Influence of fold-stability on allergenicity and immunogenicity

In the past decade, immunology research made great progress in understanding the interplay between innate and adaptive immunity. Especially the mechanisms of sensing pathogens and the resulting promotion of T helper 1/T helper 17 responses have been elucidated in great detail. Much less is known about the underlying mechanisms concerning activation of regulatory T cells, and even less about that of T helper 2 cells, which represent the key players of the allergic immune response. Initiation of T helper 2 polarization occurs at different levels. Cells and receptors of the innate immune system play an important role, dendritic cells either act as translators of the T helper 2-driving stimuli from the tissues or as direct sensors. In principle, T helper 2 polarization seems to be as complex as T helper 1, T helper 17 and regulatory T cell induction and the allergenicity of the various allergens cannot be defined by one general rule. Recent publications clearly indicate that, besides polarization via bystander effects, inherent features of the protein itself such as protease activity, receptor/ligand mimicry, and dimerization can contribute to the T helper 2-polarizing effect. In addition, accumulating data also point to a possible role of the protein fold concerning immunogenicity and response type modulation. This project will focus on the latter aspect. Based on known crystal structures, point mutations which decrease or increase fold stability (compared to the wild type protein) can be identified by an in silico approach based on knowledge-based potentials and the use of molecular dynamics simulations. Selected molecules of both categories will be expressed as recombinant proteins, biochemically and biophysically characterized, and their uptake and degradation by dendritic cells will be analysed, including investigation of the peptide patterns presented on MHC-II. Furthermore, in silico-generated data will be correlated with empirical structural data using X-ray diffractometry and nuclear magnetic resonance spectroscopy. The fold derivatives will be tested employing in vivo mouse models, using adjuvant-free modes of sensitization, mimicking natural routes of allergen exposure. Antibody subclass analysis, assessment of T-cell proliferation, cytokine profiling, and T-cell epitope mapping will serve to test the influence of protein fold on immunogenicity, allergenicity, and immune polarization. The project will be focused on two clinically relevant pollen allergens, Bet v 1 and Phl p 6. Additionally, we will use hen egg lysozyme and ovalbumin, two immunologically well characterized proteins, as surrogate molecules. Transgenic mouse models providing T- cells specific for these proteins will allow us to study the early events of T cell activation and immune polarization. Together, the results will provide detailed understanding and a comprehensive picture of how protein fold stability acts on critical steps of the immune response and allergic sensitization.

Why some ubiquitous proteins act as allergens, whereas the majority of proteins from the same sources do not have the potential to induce allergic sensitization, remains an unsolved question. In our project we wanted to clarify whether the structural stability of protein antigens contributes to their allergenicity. For this purpose, we employed an algorithm, which predicts the effect of point mutations on the stability of a certain protein. Selected candidate molecules with either increased or decreased structural stability were recombinantly expressed and underwent an in-depth physico-chemical characterization. Additionally, we analyzed these protein variants in vitro for properties such as protease resistance, processing, epitope usage, and T cell activation and polarization. Finally, we evaluated the newly generated molecules for their in vivo immunogenicity and allergenicity by immunization of mice and analysis of the induced immune responses. 10 years ago, a paradigm was established that increasing the stability of a protein also increases its immunogenicity in terms of antibody production, but only to a certain threshold. Above this threshold, immunogenicity has been postulated to become diminished. In contrast, we could demonstrate that also proteins with a stability far above this threshold can be highly immunogenic and that not solely the thermodynamic stability of a protein, but rather its pH stability during the endolysosomal pathway is decisive for its immunogenicity. We suggest that to be highly immunogenic, a protein has to be stable enough to survive within the early endosome, whereas in the late endosome it has to be unfolded to become accessible for proteolytic digest, to enable efficient processing of peptides in the MHC-II loading compartment. So far, the pattern of peptides actually presented by antigen presenting cells has been analyzed by purifying peptides from the MHC II of in vitro generated dendritic cells followed by mass spectrometry. In our study, we could only detect the endogenous peptide pool from the respective dendritic cells, but not peptides from the exogenous protein of interest. As this indicates a lack of sensitivity of this method, we alternatively generated T cell hybridomas specific for immunodominant peptides, which produce IL-2 upon cognate interaction with their respective peptide:MHCII complex in a dose dependent manner. Using this method, we could demonstrate for the first time that depending on the fold stability of the surrounding protein, a certain peptide can be processed and presented with extremely different efficacy. Additionally, we could show that changing the stability of a protein by introducing a single point mutation can lead to major changes in T helper cell polarization. These findings are not only relevant for development of tailor-made therapeutics against allergic diseases, but might also contribute to the design of improved vaccines in general.