Virus-like Particles Derived from Bacteriophage Beihai32 as a Versatile Carrier for Displaying Large Peptide Antigens

<jats:p>Virus-like particles (VLPs) based on the capsid protein (CP) of the ssRNA bacteriophage Beihai32 represent a promising nanoscale platform for the presentation of heterologous peptides. Previous studies have shown that the C-terminus of the CP tolerates long insertions without compromising VLP assembly. Here, we demonstrate that the N-terminus is similarly permissive to extended insertions. Hybrid CPs with one to four copies of the influenza A virus M2e peptide fused to the N-terminus were expressed in Escherichia coli. Fusion proteins containing four copies of M2e self-assembled into spherical VLPs, displaying the inserted peptides on the surface. Subcutaneous immunization of mice with chimeric VLPs induced high titers of M2e-specific antibodies. Unlike C-terminal fusions, the N-terminal insertion prevented the induction of anti-carrier antibody response indicting masking of the carrier protein in the chimeric VLP. To evaluate the capacity of the N-terminus for larger inserts, green fluorescent protein (GFP, 238 a.a.) was attached to the N-terminus of CP. The hybrid protein was expressed in Escherichia coli and formed VLPs. GFP was displayed on the particle surface and retained fluorescent activity. Overall, the phage Beihai32 CP is a versatile platform for the presentation of peptide antigens, supporting its potential application in VLP-based vaccine design.</jats:p>