Bioengineering to harness the immune adjuvanting properties of reactive carbonyls

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.

Our preliminary data strongly support a model in which the RC adduct on the flanking regions of a peptide epitope interacts with a positively charged residue on the flank of an MHC class II molecule. This interaction is predicted to stabilise peptide-MHC binding, increasing T cell activation. To interrogate this peptides will be synthesised with RCs incorporated at specified positions using orthogonally protected lysines. Peptides will be C-terminal labelled allowing direct binding studies, allowing us to measure peptide affinity with and without targeted RC groups and relate this to T cell affinity. This approach will be extended to intact protein by site-specific RC insertion using a chemoenzymatic approach by incorporating a formylglycine-generating enzyme motif into the protein. MHC class II will be mutated in the putative interacting residue (alpha-chain lysine50) to disrupt RC-lysine interactions, and peptide binding and T cell activation will be measure to provide further evidence supporting this interaction. Peptide RC groups may interact with lysine on MHC class II by electrostatic interactions or by Schiff base formation. We will investigate possible the formation of covalent links between peptide-RC and MHC using cyanoborohydride reduction and deuterium addition followed by digestion, LC, and mass spectrometric analysis. A homogenous RC-containing peptide-MHC class II complex will be used for X-ray crystallography. Binding and structural studies will be carried out in parallel with immunological readouts to correlate biochemical and immunological outcomes. Antigen presentation analyses will be carried out in vitro using T cell hybridoma activation and in vivo by immunization of mice and subsequent analysis of the immune responses. Finally, RC groups will be incorporated into influenza HA and evidence for epitope specific enhancement of T cell responses evaluated by peptide specific proliferative responses and cytokine secretions.

The implications of the finding that RCs have adaptive immune modulating effects will be of interest to academic researchers in a wide variety of fields including basic immunology, biomedical research into oxidative stress-related diseases and vaccinology. Mechanistic insight into how a post-translational protein modification, such as RC adduction, influences antigen presentation will be of particular interest to those working on antigen presentation and T cell activation. Given the novelty of this phenomenon, it will boost a new research into other examples of RCs or related modifications might be adducted to influence immune responses. Another relevant area would be antimicrobial research. Since respiratory burst and radical release is a central anti-microbial property of the innate immune system, it will be of particular interest to researchers in these areas to investigate the role of radical-mediated oxidised microbial protein in adaptive immune activation. Our finding should stimulate biomedical research into the disease potential of RCs. For example, study of the potential role of RCs in the oxidised apolipoprotein constituent of LDL in atherosclerosis, and protein glycation in the context of diabetes. Likewise, oxidative stress is widely implicated as a driver of, or contributor to, the etiology of autoimmunity. Here, the epitope-specific enhancing properties of RCs could set a highly relevant example to explain how an otherwise cryptic epitope within a self-protein could undergo oxidative changes that making it 'presentable' to the adaptive immune system, breaking tolerance to induce autoimmune responses. Similarly, a growing area of allergy research encompasses aberrant immune responses to oxidised environmental antigens. Again, the role of RCs can now be tested as relevant to this type of immune dysfunction. Finally, vaccinologists will be interested in the potential for RC incorporation to modulate immunity in an epitope-specific manner in the absence of inflammatory exogenous adjuvants. There are several potential technological innovation outcomes to our proposal. First, RCs might be used as intrinsic adjuvants that could be designed into the structure of subunit protein vaccines to enhance targeted epitope immunogenicity. Second, the possibility to modulate immunogenicity in the absence of extrinsic adjuvants would simplify vaccine formulation and avoid inflammatory adjuvants. Third, understanding the mechanistic nature of some oxidative-stress-related diseases might lead to anti-oxidant interventions designed to specifically eliminate RC adduction in vivo.