Amyloidogenic propensity of self-assembling peptides and their adjuvant potential for use as DNA vaccines

De novo designed peptides that self-assemble into cross-beta rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant-and inflammation-free vaccines. However, they share structural and morphological properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their successful translation. Here, we comprehen-sively characterize the amyloidogenic character of the amphipathic self-assembling cross-beta peptide KFE8, compared to pathological amyloid and amyloid-like proteins alpha-synuclein (alpha-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE8 backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE8 is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE8 fibrils do not cross-seed amyloid for-mation of alpha-syn in mammalian cells compared to alpha-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE32-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8 + T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays in-dicate a balanced Th1/Th2 response. These findings illustrate that cross-beta-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attrac-tive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles. Statement of significance Biomaterials comprised of self-assembling peptides hold great promise for the development of new vac-cines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-beta rich fibrils that are structurally similar to amyloid species implicated in disease. Here, we comprehensively study the properties of these biomaterials. We demonstrate that they have distinct properties from pathological proteins. They are non-toxic and do not trigger amyloidogenesis. Vaccination of these materials in mice elicited a robust immune response. Most excitingly, our work sug-gests that this platform could be used to develop DNA-based vaccines, which have few storage require-ments. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification.(c) 2023 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )