Molecular Adjuvant C3d3 Improves Humoral Immune Response Of HCGβ DNA And Mucosal Vaccination Through Promoting ASC Trafficking And Homing

Research Background Immuno-contraception based on hCGβwas the only one thathas accomplished clinicalⅡtrial. But the most shortcomings of this approach werethat the antibody responses were variable from recipient to recipient, and those withinadequate antibody levels, especially in reproductive tract, could not be protectedfrom pregnancy. Thus it is imperative to probe into an optimal strategy to improve theantibody response to the hCGβcontraceptive vaccine. C3d as a molecular adjuvantdiminished the individual differences in population, and enhanced humoral immuneresponse. In our previous study, we have successfully constructed hCGβDNAvaccination pCMV4-hCGβ-C3d3 and hCGβmucosal vaccination Lb.hCGβ-C3d3, anddemonstrated that the molecular adjuvant C3d3 increases humoral immune responseagainst hCG antigen in mouse in vivo and human in vitro. However, the preciseefficient mechanisms of hCGβDNA and mucosal vaccination as well as molecularadjuvant C3d3 are to be elucidated further.Objective In the present study, BALB/c mice were inoculated with hCGβDNA andhCGβmucosal vaccination via vagina, respectively. The anti-hCGβhumoralresponses and the efficiency of molecular adjuvant C3d3 were investigated. Theexpression of chemokine receptors/chemokines and adhesion molecules in ASC, andsplenic and uterine tissue were analyzed. This study is to elucidate the mechanisms ofthe ASC trafficking and homing to genital tract, to explain the potential anti-fertilityefficiency of hCGβDNA and hCGβmucosal vaccination.Molecular adjuvant C3d3 promotes trafficking of the ASCs and theantigen-specific humoral immune response through up-regulation of CXCR4and CD62L expression on ASCs in hCGβDNA vaccinationMethods BALB/c mice were inoculated intramuscularly with pCMV4-hCGβ-C3d3,pCMV4-hCGβand pCMV4, respectively. The titers of anti-hCGβIgG and IgA antibody in serum and vaginal douche were determined by indirect ELISA. The IgGand IgA ASC levels were evaluated by ELISPOT. The expressions of chemokinereceptors on B cells were analyzed by RT-PCR, and CXCR4 expressions wereanalyzed respectively by RT-PCR and FCM. The expressions of CXCL12 in spleenwere investigated respectively by RT-PCR and ELISA. Chemotaxis assay was tocheck the chemotactic effects of SDF-1 on peripheral B cell and local B cell from thedifferent immunized groups, and ELISPOT was to analyze the numbers of themigrated ASC. CD62L/VLA/LFA expressions on B cell were analyzed by FCM, andMAdCAM/VCAM/ICAM expressions in spleen were analyzed by IHC.Results In DNA immunization, we found that anti-hCGβIgG antibody titer in serumafter pCMV4-hCGβ-C3d3 immunization was significantly higher than that ofpCMV4-hCGβimmunization, but the anti-hCGβIgA antibody titers in sera andanti-hCGβIgG and IgA antibody titer in douche appeared no difference between thetwo groups. The level of IgG ASC in spleen of pCMV4-hCGβ-C3d3 immunizationwas significantly higher than that of pCMV4-hCGβimmunization, but the levels ofIgA ASC in spleen and IgG and IgA ASC in uterus appeared no difference betweentwo groups. The DNA immunization induced principally systemic humoral response.The transcriptions of CXCR4 in B cells increased after pCMV4-hCGβ-C3d3immunization, and significantly higher than that of pCMV4-hCGβimmunization.CXCL12 production increased after pCMV4-hCGβand pCMV4-hCGβ-C3d3immunization, but appeared no difference between the two groups, which suggestedthat C3d3 up-regulate the expression of CXCR4 on B cell, rather than the secretion ofCXCL12 in spleen. It was showed in chemotaxis assay that the splenic supernatants ofpCMV4-hCGβand pCMV4-hCGβ-C3d3 had effect on peripheral B cell chemotaxis,and anti-SDF-1 antibody could block it partly, there was no difference in chemotaxisbetween pCMV4-hCGβand pCMV4-hCGβ-C3d3 groups. Splenic B cells ofpCMV4-hCGβand pCMV4-hCGβ-C3d3 groups migrated to SDF-1, which could beblocked by anti-CXCR4 antibody, and the number of B cell of pCMV4-hCGβ-C3d3immunization was more significantly than that of pCMV4-hCGβ. The number of themigrated ASC of pCMV4-hCGβ-C3d3 immunization was more significantly than thatof pCMV4-hCGβ. It is suggested that C3d3 can enhance the