Journal: bioRxiv

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

doi: 10.64898/2026.06.01.729406

Figure Lengend Snippet: ( a ) Schematics depicting ( left ) the Am-S genetic construct encoding the AmEnc subunit (grey) fused at the C-terminus to SpyCatcher (orange) via a linker comprising a His-tag (green) flanked by flexible (GGGS) n spacers (white); and ( right ) covalent coupling of SpyTagged antigens to the surface of the self-assembled Am-S scaffold, generating an antigen-displaying nanovaccine. ( b ) PAGE analysis of Am-S purified by sequential IMAC and SEC: ( left ) SDS-PAGE showing the Am-S subunit (∼44 kDa); ( right ) native PAGE verifying nanocage assembly. ( c ) DLS analysis demonstrating monodisperse Am-S nanocages with a mean hydrodynamic diameter of 36.4 ± 9.4 nm. ( d ) ( left ) Cryo-EM micrograph showing Am-S self-assembly into nanocage structures (scale bar = 100 nm) ( right ) 3D reconstruction at 2.58 Å resolution (external view) confirming high-fidelity assembly into 21.2 nm particles with T = 1 icosahedral symmetry. ( e–j ) Storage stability of Am-S. ( e ) Solubility after 1 and 4 freeze-thaw cycles; untreated material (0) was defined as 100% soluble. ( f ) DLS analysis of samples in (e). ( g ) Solubility after storage at the indicated temperatures for 6 weeks; samples stored at −80 °C were defined as 100% soluble. ( h ) DLS analysis of samples in (g). ( i ) Solubility before and after lyophilisation and storage at ambient temperature for 1 day; pre-lyophilisation material was defined as 100% soluble. ( j ) DLS analysis of samples in (i). Soluble Am-S fractions were isolated by centrifugation and quantified by SDS-PAGE densitometry (mean ± SD, n = 3).

Article Snippet: The E. coli codon optimised AmEnc-SpyCatcher (Am-S) sequence ( Table S2 ) was synthesised by Twist Bioscience into the pET-24(+) expression vector and transformed into E. coli BL21(DE3) competent cells (New England Biolabs).

Techniques: Construct, Purification, SDS Page, Clear Native PAGE, Cryo-EM Sample Prep, Solubility, Isolation, Centrifugation

Journal: bioRxiv

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

doi: 10.64898/2026.06.01.729406

Figure Lengend Snippet: ( a ) Schematic of peptide antigens with an N-terminal SpyTag (orange) linked via a flexible (GS)n spacer (black) to peptide antigens derived from pTau (S-pTau; green) or Aβ (S-Aβ; purple). ( b ) Conceptual illustration of unconjugated and conjugated Am-S, including the bare nanoscaffold (Am-S), monovalent nanocage formats bearing S-pTau (Am-S-pTau) or S-Aβ (Am-S-Aβ), and a multivalent “mosaic” nanocage bearing both antigens. ( c ) PAGE assessment of SpyTag/SpyCatcher-mediated conjugation, with ( left ) SDS-PAGE showing covalent coupling of antigen(s) to the Am-S subunit, and ( right ) non-denaturing native PAGE indicating antigen (co-)display on the assembled Am-S nanocage. ( d ) DLS characterisation of hydrodynamic diameter and dispersity of the (co-)conjugated nanocages. ( e ) Negatively stained TEM images of Am-S (orange), Am-S-pTau (green), Am-S-Aβ (purple), and mosaic (blue) nanocage formats; Scale bars = 200 nm.

Article Snippet: The E. coli codon optimised AmEnc-SpyCatcher (Am-S) sequence ( Table S2 ) was synthesised by Twist Bioscience into the pET-24(+) expression vector and transformed into E. coli BL21(DE3) competent cells (New England Biolabs).

Techniques: Derivative Assay, Conjugation Assay, SDS Page, Clear Native PAGE, Staining

Journal: bioRxiv

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

doi: 10.64898/2026.06.01.729406

Figure Lengend Snippet: ( a ) Immunisation schedule for C57BL/6J mice ( n = 4 per group) with end-point sera collection. ( b ) ELISA measurement of anti-pTau total IgG levels in terminal sera from mice receiving Am-S-pTau and controls. ( c ) Body-weight change of mice over time-course in (b), expressed as normalised area under the curve (AUC). ( Right panel ) AddaVax™ and Alhydrogel®. ( d ) Immunisation and sera collection schedule for mice receiving Am-S-pTau formulated with ADV or ALH ( n = 4 per group). ( e ) Anti-pTau IgG titres in sera over time. Black arrows indicate immunization days. ( f ) Body-weight change over time in (e). Data are mean ± SD. ****P < 0.0001; ns, non-significant (> 0.05). Statistical analyses: one-way ANOVA with Tukey’s multiple comparisons for (b,c,f); two-way mixed-effects ANOVA with time and adjuvant as factors for (e).

Article Snippet: The E. coli codon optimised AmEnc-SpyCatcher (Am-S) sequence ( Table S2 ) was synthesised by Twist Bioscience into the pET-24(+) expression vector and transformed into E. coli BL21(DE3) competent cells (New England Biolabs).

Techniques: Enzyme-linked Immunosorbent Assay, Adjuvant

Journal: bioRxiv

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

doi: 10.64898/2026.06.01.729406

Figure Lengend Snippet: Representative immunohistochemical staining of (Left panel) amygdala sections from tauopathy TAU58/2 mice with the corresponding region from wild-type mice controls ( n = 1); and (Right panel) hippocampal sections from amyloidogenic APP/PS1 mice with the corresponding region from wild-type controls ( n = 1). As indicated, ex vivo brain sections were incubated with sera from C57BL/6J mice immunised with single-targeting nanovaccines (Am-S-pTau or Am-S-Aβ), or dual-targeting mosaic or cocktail formulations. Positive control antibodies were included: PHF-1 that recognizes pTau (pSer396/404); or 6E10 that binds Aβ (residues 1-16/17). Pathology-bound IgG was detected using Alexa Fluor 488 (green) and Alexa Fluor 568 or 647 (orange). Cell nuclei were counterstained with DAPI (blue). Scale bars = 200 µm (TAU58/2); or 500 µm (APP/PS1) and 200 µm (zoomed-in region, APP-PS1).

Article Snippet: The E. coli codon optimised AmEnc-SpyCatcher (Am-S) sequence ( Table S2 ) was synthesised by Twist Bioscience into the pET-24(+) expression vector and transformed into E. coli BL21(DE3) competent cells (New England Biolabs).

Techniques: Immunohistochemical staining, Staining, Ex Vivo, Incubation, Positive Control