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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

Stabilization of MHC class I molecules with binding of HCV SLPs. To induce MHC class I expression on cell surface, RMA-S cells were cultured at 26 °C for 48 h prior to the incubation with SLPs. Cells were then incubated with 10 μM of SLPs at 26 °C for 4 h, followed by a 1-h cultured at 37 °C. The expression levels of surface MHC class I molecules, (a) H-2Db and (b) H-2Kb, were analyzed using flow cytometry. HPV E749–57 and OVA257–264 short peptides were positive controls for binding to H-2Db and H-2Kb molecules, respectively. Dash lines indicate the cutoff values for H-2Db (0.2–0.5: weak binders (+); 0.5–1.5: intermediate binders (++); >1.5: strong binders (+++)) and H-2Kb (0.2–2: weak binders (+); 2–8: intermediate binders (++); >8: strong binders (+++)).
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vaccines-03-00203-f001: Stabilization of MHC class I molecules with binding of HCV SLPs. To induce MHC class I expression on cell surface, RMA-S cells were cultured at 26 °C for 48 h prior to the incubation with SLPs. Cells were then incubated with 10 μM of SLPs at 26 °C for 4 h, followed by a 1-h cultured at 37 °C. The expression levels of surface MHC class I molecules, (a) H-2Db and (b) H-2Kb, were analyzed using flow cytometry. HPV E749–57 and OVA257–264 short peptides were positive controls for binding to H-2Db and H-2Kb molecules, respectively. Dash lines indicate the cutoff values for H-2Db (0.2–0.5: weak binders (+); 0.5–1.5: intermediate binders (++); >1.5: strong binders (+++)) and H-2Kb (0.2–2: weak binders (+); 2–8: intermediate binders (++); >8: strong binders (+++)).

Mentions: Selection of synthetic long peptides containing CTL epitopes from HCV nsPs by prediction algorithms. Sequences of the long synthetic peptides, for which MHC binding affinity was shown in Figure 1, are shown. Strong binders are depicted in bold and highlighted in grey. The cut off score of SYFPEITHI is set at 20; a high score indicates a strong binder. The cut off score of NetMHCpan 2.8 and IEDB is set at 0.5 (% rank); a low score indicates a strong binder.


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

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

Stabilization of MHC class I molecules with binding of HCV SLPs. To induce MHC class I expression on cell surface, RMA-S cells were cultured at 26 °C for 48 h prior to the incubation with SLPs. Cells were then incubated with 10 μM of SLPs at 26 °C for 4 h, followed by a 1-h cultured at 37 °C. The expression levels of surface MHC class I molecules, (a) H-2Db and (b) H-2Kb, were analyzed using flow cytometry. HPV E749–57 and OVA257–264 short peptides were positive controls for binding to H-2Db and H-2Kb molecules, respectively. Dash lines indicate the cutoff values for H-2Db (0.2–0.5: weak binders (+); 0.5–1.5: intermediate binders (++); >1.5: strong binders (+++)) and H-2Kb (0.2–2: weak binders (+); 2–8: intermediate binders (++); >8: strong binders (+++)).
© Copyright Policy
Related In: Results  -  Collection

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

vaccines-03-00203-f001: Stabilization of MHC class I molecules with binding of HCV SLPs. To induce MHC class I expression on cell surface, RMA-S cells were cultured at 26 °C for 48 h prior to the incubation with SLPs. Cells were then incubated with 10 μM of SLPs at 26 °C for 4 h, followed by a 1-h cultured at 37 °C. The expression levels of surface MHC class I molecules, (a) H-2Db and (b) H-2Kb, were analyzed using flow cytometry. HPV E749–57 and OVA257–264 short peptides were positive controls for binding to H-2Db and H-2Kb molecules, respectively. Dash lines indicate the cutoff values for H-2Db (0.2–0.5: weak binders (+); 0.5–1.5: intermediate binders (++); >1.5: strong binders (+++)) and H-2Kb (0.2–2: weak binders (+); 2–8: intermediate binders (++); >8: strong binders (+++)).
Mentions: Selection of synthetic long peptides containing CTL epitopes from HCV nsPs by prediction algorithms. Sequences of the long synthetic peptides, for which MHC binding affinity was shown in Figure 1, are shown. Strong binders are depicted in bold and highlighted in grey. The cut off score of SYFPEITHI is set at 20; a high score indicates a strong binder. The cut off score of NetMHCpan 2.8 and IEDB is set at 0.5 (% rank); a low score indicates a strong binder.

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