附睾蛋白酶抑制蛋白的B细胞表位分析及抗生育效应研究
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摘要
世界人口持续增长的压力和非计划妊娠对生殖健康的危害使人们深刻认识到进一步完善避孕措施的迫切性。目前针对男性的避孕措施仅限于安全套和输精管结扎,前者不够方便,后者不可逆,因而远远不能满足需求。开发更方便、高效、安全、可逆的男性避孕方法成为计划生育研究的重要课题。迄今,男性避孕疫苗候选分子很多,但尚面临避孕效果不理想以及T细胞介导的免疫损伤等局限。针对这两个局限,设计新的疫苗形式和免疫策略实现突破,对开发安全、有效的避孕疫苗具有重要意义。
附睾蛋白酶抑制蛋白(Epidydimal protease inhibitor, Eppin)基因的发现为男性避孕疫苗的开发带来了曙光。Eppin蛋白特异表达于睾丸和附睾,在精子成熟中具有重要作用。抗Eppin抗体可直接破坏Eppin-精囊凝固蛋白(Semenogelin, Sg)复合体,干扰Eppin调控前列腺特异性抗原对Sg的水解作用,影响精子活动力,进而抑制生育力。利用重组Eppin蛋白免疫雄猴获得了较理想的避孕效果(78%),停止免疫后,71%的受试雄猴恢复了生育力。由此可见,Eppin可望作为男性避孕的有效靶点,但仍需针对男性避孕疫苗存在的上述局限寻求突破。筛选获得Eppin优势中和B细胞表位,并通过免疫小鼠研究其抗生育有效性和安全性是本研究的目的。
出于安全考虑,全长蛋白不适于用作疫苗,找出真正抑制生育的关键性表位并增强其免疫原性和避孕效果是关键。基于免疫学基本理论,我们设想,用全长Eppin蛋白免疫,将会产生针对其各个表位的记忆细胞克隆,当只用某个表位肽再次免疫时,则只有针对该表位的记忆细胞被激活、增殖进而分泌抗体。如果初次免疫采用重组人Eppin蛋白(recombinant human Eppin, rhEppin),加强免疫分别采用各候选表位肽,则可以通过所产生的抗体滴度高低来判断该表位是否优势表位;而通过分析被免疫小鼠的生育力抑制效果及其抗血清对精子活力的体外抑制能力,则可判断该表位是否中和性表位。因此,我们拟采用该策略来筛选Eppin的优势中和B细胞表位并研究其抗生育潜能。
本研究首先通过RT-PCR法从人新鲜睾丸组织扩增获得Eppin cDNA,构建入原核表达载体,诱导表达并经纯化获得重组人Eppin蛋白(第一部分)。同时,通过计算机软件分析Eppin蛋白二级结构及表面特性,预测出Eppin蛋白的B细胞表位所在区域;并通过与抗血清的结合试验进行初步筛选,得出Eppin候选优势B细胞表位区段(第二部分)。最后,采用rhEppin初次免疫,上述候选表位肽加强免疫的策略,观察各个表位肽刺激抗体产生、抗体对精子活力的抑制能力,以此进一步筛选出其优势中和B细胞表位,并以rhEppin重复免疫为对照,分析其抗生育潜能及安全性(第三部分)。
结果发现:①通过分析Eppin蛋白二级结构及表面特性(包括蛋白质的理化性质、亲水性、可塑性、溶剂可及性及免疫原性等),初步预测出Eppin蛋白的B细胞表位区域在第19～31、30～44、58～70、73～85、94～102、103～115、113～124、125～132区段;与抗血清的结合试验发现第58～70、19～31、103～115、113~124区域的抗原性相对较强,从而初步筛选得出了Eppin候选优势B细胞表位(前述四个肽分别命名为肽A～D)。②进一步采用rhEppin初次免疫-候选表位肽加强免疫的筛选策略,发现用四个候选表位肽加强免疫时都能激发产生高滴度的抗体,并维持至8~9个月,而仅B肽、C肽和D肽加强免疫时所获得的抗血清能与人精子天然Eppin蛋白结合,其中仅C肽加强免疫获得的抗血清能显著抑制人精子活力。从而获知Eppin优势中和B细胞表位所在区段为Eppin103-115。③接受rhEppin初次免疫-表位肽C加强免疫的雄鼠(EC组),只能使26.6%的雌鼠受孕,与PBS组(88.9%)相比显著降低,说明雄鼠的生育力受到抑制;而免疫8～9个月后,伴随抗体滴度下降,该组雄鼠能使雌鼠的受孕率达80%,与PBS组(87%)无显著差别,说明避孕作用具有可逆性。④接受rhEppin重复免疫的雄鼠(Eppin组),产生的抗体滴度虽略高于EC组,而抗生育效果两组相当。Eppin组和EC组都只是显著抑制了附睾精子的活力,而性激素水平、精子生成等均未受影响。各组织器官的病理检查也未发现异常。但理论上,采用rhEppin初次免疫-候选表位肽加强免疫策略,产生的抗体更多针对该表位肽,从而更具有安全性。
本研究结果表明计算机表位预测结合蛋白初免-表位肽加强免疫策略是筛选蛋白优势中和B细胞表位的有效方法和又一选择;Eppin蛋白的第103-115区段是Eppin优势中和B细胞表位所在区域;rhEppin初次免疫-表位肽C加强免疫策略用于免疫避孕具有较好的安全性和有效性。鉴于Eppin蛋白在人和鼠的遗传差异,尚需在灵长类动物进一步研究证实。
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.
