DEVELOPMENT OF THE HBcAG AS A VACCINE CARRIER PLATFORM

描述（由申请人提供）：本申请是R 01 AI 49730 -03的竞争延续。使用肽和小的定义明确的寡糖（OS）抗原用于亚单位疫苗设计有许多优点。例如，化学纯度和安全性，易于生产，成本，稳定性和可变性。然而，肽和OS抗原需要与T细胞载体蛋白缀合以有效递送至免疫系统。肝炎核心平台是一种颗粒载体，能够引发对掺入核心颗粒上的弱“半抗原样”肽和OS抗原的高滴度抗体应答。在该资助的过程中，我们使用了乙型肝炎B核心（HBcAg）作为恶性疟原虫疟疾环子孢子（CS）中和重复序列的载体平台（即，NANPn）。杂交HBcAg-CS重复颗粒在小鼠中具有高度免疫原性，目前正处于1/11期临床试验中。虽然HBcAg具有高度免疫原性，但我们已经确定了使用HBcAg作为疫苗平台的许多理论和实践限制。为了解决这些局限性，我们开发了来自土拨鼠嗜肝DNA病毒（WHcAg）的核心蛋白作为颗粒载体平台，原因如下：（1）WHcAg在B细胞和T细胞水平上与HBcAg具有相同或更高的免疫原性;（2）WHcAg的使用不损害抗-HBc诊断测定，因为WHcAg和HBcAg在抗体水平上不交叉反应;（3）与HBcAg不同，不存在针对WHcAg的预先存在的抗体;（4）慢性HBV携带者对HBcAg的免疫耐受（全球3 - 4亿）可以通过使用WHcAg平台来规避，因为WHcAg和HBcAg在CD 4 + T细胞水平上仅具有部分交叉反应性;和（5）我们已经开发了一种基于WHcAg的组合技术，该技术在适应更多种类的外源表位的插入方面比现有的HBcAg技术更通用。例如，我们已经修饰了WHcAg基因，以创建17个插入位点和22个C末端修饰的文库。结合这些文库使我们能够成功地将24个尝试的外源表位中的22个插入WHcAg平台。我们建议继续扩展WHcAg组合技术，并对杂交-WHcAg颗粒进行生物化学/结构分析，以尝试将结构和功能相关联（目标1）;在小动物模型中表征体外和体内对杂交-WHcAg颗粒的免疫应答，包括攻毒实验，以确定杂交-WHcAg疫苗候选物的保护效力（如果需要）（目标2）;并扩展WHcAg平台以适应非线性较大蛋白质和碳水化合物抗原（Aim 3）。迄今为止产生的WHcAg和混合WHcAg颗粒的许多独特的免疫学特征表明，这种颗粒平台将可用作将各种医学相关抗原递送至免疫系统的手段。

DESCRIPTION (provided by applicant): This application is a competitive continuation of R01 AI49730-03. There are a number of advantages to the use of peptide and small well-defined oligosaccharide (OS) antigens for subunit vaccine design. For example, chemical purity and safety, ease of production, cost, stability and mutability. However, peptidic and OS antigens require conjugation to T cell carrier proteins for efficient delivery to the immune system. The hepatitis core platform is a particulate carrier capable of eliciting high titer antibody responses to weak "hapten-like" peptidic and OS antigens incorporated onto core particles. During the course of this grant we have used the hepatitis B core (HBcAg) as a carrier platform for P. falciparum malaria circumsporozoite (CS) neutralizing repeat sequences (i.e., NANPn). The hybrid HBcAg-CS repeat particles are highly immunogenic in mice and are currently in phase 1/11clinical trials. Although the HBcAg is highly immunogenic, we have identified a number of theoretical and practical limitations to the use of the HBcAg as a vaccine platform. To address these limitations we are developing the core protein from the woodchuck hepadna virus (WHcAg) as a particulate carrier platform for several reasons: (1) The WHcAg is equally or more immunogenic than the HBcAg at the B cell and T cell levels; (2) the use of the WHcAg does not compromise the anti-HBc diagnostic assay because WHcAg and HBcAg are not crossreactive at the antibody level; (3) no pre-existing antibody to the WHcAg is present unlike for the HBcAg; (4) immune tolerance to HBcAg in chronic HBV carriers (300-400 million worldwide) can be circumvented by the use of the WHcAg platform because WHcAg and HBcAg are only partially crossreactive at the CD4+ T cell level; and (5) we have developed a WHcAg-based combinatorial technology that is more versatile than the existing HBcAg technology in terms of accommodating the insertion of a greater variety of foreign epitopes. For example, we have modified the WHcAg gene to create libraries of 17 insertion sites and 22 C-terminal modifications. Combining these libraries has allowed us to successfully insert 22 of 24 attempted foreign epitopes into the WHcAg platform. We propose to continue to expand the WHcAg combinatorial technology and perform biochemical/structural analysis of hybrid-WhcAg particles in an attempt to correlate structure and function (Aim 1); characterize the immune response to hybrid-WHcAg particles in vitro and in vivo in small animal models including challenge experiments to determine the protective efficacy of hybrid-WHcAg vaccine candidates if warranted (Aim 2); and extend the WHcAg platform to accommodate non-linear larger protein and carbohydrate antigens (Aim 3). A number of unique immunologic characteristics of the WHcAg and of the hybrid-WHcAg particles produced to date suggest that this particulate platform will be useful as a means of delivering a variety of medically relevant antigens to the immune system.