Evaluation of lumazine synthase from Bacillus anthracis as a presentation platform for polyvalent antigen display

Abstract

Polyvalent antigen display is an effective strategy to enhance the immunogenicity of subunit vaccines by clustering them in an array-like manner on a scaffold system. This strategy results in a higher local density of antigens, increased high avidity interactions with B cells and other antigen presenting cells, and therefore a more effective presentation of vaccine antigens. In this study, we used lumazine synthase (LS), an icosahedral symmetry capsid derived from Bacillus anthracis, as a scaffold to present 60 copies of a linear B cell epitope (PB10) from the ricin toxin fused to the C terminus of LS via four different linkers. We then investigated the effects of linker length, linker rigidity and formaldehyde crosslinking on the protein assembly, conformational integrity, thermal stability, in vitro antibody binding, and immunogenicity in mice. Fusion of the PB10 peptide onto LS, with varying linker lengths, did not affect protein assembly, thermal stability or exposure of the epitope, but had a minor impact on protein conformation. Formaldehyde crosslinking considerably improved protein thermal stability with only minor impact on protein conformation. All LS_PB10 constructs, when administered to mice by injection without adjuvant, elicited measurable anti-ricin serum IgG titers, although the titers were not sufficient to confer protection against a 10× lethal dose ricin challenge. This work sheds light on the biophysical properties, immunogenicity and potential feasibility of LS from B. anthracis as a scaffold system for polyvalent antigen display.

Improved Fluorescence Methods for High-Throughput Protein Formulation Screening

“…Moment can also be described as the mean spectral center of mass (MSM) in intrinsic Trp fluorescence studies, as previously described. 12,13 Plotting intensity or moment versus temperature generates a melting curve to describe a thermal unfolding event. Each melting curve was further processed to obtain T m values employing two commonly used methods: a six-parameter fitting (fit) method ( Fig.…”

Protein Cages: From Fundamentals to Advanced Applications

“…The dengue virus spike protein and the linear B cell epitope (PB10) from the ricin toxin have also been successfully displayed. 545 Other natural capsids have been analogously adapted to develop vaccines for malaria, 546−549 borrelia, 550 and influenza 551,552 as well as to protect against bacterial and parasitic pathogens 553−557 and treat cancer 558−572 and other human 573−584 and animal diseases. 585−598 Seamless integration of the antigen into the capsid protomer by genetic fusion is advantageous for vaccine production, as both the antigen and the scaffold are expressed together, but appending peptides or proteins to the cage sometimes reduces the yield or disrupts cage assembly.…”

Second career of a biosynthetic enzyme: Lumazine synthase as a virus-like nanoparticle in vaccine development

“…Differential calorimetric measurements of LS capsids from Bacillus anthracis (BaLS) have shown two major thermal transitions (52.0° and 93.6 °C). The first thermal transition has been interpreted by the thermal dissociation of a phosphate ion from the LS/phosphate complex, the second transition likely corresponds to the dissociation of LS oligomers and unfolding of the monomers [ 16 ].…”

“…Icosahedral LS (60 identical subunits, T = 1, diameter 16 nm) has in recent years become an attractive nanoparticle for display and presentation of polyvalent antigens [ 16 , 29 , 30 ]. The N- and C- termini of all LS monomer forms are exposed on the surface and point outwards [ 9 , 10 ].…”