Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, University of Groningen, PO Box 30 001, HPC EB88, 9700RB Groningen, the Netherlands.
Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, University of Groningen, PO Box 30 001, HPC EB88, 9700RB Groningen, the Netherlands.
Vaccine. 2024 Sep 17;42(22):126032. doi: 10.1016/j.vaccine.2024.05.080. Epub 2024 Jul 3.
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.
为了合理设计表位特异性疫苗，确定可加工和呈递的表位至关重要。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 CD8 T 细胞引发方案，以识别真正和功能表达的 HLA Ⅰ类表位。该测定法用于鉴定丙型肝炎病毒 (HCV) 感染细胞呈递的表位，并进行了建立和验证。通过在存在特定的生长因子和细胞因子混合物的情况下培养未分馏的 PBMCs，来实现幼稚 CD8 T 细胞的体外引发，然后使细胞暴露于表达 HCV NS3 蛋白的肝细胞中。在 10 天的共培养后，根据 IFN-γ ELISpot 分析鉴定 HCV 特异性 T 细胞反应。为此，用跨越整个 NS3 蛋白序列的长合成肽 (SLP) 再次刺激 T 细胞，以鉴定 HCV 特异性。我们证明，该方案导致针对 98 个 SLP 中 11 个的幼稚前体在体外被引发，成为针对抗原的经验丰富的 T 细胞，这些 SLP 包含 12 个不同的 HLA-A*02:01 限制的表位，这是通过三种表位预测算法的组合预测的。此外，我们鉴定了针对 3 个未预测含有任何免疫原性 HLA Ⅰ类表位的肽的反应，但在体外显示出 HCV 特异性反应。从体外引发的 PBMCs 中分离 CD8 和 CD8 T 细胞，仅在用短肽再次刺激时才显示出反应。我们建立了一种体外方法，可鉴定源自交叉呈递抗原的 HLA Ⅰ类表位，该方法可交叉引发 T 细胞，并有效选择用于针对持续性病毒感染和肿瘤抗原的定制治疗性疫苗的功能性免疫原性表位，也包括免疫原性较低的表位。
