Surface-bioengineering of bacteriophage AP205 and MS2 virus-like particles with novel Spytag003 for antigen conjugation.

The insertion of foreign antigens on the coat protein of viruses sometimes interferes with the ability of the coat proteins to assemble into virus-like particles (VLPs). To overcome this limitation, Spytag/Spycatcher bio-conjugation system was developed; however, the conjugation system has pre-existing antibodies that makes it less desirable for vaccine applications. Recently, a novel bio-conjugation system, Spytag003/Spycatcher003, was developed to by-pass this problem. The ability of this new bio-conjugation system to be used to displayed foreign antigens on VLPs has never been explored. Here we assessed whether the insertion of Spytag003 on the coat proteins of AP205 and MS2 would interfere with the ability of the coat proteins to assemble into VLPs. We showed that the insertion of Spytag003 on the N-termini of coat proteins of AP205 and MS2 did not affect their ability to assemble into VLPs. Optimized purification yielded VLPs with high purity, which were successfully conjugated with Spycatcher003 and Ag2/PRA-CSA protein. Mice immunized with the conjugated Spycatcher003 protein elicited high-titer IgG antibodies compared to immunization with unconjugated protein. However, immunization with Ag2/PRA-CSA conjugated or unconjugated to Spytag003-tagged VLPs did not have a significant difference in antibody responses between the two groups. Overall, our results show that the N-termini of AP205 and MS2 are more tolerant to Spytag003 insertions and can be used to conjugate a foreign antigen; as a proof-of-concept, we conjugated a prototype antigen, Ag2/PRA-CSA, on the VLPs. The potential to conjugate diverse antigens on these novel bio-engineered platforms should be explored further.