Predicted 3D model of Encapsulin (A) and HBcAg (B) NPs displaying βbarrel into an exposed loop.

(PNG)</p

Negative staining transmission electron microscopy (NSTEM) of chimeric NPs displaying βbarrel antigen after SEC purification.

Properly assembled particles were detected for (A) βbarrel-Ferritin with a diameter of 25nm (B) βbarrel-mI3 with a diameter of 30nm (C) βbarrel-Encapsulin presents a diameter of 30nm (D) βbarrel-CP3 presents a diameter of 30nm (E) βbarrel-HBcAg with a diameter of 35nm. Scale bars inserted in the pictures correspond to 50nm (A-B-E) and 100nm (C-D).</p

S1 Raw images -

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SDS-PAGE analysis of purified monomeric antigen, naked and chimeric NPs after SEC purification, performed under denaturing conditions and stained with Coomassie blue.

First lane reports the molecular weight marker expressed in KDa. Theoretical molecular weights of each sample: βbarrel 14KDa, Ferritin 21KDa, βbarrel-Ferritin 34,8KDa, mI3 23,6 KDa, βbarrel-mI3 37,3KDa, encapsulin 32,1 KDa, βbarrel-encapsulin 45,9 KDa, CP3 15,2 KDa, βbarrel-CP3 29,5KDa, Qβ 16,1 KDa, βbarrel-Qβ 29,7KDa, HBcAg 19KDa, βbarrel-HBcAg 32,3 KDa.</p

Summary of protein-based nanoparticles and virus like particles tested in this study.

Summary of protein-based nanoparticles and virus like particles tested in this study.</p

Structural analysis and in silico design of chimeric NPs.

(A) Cartoon representation of 3D structure of fHbp antigen (pdb code 3KVD). In grey it is reported the N-terminal domain (residues 1–118) while in yellow it is shown the C-terminal βbarrel domain used in this work (residues 119–249). (B) Cartoon representation of the monomeric structure of each tested NPs. Engineerable sites explored for the genetic fusion of the antigen are highlighted: the N terminus in dark blue and the exposed loops in red. (C) Cartoon of predicted 3D models of each chimera obtained with Rosetta homology modelling. The βbarrell exposed was represented in yellow. Images were obtained with ChimeraX (panel A, C) and Pymol (panel B).</p

Biacore SPR analysis of monomeric βbarrel and βbarrel-NPs for evaluation of binding avidity with cross-bactericidal 4B3 hmAb.

The interaction between the sample and the antibody has been observed during a time frame of 1500s.</p

Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

<div><p>Group B <i>Streptococcus</i> (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.</p></div

Dot Blot analysis of different chimeric NPs displaying βbarrel for the detection of exposed antigen.

(1) βbarrel-ferritin, (2) βbarrel-mI3, (3) βbarrel-Encapsulin, (4) βbarrel-CP3, (5) βbarrel-HBcAg, (NC) Negative control represented by naked ferritin, (PC) Monomeric βbarrel used as positive control.</p

TEM analysis of unstructured NPs.

(A) βbarrel-Qbeta (B) βbarrel-encapsulin. Only aggregates or monomers were detected after affinity and size exclusion chromatography. (TIF)</p