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Epitope Prediction Assays Combined with Validation Assays Strongly Narrows down Putative Cytotoxic T Lymphocyte Epitopes.

Ip PP, Nijman HW, Daemen T - Vaccines (Basel) (2015)

Bottom Line: However, their results do not reflect a one-to-one correlation with experimental data.This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes.This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, HPC EB88, 9700 RB Groningen, The Netherlands. p.p.ip@umcg.nl.

ABSTRACT
Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different algorithms and are instrumental for a first screening of possible epitopes. However, their results do not reflect a one-to-one correlation with experimental data. We combined several in silico prediction methods to unravel the most promising C57BL/6 mouse-restricted Hepatitis C virus (HCV) MHC class I epitopes and validated these epitopes in vitro and in vivo. Cytotoxic T lymphocyte (CTL) epitopes within the HCV non-structural proteins were identified, and proteasomal cleavage sites and helper T cell (Th) epitopes at close proximity to these CTL epitopes were analyzed using multiple prediction algorithms. This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes. This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

No MeSH data available.


Related in: MedlinePlus

Induction of peptide-specific effector CD8+ T cells in vivo. Mice were intramuscularly immunized thrice with rSFV expressing HCV nsPs (rSFVeNS2'-5B', rSFVeNS3/4A or rSFVeNS5A/B') or PBS control with a one-week interval. Mice were sacrificed 1 to 3 weeks after the boost immunization. Splenocytes were isolated and cultured with 10 μg/mL of peptides for 5 h before surface and intracellular staining. Background (splenocytes incubated with an equivalent concentration of DMSO) subtraction was applied and values above background are shown. Data represent combined results from three independent experiments, showing the mean +SEM (3–5 mice per group).
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vaccines-03-00203-f003: Induction of peptide-specific effector CD8+ T cells in vivo. Mice were intramuscularly immunized thrice with rSFV expressing HCV nsPs (rSFVeNS2'-5B', rSFVeNS3/4A or rSFVeNS5A/B') or PBS control with a one-week interval. Mice were sacrificed 1 to 3 weeks after the boost immunization. Splenocytes were isolated and cultured with 10 μg/mL of peptides for 5 h before surface and intracellular staining. Background (splenocytes incubated with an equivalent concentration of DMSO) subtraction was applied and values above background are shown. Data represent combined results from three independent experiments, showing the mean +SEM (3–5 mice per group).

Mentions: Induction of in vivo T-cell response depends on the presentation of peptides and the availability, avidity and affinity of precursor CD8+ T cells [24,25]. Here, we investigated the induction of T-cell response against the predicted CTL epitopes in mice immunized three times with the rSFV particles expressing all HCV nsPs, NS3/4A or NS5A/B' (rSFVeNS2'-5B', rSFVeNS3/4A or rSFVeNS5A/B'). Splenocytes were isolated 1 to 3 weeks after the last immunization and re-stimulated with selected short peptides in order to induce degranulation (surface expression of CD107a/b) and secretion of IFN-γ by peptide-specific CD8+ T cells (Figure 3). rSFV immunizations induced functional effector CD8+ T cells (CD107a/b+IFN-γ+) against NS2139–147, NS3603–611, NS5B2–10 and NS5B157–165. When no peptides were added to the splenocytes, we already observed the presence of endogenous CD107a/b+IFN-γ+CD8+ cells from mice immunized with any rSFV particles but not from mice immunized with PBS. This indicates that rSFV immunizations in general induced functional CD8+ T-cell responses. To check for a specific response against peptide, background (without peptide re-stimulation) subtraction was applied. Frequencies above zero indicate specific response against the re-stimulating peptide. CD8+ T-cell response against NS3603–611 [26] was the highest among all selected peptides and was detected in mice immunized with rSFVeNS2'-5B' or rSFVeNS3/4A. Responses against NS5B2–10 and NS5B157–165 were low and induced in mice immunized with rSFVeNS5A/B' or rSFVeNS2'-5B'. A very low response against NS2139–147 was observed only in mice immunized with rSFVeNS2'-5B', not in mice with other immunizations. Of note, all responding peptides were predicted by at least one algorithm for MHC class I prediction. Proteasomal cleavages sites were presented in the carboxyterminals of all four peptides. Three peptides (NS2139–147, NS3603–611 and NS5B2–10) contained predicted MHC class II epitopes in close proximity to their CTL epitopes with relatively high rankings (Table 2).


Epitope Prediction Assays Combined with Validation Assays Strongly Narrows down Putative Cytotoxic T Lymphocyte Epitopes.

