Anti-fertile Potential And Humoral Immune Reaction Of Polypeptide Vaccine Based On Fusogenic Trophoblast Epitope

Objective:Based on fusogenic epitope mimic epeptid of trophoblastic cell(P09-04), antigenic peptides for immunocontraception were designed and synthesized to immune female C57BL/6 mice. Then we observed the antigenic peptides'humoral immune response and evaluate their immunogenicity to explore how to obtain an effective epitope polypeptide vaccine from epitope mimic peptide. Anti-fertile potential of the polypeptide vaccine was evaluated by studying the cross-reactivity between anti-serum induced by the epitope polypeptide and natural antigens, and anti-serum's effect on inhibiting intercellular fusion of BeWo choriocarcinoma cells in vitro.Methods:1. Based on fusogenic epitope mimic peptide and synthetic non-natural Pan DR T Helper Epitope(PADRE), both Multiple Antigenic Peptide (PADRE-P09-04)8-MAP and linear structural peptide (PADRE-P09-04) were designed and subsequently synthesized on the peptide synthesizer. After the peptide was synthesized, they were purified in high performance liquid chromatography (HPLC) and identified in mass spectrometer.2. Three antigens such as MAP (P1) ,linear structural peptide(P2), and epitope mimic peptide(P3) were combined with the equivalence Freund's adjuvant to immunize the female C57BL/6 mice(100μg per mice) subcutaneouly at week 0, 2 and 4 respectively. To compare the immunogenicity of the three peptide antigens, the presence and reactivity of antibody (IgG and IgA) in serum and uterine mucous membrane washings were analyzed against epitope mimic peptide by ELISA at week 2, 5, 8, 10, and 12 after first immunization .3. Fresh chorionic tissue was obtained from female patients undergoing induced abortion during their early timester of normal pregnancy. Murine anti-serum was applied as first antibody while normal murine serum without immunization was used as negative control. The potency of human trophoblastic cell related antigen epitope was identified by the specific antibody in immunized serum. 4. Forskolin-induced choriocarcinoma BeWo cells were applied to mimic the process of differentiation and fusion of villous cytotrophoblast in vivo. Firstly, we applied MTT assay to detect the proliferation and activity of BeWo cells under the influence of murine serum. Then, we divided the cells into four groups: GroupⅠ: BeWo cells were cultured in HAM'S F12 medium; GroupⅡ: BeWo cells were cultured in medium containing 100μM forskolin ; GroupⅢ: BeWo cells were cultured medium containing 100μM forskolin and 10% unimmunized murine serum; GroupⅣ: BeWo cells were cultured medium containing 100μM forskolin and 10% murine anti-serum. After 48 hours culture, the membrane and nuclear of BeWo cells were marked by immunofluorescence., then intercellular fusion was quantified to analyze the potency of anti-serum on inhibiting the fusion of BeWo cells.Results:1. Purified synthetic peptides were demonstrated as mono peak by HPL and their purity could reach 95% after integral calculation. The mass chromatographic analysis indicated molecular weight of synthetic peptides were 23372 and 2826.3 and 1727.8 respectively, which were identical with theorical molecular weight.2. Detection of anti-epitope mimic polypeptide IgG in murie serum: synthetic peptide P1 became positive at week 5, the antibody levels were related with the numbers of immunization and time and reached maximum (1:1200) at week 10. Low levels of mimic eptipe IgG antibody (1:200)was detected at week 8 in synthetic peptide P2 group, but the levels of antibody did not show significant changes through time. In synthetic peptide P3 group, no antibody could be detected in our experiment in synthetic peptide P3 group and did not show significant difference when comparing with normal controls. In all, polypeptide induced antibody titer showed the trend to decrease from PI to P2 to P3 in five detections.3. Detection of anti-epitope mimic polypeptide IgA in murie uterine mucous membrane washings: Low levels of mimic eptipe IgA antibody could be detected at week 8 and reached maximum at week 10 in synthetic peptide P1 group . But there were no specific antibody detected in synthetic peptide P2 and P3 groups, also there were no significant differences between normal controls , synthetic peptide P2 group, and synthetic peptide P3 group. In all, polypeptide induced antibody titer showed the trend to decrease from PI to P2 to P3 in five detection.4. Immunized serum in P1 group served as first antibody. Then immunohistochemistry showed the specific binding between anit-serum, and syncytiotrophoblast as well as cytotrophoblast in chorionic tissue from female patients undergoing induced abortion during their early timester of normal pregnancy. The binding mainly happened on cell membrane and in Kytoplasma. But no reaction could be drawn between anti-serum and other tissue cells in chorionic villi. There is no reaction between normal murine serum and human chorionic villi.5. MTT showed murine serum had no effect on proliferation and activity of BeWo cells, which permitted the next step of comparing the fusion rate in all groups. Choriocarcinoma BeWo cells were applied to mimic the differentiation and fusion of villous cytotrophoblast in vivo, and the results demonstrated that , under the induce of forskolin, the fusion rate between BeWo cells increased from 1.59% (groupⅠ) to 11.45%(groupⅡ); while under the influence of 10% anti-serum, the intercellular fusion rate decreased to 6.97 % ( groupⅣ) . There is significant difference betweenⅡgroup andⅣgroup(P<0.01) .The fusion rate in group III with normal murine serum was 11.1%. There was no significant difference between group III and groupⅡ, while the results between group III and group IV was contrary(P<0.01).Conclusion:MAP constructive epitope polypeptide based on fusogenic epitope mimic epeptid of trophoblastic cell and one general Th cells epitope(PADRE) was proved to possess good immunogenicity and induce strong humoral immune reaction in female C57BL/6 mice, furthermore its antirserum could identify natural antigen epitope of human trophoblastic cell and significantly inhibited the intercellular fusion of BeWo choriocarcinoma cells. Our study demonstrated that MAP constructive peptide with fusogenic epitope mimic epeptid of human trophoblastic cell was capable of antireproduction. It is feasible to design immunocontraception vaccine based on this epitope mimic peptide. Our study provided basic theory for immunocontraception vaccine acting on trophoblastic cell.