| With the development of society and economy, the aspiration of men to participate in family planning is strongly increasing. However, the choice of contraception is limited so far. Immunocontraceptive vaccines which have multiple advantages have been considered one of the most promising strategies in the male contraception. FSH/FSHR signaling involved in activating spermatogenesis and maintaining normal sperm generation, has important roles in male reproduction. FSHR is only expressed in testis sertoli cells of male, and therefore it is the best target antigen for contraceptive research. Recently, the research on contraceptive vaccines based on FSHR has achieved exciting success. The FSHR32-44 peptide which was filtrated using the computer-based epitope prediction technology of, has been verified to be able to avoid the side effects of the reproductive organ damage, and it has anti-fertility inhibition effects. Currently, there are still some problems which limit its clinical application, such as the poor immunogenicity of FSHR epitope-based vaccine induced low antibody titer, the safety of Freund’s adjuvant used for human, the risk of immune injury caused by immune deviation of the fusion peptide carrier. Nanocarriers are safe, effective, and targeted delivery systems. They have shown unique advantages in drug delivery and tumor vaccines. Therefore, nanoparticulate vaccines based on FSHR were developed in order to solve above problems of the epitope vaccine. In this research, FSHR peptide that does not induce pathological damages was used as the model antigen. Biodegradalbe biomaterials including p H-sensitive Ac-bCD) and non-sensitive PLGA were used as carrier materials, to prepare epitope peptide nanovaccines of FSHR/Ac-bCD NP and FSHR/PLGA NP with different p H-sensitivities, respectively. The experiments were carried out to investigate the effects of immune responses and anti-fertility with these nanovaccines in vitro and in vivo. The studies can be divided into the following three sections.1. Fabrication and characterization of FSHR nanovaccine with different p H- sensitivities.Earlier, the dominant functional epitope peptide(FSHR32-44, IELRFVLTKLRVI) was derived from the follicle-stimulating hormone receptor. The solid-phase synthetic technology was used to synthesize high-purity FSHR32-44 peptide. Preparation of Ac-b CD or PLGA nanoparticles(NPs)loaded with FSHR peptide was performed by a modified emulsion technique. The results demonstrated that the purity of FSHR peptide was more than 95%. Both Ac-b CD and PLGA can successfully package FSHR peptide, resulting in NPs with particle size of about 200 nm. The encapsulation efficiency of FSHR was more than 60%. The obtained NPs were sphericalparticles, with uniform particle size distribution, as observed by TEM and SEM. Hydrolysis and release experiments in vitro confirmed that FSHR/Ac-bCD NP could be quickly hydrolyzed in pH 5.0 PBS, with rapid release of FSHR peptide. By contrast, slow release was observed in p H 7.4 PBS, which was similar to that of PLGA NPs.2. The in vitro immune effects of FSHR nanovaccinesiDCs, induced from bone marrow of mice by culture with cytokines were used for in vitro experiments. The DCs were observed by confocal laser scanning microscopy and flow cytometry techniques, after co-culture with various formulations of FSHR antigen(soluble or NPs). The results showed that NPs could be uptaken by DCs efficiently,with the time and dose dependent manners. NPs have the potential of inducing DCs maturation and up-regulating the phenotypic molecular expression of DCs, such as CD40, CD80, CD86 and MHC-II, especially the FSHR/PLGA NP. NPs could effectively stimulate the T cells proliferation, and induce secretion of large amounts of TNF-α and IFN-γ. There are no significant difference between Ac-b CD and PLGA materials.3. In vivo evaluation of FSHR nanovaccinesC57BL/6 J adult male mice were chosen for in vivo studies, including groupsof PBS, FSHR, FSHR/CFA, FSHR/Ac-b CD NP and FSHR/PLGA NP. The vaccines were administrated at the foot pads, by intracutaneous and subcutaneous multipoint injection, for three times. At the different time points,samples of blood were collected, and the ELISA method was employed to detect the special antibody titer change and the concentration of serum testosterone before and after the first inoculation. The fertility experiment was conductedto examine the effect of contraception at 9 w when the antibody reached the peak of immunization. Subsequently, the sperm quality analysis of epididymis and sperm/egg binding test were carried out. Pathological examination of liver, spleen, kidney, and reproductive organs were implemented by HE staining. The blood-testis barrier(BTB) permeability was detected by using the lanthanum tracer combined with TEM observation.The results revealed that the titers of specific antibody induced by FSHR/CFA, FSHR/Ac-bCD NP and FSHR/PLGA NP groups increased and the titers of nanovaccine groups reached the peak at the 9 w after primary immunization. The highest IgG titer was observed for the PLGA group. Reproductive inhibition and sperm quality analysis results are consistent with the antibody levels. Obvious anti-fertility was found for the three groups with high antibody levels. The lowest anti-fertility of 25% was achieved for the PLGA group. The higher the antibody titer, along with the less total number of sperm, sperm vitality was declining, the more teratospermia percentage. Detect of serum testosterone levels before or after immunization showed no significant changes in all groups. There were no obvious pathological changes in liver, spleen, kidney, testis, and epididymis as indicated by HE sections. Lanthanum nitrate deposited in the basilar membrane or around sertoli cell and was confined to permeate into the seminiferous tubule, suggesting that the blood-testosterone barrier was complete and functionally normal.In conclusion, nanocarriers based on Ac-b CD and PLGA possessed good biocompatibility and adjuvant capacity, without obvious side effects, and they could be used as the vectors of contraceptive vaccines. It is an effective approach to improve immunological effects and target dendritic cells with NPs. It should be noted that the p H-sensitive Ac-bCD NP does not show better efficacy than PLGA NP, with respect toimmune responses. This research provided a new method for optimization and design of contraception vaccines. |
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