Identification Of B-cell Epitope Of FSHR And Study On Antifertility Effect Of Its Polypeptide Vaccine

As the world's population continues to soar and men are willingness to participate in family planning, it is urgent to develop new contraceptive methods. The most properties of an ideal contraceptive should be effective, safe, inexpensive and reversible. The contraceptive vaccine (CV) can fulfill most of the properties and is an exciting proposition. Based on various targets, the CVs fall into three categories: vaccines inhibiting gamete production, gamete function and gamete outcome. The CVs for male focus on interfering spermatogenesis, inhibiting sperm motility or any process involved in sperm-egg interaction which resulted in fertility suppression.Follicle stimulating hormone is a glycoprotein hormone secreted by the pituitary gland which is essential for the initiation and maintenance of spermatogenesis in males. It exerts its biological actions through binding to its receptor (FSHR) expressed exclusively on Sertoli cells. For its importance on spermatogenesis, it is a potential target for male contraceptive. Literature showed that various discrete, discontinuous regions of the extracellular domain (ECD) are crucial for either FSH binding or subsequent signal transduction or both. In the past several decades, apart from monoclonal or polyclonal antibodies employed as probes, other approaches have been explored to understand structure-function relationship between FSH and FSHR, such as filamentous phage display, peptide challenge experiment and epitope mapping. However, the sites assessed in previous studies neither provide ideal suppressive fertility in animals nor go further to fertility test.Eppin (epididymis protease inhibitor) is a ~133 amino acid protein that specifically expressed on posterior part of perforatorium, Sertoli cells, Leydig cells, round spermatids and principal cell in epididymal epithelium. It is likely to be important for sperm maturation. Eppin presents on the surface of ejaculated sperm and becomes saturated with semenogelin (Sg), which is the major component of semen cogulum. Thus the Eppin-Sg complex provided sperm the antimicrobial activity while through the female reproductive tract. While the Macaca radiate receiced active immunization with recombinant Eppin, seven out of nine males showed inhibited fertility, and the fertilizing capacities of five out of seven males were restored when stopping immunization. For this reason, Eppin is a candidate molecular for CV.Therefore, our researches aimed at identify immunodominant neutralizing B-cell epitopes of FSHR and investigate its efficacy and safty as a CV for males. In this study, we divided the extracellular of hFSHR into two fragments on the basis of the amino acids it contained, including 1-140 amino acids and 120-350 amino acids, respectively. The gene sequences were inserted in plasmid pET 32a and the prokaryotic proteins were induced to express and purified. Four candidate peptides were selected after analysis of secondary structure of ECD, online prediction of B cell epitope and molecular docking. The four B cell peptides were in tandem with Pan DR epitope (PADRE) to enhance their immunogenicity. We employed the protein prime-peptide boost strategy to explore their antifertility efficacy on adult male mice. As vaccines based on FSHR produced only moderate fertility inhibition, we applied Eppin for combination immunization and observed its effect.Methods:1. The inductive expression of prokaryotic proteins for ECD of hFSHR and hEppinThe gene sequence of 1-140 amino acids and 120-350 amino acids of ECD were obtained by GeneSOEing PCR. The gene sequence of Eppin was amplificated by PCR. These genes were inserted in plasmid pET 32a and the prokaryotic proteins were expressed under induction of 1 mM IPTG. The recombinant proteins were purified and identified. The purified FSHR proteins were signed as F140 and F240, respectively.2. Prediction of B cell epitope of ECD of hFSHRThe ECD of hFSHR was analyzed with DNAstar software for secondary structure, hydrophilicity, accessibility, plasticity and antigen index. At meantime, the potential B cell epitope was predicted online. The molecular docking of hFSHR and hFSH was performed using the InsightⅡsoftware package. The InsightⅡ/Homology module was used to build the initial FSHR model with crystal structure of complex of human FSH and FSHR (PDB ID: 1xwd) as template.3. Synthesis of confusion peptide of hFSHR The candidate peptides were conjugated with PADRE to enhance their immunogenic- city and synthesized by GL Biochem (Shanghai) Ltd.4. The immunological effect of B cell epitopeThe sexually mature Balb/C male mice received active immunization with protein prime-peptide boost regimen. The immunogen was emulsified with same volume of Freund's adjuvant. The first boost vaccination was given at 2 weeks after the prime immunization and repeated for three times at one week interval. The specific antibody IgG was monitored using ELISA. The Western blot analysis was employed to testify whether the antisera from vaccinated mice could bind with the natural FSHR protein or not. Also, the specific antibody IgG of mice received combination immunization with FSHR and Eppin was assessed at certain times.5. Antifertility effectThe productive capacities of immunized male mice were investigated by mating one male with three randomly selected females of proven fertility for 2 weeks. The number of pregnant females and litter sizes were recorded. Two successive mating trials were carried out at 2 weeks and 6 weeks respectively after the final immunization.6. Studies on the effects of inoculation to the reproductive and endocrine system of male mice