A Single Unnatural Amino Acid Substitution In RANKL,A Vaccine Therapy Against Collagen-Induced Arthritis

Rheumatoid arthritis is a chronic systemic autoimmune disease characterized the joint disease. It main affects small joints. Synovitis lead to swelling and pain of joints, then cartilage damage and bone erosion. Finally, joints become deformity and dysfunction caused by severe bone destruction and resorption. Systematical bone loss, which lead to a high risk of fracture is the most serious rheumatoid arthritis performance. It is supposed that under the chronic inflammation stimulation, the excessive of osteoclast activity is the key pathological mechanism of bone erosion.OPG/RANKL-RANK transduction pathway is the key signaling pathways for osteoclastogenisis and resorption function. After RANKL binds to RANK, a series of intracellular signaling are activated, including of mitogen-activated protein kinase signaling pathways（MAPKs）, PI3K/AKT and NF-kB signaling pathway. They activate the downstream of osteoclast-related genes and initiate the osteoclast differentiation and maturation. Besides, osteoprotegerin, OPG as decoy receptors of RANKL, competitive blocks the combination of RANKL and RANK so that inhibits osteoclastogenisis and resorption.Recent studies have shown that anti-RANKL antibody can specifically block the combination of RANKL and its receptors, RANK. In results, the prevention of upstream signaling transduction pathways effectively lead to the disable of osteoclast differentiation and bone resorption. However, high cost of antibodies may limit its widespread. Comparing that, a vaccine which can induce the body to produce specific antibody is more economical and safety. Recent studies found that the unnatural amino acid insertion method is a new strategy to breakthrough autologous immune tolerance. Namely, incorporate a specific site in self-protein to enhance its immunogenicity, break immune tolerance, and produce high titers of polyclonal anti self-protein antibody.In this experiment, we use the method of unnatural amino acids substitution into the self-protein to produce a mutation mRANKL recombinant protein vaccine(pNO₂Phe²⁴⁰mRANKL), then test it on a type II-collagen induced arthritis disease model in mice and explore its efficacy and feasibility. In all, we can base this vaccine for further optimizing and treatment of rheumatoid arthritis. Methods and results1. By substitution the 240 locus of pET28a-mRANKL recombinant expression vector, of p-nitrobenzene alanine, we successfully engineer pNO₂Phe²⁴⁰ mRANKL mutation recombination vector. By prokaryotic expression system, we express both wild-type protein and mutation recombinant proteins, and then purify them. Sequence and protein are verified by sequencing and mass spectrometry analysis, respectively.2. Respectively, immune DBA1 mice with pNO₂Phe²⁴⁰ mRANKL recombinant proteins and wildtype mRANKL protein. 7 days after the end of the immune, take the serum of mice to analysis anti-mRANKL antibody with ELISA. Results showed that the serum is with a high titer（1:6400） of antibody.3. To test the treatment effect of recombinant protein vaccines with a single site of p-nitrophenylalanine substitution in RANKL, on mice collagen induced arthritis models. 7 days after the protein vaccine immunization, we start to induce arthritis in DBA1 mice by injection of type II collagen subcutaneous at the bottom of tail. The severity of arthritis is evaluate by the clinical arthritis scores, titers of serum anti-collagen II antibody and histopathological experiment methods. X-ray plain film and microCT scan detect the degree of local and whole body bone bone erosion. Results show that the mutation of recombinant protein in mice arthritis have significant effect against bone erosion, while it also have some effect on arthritis manifests. ConclusionIn this issue, we firstly constructed the unnatural amino acid substitution protein, pNO₂Phe²⁴⁰ mRANKL recombinant protein vaccine and validate its therapeutic effect in type-II collagen-induced arthritis mice model. The results arthritis clinical manifests and erosion confirm that pNO₂Phe²⁴⁰ mRANKL vaccine can significantly relieve the bone erosion in mice with arthritis as well as the arthritis performance, according with our expectations. This could be contribute to the further optimization and mechanism research.