附睾蛋白酶抑制蛋白(Epidydimal protease inhibitor, Eppin)基因的发现为男性避孕疫苗的开发带来了曙光。Eppin蛋白特异表达于睾丸和附睾,在精子成熟中具有重要作用。抗Eppin抗体可直接破坏Eppin-精囊凝固蛋白(Semenogelin, Sg)复合体,干扰Eppin调控前列腺特异性抗原对Sg的水解作用,影响精子活动力,进而抑制生育力。利用重组Eppin蛋白免疫雄猴获得了较理想的避孕效果(78%),停止免疫后,71%的受试雄猴恢复了生育力。由此可见,Eppin可望作为男性避孕的有效靶点,但仍需针对男性避孕疫苗存在的上述局限寻求突破。筛选获得Eppin优势中和B细胞表位,并通过免疫小鼠研究其抗生育有效性和安全性是本研究的目的。
出于安全考虑,全长蛋白不适于用作疫苗,找出真正抑制生育的关键性表位并增强其免疫原性和避孕效果是关键。基于免疫学基本理论,我们设想,用全长Eppin蛋白免疫,将会产生针对其各个表位的记忆细胞克隆,当只用某个表位肽再次免疫时,则只有针对该表位的记忆细胞被激活、增殖进而分泌抗体。如果初次免疫采用重组人Eppin蛋白(recombinant human Eppin, rhEppin),加强免疫分别采用各候选表位肽,则可以通过所产生的抗体滴度高低来判断该表位是否优势表位;而通过分析被免疫小鼠的生育力抑制效果及其抗血清对精子活力的体外抑制能力,则可判断该表位是否中和性表位。因此,我们拟采用该策略来筛选Eppin的优势中和B细胞表位并研究其抗生育潜能。
本研究首先通过RT-PCR法从人新鲜睾丸组织扩增获得Eppin cDNA,构建入原核表达载体,诱导表达并经纯化获得重组人Eppin蛋白(第一部分)。同时,通过计算机软件分析Eppin蛋白二级结构及表面特性,预测出Eppin蛋白的B细胞表位所在区域;并通过与抗血清的结合试验进行初步筛选,得出Eppin候选优势B细胞表位区段(第二部分)。最后,采用rhEppin初次免疫,上述候选表位肽加强免疫的策略,观察各个表位肽刺激抗体产生、抗体对精子活力的抑制能力,以此进一步筛选出其优势中和B细胞表位,并以rhEppin重复免疫为对照,分析其抗生育潜能及安全性(第三部分)。
结果发现:①通过分析Eppin蛋白二级结构及表面特性(包括蛋白质的理化性质、亲水性、可塑性、溶剂可及性及免疫原性等),初步预测出Eppin蛋白的B细胞表位区域在第19～31、30～44、58～70、73～85、94～102、103～115、113～124、125～132区段;与抗血清的结合试验发现第58～70、19～31、103～115、113~124区域的抗原性相对较强,从而初步筛选得出了Eppin候选优势B细胞表位(前述四个肽分别命名为肽A～D)。②进一步采用rhEppin初次免疫-候选表位肽加强免疫的筛选策略,发现用四个候选表位肽加强免疫时都能激发产生高滴度的抗体,并维持至8~9个月,而仅B肽、C肽和D肽加强免疫时所获得的抗血清能与人精子天然Eppin蛋白结合,其中仅C肽加强免疫获得的抗血清能显著抑制人精子活力。从而获知Eppin优势中和B细胞表位所在区段为Eppin103-115。③接受rhEppin初次免疫-表位肽C加强免疫的雄鼠(EC组),只能使26.6%的雌鼠受孕,与PBS组(88.9%)相比显著降低,说明雄鼠的生育力受到抑制;而免疫8～9个月后,伴随抗体滴度下降,该组雄鼠能使雌鼠的受孕率达80%,与PBS组(87%)无显著差别,说明避孕作用具有可逆性。④接受rhEppin重复免疫的雄鼠(Eppin组),产生的抗体滴度虽略高于EC组,而抗生育效果两组相当。Eppin组和EC组都只是显著抑制了附睾精子的活力,而性激素水平、精子生成等均未受影响。各组织器官的病理检查也未发现异常。但理论上,采用rhEppin初次免疫-候选表位肽加强免疫策略,产生的抗体更多针对该表位肽,从而更具有安全性。
本研究结果表明计算机表位预测结合蛋白初免-表位肽加强免疫策略是筛选蛋白优势中和B细胞表位的有效方法和又一选择;Eppin蛋白的第103-115区段是Eppin优势中和B细胞表位所在区域;rhEppin初次免疫-表位肽C加强免疫策略用于免疫避孕具有较好的安全性和有效性。鉴于Eppin蛋白在人和鼠的遗传差异,尚需在灵长类动物进一步研究证实。
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.
引文
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