In the last century, vaccines have been the most effective way to prevent deaths caused by infectious diseases. Advances in science are a key driver of vaccine development, so research in the field of vaccines is very important. Exploring different approaches to designing new vaccines is important for improvement of vaccine efficacy. Vaccines are separated in two types: live-attenuated and inactivated. The difference between the two is that the first contains attenuated pathogens that can reproduce in our body, while non-living ones contain dead pathogens or their components. In this doctoral dissertation, we focused on peptides that form coiled coils (CC-peptides) as carriers with which to combine immunologically active components into an effective inactivated vaccine. Due to their modular nature, CC-peptides have served as good building blocks for the design of a nanovaccine that allows simultaneous delivery of antigen and adjuvant to the target cell, which is important for vaccine effectiveness. We designed several sets of stable and orthogonal CC-peptides. Well-defined rules for individual position within a CC peptide were followed. Most important positions are those that form the hydrophobic core. Electrostatic interactions between oppositely charged amino acid residues are another important contribution to the stabilization of the coiled helix and crucial for selectivity. After determining the properties of synthetic peptides in vitro, they were used as dimerization domains for the assembly of split proteins. Using split proteins method, we made sure the designed peptides retained their properties in fusion with larger domains (proteins), which was crucial for further experiments. The peptide pairs with most suitable properties were used as components of the nanoscaffold, where the designed peptides were expressed in fusion with fluorescent proteins and cell delivery domains. The fluorescent protein added to the nanostructure allowed us to monitor cell delivery by confocal microscopy or flow cytometry. In addition to selecting the appropriate antigen and adjuvant, target delivery into cells is also important for vaccine efficacy. We thus focused on several delivery domains. After selecting the most efficient delivery domain, the nano framework was used as a nanovaccine to target the antigen and adjuvant (short flagellin) to the model antigen-presenting cell. We have shown that CC-mediated delivery of immunologically active molecules improves the inflammatory response.
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