Abstract:
For the rational design of epitope-specific vaccines, identifying epitopes that can be processed and presented is essential. As algorithm-based epitope prediction is frequently discordant with actually recognized CD8+ T-cell epitopes, we developed an in vitro CD8 T-cell priming protocol to enable the identification of truly and functionally expressed HLA class I epitopes. The assay was established and validated to identify epitopes presented by hepatitis C virus (HCV)-infected cells. In vitro priming of naïve CD8 T cells was achieved by culturing unfractionated PBMCs in the presence of a specific cocktail of growth factors and cytokines, and next exposing the cells to hepatic cells expressing the NS3 protein of HCV. After a 10-day co-culture, HCV-specific T-cell responses were identified based on IFN-γ ELISpot analysis. For this, the T cells were restimulated with long synthetic peptides (SLPs) spanning the whole NS3 protein sequence allowing the identification of HCV-specificity. We demonstrated that this protocol resulted in the in vitro priming of naïve precursors to antigen-experienced T-cells specific for 11 out of 98 SLPs tested. These 11 SLPs contain 12 different HLA-A*02:01-restricted epitopes, as predicted by a combination of three epitope prediction algorithms. Furthermore, we identified responses against 3 peptides that were not predicted to contain any immunogenic HLA class I epitopes, yet showed HCV-specific responses in vitro. Separation of CD8+ and CD8- T cells from PBMCs primed in vitro showed responses only upon restimulation with short peptides. We established an in vitro method that enables the identification of HLA class I epitopes resulting from cross-presented antigens and that can cross-prime T cells and allows the effective selection of functional immunogenic epitopes, but also less immunogenic ones, for the design of tailored therapeutic vaccines against persistent viral infections and tumor antigens.
摘要:
为了合理设计表位特异性疫苗,鉴定可以加工和呈递的表位是必不可少的。由于基于算法的表位预测经常与实际识别的CD8+T细胞表位不一致,我们开发了一种体外CD8T细胞引发方案,以鉴定真正和功能表达的HLAI类表位。建立并验证该测定以鉴定由丙型肝炎病毒(HCV)感染的细胞呈递的表位。通过在生长因子和细胞因子的特定混合物存在下培养未分级的PBMC,可以实现初始CD8T细胞的体外引发。然后将细胞暴露于表达HCV的NS3蛋白的肝细胞。经过10天的共同培养,基于IFN-γELISpot分析鉴定HCV特异性T细胞应答。为此,用跨越整个NS3蛋白序列的长合成肽(SLP)再刺激T细胞,从而鉴定HCV特异性.我们证明了该方案导致初始前体对所测试的98个SLP中的11个具有特异性的抗原经历T细胞的体外引发。这11个SLP包含12个不同的HLA-A*02:01限制性表位,通过三种表位预测算法的组合预测。此外,我们确定了针对3种预测不包含任何免疫原性HLAI类表位的肽的反应,但在体外显示HCV特异性应答。从体外引发的PBMC中分离CD8+和CD8-T细胞仅在用短肽再刺激时显示出应答。我们建立了一种体外方法,该方法能够鉴定由交叉呈递抗原产生的HLAI类表位,并且可以交叉引发T细胞,并允许有效选择功能性免疫原性表位。但免疫原性也较低,用于设计针对持续性病毒感染和肿瘤抗原的定制治疗性疫苗。