De novo designed peptides that self-assemble into cross-β 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 comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-β peptide KFE, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE fibrils do not cross-seed amyloid formation of α-syn in mammalian cells compared to α-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-β-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attractive 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 vaccines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-β 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 suggests that this platform could be used to develop DNA-based vaccines, which have few storage requirements. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification.