Search

Introduction:Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods:MAESTRO software was used forin silicoprediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed inE. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferationin vitro, as well as antibody responses and T cell polarizationin vivoin an adjuvant-free BALB/c mouse model. Results:In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14 degrees. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processingin vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant.In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-alpha, IFN-gamma, IL-17, and IL-21. Conclusion:MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cellsin vitro. This change in processing kinetics significantly influenced the polarization of T cell responsesin vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from 5 to +14. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-, IFN-, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines. (VLID)5400402

Even with the widespread use of nanomaterials (NMs) in everyday life, consumer knowledge about the functionality, benefits, and possible dangers of nanotechnology (NT) is still modest. As with any developing technology, its public perception has direct implications on future policies and has to be taken into account by academia and industry alike. As part of the “Nan-O-Style” interdisciplinary research project, an online survey was conducted using a Citizen Science-guided approach. The main goal was to evaluate the current levels of knowledge and the attitude towards NT among the general Austrian public and to determine how differing sociodemographic factors may affect these. Over the course of 17 months, a total of 1067 responses were collected and quantitatively analysed. We found that while Austrians display a generally optimistic view and a positive attitude towards NT, there are still remaining concerns about its safety and possible risks. Participants expressed great desire for more information about NT and its applications, as well as for clear labelling and transparency of products containing NMs. Notably, we found that age did not affect the general attitude towards NT nor the levels of NT awareness. While participants with a university degree were generally more knowledgeable on this specific topic, surprisingly, there were no significant differences in the attitude towards NT among people from different educational backgrounds. Similar to previous studies, we observed that male participants demonstrated a more positive attitude towards NT and scored slightly higher in our NT quiz compared to female participants. However, female participants voiced greater desire for more information and transparency regarding NMs. Interestingly, while participants with a negative attitude towards NT scored lowest on the NT quiz, they also expressed the least interest in receiving more NT-related information. This illustrates a difficulty in mitigating public aversion solely by providing more information. (VLID)4701856

submitted by Isabella Anna Joubert Literaturverzeichnis: Blatt 102-112 Universität Salzburg, Masterarbeit, 2019 (VLID)5028752