Rational design of structure-based vaccines targeting misfolded alpha-synuclein conformers of Parkinson's disease and related disorders

Background: Synucleinopathies, including Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies, are neurodegenerative disorders caused by the accumulation of misfolded alpha-synuclein protein. Developing effective vaccines against synucleinopathies has been challenging due to the difficulty of stimulating an immune-specific response against alpha-synuclein, conferring neuroprotection without causing harmful autoimmune reactions, and selectively targeting only pathological forms of alpha-synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure for immunization failed in clinical trials. The immune system was unable to distinguish between the native alpha-synuclein and its amyloid form. Results: The prion domain of the fungal HET-s protein was selected as a scaffold to introduce select epitopes from the surface of alpha-synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein in regions that showed structural similarity to alpha-synuclein fibril structures. Each vaccine candidate had unique amino acid substitutions that imitated a specific epitope from alpha-synuclein amyloid fibrils. The approach successfully mimicked the stacking of the parallel in-register beta-sheet structure seen in alpha-synuclein fibrils as the vaccine candidates were found to be structurally stable and self-assembling into the desired conformations. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha-synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the resulting anti-sera recognized pathological alpha-synuclein aggregates in brain lysates from patients who died from dementia with Lewy bodies, multiple system atrophy, or Parkinson's disease, but did not recognize linear alpha-synuclein peptides. Each vaccine candidate induced a unique pattern of reactivity toward alpha-synuclein aggregates contained in distinct disease pathologies. Conclusions: This new approach, based on the rational design of vaccines using the secondary and tertiary structure of alpha-synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha-synuclein, provides a promising avenue towards developing effective vaccines against alpha-synuclein fibrils, which may be crucial for the prevention and treatment of synucleinopathies. ### Competing Interest Statement The authors have declared no competing interest.