Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA, USA.
Immunotope, Inc., Pennsylvania Institute for Biotechnology, Doylestown, PA, USA.
Vaccine. 2018 Aug 9;36(33):5046-5057. doi: 10.1016/j.vaccine.2018.07.002. Epub 2018 Jul 10.
Human T-cell leukemia virus type 1 (HTLV-1) has infected as many as 10 million people worldwide. While 90% are asymptomatic, 5% develop severe diseases including adult T-cell leukemia/lymphoka (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). No vaccine against HTLV-1 exists, and screening programs are not universal. However, patients with chronic HTLV-1 infection have high frequencies of HTLV-1-activated CD8+ T cells, and the two main HLA alleles (A2, A24) are present in 88% of infected individuals. We thus utilized an immunoproteomics approach to characterize MHC-I restricted epitopes presented by HLA-A2+, A24+ MT-2 and SLB-1 cell lines. Unlike traditional motif prediction algorithms, this approach identifies epitopes associated with cytotoxic T-cell responses in their naturally processed forms, minimizing differences in antigen processing and protein expression levels. Out of nine identified peptides, we confirmed six novel MHC-I restricted epitopes that were capable of binding HLA-A2 and HLA-A24 alleles and used in vitro and in vivo methods to generate CD8+ T cells specific for each of these peptides. MagPix MILLIPLEX data showed that in vitro generated epitope-specific CD8+ T cells secreted IFN-ɣ, granzyme B, MIP-1α, TNF-α, perforin and IL-10 when cultured in the presence of MT-2 cell line. Degranulation assay confirmed cytotoxic response through surface expression of CD107 on CD8+ T cells when cultured with MT-2 cells. A CD8+ T-cell killing assay indicated significant antiviral activity of CD8+ T cells specific against all identified peptides. In vivo generated CD8+ T cells similarly demonstrated immunogenicity on ELISpot, CD107 degranulation assay, and MagPix MILLIPLEX analysis. These epitopes are thus candidates for a therapeutic peptide-based vaccine against HTLV-1, and our results provide preclinical data for the advancement of such a vaccine.
人类 T 细胞白血病病毒 1 型（HTLV-1）已感染全球多达 1000 万人。虽然 90%的人无症状，但 5%的人会发展为严重疾病，包括成人 T 细胞白血病/淋巴瘤（ATLL）和 HTLV-1 相关脊髓病/热带痉挛性截瘫（HAM/TSP）。目前尚无针对 HTLV-1 的疫苗，且筛查计划并非普遍适用。然而，慢性 HTLV-1 感染者体内存在高频率的 HTLV-1 激活的 CD8+T 细胞，而两种主要的 HLA 等位基因（A2、A24）存在于 88%的感染者中。因此，我们利用免疫蛋白质组学方法来鉴定 HLA-A2+、A24+MT-2 和 SLB-1 细胞系中由 MHC-I 限制的表位。与传统的基序预测算法不同，该方法鉴定了与细胞毒性 T 细胞反应相关的表位，这些表位以其天然加工形式呈现，最大限度地减少了抗原加工和蛋白表达水平的差异。在鉴定出的 9 种肽中，我们验证了 6 种新的 MHC-I 限制表位，这些表位能够与 HLA-A2 和 HLA-A24 等位基因结合，并使用体外和体内方法生成针对这些肽的特异性 CD8+T 细胞。MagPix MILLIPLEX 数据显示，在体外生成的针对表位的 CD8+T 细胞在与 MT-2 细胞系共培养时，可分泌 IFN-γ、颗粒酶 B、MIP-1α、TNF-α、穿孔素和 IL-10。脱颗粒测定通过共培养时 CD8+T 细胞表面表达 CD107 证实了细胞毒性反应。CD8+T 细胞杀伤测定表明，针对所有鉴定出的肽的 CD8+T 细胞具有显著的抗病毒活性。体内生成的 CD8+T 细胞在 ELISpot、CD107 脱颗粒测定和 MagPix MILLIPLEX 分析中也表现出免疫原性。这些表位是针对 HTLV-1 的治疗性肽疫苗的候选者，我们的结果为推进这种疫苗的发展提供了临床前数据。
