Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran.
Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
Curr HIV Res. 2019;17(6):408-428. doi: 10.2174/1570162X17666191121114522.
An effective vaccine against human immunodeficiency virus 1 (HIV-1) is an important global health priority. Despite many efforts in the development of the HIV-1 vaccine, no effective vaccine has been approved yet. Recently, polyepitope vaccines including several immunogenic and conserved epitopes of HIV-1 proteins have received special attention.
In this study, HIV-1 Nef, Tat, Gp160 and P24 proteins were considered for selection of immunodominant and conserved epitopes due to their critical roles in the viral life cycle and pathogenesis. At first, the Nef60-84-Nef126-144-Tat29-49-Gp16030-53-Gp160308-323-P248-151 DNA construct was designed using in silico studies. Then, the DNA construct was subcloned in pEGFP-N1 and pET- 24a (+) expression vectors and the rNef-Tat-Gp160-P24 polyepitope peptide was generated in E.coli expression system for in vitro delivery using novel cell-penetrating peptides (CPPs), LDP-NLS and CyLoP-1, in a non-covalent manner. Also, the HR9 and MPG CPPs were used to transfer the DNA construct.
Our results showed that the recombinant polyepitope peptide generated in Rosetta strain migrated as a clear band of ~31 kDa in SDS-PAGE. The SEM data confirmed the formation of stable nanoparticles with a size below 250 nm. MTT assay revealed that the complexes did not represent any considerable cytotoxic effect compared to untreated cells. The results of fluorescence microscopy, flow cytometry and western blotting indicated that these CPPs successfully delivered polyepitope constructs into HEK-293T cell line.
These data suggested that these CPPs can be used as a promising approach for the development of the HIV-1 vaccine.
针对人类免疫缺陷病毒 1 (HIV-1) 的有效疫苗是全球健康的重要优先事项。尽管在 HIV-1 疫苗的开发方面做出了许多努力，但尚未批准任何有效的疫苗。最近，多表位疫苗，包括 HIV-1 蛋白的几个免疫原性和保守表位，受到了特别关注。
在这项研究中，由于其在病毒生命周期和发病机制中的关键作用，选择了 HIV-1 Nef、Tat、Gp160 和 P24 蛋白来选择免疫优势和保守表位。首先，使用计算机模拟研究设计了 Nef60-84-Nef126-144-Tat29-49-Gp16030-53-Gp160308-323-P248-151 DNA 构建体。然后，将 DNA 构建体亚克隆到 pEGFP-N1 和 pET-24a(+)表达载体中，并在大肠杆菌表达系统中生成 rNef-Tat-Gp160-P24 多表位肽，使用新型细胞穿透肽（CPPs）LDP-NLS 和 CyLoP-1 以非共价方式进行体外递呈。此外，还使用 HR9 和 MPG CPP 转染 DNA 构建体。
我们的结果表明，在 Rosetta 菌株中生成的重组多表位肽在 SDS-PAGE 中迁移为约 31 kDa 的清晰条带。SEM 数据证实形成了尺寸小于 250nm 的稳定纳米颗粒。MTT 测定表明，与未处理的细胞相比，这些复合物没有表现出任何明显的细胞毒性作用。荧光显微镜、流式细胞术和 Western blot 结果表明，这些 CPP 成功地将多表位构建体递送至 HEK-293T 细胞系。
这些数据表明，这些 CPP 可以作为开发 HIV-1 疫苗的有前途的方法。