expression of CXCR4 onASC, with which ASC responded to CXCL12, and their trafficking was promoted.LFA and VLA expressions were low, and there is no difference among all the immunized groups. CD62L expression increased after DNA immunization, theexpression of CD62L of pCMV4-hCGβ-C3d3 was significantly higher than that ofpCMV4-hCGβ. The adhesion molecules MAdCAM/VCAM/ICAM were expressedin all the immunization groups, but there was no difference among groups.Conclusions DNA immunization induced mainly systemic humoral response. Themolecular adjuvant C3d3 up-regulates expression of CXCR4 and CD62L on ASC inspleen, promotes ASC trafficking through CXCR4/CXCL12 and CD62L/MAdCAMinteraction, and enhances systemic humoral response in DNA immunization..Molecular adjuvant C3d3 promoted ASC homing and antigen-specific mucosalimmune response through up-regulation of CCR9 and VLA expression on ASCin hCGβmucosal vaccination via vaginaMethods BALB/c mice were inoculated intravaginally with Lb.hCGβ-C3d3,Lb.hCGβ, and Lb.pIlac, respectively. The titers of anti-hCGβIgG and IgA antibody inserum and vaginal douche were determined by indirect ELISA. The IgG and IgAASCs levels were evaluated by ELISPOT. The expressions of chemokine receptors onB cells were analyzed by RT-PCR, and CCR9 expressions were analyzed by RT-PCRand FCM, respectively. The expression of CCL25 was investigated by RT-PCR andELISA, respectively. Chemotaxis assay was used to check the chemotactic effects ofTECK on the peripheral B cell and local B cell from different immunized groups, andELISPOT was used to analyze the numbers of the migrated ASC. Expressions ofCD62L/VLA/LFA on B cell were analyzed by FCM, and expressions ofMAdCAM/VCAM/ICAM in uterus were analyzed by IHC.Results In mucosal immunization, we found that anti-hCGβIgG antibody titer inserum of pIlac-hCGβ-C3d3 immunization was significantly higher than that ofpIlac-hCGβimmunization, but the anti-hCGβIgA antibody titers appeared nodifference between the two groups. The anti-hCGβIgG and IgA titers in douche ofpIlac-hCGβ-C3d3 were higher significantly than that of pIlac-hCGβ. The levels ofIgG and IgA ASC in uterus of pIlac-hCGβ-C3d3 immunization were significantlyhigher than that of pIlac-hCGβimmunization, but the levels of IgG and IgA ASC inspleen appeared no difference between two groups. The transcriptions of CCR9 in B cell increased after pIlac-hCGβ-C3d3immunization, and significantly higher than that of pIlac-hCGβimmunization, and itsligand CCL25 production increased after pllac-hCGβand pIlac-hCGβ-C3d3immunization, but appeared no difference between two groups. It was suggested thatC3d3 increased the expression of CCR9 on B cell, rather than the secretion of CCL25in the genital tissue. It was showed by chemotaxis assay that the uterine supernatantsafter the pIlac-hCGβand pIlac-hCGβ-C3d3 inoculation in vagina had effect onperipheral B cell chemotaxis, and anti-TECK antibody could block it partly, there wasno difference in this chemotactic effect between pIlac-hCGβand pIlac-hCGβ-C3d3groups. The uterine B cells of pIlac-hCGβand pIlac-hCGβ-C3d3 groups migrated toTECK, and the number of migrated B cell of pIlac-hCGβ-C3d3 was moresignificantly than pIlac-hCGβ. The number of migrated ASC of pIlac-hCGβ-C3d3was more significantly than that of pIlac-hCGβ. It was suggested that C3d3 couldenhance the expression of CCR9 on ASC, with which ASC responded to CCL25, andhomed more into the genital tract. The LFA and CD62L expressions were low, andthere is no difference between all the immunized groups. The VLA expressionincreased after the mucosal immunization, and the expression of VLA ofpIlac-hCGβ-C3d3 immunization was higher significantly than that of pIlac-hCGβ. NoMAdCAM was expressed in any immunized groups, VCAM was only expressed inpIlac-hCGβ-C3d3 group, ICAM was expressed in all the immunized groups, and therewas no difference among these inoculated groups.Conclusions The hCGβmucosal vaccination via vagina could induce not onlysystemic but also mucosal humoral immune response in genital tract. The molecularadjuvant C3d3 could enhance the antigen-specific humoral response in mucosalimmunization, and increased the expression of CCR9 and VLA on ASC, facilitatedASC homing to genital tract through CCR9/CCL25 and VLA/VCAM interaction.