Investigation of Synthetic DNA-based Viral Particles for Spatially Controlled Antigen Presentation

项目总结 对亚单位反应增强抗原性、抗体亲和力成熟和记忆诱导的策略 疫苗对于设计有效的传染病疫苗具有广泛的相关性，并可能 对艾滋病毒等难以中和的病原体尤其重要。提高药效的一种方法 亚单位疫苗是以多价、纳米颗粒的形式形成抗原，这促进了几个方面 增强体液免疫，最显著的是增强B细胞受体(BCR)的交联性。这种方法具有 在获得许可的疫苗(例如，HPV和乙肝疫苗)和各种疫苗中都得到了利用 在临床前和临床开发方面。然而，到目前为止，人们仍然不清楚什么是理想的特征 纳米颗粒抗原展示。在这个项目中，我们使用了独特的支架DNA折纸技术来工程 10-100纳米尺度的纳米颗粒，能够研究支架尺寸的影响， 抗原拷贝数高达100多个，抗原与bcr亲和力强，以及纳米尺度的空间组织 BCR活化的抗原提呈维度。具体地说，我们测试了 这些关于B细胞激活的参数，对于成功的亚单位的发展是至关重要的 使用种系靶向HIV-1 gp120工程外区的疫苗，称为EOD-GT8，及其 不同亲和力的变种，如试验床。早期B细胞信号转导和通路激活的体外研究 将被描述为特征，并与组织在 蛋白质支架。用单细胞荧光成像技术研究BCR结合的详细机制 以及基于找到的最佳免疫原提呈物激活B细胞。然后使用这些构造来 用小鼠体内检测这些优化的HIV DNA-NP结构对T细胞和B细胞应答的影响 模特们。综上所述，我们的结果将为最佳免疫原呈递参数的阐明提供依据。 在开发更有效的亚单位疫苗中进行有效的免疫细胞反应，主要是翻译 有可能感染艾滋病毒和其他传染病。

PROJECT SUMMARY Strategies to enhance antigenicity, antibody affinity maturation, and memory induction in response to subunit vaccines are of broad relevance for the design of effective vaccines against infectious diseases, and may be especially important for difficult-to-neutralize pathogens such as HIV. One approach to enhance the efficacy of subunit vaccines is to formulate antigens in a multivalent, nanoparticulate form, which promotes several aspects of humoral immunity, and most notably enhances crosslinking of B cell receptors (BCRs). This approach has been exploited both in licensed vaccines (e.g., the HPV and HBV vaccines), and in a great variety of vaccines in preclinical and clinical development. However, to date it remains unclear what are the ideal characteristics of nanoparticle antigen display. In this project, we use the unique technology of scaffolded DNA origami to engineer nanoparticles on the 10–100 nanometer scale that offer the ability to investigate the impact of scaffold size, antigen copy number up to more than 100, antigen-BCR affinity, as well as the nanoscale spatial organization and dimensionality of antigen presentation on BCR activation. Specifically, we test the relative importance of these parameters on B-cell activation, which are of central importance to the development of a successful subunit vaccines, using the germline targeting engineered outer domain of HIV-1 gp120, termed eOD-GT8, and its variants with different affinities, as a testbed. In vitro evaluation of early B-cell signaling and pathway activation will be characterized, and contrasted with the benchmark strongly activating 60-mer control organized on a protein scaffold. Single-cell fluorescence imaging is used to investigate the detailed mechanism of BCR-binding and B-cell activation based on the optimal immunogen presentation found. These constructs are then used to test the impact of these optimal HIV DNA-NP constructs on T-cell and B-cell response in vivo using mouse models. Taken together, our results will offer the elucidation of the optimal immunogen presentation parameters for effective immune cell response in the development of more effective subunit vaccines, with major translational potential for HIV and other infectious diseases.