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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

Binding affinity of HCV short peptides to MHC class I molecules. Short synthetic HCV peptides were serial diluted and incubated with RMA-S cells as described in Figure 1. (a) H-2Db; (b) H-2Kb. 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-f002: Binding affinity of HCV short peptides to MHC class I molecules. Short synthetic HCV peptides were serial diluted and incubated with RMA-S cells as described in Figure 1. (a) H-2Db; (b) H-2Kb. 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: Binding affinity of selected short synthetic peptides on MHC class I molecules was determined by MHC I stabilization assay with various concentrations of peptide (Figure 2). As expected, stabilization of both H-2Db and H-2Kb molecules increased with increasing concentration of peptide. The kinetic of the stabilization of H-2Db molecules was similar for all selected short peptides. The plateau of stabilization was reached at approximately 30 μM of peptides (Figure 2a). Two kinetic patterns of stabilizations of H-2Kb molecules were observed. For strong binders (at 100 μM, FI > 8; OVA257–264, NS5B2–10, NS3265–273, NS5B157–165, NS2139–147 and NS5B52–60), there was a dose dependent increase of FI and the plateau of stabilization was not detectable even at high peptide concentration (100 μM). For intermediate binders (NS3514–522, NS5B425–433 and NS5A280–287), stabilization of H-2Kb molecules was modest at low peptide concentration (0.3–10 μM). However, the stabilization effect increased exponentially once the concentration was above 30 μM. This observation may explain why we did not see strong H-2Kb binders when SLPs at 10 μM were used in the MHC I stabilization assay (Figure 1). Results of stabilization of MHC class I molecules with short peptides matched the result from all of the MHC class I prediction algorithms used (H-2Db: >90% matched; H-2Kb: 100% matched) (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)

Binding affinity of HCV short peptides to MHC class I molecules. Short synthetic HCV peptides were serial diluted and incubated with RMA-S cells as described in Figure 1. (a) H-2Db; (b) H-2Kb. 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-f002: Binding affinity of HCV short peptides to MHC class I molecules. Short synthetic HCV peptides were serial diluted and incubated with RMA-S cells as described in Figure 1. (a) H-2Db; (b) H-2Kb. 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: Binding affinity of selected short synthetic peptides on MHC class I molecules was determined by MHC I stabilization assay with various concentrations of peptide (Figure 2). As expected, stabilization of both H-2Db and H-2Kb molecules increased with increasing concentration of peptide. The kinetic of the stabilization of H-2Db molecules was similar for all selected short peptides. The plateau of stabilization was reached at approximately 30 μM of peptides (Figure 2a). Two kinetic patterns of stabilizations of H-2Kb molecules were observed. For strong binders (at 100 μM, FI > 8; OVA257–264, NS5B2–10, NS3265–273, NS5B157–165, NS2139–147 and NS5B52–60), there was a dose dependent increase of FI and the plateau of stabilization was not detectable even at high peptide concentration (100 μM). For intermediate binders (NS3514–522, NS5B425–433 and NS5A280–287), stabilization of H-2Kb molecules was modest at low peptide concentration (0.3–10 μM). However, the stabilization effect increased exponentially once the concentration was above 30 μM. This observation may explain why we did not see strong H-2Kb binders when SLPs at 10 μM were used in the MHC I stabilization assay (Figure 1). Results of stabilization of MHC class I molecules with short peptides matched the result from all of the MHC class I prediction algorithms used (H-2Db: >90% matched; H-2Kb: 100% matched) (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