Bioengineering to harness the immune adjuvanting properties of reactive carbonyls

蛋白质氧化广泛存在，具有生理功能，如在白细胞被氧化爆发的细菌杀死期间，但如果不加控制也会导致病理，如糖尿病和动脉粥样硬化中出现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，但其潜在的机制仍不清楚。我们的小组是最先提出蛋白质氧化的化学标志之一，一种被称为活性羰基(RC)的活性化学基团，当被引入蛋白质中时，作为一种常见的免疫增强剂。我们已经证明了在不同的氧化条件下将RC基团添加到各种蛋白质中可以增强它们的免疫刺激活性，这表明这适用于许多常见的蛋白质。我们最近证明，RC的添加激活了一种重要的免疫细胞，称为辅助T细胞(Th)。当他们遇到抗原提呈细胞(APC)提供给他们的蛋白质时，TH就会被激活。APC通过一种名为MHC第二类的分子与Th细胞相互作用，该分子包含一段T细胞可以看到的蛋白质的多肽片段。我们的结果表明，在多肽中加入RC基团可以使多肽与MHC II类分子更紧密地结合，从而导致Th细胞活化和抗体反应增强。这项建议的目的是研究作为其氧化结果的RC的蛋白质如何更具免疫原性的机制基础。我们着手通过蛋白质和多肽的化学修饰、免疫学分析、肽-MHC II类结合分析和X射线结晶学来研究这种相互作用的生化性质，以解决肽-MHC II类复合体的原子结构。然后，这些信息将被用来试图修改整个蛋白质而不是多肽的免疫激活特性。如果成功，我们将把这项技术应用于基于流感血凝素(HA)的实验性疫苗的设计，HA是病毒上保护性中和抗体的目标蛋白。这将为在没有炎症佐剂的情况下针对特定表位的适应性免疫反应的疫苗设计提供原则证据。对蛋白质氧化介导的免疫增强的主要机制的表征，加上疫苗佐剂的新概念，使这一提议具有很高的原创性，在生物化学和免疫学领域具有很大的影响。该项目受益于一个由化学家、蛋白质组学专家、结晶学家和免疫学家组成的合作联盟，该联盟将确保在拟议的时间范围内获得最高的成功机会。

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.