Engineering a Novel Bacterial Encapsulin for Programmable Surface Functionalization: From Single-Target to Mosaic...

Area of Science:

• Biochemistry and Structural Biology
• Nanotechnology and Materials Science
• Immunology and Vaccinology

Background:

• Encapsulins are prokaryotic protein nanocages with potential as nanovaccine scaffolds.
• Effective nanovaccine platforms require modularity, high-yield production, stability, and controlled antigen display.
• Previous encapsulin systems faced limitations in surface engineering and antigen co-display.

Purpose of the Study:

• To engineer a previously uncharacterized encapsulin from Alkaliphilus metalliredigens into a versatile nanoscaffold for antigen display.
• To assess the structural integrity, production yield, and stability of the engineered nanoscaffold.
• To demonstrate the scaffold's utility for single- and multi-antigen display and its immunogenicity in vivo.

Main Methods:

• Engineering of an Alkaliphilus metalliredigens encapsulin with a C-terminal SpyCatcher fusion (Am-S).
• Cryo-electron microscopy (Cryo-EM) for structural analysis of the native and engineered nanocages.
• Production in Escherichia coli, formulation stability testing (freeze-thaw, storage), and SpyTagged peptide conjugation.
• Immunization studies in mice using Am-S functionalized with Alzheimer's disease-associated epitopes (amyloid-β and hyperphosphorylated tau).

Main Results:

• The engineered Am-S nanocage maintained native encapsulin structure (T=1 icosahedral symmetry) and integrity.
• High-yield soluble production in E. coli and excellent monodisperse stability after freeze-thaw and storage were achieved.
• Efficient conjugation of SpyTagged antigens enabled single- and multi-antigen display, including mosaic nanocages co-displaying amyloid-β and tau epitopes.
• In mice, Am-S nanovaccines enhanced antigen-specific IgG responses and induced predominantly IgG1-biased immunity.
• Mosaic nanocages elicited antibodies against both targets, which recognized Alzheimer's disease pathology in ex vivo mouse brain sections.

Conclusions:

• The Am-S nanoscaffold is structurally sound, manufacturable, and stable, overcoming key limitations of previous systems.
• It enables controlled single- and multi-antigen display for potential nanovaccine development.
• Am-S functionalized with disease-specific epitopes elicits robust humoral immunity and demonstrates potential for targeting complex diseases like Alzheimer's.