PEPTIDE BASED DELIVERY OF HIV IMMUNOGEN

基于肽的 HIV 免疫原递送

• 批准号: 2887876
• 负责人: LEE W RILEY
• 金额: $ 22.56万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887876
• 关键词: AIDS vaccines; CHO cells; Escherichia coli; HIV envelope protein gp120; HeLa cells; MHC class I antigen; MHC class II antigen; Mycobacterium tuberculosis; antigen presentation; antigen presenting cell; bacterial proteins; chimeric proteins; clone cells; cytotoxic T lymphocyte; human immunodeficiency virus 1; ovalbumin; transmission electron microscopy; vaccine development; vaccinia virus; vector vaccine

This project will examine a novel method of delivery of proteins into mammalian cells, and it specifically targets the area of investigation related to the HIV envelope protein gp120. Eliciting a strong CD4 T cell stimulation and cytotoxic T lymphocyte (CTL) response against HIV-1 and HIV-1 infected cells should be a major objective of any HIV vaccine development work. One way to achieve this objective is to develop a more efficient but safe antigen delivery system. The current approaches to deliver HIV immunogens using live vector systems are not adequate. We have developed a peptide-based delivery system that may overcome the limitations of existing delivery vectors. This project will serve as a proof-of-the-concept project using gp120 to study the efficiency of protein delivery, intracellular processing, and antigen presentation mediated by a 22-amino acid peptide derived from a surface protein of Mycobacterium tuberculosis. The peptide called Inv3 was derived from a sequence based on the most active domain of a protein called mycobacterium cell entry protein or Mcep. Mcep and Inv3 are able to deliver materials (microspheres, colloidal gold particles) complexed to them into nonphagocytic cells with very high efficiency. We have constructed an expression plasmid (pInv3) that facilitates expression of any protein with Inv3 peptide fused to the N-terminus. We have already shown that beta-galactosidase, green fluorescent protein, and ovalbumin expressed in fusion with Inv3 are able to abundantly associate with HeLa cells. We plan to express gp120 using pInv3 to deliver the protein into mammalian cells to study the efficiency of its delivery and the intracellular location of the protein. The two specific aims are as follows: 1) study the efficiency of cell delivery of Inv3-gp120 fusion protein and its final destination inside nonphagocytic cells and 2) evaluate the presentation of peptides derived from a known protein (ovalbumin) expressed in fusion with Inv3 by antigen presenting cells (APCs) via MHC class I and class II pathways, using APCs that have been specifically designed to express a) Kb allele in HeLa cells for MHC class I antigen presentation, and b) Ak specific L cells for MHC class II antigen presentation. The intracellular delivery, processing, and antigen presentation facilitated by Inv3-ovalbumin will be compared to those associated with a vaccinia vector delivered ovalbumin. If Inv3 is demonstrated to enhance the delivery of gp120 and facilitate ovalbumin peptide presentation by APCs superior to that mediated by the vaccinia vector system, we will enter a full collaboration with the HIVNET program at the California Department of Health Services to apply this technology to human cells as part of our long-term goal, for which we will seek additional funding support. The successful outcome of this project may not only identify potential vaccine candidates for HIV-1, but may serve as a proof-of-concept study for a completely novel approach to vaccine development. This ability to deliver any polypeptide into cells enables one to screen the entire coding capacity of a genome of a pathogen, without any preconceived bias, for immunodominant or immunoprotective peptide sequences. The ability to identify such domains may open a completely new direction in identifying peptides that may be able to elicit protective CTL response against not just intracellular pathogens, but against tumor cells. While this may be a high risk project, the yield, if shown to work, even as a method to enhance mammalian cell delivery of proteins, should have a significant impact.

该项目将研究一种新颖的蛋白质输送方法 哺乳动物细胞，并专门针对研究区域 与HIV包膜蛋白GP120有关。引起强CD4 T细胞 刺激和细胞毒性T淋巴细胞（CTL）反应针对HIV-1和 HIV-1感染的细胞应成为任何HIV疫苗的主要目标 发展工作。实现这一目标的一种方法是开发更多 有效但安全的抗原输送系统。 当前的方法 使用活媒介系统输送艾滋病毒免疫原是不够的。 我们 已经开发了一种基于肽的输送系统，该系统可能会克服 现有交付向量的局限性。 这个项目将作为 使用GP120的概念证明项目研究的效率 蛋白质递送，细胞内加工和抗原表现 由22个氨基酸肽介导，该肽是从表面蛋白的 结核分枝杆菌。 称为Inv3的肽是从 基于称为蛋白质的最活跃结构域的序列 分枝杆菌细胞进入蛋白或MCEP。 MCEP和Inv3能够 交付材料（微球，胶体金颗粒） 它们以非常高的效率进入非晶状细胞。 我们有 构建了促进表达的表达质粒（PINV3） 与N末端融合的Inv3肽的任何蛋白质的。我们有 已经表明β-半乳糖苷酶，绿色荧光蛋白和 与Inv3融合表达的椭圆蛋白能够大量关联 与HeLa细胞。 我们计划使用PINV3表达GP120以交付 蛋白质进入哺乳动物细胞，以研究其递送效率和 蛋白质的细胞内位置。 两个具体目标是 如下：1）研究Inv3-GP120的细胞递送的效率 融合蛋白及其在非孢子细胞内的最终目的地和 2）评估源自已知蛋白质的肽的表现 （椭圆蛋白）与抗原呈现细胞在Inv3中表达 （APC）通过MHC I类和II类途径，使用已经 专门设计用于在HeLa细胞中表达A）MHC的KB等位基因 I类抗原表现，b）MHC类的AK特异性L细胞 II抗原表现。 细胞内交付，处理和 将Inv3-overBumin促进的抗原表现与 与离甲酸载体相关的那些递送卵蛋白。 如果Inv3 被证明可以增强GP120的交付并促进 APC比由APC介导的椭圆蛋白肽表现优于由 Vaccinia矢量系统，我们将与 加利福尼亚卫生服务部的HIVNET计划申请 作为我们长期目标的一部分，该技术向人类细胞进行了 我们将寻求额外的资金支持。 该项目的成功结果不仅可以确定潜力 HIV-1的疫苗候选物，但可以作为概念证明研究 用于疫苗开发的完全新颖的方法。 这个能力 要将任何多肽输送到细胞中，都可以筛选整个多肽 病原体的基因组的编码能力，没有任何先入为主 偏置，用于免疫主导或免疫保护肽序列。 这 识别此类域的能力可能会在 识别可能能够引起保护性CTL响应的肽 不仅针对细胞内病原体，还针对肿瘤细胞。 虽然这可能是一个高风险项目，但收益率，如果证明是有效的，甚至 作为增强蛋白质哺乳动物细胞递送的一种方法，应具有 重大影响。