Abstract：
Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis and develops resistance to many of the existing drugs. The sole licensed TB vaccine, BCG, is unable to provide a comprehensive defense. So, it is crucial to maintain the immunological response to eliminate tuberculosis. Our previous in silico study reported five uncharacterized proteins as potential vaccine antigens. In this article, we considered the uncharacterized Mtb H37Rv regions of difference (RD-2) Rv1987 protein as a promising vaccine candidate. The vaccine quality of the protein was analyzed using reverse vaccinology and immunoinformatics-based quality-checking parameters followed by an ex vivo preliminary investigation. In silico analysis of Rv1987 protein predicted it as surface localized, secretory, single helix, antigenic, non-allergenic, and non-homologous to the host protein. Immunoinformatics analysis of Rv1987 by CD4 + and CD8 + T-cells via MHC-I and MHC-II binding affinity and presence of B-cell epitope predicted its immunogenicity. The docked complex analysis of the 3D model structure of the protein with immune cell receptor TLR-4 revealed the protein\'s capability for potential interaction. Furthermore, the target protein-encoded gene Rv1987 was cloned, over-expressed, purified, and analyzed by mass spectrometry (MS) to report the target peptides. The qRT-PCR gene expression analysis shows that it is capable of activating macrophages and significantly increasing the production of a number of key cytokines (TNF-α, IL-1β, and IL-10). Our in-silico analysis and ex vivo preliminary investigations revealed the immunogenic potential of the target protein. These findings suggest that the Rv1987 be undertaken as a potent subunit vaccine antigen and that further animal model immuno-modulation studies would boost the novel TB vaccine discovery and/or BCG vaccine supplement pipeline.
摘要：
结核分枝杆菌（Mtb）是引起结核病的病原体，并对许多现有药物产生耐药性。唯一获得许可的结核病疫苗，BCG,无法提供全面的防御。所以,维持免疫反应对消除结核病至关重要。我们先前的计算机模拟研究报道了五种未表征的蛋白质作为潜在的疫苗抗原。在这篇文章中,我们认为未表征的MtbH37Rv差异区（RD-2）Rv1987蛋白是有希望的疫苗候选物。使用反向疫苗学和基于免疫信息学的质量检查参数，然后进行离体初步研究，分析了蛋白质的疫苗质量。Rv1987蛋白的计算机模拟分析预测它是表面定位的，分泌，单螺旋,抗原性,非过敏性，并且与宿主蛋白非同源。通过CD4+和CD8+T细胞通过MHC-I和MHC-II结合亲和力和B细胞表位的存在对Rv1987进行的免疫信息学分析预测了其免疫原性。蛋白质与免疫细胞受体TLR-4的3D模型结构的对接复合物分析揭示了蛋白质潜在相互作用的能力。此外，克隆了靶蛋白编码基因Rv1987，过度表达,纯化,并通过质谱(MS)分析以报告目标肽。qRT-PCR基因表达分析表明，它能够激活巨噬细胞并显着增加许多关键细胞因子（TNF-α，IL-1β,和IL-10）。我们的计算机内分析和离体初步研究揭示了靶蛋白的免疫原性潜力。这些发现表明Rv1987被用作有效的亚单位疫苗抗原,并且进一步的动物模型免疫调节研究将促进新的TB疫苗发现和/或BCG疫苗补充管道。
