Synthesizing Of Eppin Peptide Self-assemblies And Study On Its Immunocontraceptive Effects

In recent years, the researches indicate that the development of contraceptive vaccinebased on epididymal protease inhibitor (Eppin) has made some progress, but still have thefollowing problems:(1) the contraceptive vaccine based on the dominant functional epitopeof Eppin can reduce the adverse reactions, but poorly immunogenic;(2) epitopecross-linked carrier protein can enhance immunogenicity, but the specificity of the antibodyreduced;(3) in animal experiment, the contraceptive vaccine based on Eppin always needimmune adjuvants, such as Freund’s adjuvant, but its not suitable for human. Therefore, thekey step for developing a contraceptive vaccine is to develop a safer, more effectivedelivery system. Previous studies have also shown that the dominant functional epitope ofEppin can induce a specific antibody response and suppress fertility. For this reason, aself-assembled peptide nanofibers vaccine strategy (E-Q11) was developed and carried outto improve upon the immune potential of this functional epitope as a vaccine immunogen.Our research used C57BL/6mice as the study object and investigated the immune response,anti-fertility effect and its mechanism of dendritic cell (DC) induced by self-assembledpeptide (E-Q11). The research was divided into three sections.1. Preparation and characterization of E-Q11self-assembled peptidePreviously, the dominant functional B cell epitope from Eppin was screened andidentified in our laboratory. Herein, a self-assembled peptide material (QQKFQFQFEQQ,Q11) with their significant degree of multivalency which enabled the repetitive display ofepitopes was chosen as the effective delivery system for Eppin. The Eppin dominantfunctional peptide epitope CSMFVYGGCQGNNNNFQSKANC (Eppin102-123) wasconjugated to Q11and synthesized using standard Fmoc chemistry. Then, the fibrillizationbehavior and secondary structure were investigated. The results showed that using ofchemical synthesis method can obtain the fusion polypeptide E-Q11with the purity greaterthan90%. By TEM, it was observed that E-Q11self-assembled into fibrous structures similar to Q11. Secondary structure analysis of E-Q11indicated a predominant β-sheetcharacter similar to Q11. E-Q11showed positive results by peptide combination assay.2. Dendritic cell responses to E-Q11self-assembled peptideMouse bone marrow cell suspensions were prepared with aseptic, and monocytes wereisolated and induced by GM-CSF and IL-4. FITC-labeled Eppin (FITC-E) andFITC-labeled E-Q11(FITC-E-Q11) were used to evaluate the uptake of self-assembledpeptide nanofibers by immature dendritic cells (iDCs), and iDCs were incubated with5and50μg/mL of E and E-Q11peptide to investigate the influence of antigens uptake on DCmaturation. Then the antigen loaded DCs were incubated with CD4+cells from spleen ofC57BL/6mouse in the allo MLR to determine their cytokine levels in supernatants. Theresults show that the CD11c expression rate of DCs cultured in vitro more than70%; E-Q11were efficiently taken up by iDCs, DCs upregulate the maturation marker CD40and CD80in response to incubation with E-Q11. Compared with rhEppin and LPS group, the levels ofIFN-γsecreted by CD4+T cells were deceased in E-Q11group, and the levels of IL-4andIL-5were higher in E-Q11group. In conclusion, E-Q11could induce Th2cell mediatedimmune response.3. Microneedle-based transcutaneous immunization in mice with E-Q11self-assembled peptideCommercially available MNs coated with rhEppin or E peptides or E-Q11peptideswere prepared for vaccine delivery to the skin. Groups of C57BL/6mice were immunizedwith MNs coated with10μg three antigens for delivery to the skin or immunizedsubcutaneously using conventional hypodermic needles with three antigens (80μg or10μg/100μL). The follow-up consisted of immunological features and the contraceptiveeffect.The results showed that:(1) the500-μm MNs were suitable for transcutaneousimmunization in mice. The MNs coated with fluorescent antigens inserted into the mouseskin deposited78%of the coating within60s of insertion. By using the dip-coating method,the three antigens were originally coated onto the500-μm MNs.(2) In addition to the E peptide group (E80-SC, E10-SC, E10-MN) and low-doserhEppin group (r10-SC), the rest of the groups induced high antibody titers whichmaintained8~9months after immunization. We found that low-dose E-Q11and rhEppin vaccination using microneedles were more immunogenic than low-dose subcutaneous (SC)vaccination and similarly immunogenic as high-dose SC vaccination in a mouse model.(3) rhEppin SC immunized mice generated high levels of IgG1and IgG2a. However,mice from both groups MN immunized with E-Q11and rhEppin induced predominantlyIgG1isotype. The IgG1/IgG2a ratio for E-Q11was about2times greater than that forrhEppin, and E-Q11MN immunized was about3times greater than that SC immunized.E-Q11MN immunized mice induced the highest IL-4response, whereas no difference wasseen when compared with that of rhEppin immunized mice. These data suggest thatrhEppin and E-Q11immunized by using MNs induced enhanced Th2-polarized immuneresponses in male mice.(4) There were significant reductions in the average viable fetuses of the80μg-E-Q11-SC,80μg-rhEppin-SC and10μg-rhEppin-MN-immunized groups compared withthat of the PBS-control and E peptide mice. Likewise, the pregnancy rate of the femalesmating with males of80μg-rhEppin-SC,10μg-rhEppin-MN and80μg-E-Q11-SCimmunized groups were13.3%,26.6%and20.0%, respectively, which were significantlylower than the PBS-control and E peptide groups. The HOS test and total sperm viabilitywere greatly inhibited in group rhEppin and E-Q11compared with the PBS-control group.There showed no significant difference in sperm count among groups.