Fusion of hC3d3 to hCGbeta enhances responsiveness in vitro of human peripheral immunocompetent cells upon the antigen primary challenge.
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.
Li
Hua-Ping
HP
Laboratory for Reproductive Immunology, Hospital and Institute of Obstetrics & Gynecology, Fudan University Shanghai Medical College, Shanghai, China.
He
Xiao-Ju
XJ
Tang
Chuan-ling
CL
Yao
Xiao-Ying
XY
Li
Da-Jin
DJ
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
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