title: Fusion of hC3d3 to hCGbeta enhances responsiveness in vitro of human peripheral immunocompetent cells upon the antigen primary challenge.
abstract: Contraceptive vaccines based on hCGbeta have not met clinical application because of poor immunogenicity. In the present study, the eukaryotic expression vectors pCI-gs-signal-6His-hCGbeta and pCI-gs-signal-6His-hCGbeta-hC3d3 were constructed, and transfected into CHO cells with aid of Lipofectaine 2000 reagent to gain the secretory recombinant protein. Isolated B cells from human peripheral blood, combined B cells with T cells, and PBMC were treated in vitro, respectively, with 1 nM, 10 nM, 100 nM hCGbeta, hCGbeta-hC3d3 or PWM for 12 days. Immunoglobulin (Ig) and anti-hCG antibody levels in the supernatant were measured by an indirect enzyme-linked immunosorbent assay (ELISA). The expressions of CD80/CD86 on B cells, and CD154/CD25 on T cells, were analyzed by flow cytometry (FCM), and IL-2 production was assayed by ELISA. It was found that the Ig levels in the B-cell supernatants, the combined B with T cells, and PBMC treated with 100 nM hCGbeta-C3d3 fusion protein were 4-fold, 10-fold and 10.9-fold more, respectively, than that of hCGbeta. The anti-hCG antibody could be produced in the combined B cells with T cells, as well as PBMC challenged with 100 nM hCGbeta-C3d3, but no anti-hCG antibody was produced in the challenge with hCGbeta. The hCGbeta-hC3d3 fusion protein enhanced the expression of CD80 and CD86 on B cells, especially CD86 (P<0.05), and significantly increased the expression of CD154 and CD25 molecules on T cells compared to that of hCGbeta (P<0.05). The hCGbeta-hC3d3 promoted human PBMC producing more IL-2 than hCGbeta. These findings indicate that the fusion of hC3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may contribute to a more efficient humoral immune response, and might provide a potential application of protein vaccine strategies in humans in the future.
authors.affiliation: Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Hua-Ping
authors.initials: HP
authors.lastname: Li
authors.forename: Xiao-Ju
authors.initials: XJ
authors.lastname: He
authors.forename: Chuan-ling
authors.initials: CL
authors.lastname: Tang
authors.forename: Xiao-Ying
authors.initials: XY
authors.lastname: Yao
authors.forename: Da-Jin
authors.initials: DJ
authors.lastname: Li
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018122:B7-1 Antigen / Q000276:immunology / Q000378:metabolism; D051940:B7-2 Antigen / Q000276:immunology / Q000378:metabolism; D023201:CD40 Ligand / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000276:immunology* / Q000378:metabolism; D015933:Complement C3d / Q000276:immunology* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology / Q000378:metabolism; D008211:Lymphocyte Cooperation; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014162:Transfection; D022642:Vaccines, Contraceptive / Q000276:immunology
chemical_list: D018122:B7-1 Antigen; D051940:B7-2 Antigen; D018997:Chorionic Gonadotropin, beta Subunit, Human; C508594:IL2 protein, human; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins; D022642:Vaccines, Contraceptive; D023201:CD40 Ligand; D015933:Complement C3d