Analysis Of B-cell Epitope Of Eppin And Study On Its Anti-fertility Potential

The world's population is growing at a tremendous rate and despite of the availability of various contraceptive modalities there is an urgent requirement for a better contraception method, which is reversible, safe and highly effective, easily obtained, inexpensive and suitable for all stages of reproductive life. Contraceptive vaccines can fulfill most of the properties of an ideal contraceptive. Up to now, there has been no indication of an ideal contraceptive vaccine being developed probably due to the lack of an ideal target antigen. The target antigen applied in contraceptive vaccine must meet at least two conditions. First, it should be expressed specifically in the reproductive system; second, it should act as an important component in the reproductive process.Recently, epididymal protease inhibitor (Eppin), a sperm specific antigen, was identified as an ideal and promising target for the development of a series of safer and more effective contraceptive vaccines. Human-derived Eppin is specifically expressed in epididymis and testis. Eppin was detected in Sertoli cells in 12 day-old mice and afterwards expresses during the maturation process of spermatids. When used for active immunization of adult male Macaca monkeys, seven out of nine males (78%) developed high titers to recombinant Eppin, and all these high titer monkeys were infertile. Five out of seven (71%) high anti-Eppin titer males recovered fertility when immunization was stopped. This study revealed that Eppin is a promising target for male contraception.However, previous studies showed that the contraceptive effect of the full-length protein was not sufficient to satisfy clinical demand. This may ascribe to: Eppin protein not only has B-cell and T cell epitopes providing immune recognition, but also has toxic or inhibitory epitopes, non-neutralized dominant epitopes, pathological epitopes and cross-reactive epitopes with auto-antigen. Therefore, in order to induce an immune response with stronger specificity and an increased immune effect, we must effectively avoide the former and modify the latter, allowing more ideal vaccine molecules to be developed. Epitope vaccine, synthesized with antigen epitope, is a newly developed vaccine technique with the advantages of a simple manufacturing processes, relatively low cost, stable chemical property and strong immune specificity with minimal toxicity or side-effects. Screening as well as identification of epitopes is the primary and most important task during preparation of an epitope vaccine.Eppin cDNA was obtained by RT-PCR from fresh human testis, and then cloned into expression vector pET-32a, the recombinant vector was transformed into BL21 (DE3) and induced by IPTG. The recombinant protein was purified by Ni2+-NTA-column.The antigenic activity of purified recombinant protein was assessed with Western blot (Part I).The epitope prediction was performed according to the correlation of B-cell epitope with physico- chemical property or secondary structures. After reviewing references, analyzing the homology and rejecting the crossing part of Eppin,8 sequential overlapping decapeptides (six amino acids overlap) targeting the Kunitz domain were designed and synthesized by solid-phase synthesis using a synthesis peptide technique.The dominant cellular epitopes were screened and identified by further experiments in vitro and in vivo. It was theoretically confirmed that the antigens had strong antigenicity and prevented potentially adverse immune reactions caused by pathological antigen and cross-reactive epitopes (Part II).Finally, the two branches of multiple antigen peptide (MAP) was designed and synthesized by solid-phase synthesis using a synthesis peptide technique. The sexually mature male rhesus monkeys were immunized with MAP by abdominal subcutaneous injection in Freund's adjuvant. The presence and reactivity of antibody (IgG) against peptides in the serum were analyzed using ELISA. The levels of Th1/Th2 cytokine in serum were detected by non-human primate Th1/Th2 cytokine kit (BDTM cytometric bead array) (Part III).The results showed that:①Enzyme digestion analysis and sequencing showed that the target fragment was successfully inserted into the vector pET-32a (+).Soluble Eppin protein possessing specific antigenicity was expressed and purified successfully.②B-cell epitope prediction of the human testis and epididymis specific sequence of Eppin was made based on the characteristics of hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface and turns, using DNASTAR'S Lasegene software (DNAStar Inc.). Eight B-cell epitopes corresponding to 89-133 sites amino acids domain were selected, synthesized and coupled with keyhole limpet hemocyanin (KLH), or bovine serum albumin (BSA) by Xi'an Huachen Bio-technology Co., Ltd, China. Homology analysis using the BLAST function of NCBI didn't show any peptide sequences which share significant homology with other proteins with the exception of Eppin protein itself. The purity of each peptide was above 90% as analyzed by reverse phase high-performance liquid chromatography (HPLC).③All eight peptides can elicit similar specific antibody titers, however,only the antisera from F5 showed the capacity to combined with natural Eppin in semen,and significantly decreased the rate of pregnancy in mice. Therefore the immunodominant neutralizing B-cell epitope of Eppin is F5: FVYGGCQGNN (Eppin 105-114).④High–anti-Eppin titers of 1:400 ~ 1600 were detected in eight of the ten monkeys immunized with Eppin MAP; two monkeys were low responders with titers <1:400 on postimmunization day 120. Because the purpose of this study was to test the efficacy of antibodies to Eppin MAP on fertility, the two low-titer monkeys were dropped from the study group and their fertility was not tested. Each male monkey in the immune and control groups were subjected to fertility testing by cohabiting with a proven fertile female during her two ovulatory cycles. Three out of eight males in the immune group were infertile and one out of five males in the control group was infertile. This revealed the MAP of Eppin had an affirmative contraceptive effect. The electron microscope examination of testis biopsy also showed no obvious abnomality. But in the detecting of Th1/Th2 cytokines, there was no significant difference in the males of immune and control groups.The results of this research indicates that epitope prediction by bioinformatics method combinated with synthetic peptide coupling carrier protein immunization strategy is an an effective way and a beneficial alternative to analysis B-cell epitope.The immunodominant neutralizing B-cell epitope of Eppin located at the 105-114 amnio acids.Epitope peptide of Eppin is an effective and safe modality for male contraception.However contraceptive vaccine based on Eppin dominant epitope needs further modification on the level of epitope to meet the requirements of contraceptive vaccine for human use.