鉴定传染性支气管炎病毒 N 蛋白上新的 T 细胞表位并开发多表位疫苗。

Identification of Novel T-Cell Epitopes on Infectious Bronchitis Virus N Protein and Development of a Multi-epitope Vaccine.

机构信息

Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, People's Republic of China.

出版信息

J Virol. 2021 Aug 10;95(17):e0066721. doi: 10.1128/JVI.00667-21.

Abstract

Cellular immune responses play a key role in the control of viral infection. The nucleocapsid (N) protein of infectious bronchitis virus (IBV) is a major immunogenic protein that can induce protective immunity. To screen for potential T-cell epitopes on IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. Four T-cell epitope peptides were identified by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot), intracellular cytokine staining, and carboxyfluorescein succinimidyl ester (CFSE) lymphocyte proliferation assays; among them, three peptides (N, N, and N) were cytotoxic T lymphocyte (CTL) epitopes, and one peptide (N) was a dual-specific T-cell epitope, which can be recognized by both CD8 and CD4 T cells. Multi-epitope gene transcription cassettes comprising four neutralizing epitope domains and four T-cell epitope peptides were synthesized and inserted into the genome of Newcastle disease virus strain La Sota between the P and M genes. Recombinant IBV multi-epitope vaccine candidate rLa Sota/SBNT was generated via reverse genetics, and its immune protection efficacy was evaluated in specific-pathogen-free chickens. Our results show that rLa Sota/SBNT induced IBV-specific neutralizing antibody and T-cell responses and provided significant protection against homologous and heterologous IBV challenge. Thus, the T-cell epitope peptides identified in this study could be good candidates for IBV vaccine development, and recombinant Newcastle disease virus-expressing IBV multi-epitope genes represent a safe and effective vaccine candidate for controlling infectious bronchitis. T-cell-mediated immune responses are critical for the elimination of IBV-infected cells. To screen conserved T-cell epitopes in the IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. By combining IFN-γ ELISpot, intracellular cytokine staining, and CFSE lymphocyte proliferation assays, we identified three CTL epitopes and one dual-specific T-cell epitope. The value of T-cell epitope peptides identified in the N protein was further verified by the design of an IBV multi-epitope vaccine. Results show that IBV multi-epitope vaccine candidate rLa Sota/SBNT provided cross protection against challenges with a QX-like or a TW-like IBV strain. So, T-cell-mediated immune responses play an important role in the control of viral infection, and conserved T-cell epitopes serve as promising candidates for use in multi-epitope vaccine construction. Our results provide a new perspective for the development of a safer and more effective IBV vaccine.

摘要

细胞免疫反应在控制病毒感染中起着关键作用。传染性支气管炎病毒 (IBV) 的核衣壳 (N) 蛋白是一种主要的免疫原性蛋白,可诱导保护性免疫。为了筛选 IBV N 蛋白上的潜在 T 细胞表位,设计并合成了 40 个覆盖 N 蛋白全长的重叠肽。通过γ干扰素 (IFN-γ) 酶联免疫斑点 (ELISpot)、细胞内细胞因子染色和羧基荧光素琥珀酰亚胺酯 (CFSE) 淋巴细胞增殖试验,鉴定出 4 个 T 细胞表位肽;其中 3 个肽 (N 、 N 、 N) 是细胞毒性 T 淋巴细胞 (CTL) 表位,1 个肽 (N) 是双特异性 T 细胞表位,可被 CD8 和 CD4 T 细胞识别。包含四个中和表位域和四个 T 细胞表位肽的多表位基因转录盒被合成并插入到新城疫病毒株 La Sota 的 P 和 M 基因之间的基因组中。通过反向遗传学生成了重组 IBV 多表位疫苗候选物 rLa Sota/SBNT,并在无特定病原体鸡中评估了其免疫保护效力。我们的结果表明,rLa Sota/SBNT 诱导了 IBV 特异性中和抗体和 T 细胞反应,并对同源和异源 IBV 攻击提供了显著保护。因此,本研究中鉴定的 T 细胞表位肽可能是 IBV 疫苗开发的良好候选物,而表达 IBV 多表位基因的重组新城疫病毒代表了控制传染性支气管炎的安全有效的疫苗候选物。T 细胞介导的免疫反应对于清除 IBV 感染细胞至关重要。为了筛选 IBV N 蛋白中的保守 T 细胞表位,设计并合成了 40 个覆盖 N 蛋白全长的重叠肽。通过 IFN-γ ELISpot、细胞内细胞因子染色和 CFSE 淋巴细胞增殖试验相结合,我们鉴定出三个 CTL 表位和一个双特异性 T 细胞表位。通过 IBV 多表位疫苗的设计进一步验证了 N 蛋白中鉴定的 T 细胞表位肽的价值。结果表明,IBV 多表位疫苗候选物 rLa Sota/SBNT 对 QX 样或 TW 样 IBV 株的攻击提供了交叉保护。因此,T 细胞介导的免疫反应在控制病毒感染中起着重要作用,保守 T 细胞表位是构建多表位疫苗的有前途的候选物。我们的结果为开发更安全有效的 IBV 疫苗提供了新视角。

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