A loop-mimetic SOD1 antigen elicits antibodies to a mutant-associated conformational epitope and ameliorates disease in SOD1G93A mice

Misfolded Cu/Zn-superoxide dismutase (SOD1) contributes to amyotrophic lateral sclerosis (ALS), but effective immunization against SOD1 depends on identifying a pathogenically relevant neo-epitope selectively exposed in misfolded SOD1. Here, we developed Ag2, a multivalent antigen based on a highly stable six-helix scaffold (6-Helix) engineered to display the SOD1 β6/β7 loop, a neo-epitope exposed in pathogenic SOD1 conformers and implicated in their prion-like activity, together with helper T-cell epitopes arranged in alternating tandem repeats to enhance humoral immunogenicity. In SOD1 G93A ALS mouse model, Ag2 induced an adjuvant-tunable antibody response and generated high-titer conformational antibodies that recognized multiple ALS-linked SOD1 mutants, but not SOD1 WT . Ag2-antibody binding was not reproduced in SOD1 WT under the misfolding-promoting conditions examined here, indicating that exposure of the targeted epitope depends on more than conversion to a generic non-native state and instead requires a conformational context associated with pathogenic SOD1 mutations. Vaccination delayed disease onset and attenuated spinal misfolded SOD1 accumulation, motor neuron loss, and gliosis. These findings show that structural loop mimicry can focus humoral immunity on disease-relevant SOD1 conformations while sparing the native protein in vivo. More broadly, the 6-Helix scaffold may provide a useful antigenic platform for active immunization against loop-like pathogenic neo-epitopes in other proteopathies.