Surface-Functionalization of PP7 Virus-Like Particles with Spytag for Bioconjugation Applications

Area of Science:

• Biotechnology
• Immunology
• Structural Biology

Background:

• Virus-like particles (VLPs) are non-infectious, highly immunogenic protein assemblies used as scaffolds for displaying foreign antigens.
• Current methods for antigen display on VLPs face limitations due to the inability of some viral coat proteins to tolerate foreign insertions.

Purpose of the Study:

• To assess the tolerability of bacteriophage Qβ and PP7 coat proteins to insertions of Spytag peptides.
• To determine if these peptide insertions affect VLP assembly.
• To establish a VLP platform for antigen conjugation in vaccine studies.

Main Methods:

• Genetic insertion of Spytag003 and Spytag peptides into Qβ and PP7 coat proteins.
• Analysis of protein expression, solubility, and VLP assembly.
• Conjugation of a fungal antigen to PP7-Spytag VLPs using SpyCatcher003.
• Immunization studies to assess antibody titers.

Main Results:

• Insertion of Spytag003 into Qβ and PP7 coat proteins resulted in insoluble proteins.
• Insertion of the shorter Spytag peptide into PP7 coat proteins (N terminus or AB-loop) yielded soluble proteins that assembled into VLPs.
• PP7-Spytag VLPs successfully conjugated with SpyCatcher003 and a fungal antigen.
• Immunization with conjugated SpyCatcher003 on PP7-SpyTag VLPs significantly increased anti-SpyCatcher003 antibody titers compared to unconjugated SpyCatcher003.

Conclusions:

• PP7 coat protein tolerates specific peptide insertions, allowing for the creation of functional VLPs.
• PP7-Spytag VLPs serve as a viable platform for conjugating foreign antigens, enhancing immunogenicity.
• This study provides proof of concept for utilizing PP7-Spytag VLPs in vaccine development.