A new vaccine targeting RANKL, prepared by incorporation of an unnatural Amino acid into RANKL, prevents OVX-induced bone loss in mice

Bone homeostasis is maintained by a dynamic balance between osteoblastic bone formation and osteoclastic bone resorption. The receptor activator of nuclear-κB ligand (RANKL) is essential for the function of the bone-resorbing osteoclasts, and targeting RANKL has been proved highly successful in osteoporosis patients. This study aimed to design a novel vaccine targeting RANKL and evaluate its therapeutic effects in OVX-induced bone loss model. Anti-RANKL vaccine was generated by incorporating the unnatural amino acid p-nitrophenylalanine (pNO₂Phe) into selected sites in the murine RANKL (mRANKL) molecule. Specifically, mutation of a single tyrosine residue Tyr²³⁴ (Y234) or Tyr²⁴⁰ (Y240) of mRANKL to pNO₂Phe (thereafter named as Y²³⁴pNO₂Phe or Y²⁴⁰pNO₂Phe) induced a high titer antibody response in mice, whereas no significant antibody response was observed for the wild type mRANKL (WT mRANKL). The antiserum induced by Y²³⁴pNO₂Phe or Y²⁴⁰pNO₂Phe could efficiently prevent osteoclastogenesis in vitro. Moreover, immunization with Y²³⁴pNO₂Phe or Y²⁴⁰pNO₂Phe could also prevent OVX-induced bone loss in mice, suggesting that selected pNO₂Phe-substituted mRANKL may pave the way for creating a novel vaccine to treat osteoporosis.