DEVELOPMENT OF THE HBcAG AS A VACCINE CARRIER PLATFORM

说明(申请人提供)：本申请是R01 AI49730-03的竞争性延续。在亚单位疫苗设计中使用多肽和定义明确的小分子寡糖(OS)抗原有许多优点。例如，化学纯度和安全性、生产简易性、成本、稳定性和易变性。然而，多肽和OS抗原需要与T细胞载体蛋白结合才能有效地传递到免疫系统。肝炎核心平台是一种颗粒载体，能够对结合在核心颗粒上的弱的“半抗原样”多肽和OS抗原产生高滴度抗体反应。在这笔赠款的过程中，我们使用乙肝核心蛋白(HBcAg)作为恶性疟原虫环子孢子(CS)中和重复序列(即NANPn)的载体平台。混合HBcAg-CS重复颗粒在小鼠中具有高度的免疫原性，目前正处于1/11期临床试验。尽管HBcAg具有高度的免疫原性，但我们已经确定了将HBcAg用作疫苗平台的一些理论和实践限制。为了解决这些局限性，我们正在开发土拨鼠庚型肝炎病毒(WHcAg)的核心蛋白作为颗粒载体平台，原因有几个：(1)WHcAg在B细胞和T细胞水平上与HBcAg具有同等或更强的免疫原性；(2)WHcAg的使用不损害抗HBc诊断分析，因为WHcAg和HBcAg在抗体水平上没有交叉反应；(3)与HBcAg不同，WHcAg不存在预先存在的抗体；(4)慢性乙肝病毒携带者(全球3-4亿人)对HBcAg的免疫耐受可以通过使用WHcAg平台来规避，因为WHcAg和HBcAg在CD4+T细胞水平上只有部分交叉反应；以及(5)我们开发了一种基于WHcAg的组合技术，在适应更多种类的外来表位插入方面比现有的HBcAg技术更具通用性。例如，我们已经修改了WHcAg基因，创建了包含17个插入位点和22个C末端修饰的文库。结合这些文库，我们已经成功地将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颗粒进行生化/结构分析，试图将结构和功能联系起来(目标1)；在体外和体内小动物模型中表征对杂交-WHcAg颗粒的免疫反应，包括挑战实验，以确定杂交-WHcAg候选疫苗的保护效力(目标2)；并扩展WHcAg平台，以容纳非线性的更大的蛋白质和碳水化合物抗原(目标3)。到目前为止生产的WHcAg和混合WHcAg颗粒的一些独特的免疫学特征表明，这种颗粒平台将作为一种将各种医学相关抗原输送到免疫系统的手段。

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.