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 KFE8, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-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 formation of α-syn in mammalian cells compared to α-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 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.

Parkinson disease (PD) is closely linked to the misfolding and accumulation of α-synuclein (α-syn) into Lewy bodies. HtrA1 is a PDZ serine protease that degrades fibrillar tau, which is associated with Alzheimer disease (AD). Further, inactivating mutations to mitochondrial HtrA2 have been implicated in PD. Here, we establish that HtrA1 inhibits the aggregation of α-syn as well as FUS and TDP-43, which are implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We demonstrate that the protease domain of HtrA1 is necessary and sufficient for inhibition of aggregation, yet this activity is independent of HtrA1 proteolytic activity. Further, we find that HtrA1 also disaggregates preformed α-syn fibrils, which may promote their clearance. Treatment of α-syn fibrils with HtrA1 renders α-syn incapable of seeding the aggregation of endogenous α-syn in mammalian biosensor cells. We find that HtrA1 remodels α-syn by specifically targeting the NAC domain, which is the key domain that catalyzes α-syn oligomerization and fibrillization. Finally, in a primary neuron model of α-syn aggregation, we show that HtrA1 and its proteolytically inactive form both detoxify α-syn and prevent the formation of hyperphosphorylated α-syn accumulations. Our findings suggest that HtrA1 prevents aggregation and promotes disaggregation of multiple disease-associated proteins, and may be a therapeutic target for treating a range of neurodegenerative disorders.