A Single Unnatural Amino Acid Substitution In Sclerostin,a Vaccine Therapy Against Osteoporosis

【Background】Osteoporosis,mostly in the elderly and postmenopausal female,is a systemic skeletal disease characterized by a progressive loss of bone mass and microarchitectural deterioration of bone tissue,and therefore leads to increased fragility fractures.The balance of bone metabolic homeostasis is mainly dependent on the osteoblast osteogenesis and osteoclast bone resorption.And the unbalance caused by any factors results in bone disorders.Among all the causes,the inefficiency of osteoblast activity and function plays an important role in this process.Sclerostin is a secretory glycoprotein programed by the SOST gene and secreted by osteocytes,which binds to the low density lipoprotein-associated protein complex receptor(LRP 5/6)on the surface of osteoblasts to inhibit the Wnt signaling pathway,thereby inhibiting osteoblast differentiation and maturation.The monoclonal antibody against Sclerostin can effectively promote osteoblast differentiation and maturation through the specific binding to Sclerostin,thus promoting the bone formation and preventing osteoporosis.However,the sophisticated technological process,high price and some adverse side effects of the monoclonal antibody limit its clinical applications widely.In contrast,the vaccine as an active immunization by inducing the body to produce specific polyclonal antibodies against endogenous antigens may be a better strategy.However the inability to selectively induce immune response against self-proteins leading to diseases restricts the fulfillment of the idea.In this study,we show that site-specific incorporation of an immunogenic artificial amino acid into a protein of interest produces relatively high-affinity antibodies that cross-react with natural protein.Specifically,replacement of the single amino acid of murine Sclerostin with p-nitrophenylalanine(pNO2Phe)induced an efficient antibody induction in mice.The antibodies generated against the pNO2Phe-Sclerostin are cross-reactive with native Sclerostin and protect mice from osteoporosis in OVX model.In this study,we show that site-specific incorporation of an immunogenic artificial amino acid into Sclerostin and detected the antibody titer and activity of the antiserum.Then we analyzed the efficacy and feasibility of vaccine derived from the purified Sclerostin with an unnatural amino acid mutant in the mouse osteoporosis model(OVX),which may provide a general strategy for inducing efficient antibody response to disease related self-antigens,and thus producing neutralized antibodies for osteoporosis treatment.【Methods and Results】1.pET28a-mSOST recombinant expression vector constructed by the codon-optimized mouse mSOST gene was mutated into amber codons(TAG)to encode p-nitrophenylalanine in the codons encoding the amino acids 115,121 and 126.Then we constructed and sequenced the pET28a-pNO2 Phe mSOST recombinant expression vector.2.Prokaryotic expression and purification of wild type and mutant Sclerostin were testified by SDS-PAGE and Western Blot.And the successful insertion of p-nitrophenylalanine was detected by HPLC combined with mass spectrometry.3.C57/BL female mice were inoculated with concentrated and purified wild type and mutant Sclerostin.Blood samples were taken from the tail vein at the end of the last immunization.The antibody activity and titer of the antiserum were detected by Western Blot and indirect ELISA respectively.The results show that mutant Sclerostin can break through autoimmune tolerance to produce high titers,high specific antibodies,which not only combined with mutant Sclerostin but also endogenous Sclerostin.4.To evaluate the therapeutic effect of mutant Sclerostin vaccine on mice OVX model,we detect the antibody titer by ELISA during the whole experiment.Then,osteocalcin was measured by serological examination and bone formation was detected by micro-CT technique.The results showed that the Sclerostin vaccine modified by unnatural amino acid had obvious therapeutic effect on osteoporosis in mice OVX model.【Conclusions】In this study,we successfully expressed and purified mouse Sclerostin with p-nitrophenylalanine mutant.And pNO2Phe126 Sclerostin mediated vaccine generates expected antibodies highly cross-reactive with the native Sclerostin and thus protects mice from osteoporosis through promoting the function of osteoblasts,thus laying a solid foundation for the next step to evaluate vaccine safety and optimize vaccine therapy.