Ip PP, Nijman HW, Daemen T - Vaccines (Basel) (2015)

Induction of peptide-specific effector CD8+ T cells in vivo. Mice were intramuscularly immunized thrice with rSFV expressing HCV nsPs (rSFVeNS2'-5B', rSFVeNS3/4A or rSFVeNS5A/B') or PBS control with a one-week interval. Mice were sacrificed 1 to 3 weeks after the boost immunization. Splenocytes were isolated and cultured with 10 μg/mL of peptides for 5 h before surface and intracellular staining. Background (splenocytes incubated with an equivalent concentration of DMSO) subtraction was applied and values above background are shown. Data represent combined results from three independent experiments, showing the mean +SEM (3–5 mice per group).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494349&req=5

vaccines-03-00203-f003: Induction of peptide-specific effector CD8+ T cells in vivo. Mice were intramuscularly immunized thrice with rSFV expressing HCV nsPs (rSFVeNS2'-5B', rSFVeNS3/4A or rSFVeNS5A/B') or PBS control with a one-week interval. Mice were sacrificed 1 to 3 weeks after the boost immunization. Splenocytes were isolated and cultured with 10 μg/mL of peptides for 5 h before surface and intracellular staining. Background (splenocytes incubated with an equivalent concentration of DMSO) subtraction was applied and values above background are shown. Data represent combined results from three independent experiments, showing the mean +SEM (3–5 mice per group).
Mentions: Induction of in vivo T-cell response depends on the presentation of peptides and the availability, avidity and affinity of precursor CD8+ T cells [24,25]. Here, we investigated the induction of T-cell response against the predicted CTL epitopes in mice immunized three times with the rSFV particles expressing all HCV nsPs, NS3/4A or NS5A/B' (rSFVeNS2'-5B', rSFVeNS3/4A or rSFVeNS5A/B'). Splenocytes were isolated 1 to 3 weeks after the last immunization and re-stimulated with selected short peptides in order to induce degranulation (surface expression of CD107a/b) and secretion of IFN-γ by peptide-specific CD8+ T cells (Figure 3). rSFV immunizations induced functional effector CD8+ T cells (CD107a/b+IFN-γ+) against NS2139–147, NS3603–611, NS5B2–10 and NS5B157–165. When no peptides were added to the splenocytes, we already observed the presence of endogenous CD107a/b+IFN-γ+CD8+ cells from mice immunized with any rSFV particles but not from mice immunized with PBS. This indicates that rSFV immunizations in general induced functional CD8+ T-cell responses. To check for a specific response against peptide, background (without peptide re-stimulation) subtraction was applied. Frequencies above zero indicate specific response against the re-stimulating peptide. CD8+ T-cell response against NS3603–611 [26] was the highest among all selected peptides and was detected in mice immunized with rSFVeNS2'-5B' or rSFVeNS3/4A. Responses against NS5B2–10 and NS5B157–165 were low and induced in mice immunized with rSFVeNS5A/B' or rSFVeNS2'-5B'. A very low response against NS2139–147 was observed only in mice immunized with rSFVeNS2'-5B', not in mice with other immunizations. Of note, all responding peptides were predicted by at least one algorithm for MHC class I prediction. Proteasomal cleavages sites were presented in the carboxyterminals of all four peptides. Three peptides (NS2139–147, NS3603–611 and NS5B2–10) contained predicted MHC class II epitopes in close proximity to their CTL epitopes with relatively high rankings (Table 2).

Bottom Line: However, their results do not reflect a one-to-one correlation with experimental data.This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes.This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, HPC EB88, 9700 RB Groningen, The Netherlands. p.p.ip@umcg.nl.

ABSTRACT
Tumor vaccine design requires prediction and validation of immunogenic MHC class I epitopes expressed by target cells as well as MHC class II epitopes expressed by antigen-presenting cells essential for the induction of optimal immune responses. Epitope prediction methods are based on different algorithms and are instrumental for a first screening of possible epitopes. However, their results do not reflect a one-to-one correlation with experimental data. We combined several in silico prediction methods to unravel the most promising C57BL/6 mouse-restricted Hepatitis C virus (HCV) MHC class I epitopes and validated these epitopes in vitro and in vivo. Cytotoxic T lymphocyte (CTL) epitopes within the HCV non-structural proteins were identified, and proteasomal cleavage sites and helper T cell (Th) epitopes at close proximity to these CTL epitopes were analyzed using multiple prediction algorithms. This combined in silico analysis enhances the precision of identification of functional HCV-specific CTL epitopes. This approach will be applicable to the design of human vaccines not only for HCV, but also for other antigens in which T-cell responses play a crucial role.

No MeSH data available.


Related in: MedlinePlus