Agricultural Research Council-Onderstepoort Veterinary Research, Private Bag X5, Onderstepoort, 0110, South Africa; Department of Veterinary Tropical Diseases, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa.
Agricultural Research Council-Onderstepoort Veterinary Research, Private Bag X5, Onderstepoort, 0110, South Africa.
Mol Immunol. 2019 Mar;107:106-114. doi: 10.1016/j.molimm.2018.12.012. Epub 2019 Jan 31.
Several studies have shown that cytotoxic T lymphocytes (CTL) require CD4 + Th1 epitopes to generate strong immune responses to intracellular pathogens. However, not much is known about Ehrlichia ruminantium epitopes, particularly those that can be considered potential candidates for inclusion in a multi-epitope vaccine. In order to identify CD4 Th1 epitopes that induce IFNγ, a number of proteins previously identified as immunogenic were first screened to determine if they induce cellular immunity in tick infected immune sheep PBMC. Significant IFN-γ production and other Th1 cytokines were evident for 10 recombinant proteins in all sheep tested. Secondly, peptides (n = 246) derived from the top 10 E. ruminantium vaccine candidate proteins were assayed using enzyme linked immunospot (ELISPOT) assay, quantitative real-time PCR and flow cytometry. Of the 246 peptides, 23 peptides, Erum0660 (p0660-42), Erum1150 (p1150-18, p1150-19), Erum2540 (p2540-6, p2540-16, p2540-19, p2540-20, p2540-21), Erum5420 (p5420-13, p5420-14), Erum7140 (p7140-6, p7140-7, p7140-12, p7140-13, p7140-20), Erum7320 (p7320-8, p7320-9, p7320-21), Erum7350 (p7350-9), Erum7360 (p7360-8), Erum7620 (p7620-2, p7620-12) and Erum8010 (p8010-8) were identified that stimulate the best and different cell mediated immune responses. Amino acid sequences of these peptides except for p7140-12, p7140-13, p7140-20, and p7350-9 were conserved between 13 different local strains. These peptides could efficiently induce memory CD4 T cells to rapidly proliferate and significantly increase IFN-γ production in immune sheep PBMC. The upregulation of pro-inflammatory cytokines, which include, IL-1α, IL-2, IL-12p40, TNF-α, IFN-γ, inducible nitric oxide synthase (iNOS) and granulocyte-macrophage colony stimulating factor (GM-CSF) was also detected. Our results show that these peptides could serve as promising candidates for a multi-epitope vaccine against E. ruminantium.
已有多项研究表明，细胞毒性 T 淋巴细胞（CTL）需要 CD4+Th1 表位才能对细胞内病原体产生强烈的免疫反应。然而，对于埃立克体属的表位，特别是那些可以被认为是潜在的候选多表位疫苗的表位，我们了解甚少。为了鉴定诱导 IFNγ 的 CD4 Th1 表位，我们首先筛选了先前鉴定为免疫原性的多种蛋白，以确定它们是否在感染 tick 的免疫绵羊 PBMC 中诱导细胞免疫。在所有测试的绵羊中，10 种重组蛋白均明显诱导产生 IFN-γ和其他 Th1 细胞因子。其次，使用酶联免疫斑点（ELISPOT）测定法、定量实时 PCR 和流式细胞术检测了源自前 10 种候选埃立克体属疫苗蛋白的 246 个肽段。在 246 个肽段中，有 23 个肽段，Erum0660（p0660-42）、Erum1150（p1150-18，p1150-19）、Erum2540（p2540-6，p2540-16，p2540-19，p2540-20，p2540-21）、Erum5420（p5420-13，p5420-14）、Erum7140（p7140-6，p7140-7，p7140-12，p7140-13，p7140-20）、Erum7320（p7320-8，p7320-9，p7320-21）、Erum7350（p7350-9）、Erum7360（p7360-8）、Erum7620（p7620-2，p7620-12）和 Erum8010（p8010-8）被鉴定为能够刺激最佳和不同的细胞介导免疫反应的肽段。这些肽段的氨基酸序列除了 p7140-12、p7140-13、p7140-20 和 p7350-9 外，在 13 个不同的本地株之间是保守的。这些肽段能够有效地诱导记忆性 CD4 T 细胞快速增殖，并显著增加免疫绵羊 PBMC 中 IFN-γ的产生。还检测到促炎细胞因子的上调，包括白细胞介素-1α、白细胞介素-2、白细胞介素-12p40、肿瘤坏死因子-α、IFN-γ、诱导型一氧化氮合酶（iNOS）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）。我们的研究结果表明，这些肽段可能是针对埃立克体属的多表位疫苗的有前途的候选物。
