Improving phage-based medicine with immunoengineering

项目总结/摘要 对噬菌体（感染细菌的病毒）的研究兴趣的复苏正在推动 生物医学应用的工程菌，包括抗菌治疗和基于噬菌体的疫苗接种。 作为抗微生物剂，抗生素具有优于常规抗生素的显著优势：它们是良好的- 耐受性、病原体特异性并在感染部位复制。噬菌体作为疫苗也很有前途 因为它们可以被精确地工程化以递送多种外源抗原。然而， 免疫原性，并且两种应用都易受干扰免疫应答的影响。抗菌剂，用于 例如，可以引发防止细菌靶标感染的中和抗体。这种免疫原性 自然界对疫苗的开发是有利的，因为噬菌体本身就像佐剂一样，但它的作用是， 价格：对免疫显性噬菌体抗原的脱靶反应可能会分散预期的保护性反应 外来抗原。不幸的是，噬菌体免疫学的数据是有限的，混淆了常规的生物医学， phage的应用该提案概述了阐明噬菌体基础的基本实验 免疫原性和评价调节它方法。 目的1建立与三种治疗性分枝杆菌噬菌体结合的抗体结构图。结构- 引导工程和定向进化将用于产生逃避抗体的突变体 约束力这些突变体逃避对野生型对应物的既定免疫应答的能力在 将在小鼠模型中评价体内。在目标2中，相同的药物将用作治疗药物的平台。 细菌疫苗利用噬菌体展示和噬菌体DNA载体技术， 将通过三种机制防御和保护细菌：1）噬菌体感染和杀死，2）产生 细菌结合抗体，和3）辅助和细胞毒性T细胞的活化。目标3将评估一种方法， 抑制干扰性免疫应答：噬菌体与装载雷帕霉素纳米颗粒共同给药。这 将下调噬菌体特异性辅助性T细胞，上调调节性T细胞，训练免疫系统 将噬菌体识别为“自我”。这些目的将扩大我们对噬菌体免疫原性的理解，并评估 利用免疫工程原理改进噬菌体药物的潜力。此外，目标1是 为候选人提供培训工具，候选人在实验室中具有免疫学经验，但需要 提供指导培训和正规教育，以在这一领域建立独立性。Aim 1还提供额外的 结构生物学的指导培训，特别是噬菌体-抗体复合物的不对称重建。 有了计划的科学培训，出版和资助写作的实践，以及导师的支持， 在整个学术求职过程中，候选人应建立并保持独立的 研究生涯主要集中在免疫工程噬菌体，以提高其生物医学应用。

PROJECT SUMMARY/ABSTRACT A resurgence of research interest in bacteriophages, viruses that infect bacteria, is driving the development of engineered phages for biomedical applications, including antimicrobial therapy and phage-based vaccination. As antimicrobial agents, phages have significant advantages over conventional antibiotics: they are well- tolerated, pathogen-specific, and replicate at the site of infection. Phages are also promising as vaccine platforms because they can be precisely engineered to deliver multiple foreign antigens. However, phages are immunogenic and both applications are susceptible to interfering immune responses. Antimicrobial phages, for example, can elicit neutralizing antibodies that prevent infection of the bacterial target. This immunogenic nature is advantageous for vaccine development, since the phage acts as its own adjuvant, but it comes at a price: off-target responses to immunodominant phage antigens can distract from intended protective responses to foreign antigens. Unfortunately, data on phage immunology are limited, confounding routine biomedical applications of phage. This proposal outlines basic experiments to elucidate the basis of phage immunogenicity and evaluate methods of modulating it. Aim 1 will develop a structural map of antibody binding to three therapeutic mycobacteriophages. Structure- guided engineering and directed evolution will be used to generate mutant phages that escape antibody binding. The ability of these mutants to evade established immune responses to their wild-type counterparts in vivo will be evaluated in a mouse model. In Aim 2 the same phages will be used as platforms for a therapeutic bacterial vaccine. Leveraging both phage display and phage DNA vector technologies, the vaccine candidates will defend and protect against bacteria with three mechanisms: 1) phage infection and killing, 2) generation of bacterium-binding antibodies, and 3) activation of helper and cytotoxic T cells. Aim 3 will evaluate a method to suppress interfering immune responses: co-administration of phage with rapamycin-loaded nanoparticles. This will down-regulate phage-specific helper T cells and upregulate regulatory T cells, training the immune system to recognize phage as ‘self’. These Aims will expand our understanding of phage immunogenicity and assess the potential to improve phage-based medicine with principles from immunoengineering. Furthermore, Aim 1 is a training vehicle for the candidate, who has developed immunology experience in the lab but requires mentored training and formal education to establish independence in this field. Aim 1 also provides additional mentored training in structural biology, specifically asymmetric reconstructions of phage-antibody complexes. With the planned scientific training, practice in publishing and grant writing, and the support of her mentors throughout an academic job search, the candidate is expected to establish and sustain an independent research career focused on immunoengineering phage to improve their biomedical applications.