Objectives A potent HIV vaccine should overcome some limitations such as polymorphism of human HLA, the diversity of HIV-1 virus, and the lack of an effective delivery system. In this study, a DNA construct encoding Nef 60–84 , Nef 126–144 , Vpr 34–47 , Vpr 60–75 , Gp160 30–53 , Gp160 308–323, and P24 8–151 epitopes was designed using bioinformatics tools. The pcDNA3.1- nef-vpr-gp160-p24 and pcDNA3.1- nef constructs were prepared in large scale as endotoxin-free form. Moreover, the recombinant Nef-Vpr-Gp160-p24 polypeptide and Nef protein were generated in E. coli . These constructs were delivered using cell penetrating peptides (CPPs) in vivo, and immune responses were assessed for different modalities in BALB/c mice. Results The recombinant DNA constructs were confirmed as the ~ 867 bp and ~ 648 bp bands related to nef-vpr-gp160-p24 and nef genes on agarose gel. Moreover, the purified Nef-Vpr-Gp160-p24 polypeptide and Nef protein showed the ~ 32 kDa and ~ 30 kDa bands on SDS-PAGE, respectively. The results of immune responses indicated that the heterologous prime/boost regimens using both Nef-Vpr-Gp160-P24 and Nef antigens induced significantly the secretion of IgG2a, IgG2b, IFN-γ and Granzyme B compared to other groups. The levels of Granzyme B in mice immunized with Nef antigen were higher than those immunized with Nef-Vpr-Gp160-P24 antigen. The CPPs showed the same potency with Montanide adjuvant for eliciting immune responses. Conclusions The heterologous prime/boost regimens for both antigens could significantly direct immune responses toward Th1 and CTL activity compared to other regimens. Comparing the efficiency of Nef-Vpr-Gp160-P24 and Nef constructs, the Nef-Vpr-Gp160-P24 constructs delivered by CPPs showed promising results as an HIV vaccine candidate.

中文翻译：

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使用新型细胞穿透肽在体内递送多表位肽和 Nef 蛋白以开发 HIV-1 候选疫苗

目的 有效的 HIV 疫苗应克服一些限制，例如人类 HLA 的多态性、HIV-1 病毒的多样性以及缺乏有效的传递系统。在本研究中，使用生物信息学工具设计了编码 Nef 60-84、Nef 126-144、Vpr 34-47、Vpr 60-75、Gp160 30-53、Gp160 308-323 和 P24 8-151 表位的 DNA 构建体。pcDNA3.1-nef-vpr-gp160-p24和pcDNA3.1-nef构建体以无内毒素形式大规模制备。此外，在大肠杆菌中产生了重组的Nef-Vpr-Gp160-p24多肽和Nef蛋白。这些构建体是在体内使用细胞穿透肽 (CPP) 递送的，并评估了 BALB/c 小鼠中不同方式的免疫反应。结果重组DNA构建体在琼脂糖凝胶上确认为与nef-vpr-gp160-p24和nef基因相关的~867 bp和~648 bp条带。此外，纯化的 Nef-Vpr-Gp160-p24 多肽和 Nef 蛋白在 SDS-PAGE 上分别显示 ~ 32 kDa 和 ~ 30 kDa 条带。免疫反应的结果表明，与其他组相比，使用 Nef-Vpr-Gp160-P24 和 Nef 抗原的异源引发/加强方案显着诱导了 IgG2a、IgG2b、IFN-γ 和粒酶 B 的分泌。Nef抗原免疫小鼠体内颗粒酶B水平高于Nef-Vpr-Gp160-P24抗原免疫小鼠。CPP 与 Montanide 佐剂在引发免疫反应方面表现出相同的效力。结论 与其他方案相比，两种抗原的异源初免/加强方案可以显着引导针对 Th1 和 CTL 活性的免疫反应。比较 Nef-Vpr-Gp160-P24 和 Nef 构建体的效率，CPP 提供的 Nef-Vpr-Gp160-P24 构建体作为 HIV 疫苗候选物显示出有希望的结果。