Bioengineering to harness the immune adjuvanting properties of reactive carbonyls

蛋白质氧化是普遍存在的，并且发挥生理功能，例如在白细胞进行氧化爆发杀死细菌的过程中，但如果不加以控制，也可能导致病理学，例如在糖尿病和动脉粥样硬化中看到的不必要的免疫反应。虽然蛋白质氧化的免疫增强作用已被很好地描述，但其潜在机制仍不清楚。我们的小组是最早提出蛋白质氧化的化学标志之一，这是一种被称为反应性羰基（RC）的反应性化学基团，当引入蛋白质时，它是一种常见的免疫增强剂。我们已经证明，在不同的氧化条件下添加RC基团的各种蛋白质增强其免疫刺激活性，表明这适用于许多常见的蛋白质。我们最近已经证明，RC除了激活一种重要类型的免疫细胞称为辅助T细胞（Th）。Th在遇到抗原呈递细胞（APC）呈递给它们的蛋白质时被激活。APC通过一种称为MHC II类的分子与Th细胞相互作用，该分子包含T细胞“看到”的蛋白质的肽片段。我们的研究结果表明，添加RC基团的肽允许肽更紧密地结合到MHC II类，这导致增加Th细胞活化和增强的抗体应答。本建议的目的是研究如何承担RC作为其氧化的结果，蛋白质的机制基础，是更免疫原性。我们开始研究这种相互作用的生化性质，使用蛋白质和肽的化学修饰，免疫学测定，肽-MHC II类结合测定和X射线晶体学的组合来解决肽-MHC II类复合物的原子结构。然后，该信息将用于尝试修改整个蛋白质而不是肽的免疫激活特性。如果成功，我们将应用这项技术设计一种基于流感血凝素（HA）的实验性疫苗，HA是保护性中和抗体病毒上的靶蛋白。这将为在不存在炎性佐剂的情况下靶向针对特定表位的适应性免疫应答的疫苗的设计提供原理证明。蛋白质氧化介导的免疫增强的主要机制的表征加上疫苗佐剂化的新概念，使其成为一个高度原创的建议，在生物化学和免疫学领域具有很大的影响力。该项目受益于化学家，蛋白质组学专家，晶体学家和免疫学家的合作联盟，这将确保在拟议的时间表内获得最大的成功机会。

Protein oxidation is widespread and serves physiological functions such as during bacterial killing by leukocytes undergoing oxidative burst, but can also result in pathology when unchecked, such as the unwanted immune responses seen in diabetes and atherosclerosis. Although the immune enhancing effects of protein oxidation are well described, their underlying mechanism remains unknown. Our group is among the first to suggest a chemical hallmark of protein oxidation, a reactive chemical group termed a reactive carbonyl (RC), as a common immune enhancing agent when introduced into to protein. We have demonstrated that addition of RC groups to various proteins under different oxidative conditions enhances their immune stimulating activity, showing that this applies to many common proteins. We have recently demonstrated that RC addition activates an important type of immune cell called a helper T cell (Th). Th are activated when they encounter protein presented to them by an antigen presenting cell (APC). APCs interact with Th cells via a molecule called MHC class II, which contains a peptide fragment of the protein that is 'seen' by the T cell. Our results show that the addition of an RC group to a peptide allows the peptide to bind more tightly to MHC class II, which results in increased Th cell activation and enhanced antibody responses. The purpose of this proposal is to investigate the mechanistic basis of how proteins that bear RC as a consequent of their oxidation, are more immunogenic. We set out to examine the biochemical nature of this interaction using a combination of chemical modification of proteins and peptides, immunological assays, peptide-MHC class II binding assays and X-ray crystallography to solve the atomic structure of the peptide-MHC class II complex. This information will then be used to attempt to modify the immune activating properties of a whole protein rather than a peptide. If successful we will then apply this technology to the design of an experimental vaccine based on influenza haemagglutinin (HA), the target protein on the virus of protective neutralizing antibodies. This will provide proof of principle for the design of vaccines that target the adaptive immune response to specific epitopes in the absence of inflammatory adjuvants. The characterization of a major mechanism underlying protein oxidation-mediated immune enhancement coupled with a novel concept for vaccine adjuvantation makes this a highly original proposal with the potential for high impact in the fields of biochemistry and immunology. The project benefits from an established collaborative consortium of chemists, proteomics experts, crystallographers and immunologists that will ensure the highest chance of success within the proposed timescale.