| Contraceptive vaccine, based on gamete production [luteinizing hormone-releasing hormone (LHRH)/GnRH, FSH], gamete function [sperm antigens and oocyte zona pellucida (ZP)], and pregnancy outcome (HCG), has showed different degrees of efficacy. Among them, the hCGβcontraceptive vaccine is the first and only birth control vaccine to go through PhaseⅡefficacy trials successfully, which can induce humoral response to the hCGβand protect sexually active women from becoming pregnant via blocking hCG bioactivity. The anti-fertility potential of hCGβcontraceptive vaccine can be improved if more lymphocytes are recruited into the genital tract to generate adequate antibodies. In our previous work, a recombinant Lactobacilli live vaccine, Lb.hCGβ-C3d3 were successfully constructed and could induce a high level of systemic and local mucosal immune response. However, the anti-fertility mechanisms of the Lb.hCGβ-C3d3 live vaccine is still need to be evaluated. Therefore, in the present study, we analyzed the anti-fertility potential and the lymphocyte homing mechanism by inoculating via vagina with the recombinant live Lactobacilli expressing hCGβ-C3d3 fusion protein, in order to improve the efficacy of the vaccine, which makes it more feasible for clinical application.PartⅠ. The anti-fertility potential of recombinant live Lactobacilli Lb. hCGβ-C3d3 vaccination via vaginaObjective:To evaluate the anti-fertility potential of vaccination with the recombinant live Lactobacilli expressing hCG(3-C3d3 fusion protein via vagina.Methods:Female BALB/c mice of 6-8 weeks were immunized with 1010,109 Lb, Lb.hCGβor Lb.hCGβ-C3d3, respectively, via vagina, and boosted three weeks later. The anti-serum was collected respectively at the 6-8th week after the primary inoculation. MLTC-1, BeWo cell lines and primary human villous trophoblast cells were respectively treated with the anti-serum, and then the supernatant was collected, respectively. Progesterone and hCGβin the supernatant were determined by Chemoluminescence assay. hPL was detected by ELISA. Fusion of the BeWo cells was investigated by immunofluorescence microscopy.Results:The anti-serum induced by the Lb.hCGβ-C3d3 inoculation with the dose of 1010 could significantly inhibit the progesterone production in MLTC-1 cells via neutralizing hCG bioactivity. The secreting of hCGβand hPL by BeWo cell and primary human villous trophoblast cells were suppressed, and the cell fusion of BeWo was inhibited in vitro by the anti-serum.Conclusion Vaccination with the Lb.hCGβ-C3d3 live Lactobacilli is effective in blocking hCG bioactivity, which appears anti-fertility potential.PartⅡ. The molecular adjuvant C3d3 in the recombinant live Lactobacilli Lb. hCGβ-C3d3 vaccination via vagina promotes B lymphocyte homing into genital tract by up-regulating CCL25 secretionObjective:To investigate the lymphocyte homing mechanism mediated by the molecular adjuvant C3d3 after the vaccination with recombinant live Lactobacilli expressing hCGβ-C3d3 fusion protein via vagina.Methods:Female BALB/c mice of 6-8 weeks were immunized with 1010 Lb, Lb.hCGβor Lb.hCGβ-C3d3, respectively, via vagina, and then boosted three weeks later. The CD19+B, CD4+T cells were purified from splenocytes of the BALB/c mice immunized with Lb.hCGβ-C3d3 by magnetic beads, and then labeled with CFSE. The mice immunized above were adoptively transferred with the labeled CD19+B, CD4+T cells via tail vein, respectively. The mice transferred by the immuno-competent cells were injected intraperitoneally with a monoclonal antibody (mAb) against CCL25 at the same time. In 96 hours after the adoptive transfer, two-photon confocal microscopy was used to image the positioning of CFSE-labeled cells within the host uterus and vagina. Western blot was used to analyze the expression of E-cadherin and E-selectin in the genital tract.Results:In the Lb.hCGβ-C3d3 immunized mice, more T and B cells were in the uterus than that of vaccination without C3d3. Both the labeled T, B cells were found in uterus in 24 hours after the adoptive transfer, and reached the highest in the 96h, and anti-CCL25 monoclonal antibodies inhibited significantly the C3d3-induced B lymphocyte homing, but no change in T cell homing. In the Lb.hCGβ-C3d3 immunized mice, both the expression of E-cadherin and E-selectin in the genital tract were significantly higher than that of immunization without C3d3.Conclusion The Lb.hCGβ-C3d3 mucosal immunization via vagina can promote lymphocytes homing into the uterus and increase the expression of E-cadherin and E-selectin in genital tract. The chemokine CCL25 is important for the B cells homing into the genital tract.
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