Experimental Study Of An Anti-postmenopausal Osteoporosis Protein Vaccine

Osteoporosis, a metabolic bone disease, threatens postmenopausal women globally with fracture rates. Osteoporotic bone fragility fractures, severe impact on the quality of life of the elderly, can easily result in high morbidity and mortality. The incidence of osteoporosis far exceeds the combined incidence of breast cancer, stroke, and heart attacks. In China, there are nearly70million people suffer from osteoporosis, and210million people below the normal standards of bone mass. Furthermore, the aging of the population has increased the incidence of osteoporosis.For years, hypogonadal bone loss has been attributed to declining estrogen levels, and the activation of the estrogen receptor can actually protect bone mass after menopause. As a result, estrogen replacement therapy (ERT) or selective estrogen receptor modulator (SERM) therapy has been used widely in postmenopausal osteoporosis therapy in the clinic. However, the underlying molecular mechanism of how estrogen affects the bone mass is unclear. In2002, McCauley et al. reported that mice with both hypogonadal a and β estrogen receptors deleted were only mildly osteopenic. Thereafter, several studies demonstrated that hypogonadal mice, with deletion of either the α or β estrogen receptor, have a normal bone mass. All these results indicated that estrogen may not be the only mediator of postmenopausal osteoporosis.In the hypothalamic-pituitary-ovarian axis, hypothalamus-derived gonadotropin releasing hormone (GnRH) stimulates the pituitary-secreted follicle-stimulating hormone (FSH), and FSH stimulates the production and secretion of estrogen from ovarian follicle cells. FSH is negative-feedback controlled by the estrogen levels. Thus, a decrease in the estrogen levels results in increases in the FSH levels of ten or more times. However, FSH is not related to causing hypogonadal bone loss and is only regarded as a marker for the beginning of menopause. However, Mone ZaiDi et al. found that FSH can directly regulate the bone mass independent of estrogen. High levels of FSH can directly act on osteoclasts via Gi2α-coupled FSHR and activate the MEK/Erk, NF-κB and Akt pathways to stimulate osteoclastogenesis and bone resorption.In this study, we designed a novel FSH-based therapeutic vaccine to treat postmenopasaul osteoporosis.The effect of exogenous FSH on cell differentiation. The differentiation of bone marrow osteoclasts was induced with10-8M1,25VD3and a concentration series of FSH at3ng/mL, lOng/mL and30ng/mL. After culturing for7days, TRAP staining was performed, and the number of TRAP+cells was counted. Bone marrow osteoblast differentiation was induced with Ong/mL, lOng/mL and30ng/mL FSH in osteogenesis induced medium. After culturing for10days, the cells were stained with Von Kossa and Gomori calcium-cobalt.Expression of recombinant GST-hFSHβ protein in E.coli. The gene encoding the mature hFSHβ peptide was optimized with E.coli preferred codons, synthesized and cloned between the BamH I and EcoR I sites of pGEX-4T-1. The resulting plasmid, pGEX-hFSHβ, was transformed into E.coli DH5α to express the hFSHβ protein. Different isopropyl β-D-thiogalactopyranoside (IPTG) concentrations, durations and temperatures were used to optimize the expression of the GST-hFSHβ fusion protein.Immnunization of OVX rat with recombinant GST-hFSHβ protein. Three-month-old female Sprague-Dawley rats were ovariectomized. After1month, the rats were immunized subcutaneously with200μg of GST-hFSHβ (FSH immunization group) or GST (OVX group) emulsified with an equal volume of complete Freund’s adjuvant (CFA). The GST-hFSHβ protein was dissolved in PBS, and the CFA contained heat-killed Mycobacterium tuberculosis bacilli. Four weeks after the primary immunization, the first booster immunization was administered with200μg of GST-hFSHβ or GST emulsified with an equal volume of incomplete Freund’s adjuvant (IFA). Further booster immunization was administered at2week intervals with200μg of GST-hFSHβ or GST.We found that:Exogenous FSH stimulates osteoclastogenesis in a dose-dependent manner. The osteoclast differentiation experiment showed that FSH can promote osteoclast formation. There was an expected increase in the TRAP+cell number and osteoclast nuclei after FSH treatment. Additionally, the anti-FSH monoclonal antibodies or antisera produced by the immunized rats had a similar blocking effect on the FSH-induced osteoclast differentiation. Moreover, FSH had a little effects on osteoblastic bone formation, as shown by von Kossa staining and alkaline-phosphatase (ALP) staining.Highly effective expression of recombinant hFSHβ protein in E.coli. The constructed expression plasmid, pGEX-hFSHβ, was digested by BamH I and EcoR I, and the band size consistent with the expected size. SDS-PAGE showed an approximately38kDa band, which was consistent with the expected molecular weight of GST-tagged hFSHβ. The optimized studies revealed that the highest expression condition for hFSHβ was incubation at37℃with0.5mM IPTG for4h.Treatment of postmenopausal osteoporosis in OVX rat:Antibody titer. The results of the indirect ELISA revealed that positive reactions were detected at the titer of1:1280000. This result shows that the immunization of OVX rats with recombinant GST-hFSHβ protein can obtain a higher antibody titer and thus successfully block FSH.BMD analysis. The BMD in the right femur was measured. The BMD in both the FSH immunization and Sham groups was significantly higher than the OVX group (P<0.01), but there was no difference between the FSH immunization and Sham groups (P>0.05).Serum and urine analysis. The level of ALP, OC, U-Ca, U-P, DPD in OVX rat, after GST-hFSHβ immunization, were significantly reduced.Micro-CT analysis of the distal femur. The micro-CT analysis revealed significant improvements in the trabecular parameters in the FSH immunization group compared with the OVX group, including BV/TV and Tb.Sp (P<0.01). Tb.Th and Tb.N in the FSH immunization group were also increased (P<0.05). These results indicated that blocking the FSH signaling pathway can significantly improve the mechanical and structural parameters in OVX rats. The only data showing no differences were the cortical wall thickness measurements (P>0.05). The three-dimensional images of the femoral metaphysic and maximum cross-section confirmed these improvements.Three-point bending test. The results of the three-point bending test indicated that compared with the OVX group, the femurs in the FSH immunization group possessed a higher maximum load and stiffness (P<0.05) and showed significantly increases in Young’s modulus and maximum stress (P<0.01). However, the FSH immunization group showed no differences compared with the Sham group. These results indicated that GST-hFSHβ immunization significantly improved the mechanical stability of the femurs in the OVX rats.Compression test. The lumbar compression test showed that the FSH immunization group had improvements in the maximum load, stiffness, Young’s modulus and maximum stress compared with the OVX group, although these changes did not reach the normal level. These results indicated that GST-hFSHp immunization led to more stable lumbar vertebrae in the OVX rats.Histological examination. The histological observations revealed that compared with the OVX group, the Tb.N had a notable increase in the FSH immunization group. These results were consistent with the biomechanical testing and micro-CT analysis.Body weight. The mean body weight of the FSH immunization group showed little difference from that of the OVX group but was significantly higher than that of the Sham group, implying that lipogenesis was enhanced due to estrogen deficiency and that neutralization of FSH cannot inhibit lipogenesis.In conclusion, immunization with a recombinant hFSHβ fusion protein antigen can raise a high-titer anti-FSH polyclonal antibody that can neutralize FSH both in vitro and in vivo. Additionally, hFSHβ immunization can not only prevent bone loss but also enhance bone strength in a rat ovariectomy-induced osteoporosis model, demonstrating that an anti-FSHβ vaccine has great potential in treating postmenopausal osteoporosis.