Encapsulins are prokaryotic self-assembling protein nanocages with promise as nanovaccine scaffolds. Their utility as modular platforms require tolerance to surface engineering, high-yield soluble production, formulation stability, and controlled antigen (co-)display. Herein, a previously uncharacterized encapsulin from Alkaliphilus metalliredigens is engineered into a SpyCatcher-decorated nanoscaffold (Am-S) that enables controlled surface display of SpyTagged antigens. Cryo-EM confirms that the native encapsulin forms a T = 1 icosahedral nanocage, and that C-terminal SpyCatcher fusion yields Am-S without compromising nanocage assembly, symmetry, or structural integrity. Notably, Am-S exhibits high-yield soluble production in Escherichia coli, remains monodisperse after freeze-thaw and extended storage, and supports efficient SpyTagged peptide conjugation for single- and multi-antigen display. As a proof-of-concept, Am-S is functionalized with Alzheimers disease-associated beta-amyloid and/or hyperphosphorylated tau epitopes to generate single-target nanocages displaying either antigen and dual-target mosaic nanocages co-displaying both. In mice, Am-S antigen display enhances antigen-specific IgG responses relative to free antigens and induces predominantly IgG1-biased humoral immunity. Mosaic nanocages elicit antibodies against both targets, with immune sera selectively recognizing amyloid-beta and phosphorylated tau-associated pathology in ex vivo brain sections from Alzheimers disease mouse models. These findings position Am-S as a manufacturable scaffold for developing multi-targeting nanovaccines against complex diseases.
Boyton, I., Rennie, C., van der Hoven, J., Valle, M., Diaz, D., Ruan, J., Luque, D., Saunders, B., Collins-Praino, L. E., Ittner, L. M., Care, A.
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