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

解构的 HIV-1 病毒样颗粒在生物工程植物中的表达

• 批准号: 7367919
• 负责人: Nobuyuki Matoba
• 金额: $ 2.81万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-15 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367919
• 关键词: AIDS prevention; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Animal Model; Anti-Retroviral Agents; Antibodies; Biochemical; Biological Assay; Biomedical Engineering; CD4 Positive T Lymphocytes; Cells; Containment; Country; Cytoplasmic Tail; Cytotoxic T-Lymphocytes; Electron Microscopy; Epidemiologic Studies; Epithelial; Event; Gagging; Genes; Goals; HIV-1; HIV-1 vaccine; Highly Active Antiretroviral Therapy; Immune response; Immunoprecipitation; In Vitro; Income; Infection; Intestines; Investigation; Liquid substance; Low income; Membrane; Mucous Membrane; Onset of illness; Oral; Other Finding; Outcome; Pharmaceutical Preparations; Plants; Prevention program; Production; Simulate; Staging; Stomach; Suspension Culture; System; Tobacco; Tobacco Mosaic Virus; Transmembrane Domain; Vaccines; Vaginal delivery procedure; Viral; Virus-like particle; base; design; env Gene Products; env Genes; expression vector; follow-up; gag Gene Products; light scattering; mucosal vaccine; new technology; nonhuman primate; novel; pandemic disease; prevent; prophylactic; transcytosis; transmission process

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.

描述（由申请人提供）：广泛的艾滋病预防计划和高效的抗逆转录病毒疗法成功地限制了高收入国家艾滋病的传播和发病。然而，人们普遍认为，全球遏制艾滋病毒/艾滋病流行病取决于我们是否有能力研制预防性疫苗，尽量减少病毒的传播和感染。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，预防HIV-1传播和早期感染。为此，我们的努力集中在一个新的HIV-1病毒样颗粒（VLP）组成的解构HIV-1 Env蛋白（D-Env）和Gag蛋白（D-Env/Gag VLP）。为了经济生产和粘膜递送的目的，我们提出了在植物中表达D-Env/Gag VLP。D-Env由gp 41的外膜近端区域残基649-683（MPR 649 -683）、跨膜结构域和胞质尾区组成。具体的假设是，粘膜靶向的D-Env/Gag VLP可以在粘膜诱导保护性体液和细胞免疫应答，从而协同阻断HIV-1传播和感染的早期阶段。我们将该假设基于我们和其他人的发现，即（i）靶向MPR 649 -683的抗体显示出阻断HIV-1上皮转胞吞作用并潜在地中和CD 4+细胞的感染，以及（ii）Gag VLP显示出在非人灵长类动物中诱导Gag特异性细胞毒性T细胞。 作为实现长期目标的第一步，本提案的具体目的是通过在烟草细胞中共表达D-Env和Gag来生产和体外表征D-Env/Gag VLP，其是基于最流行的HIV-1亚型C设计的。为了获得高产量，gag基因将被优化用于植物表达，并且设计的基因将被从头合成。我们将采用两种表达系统，一种是基于烟草花叶病毒（TMV）的瞬时表达系统，另一种是稳定转化的烟草细胞（NT 1）悬浮培养系统。这些系统使得能够快速调查植物细胞中的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系统中共递送植物优化的d-env基因与gag基因并用D-Env表达载体超转化表达Gag的NT 1细胞来完成。为了表征植物表达的D-Env/Gag VLP，将通过免疫沉淀和下拉测定进行生化分析，通过光散射和电子显微镜进行构象分析，以及使用模拟胃液和肠液进行稳定性分析。该项目的成功结果不仅将提供一种新的HIV-1候选疫苗，并将在后续项目中使用动物模型进行评估，而且还将开发一种新的经济生产技术。