Expression of Decontructed HIV-1 Virus-Like Particles in Bioengineered Plants

描述(申请人提供)：广泛的艾滋病预防计划和高效的抗逆转录病毒疗法已经成功地限制了疾病在高收入国家的传播和发病。然而，人们普遍认为，全球遏制艾滋病毒/艾滋病大流行取决于我们是否有能力开发预防性疫苗，将病毒传播和感染降至最低。该项目的长期目标是开发一种负担得起的、有效的粘膜疫苗，防止艾滋病毒-1传播和早期感染。为此，我们的工作集中在一种新型的HIV-1病毒样颗粒(VLP)上，该VLP由解构的HIV-1包膜蛋白(D-Env)和Gag蛋白(D-Env/Gag VLP)组成。为了达到经济生产和粘膜输送的目的，我们建议在植物中表达D-env/Gag VLP。D-env由外膜近端残基649-683(MPR649-683)、跨膜区和gp41胞浆尾部组成。具体的假设是，以粘膜为靶点的D-Env/Gag VLP可以在粘膜诱导保护性体液和细胞免疫反应，从而协同阻断HIV-1的传播和早期感染。我们的假设是基于我们和其他人的发现：(I)针对MPR649-683的抗体被证明可以阻断HIV-1上皮细胞的转胞作用，并潜在地中和CD4+细胞的感染；(Ii)Gag VLP被证明在非人类灵长类动物中诱导Gag特异性的细胞毒T细胞。 作为实现长期目标的第一步，这项建议的具体目标是通过在烟草细胞中共表达D-Env和Gag来制备和体外鉴定基于最流行的HIV-1 C亚型的D-Env/Gag VLP。为了获得高产，将对Gag基因进行植物表达优化，并重新合成所设计的基因。我们将使用两种表达系统，基于改良烟草花叶病毒(TMV)的瞬时表达系统和稳定转化的烟草细胞(NT1)悬浮培养。这些系统可以快速研究植物细胞中的VLP组装。在成功表达GAG VLP后，将集中精力创建完整的D-Env/GAG VLP。这将通过在TMV系统中将植物优化的d-env基因与gag基因共传递，并用D-env表达载体超转化表达Gag的NT1细胞来实现。为了表征植物表达的D-Env/Gag VLP，将进行免疫沉淀和下拉分析的生化分析，光散射和电子显微镜的构象分析，以及模拟胃液和肠液的稳定性分析。该项目的成功不仅将提供一种新的HIV-1候选疫苗，将在后续项目中使用动物模型进行评估，而且还将为其经济生产开发一种新技术。

DESCRIPTION (provided by applicant): Extensive AIDS prevention programs and highly active anti-retroviral therapies have been successful in limiting the spread and the onset of the disease in high-income countries. However, it is generally agreed that global containment of the HIV/AIDS pandemics depends on our ability to develop prophylactic vaccines minimizing the viral transmission and infection. The long-term goal of this project is to develop an affordable, efficacious mucosal vaccine preventing HIV-1 transmission and early stages of infection. To this end, our efforts are focused on a novel HIV-1 virus-like particles (VLPs) consisting of the deconstructed HIV-1 Env protein (D-Env) and the Gag protein (D-Env/Gag VLPs). Towards the aim of economical production and mucosal delivery, we propose the expression of D-Env/Gag VLPs in plants. D-Env is composed of the external membrane proximal region residue 649-683 (MPR649-683), the transmembrane domain and the cytoplasmic tail of gp41. The specific hypothesis is that mucosally targeted D-Env/Gag VLPs can induce protective humoral and cellular immune responses at the mucosa, thereby synergistically blocking HIV-1 transmission and early stages of infection. We base that hypothesis on the findings by us and others that (i) antibodies targeting MPR649-683 were shown to block HIV-1 epithelial transcytosis and potentially neutralize the infection of CD4+ cells and (ii) Gag VLPs were shown to induce Gag-specific cytotoxic T cells in non-human primates. As an initial step to achieve the long-term goal, the specific aim of this proposal is to produce and in vitro characterize D-Env/Gag VLPs, which are designed based on the most pandemic HIV-1 subtype C, by the coexpression of D-Env and Gag in tobacco cells. In order to obtain high yield, the gag gene will be optimized for plant expression and the designed gene will be de novo synthesized. We will employ two expression systems, a modified tobacco mosaic virus (TMV)-based transient expression system and a stably transformed tobacco cell (NT1) suspension culture. These systems enable rapid investigations of the VLP assembly in plant cells. Upon successfully expressing Gag VLPs, efforts will be centered on creating the complete D-Env/Gag VLPs. This will be done by co-delivery of the plant optimized d-env gene with the gag gene in the TMV system and super-transformation of the Gag-expressing NT1 cells with D-Env expression vector. To characterize the plant-expressed D-Env/Gag VLPs, biochemical analyses by immunoprecipitation and pull-down assay, conformational analyses by light scattering and electron microscopy, and stability analysis using simulated gastric and intestinal fluids will be performed. The successful outcome of this project will not only provide a novel HIV-1 vaccine candidate that will be evaluated in the follow-up project using animal models, but also develop a new technology for its economical production.