A New Vaccine Targeting RANKL,Prepared By Incorporation Of An Unnatural Amino Acid Into RANKL,Prevents OVX-induced Bone Loss In Mice

Osteoporosis 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.It is thought to severely affect patient's quality of life with high incidence and brings a heavy economic burden to the society.Bone homeostasis is maintained by a dynamic balance between osteoblastic bone formation and osteoclastic bone resorption.Excessive activation of osteoclastic bone resorption is a common pathological mechanism of bone loss and fractures.Receptor Activator of Nuclear Factor-?B Ligand?RANKL?plays a key role on differentiation,maturation and function of osteoclast.RANKL binds to the Receptor Activator of Nuclear Factor-?B?RANK?,and then activates the downstream signal pathways such as NF-?B,P38,ERK,JNK,etc.and stimulates the differentiation,maturation and function of osteoclasts.The differentiation and maturation of osteoclasts can be inhibited by blocking the binding of RANKL and RANK.It is certified that Denosumab,an anti-RANKL monoclonal antibody,can specifically block the binding of RANKL to its receptors,which inhibits the differentiation,maturation and function of osteoclast effectively.Recent studies show that the genetic incorporation of p-nitrophenylalanine?pNO₂Phe?,one of unnatural amino acid,into the self-proteins can elicit high titer autoantibody responses to their endogenous proteins.This study was to investigate the prokaryotic expression and purification of mouse RANKL with an unnatural amino acid mutant and preparation of antiserum against mRANKL in mice,and then further to analyze the antibody titer and activity of the antiserum.And then take the pNO₂Phe-substituted mRANKL as the therapeutic vaccine,and evaluate the effects of the vaccine in mice with ovariectomy?OVX?-induced bone loss.Methods and Results?1?Total RNA was extracted from mouse bone marrow,and then the extracellular mRANKL gene was amplified by RT-PCR.pET28a-pNO₂Phe mRANKL recombinant expression vectors were constructed after mutating the 164^th,234^th,240^th or 272^thh gene codon into the amber codon?TAG?which can encode unnatural amino acid p-nitrophenylalanine?pNO₂Phe?.The recombinant expression vector was verified by sequencing.?2?The four pET28a-pNO₂Phe mRANKL recombinant expression vectors were co-transformed into E.coli BL-21?DE3?with pEVOL plasmid,respectively.Protein expression was induced with 0.2%arabinose and 1 mM IPTG and then purified(the four pNO₂Phe-substituted mRANKLs then abbreviated as F¹⁶⁴pNO₂Phe?Y²³⁴pNO₂Phe?Y²⁴⁰pNO₂Phe and Y²⁷²pNO₂Phe).SDS-PAGE and Western Blot was used to detect the protein expressed and purified.High performance liquid chromatography?HPLC?in combination with mass spectrometry was conducted to confirm the successful incorporation of an p-nitrophenylalanine?pNO₂Phe?into mRANKL.The results showed that the highly specific incorporation of the unnatural amino acid was successful.?3?Mice were immunized with the purified protein by using the RIMMS?repetitive immunization at multiple sites?protocol.The anti-mRANKL antiserum titer was determined by indirect ELISA,and its specificity was confirmed by Western Blot.The results showed that the mice antiserum against mRANKL was successfully prepared.Particularly,the titer of antiserum was shown higher in Y²³⁴pNO₂Phe and Y²⁴⁰pNO₂Phe when compared with F¹⁶⁴pNO₂Phe and Y²⁷²pNO₂Phe,and and the serum titers can remain quite robust after 168 days.Further more,the anti serum can efficiently prevents osteoclastogenesis in vitro.?4?To examine the effects of Y²³⁴pNO₂Phe and Y²⁴⁰pNO₂Phe on OVX-induced bone loss,we generated OVX mice,which had significantly smaller uterus size and lower uterus weight.As expected,?CT results for trabecular bone microarchitecture of distal femur were shown that immunization with Y²³⁴pNO₂Phe and Y²⁴⁰pNO₂Phe can prevent OVX-induced bone loss in mice.ConclusionIn this experiment,we cloned the extracellular mRANKL gene and first constructed pET28a-pNO₂Phe mRANKL recombinant expression vector.The protein of mRANKL with a p-nitrophenylalanine mutant was expressed and purified successfully which was confirmed by High performance liquid chromatography?HPLC?in combination with mass spectrometry.Then mice were immunized with the purified protein and anti-mRANKL antiserum with high titer and activity was successfully prepared.The two screened vaccine(Y²³⁴pNO₂Phe and Y²⁴⁰pNO₂Phe)can prevent OVX-induced bone loss in mice.This study established the foundation for the further research on new methods of blocking RANKL-RANK pathway and therapying osteoporosis.