and its mechanismsTwo weeks after the last fertility assay, male mice were sacrificed, blood was collected, and testis and epididymis were dissected. Sperm were collected from cauda epididymidis and assessed for sperm count, motility and hypo-osmotic swelling test (HOS). The weights of testis were taken notes and the spermatids in testis were calculated. The serum testosterone level was detected by radioimmunoassay (RIA). In addition, the inhibition effects of antiserum on normal human sperm and the IgA in epididymis lavage fluid were evaluated for animals vaccinated with combination immunization.7. Histological examination of testisAfter male mice were sacrificed, testis were removed, fixed and embedded. The slices were stained by hematoxylin-eosin (HE) and observed under light microscope.8. Fertility recovery assayForty weeks after primary immunization, one male was cohabited with three female for 2 weeks. The number of contraceptive mice and progeny size were counted. Results:1. The inductive expression of prokaryotic proteins for ECD of hFSHR and hEppinThe gene sequences of hFSHR and hEppin were amplificated and inserted in plasmid pET 32a. The prokaryotic proteins were existed as inclusion body. After purified and renaturated, the protein content was assessed by Bradford method. It was 1.26 mg/ml for F140, 1.19 mg/ml for F240 and 5.5 mg/ml for Eppin and their purity were over 95%. The Western blot analysis showed that the antiserum induced by recombinant protein could bind with 75kD or 34kD protein, which was existed in the extracted protein from human testis tissue.2. Prediction of B cell epitope of ECD of hFSHRTaken all analytic results into consideration, we selected four potential epitopes, whose location in ECD were 29~38, 32~44, 269~280 and 285~300, respectively.3. Synthesis of confusion peptide of hFSHRThe purity of confusion peptides were above 85%.4. The immunological effect of B cell epitopeAfter vaccinated with antigen based on FSHR, specific IgG antibody can be detected in serum with the highest titre above 1: 400000. The Western blot analysis displayed a band about 75kD when extracted protein from human testis tissue was probed with antisera from immunized animals. Similar antibody titres were observed in males with combination immunization. It suggested that it could elicit high specific antibody with the protein prime-peptide boost strategy.5. Antifertility effectThe fertility assay displayed that primed with hFSHR protein and boosted with conjugated peptide, partial contraceptive effects were induced in mice of group P2, P3 and P4. Especially the group P2 showed a significantly reduced pregnant rate of 26.67% and the mean litter sizes were 4.19, a litter high than that of mice immunized four times with recombinant protein. When Eppin were applied together, the inhibition effect was more obviously, mice primed with FSHR and Eppin and boosted with peptide 2 plus peptide C showed a similar higher fertility with that of combination protein prime-boost mice. In detail, the fertility rate significantly reduced to 6.67% and progeny sizes to 2.5. Therefore, peptide 2 might be a dominant B cell epitope of FSHR to produce fertility inhibition and combination vaccination was superior to single application.6. Studies on the effects of inoculation to the reproductive and endocrine system of male mice and its mechanismsFurther investigation for the mechanism of reduced fertility in vaccinated males revealed lower sperm count and motility of cauda epididymidis, smaller testis and less spermatid in testis and lower serum testosterone level. Inoculated with Eppin together, higher IgA titre in epididymis lavage was observed and the motility of sperm depressed significantly as well, this was consistent with inhibition effect of antisera on normal human sperm.7. Histological examination of testisIn fertility suppressed male mice induced by immunization with FSHR, histological examination manifested diameter of seminiferous tubule were decreased along with lack of sufficient sperm in some seminiferous tubules. In light microscope, it revealed a distorted tubular architecture in spermatogenic epithelium. The pathological change in testis did not aggravate after vaccination adding Eppin.8. Fertility recovery assayFor the pathological change existed in testis, the fertility decreased male mice showed a little high fertility rate but can not recover completely at forty weeks after primary immunization when ELISA showed antibody titres were less than 1: 5000.Conclusions:1. We amplificated the gene sequence of human FSHR and Eppin and constructed prokaryotic expression vector successfully. After inductive expression, purification and renaturation, the purity of combinant proteins were over 95%.2. The ECD of hFSHR were analyzed for secondary structure, predicted B cell epitope online and molecular docking. Taken all these results into consideration, four candidate B cell epitope peptides were selected.3. PADRE was in tandem with B cell epitope and the purity of fusion peptide was above 85%.4. The protein prime–peptide boost modality could elicit high titre of specific antibody. Peptide vaccine of FSHR could induce fertility inhibition through reduce quantity of sperm, but with side effects such as smaller testis, lower serum testosterone level and pathological change in testis.5. Combination immunization with FSHR and Eppin could induce significant decreased sperm motility accompanied with less sperm, while the side effects did not aggravated. How to enhance the immunogenicity of B cell epitope but alleviate or avoid side effects should be investigated further. But our study provided experimental ground- work for development of multivalent contraceptive vaccine.