T cells. To investigate the specificity of the identified CTL epitiope, we immunized groups of five BALB/c (H-2d), C57BL/6J × BALB/c (H-2d × H-2b) and C57BL/6J (H-2b) mice two times with 2 μg coNS3/4A-pVAX1 using transdermal delivery via gene gun. Groups of mice were also left untreated as controls. Two weeks after the last immunization, mice were killed and splenocytes were restimulated with either the H-2d peptide (AYMNTPGLPV) or the previously described H-2b peptide (GAVQNEVTL).22 Restimulated cultures were analyzed for lytic activity by a 51Cr-release assay using peptide-loaded RMA-S cells (Figures 1a–h). The results revealed that NS3/4A-specific CTLs with lytic activity were only detected in BALB/c and C57BL/6J × BALB/c cultures restimulated with the H-2d peptide (Figures 1a and c), whereas no lytic activity were detected in C57BL/6J cultures (Figure 1e). On the other hand, when splenocytes from immunized C57BL/6J mice were restimulated with the H-2b peptide, similar lytic activity was noted (Figure 1g). Hence, the H-2d-binding peptide is only presented in the context of BALB/c mice. No lytic activity was detected in the groups of non-immunized mice (Figures 1b, d, f and h).


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)

Priming of in vitro detectable CTLs against Kd- and Db-binding peptides in H-2d (BALB/c) and H-2b (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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Priming of in vitro detectable CTLs against Kd- and Db-binding peptides in H-2d (BALB/c) and H-2b (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.
Mentions: We have previously identified an NS3 major histocompatibility complex (MHC) class I CTL epitope in C57BL/6J (H-2b) mice that bound H-2Db molecules with high affinity,22 which has been used for monitoring NS3/4A-specific CTL responses. In the present study, we wanted to expand the immunological analysis of NS3/4A-specific immune responses by evaluating immune responses to NS3/4A in another mouse haplotype. To evaluate NS3/4A-based DNA vaccines in BALB/c mice, we needed to identify an H-2d-restricted NS3/4A CTL epitope. To identify new MHC class I epitopes within the NS3/4A protein, we synthesized 68 20-mer peptides (10-amino-acid (aa) overlap) spanning the entire NS3/4A protein. All 68 peptides were screened for binding to the H-2d molecule as determined by stabilization of MHC–peptide complexes on transporter associated with antigen processing 2-deficient RMA-S cells23,24 transfected with H-2Kd, H-2Dd or H-2Ld. This extensive analysis revealed one 20-mer peptide (e.g. amino-acid sequence: TVRLRAYMNTPGLPVCQDHL) that could stabilize the H-2Kd molecule. This same region has been suggested to contain a CTL epitope previously, but it has not been fine mapped.25 Hence, we decided to fine map this NS3/4 A-specific H-2d CTL epitope. To fine map the H-2d epitope, a panel of ten 10-mer peptides with 9 aa overlap was synthesized (Supplementary Figure 1A). To determine IFN-γ production in response to the 10-mer peptides, BALB/c mice were immunized once with 100 μg coNS3/4A-pVAX1 intramuscularly, and 2 weeks later the animals were killed. Spleen cells were restimulated for 4 h in vitro in the presence of the 10-mer peptides and the frequency of IFN-γ-producing CD8+ T cells was determined by flow cytometry (Supplementary Figure 1B). We could identify two peptides (numbers 3 and 5) that were able to induce high IFN-γ production by CD8+ T cells (Supplementary Figure 1B). At the same time, we performed an in vitro peptide stabilization of MHC–peptide complexes on transporter associated with antigen processing 2-deficient RMA-S cells transfected with H-2Kd, H-2Dd or H-2Ld. Our results revealed that only peptides 3, 4, 5 and 6 could bind to the H-2Kd molecule and not the other two H-2 alleles (Supplementary Figure 1B). Moreover, we analyzed the cytotoxic potential of spleen cells from BALB/c coNS3/4A-pVAX1 or peptide-immunized mice by performing a 5-day restimulation using peptides 3, 4 and 5. The cytolytic activity of the NS3/4A-specific CTLs was determined by a conventional 51Cr-release assay on peptide-loaded RMA-S target cells. Functional testing revealed that all three peptides bound to MHC class I and were recognized and eradicated by NS3/4A-specific CTLs (data not shown). Based on all the above data, peptide number 5 (amino-acid sequence: AYMNTPGLPV, positions 1541–1550 in the HCV polyprotein) was selected for all further experimental procedures as it had the highest binding affinity for the H-2Kd molecule and it also induced high levels of IFN-γ production by CD8+ T cells. To investigate the specificity of the identified CTL epitiope, we immunized groups of five BALB/c (H-2d), C57BL/6J × BALB/c (H-2d × H-2b) and C57BL/6J (H-2b) mice two times with 2 μg coNS3/4A-pVAX1 using transdermal delivery via gene gun. Groups of mice were also left untreated as controls. Two weeks after the last immunization, mice were killed and splenocytes were restimulated with either the H-2d peptide (AYMNTPGLPV) or the previously described H-2b peptide (GAVQNEVTL).22 Restimulated cultures were analyzed for lytic activity by a 51Cr-release assay using peptide-loaded RMA-S cells (Figures 1a–h). The results revealed that NS3/4A-specific CTLs with lytic activity were only detected in BALB/c and C57BL/6J × BALB/c cultures restimulated with the H-2d peptide (Figures 1a and c), whereas no lytic activity were detected in C57BL/6J cultures (Figure 1e). On the other hand, when splenocytes from immunized C57BL/6J mice were restimulated with the H-2b peptide, similar lytic activity was noted (Figure 1g). Hence, the H-2d-binding peptide is only presented in the context of BALB/c mice. No lytic activity was detected in the groups of non-immunized mice (Figures 1b, d, f and h).

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