Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Biotechnol Lett. 2021 Aug;43(8):1513-1550. doi: 10.1007/s10529-021-03143-9. Epub 2021 May 13.
Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.
Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.
The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56-60% as the bands of ~ 63 and ~ 72 kDa confirmed in western blotting, respectively.
The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.
携带高度保守和免疫优势表位的表位驱动疫苗已成为克服人类免疫缺陷病毒 1 (HIV-1) 感染的有前途的方法。
设计了两种多表位 DNA 构建体，分别编码来自 HIV-1 Gag、Pol、Env、Nef 和 Rev 蛋白的 T 细胞表位，以及与热休克蛋白 70 (Hsp70) 的免疫原性表位相连，作为免疫刺激剂。应用了包括 MHC-I 和 MHC-II 结合、MHC-I 免疫原性和抗原加工、人群覆盖率、保守性、变应原性、毒性和血液毒性的计算机分析。对预测的表位进行了肽-MHC-I/MHC-II 分子对接和细胞因子产生分析。然后，使用两种多表位融合构建体设计了选择的高免疫原性 T 细胞表位。接下来，对每个构建体进行了理化和结构特性、B 细胞表位和构建体- Toll 样受体 (TLR) 分子对接的预测。最后，生成了携带 HIV-1 蛋白 12 个细胞毒性 T 淋巴细胞 (CTL) 和 10 个辅助性 T 淋巴细胞 (HTL) 表位和 2 个 Hsp70 CTL 和 2 个 HTL 表位的真核表达质粒（即 pEGFP-N1-gag-pol-env-nef-rev），并转染到 HEK-293 T 细胞中，通过流式细胞术和 Western blot 评估多表位肽表达的百分比。
设计的 DNA 构建体可以在哺乳动物细胞中成功表达。Western blot 证实，Gag-Pol-Env-Nef-Rev-GFP 和 Hsp70-Gag-Pol-Env-Nef-Rev-GFP 的表达率约为 56-60%，分别为63 和72 kDa 的条带。
计算机模拟/体外方法的结合表明，可以产生两种多表位构建体，并可作为 HIV-1 疫苗开发的有效免疫原。
