Fusion of hC3d3 to hCGβ enhances responsiveness in vitro of human peripheral immunocompetent cells upon the antigen primary challenge

Abstract 

Contraceptive vaccines based on hCGβ have not met clinical application because of poor immunogenicity. In the present study, the eukaryotic expression vectors pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-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 1nM, 10nM, 100nM hCGβ, hCGβ-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 100nM hCGβ-C3d3 fusion protein were 4-fold, 10-fold and 10.9-fold more, respectively, than that of hCGβ. The anti-hCG antibody could be produced in the combined B cells with T cells, as well as PBMC challenged with 100nM hCGβ-C3d3, but no anti-hCG antibody was produced in the challenge with hCGβ. The hCGβ-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 hCGβ (P<0.05). The hCGβ-hC3d3 promoted human PBMC producing more IL-2 than hCGβ. These findings indicate that the fusion of hC3d3 to hCGβ, 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.

Keywords: hCGβ, Human molecular adjuvant hC3d, CD80/CD86, CD154/CD25, IL-2