Analysis Of B-cell Epitope Of Eppin And Study On Its Anti-fertility Potential In Adult Male Mice

The global population expands rapidly and the current family planning methods can't meet the needs of different people in their reproductive lives, which urge the development of additional contraceptive methods. There are several birth control methods for women; however, for men, the available choices are limited to condoms and vasectomy. Therefore, it is necessary to develop male contraceptives that are safer, more effective, economical, and reversible as well. Contraceptive vaccine (CV) has been taken for many years as a promising approach with such potential advantages. Till today, there are many candidate molecules for male contraceptive vaccine development, but none of them has been proved to be a successful contraceptive agent. The two main limitations confronted by CV development are imcomplete fertility inhibition and immune injury mediated by cytotoxic T lymphocyte (CTL). It is of great importance to design a new vaccine modality which could break through the limitations.One promising candidate for the development of immunocontraceptives is Eppin (epididymis protease inhibitor),a ~ 133 amino acid protein secreted by the epididymidis/ testis in an androgen-dependent manner, which associates with the sperm surface during epididymal transit and seems to be important for sperm maturation. Eppin on the surface of spermatozoa and in semen is bound to semenogelin, which is involved in coagulum formation in the ejaculate. Antibodies to Eppin interfere with the normal interaction of Eppin with the sperm surface and with semenogelin, which can to some degree explain the infertility due to Eppin. When used for active immunization of male monkey, eppin shows the capacity to block fertility. Seven out of nine male monkeys developed high titers specific to Eppin, and all of these high-titer monkeys were infertile. Five out of seven high-titer males recovered fertility when stopping innoculating. This study revealed that Eppin is a promising target for male contraception. However, there still needs further research to identify its immunodominant neutralizing B-cell epitopes and decide its efficacy and safety as a CV for males.However, previous studies showed that the full-length nature protein was generally not suitable for vaccine immunogen on considering of safety. This might be an obstacle of Eppin as a whole protein to be a successful contraceptive vaccine for human use. In view of this, studies should be geared towards delineating appropriate sperm-specific epitopes and enhancing the immunogenic and efficacy on the epitope peptide. We supposed theoretically that priming with full length Eppin could induce a large population of B-cells specific to a variety of epitopes, but when boosting with an epitope-based peptide, only epitope-specific memory cells undergo rapid expansion upon re-exposure to the same antigen but not the other population of B-cells. Immunodominant epitope could thus be decided by the antibody titers and the neutralizing eptitope by the bioactivity of the epitope-specific antibody. Therefore we employed the Eppin prime-epitope peptide boost strategy to identify the immunodominant neutralizing B-cell epitope of Eppin and determine its immunocontraceptive efficacy simultaneously.Eppin cDNA was obtained by RT-PCR from fresh human testis, and then cloned into prokaryotic expression plasmid which was induced by IPTG. Recombinant human Eppin (rhEppin) was purified and identified (PartⅠ). On the other hand, the B-cell epitope of Eppin was preliminarily predicted through its secondary structure and surface characteristics, then the predicted epitope peptide were scanned by peptide combination assay thus got the candidate B-cell epitopes of Eppin (PartⅡ). Finally, the modality of rhEppin prime-epitope peptide boost was employed to further analyze the four candidate B-cell epitopes (PartⅢ).The results showed that:①By preliminarily analyzing Eppin secondary structure and surface characteristics, the predicted B-cell epitope of Eppin located at 19～31,30～44,58～70,73～85,94～102,103～115,113～124,125～132 domains which were scanned by peptide combination assay and found that 58～70,19～31,103～115,113~124 domain showed positive results and thus got the candidate B-cell epitope of Eppin (the four eptitopes were designated as peptide A~D respectively).②Further analyzing the four candidate B-cell epitopes by the strategy of rhEppin prime-epitope peptide boost, the result showed that all the four candidate epitopes could induce high antibody titers which maintained 8~9 months after immunization, however, only the antisera from peptide B, C and D boosted mice showed the capacity to combined with natural Eppin in semen. Most interestingly, only the antisera from peptide C boosted mice exhibit the inhibition potential on human sperm motility. Therefore the immunodomiant neutralizing B-cell epitope of Eppin is Eppin103-115.③The mice received rhEppin prime-epitope peptide C boost (EC group) could fertilize 26.6% of the female mice caged with them, which was significantly lower than the control (88.9%). Eight to nine months later when the antibody titers reduced, the mice of EC group regained its fertility (the fertility rate of co-caged female mice is 80%), which showed no difference with the PBS group. The result indicated that the contraceptive effect was reversible.④The mice received rhEppin repeated immunization (Eppin group) had a slightly higher antibody titres, and a similar antifertility efficacy with EC group. The protein prime-peptide boost strategy is theoretically safer because the antibodies induced by protein prime-peptide boost strategy were mainly directed to epitope peptide C. However, mice from both Eppin group and EC group showed no change of sex hormones and spermatogenesis. Pathologic examination of the important organs showed no abnormality.The results of this research indicated that epitope prediction by computer combined with the protein prime-peptide boost strategy is an effective method and a beneficial alternative to analyse B-cell epitope. The immunodominant neutralizing B-cell epitope of Eppin located at the 103~115 amnio acids. And more importantly, rhEppin prime-epitope peptide C boost is an effective and safe modality for male contraception. On thinking of the genetic difference between human and mice, the research result needs further confirmation in non-human primate.