Synthesis of Chirally Chimeric Protein Nanoparticle Vaccines via Mirror‐Image Spy Chemistry

Mirror-image proteins and nucleic acids exhibit remarkable biostability and bioorthogonality, offering a promising strategy to enhance the longevity of biological therapeutics. Here, we present a modular approach for constructing complex protein architectures that integrate both natural-chirality (L-) and mirror-image (D-) motifs. Key to this strategy is the development of D-SpyStapler-a chemically synthesized, chirally inverted ligase that enables the efficient conjugation of D-SpyTag and D-BDTag. By exploiting the achirality of glycine (Gly), we used L-sortase to covalently link D-peptides (e.g., SpyTag or BDTag, bearing an N-terminal poly-Gly motif) to L-proteins (e.g., GFP, VLP-forming Mi3, or antigens) containing a C-terminal LPXTG sorting signal. The resulting D-SpyTagged and D-BDTagged proteins were further assembled via D-SpyStapler. This method enabled the construction of chirally chimeric VLP vaccines displaying antigens derived from malaria parasites and coronaviruses in various forms-recombinant proteins or synthetic peptides-providing significant flexibility and modularity for vaccine design. The resulting chirally hybrid vaccines exhibited enhanced proteolytic resistance in vitro and elicited potent immune responses in vivo. This study provides a versatile platform for developing long-acting therapeutics and vaccines.