Construction of a DNA vaccine encoding Flk-1 extracellular domain and C3d fusion gene and investigation of its suppressing effect on tumor growth

Abstract

Although the critical role of complement component C3d as a molecular adjuvant in preventing virus infection is well established, its role in cancer prophylaxis and treatment is unclear. In this study, we constructed a recombinant plasmid encoding Flk-1 and C3d3 fusion proteins and investigated its transient expression in vitro in transfected eukaryotic cells and its antibody response in immunized mice. Subsequently, we investigated the vaccine's ability to elicit an immune response leading to suppression of angiogenesis and tumor growth in mice bearing bladder transitional cell carcinoma. Using Western blotting, immunocytochemistry, and flow cytometry, we detected the expression of Flk-1 and C3d3 fusion proteins in COS-7 cells transfected with these recombinant plasmids. Further binding experiment using CR2 (C3d receptor) positive Raji cells that were incubated with transfected COS-7 supernatant indicated that C3d was successfully fused to Flk-1. Although both vaccines elicited peak antibody levels at 5 weeks, Flk-1-specific antibody titer in pSG.SS.Flk-1(ECD).C3d3.YL-immunized mice was significantly higher when compared to pSG.SS.Flk-1(ECD).YL-immunized mice. The results of experiments with bladder tumor-bearing mice showed that the vaccine inhibited tumor growth significantly. These results suggest that C3d plays a critical role in tumor immunotherapy by promoting antibody response in Flk-1-based DNA vaccines. This approach may provide a new strategy for the rational design of anti-angiogenic therapies for the treatment of solid tumors and provide a basis for the further exploitation and application of the anti-angiogenesis DNA vaccines.

Complement C5a acts as molecular adjuvant in fish by enhancing antibody response to soluble antigen

“…Development of novel vaccines based on the use of complement molecules as adjuvant is currently under investigation in mammals [7,8]. In this regard, several complement activation fragments, such as C3d and C5a have been shown to enhance antibody and antigenspecific cytotoxic T lymphocyte (CTL) responses respectively thereby acting as molecular adjuvants [7,9].…”

Genetic Immunization with Plasmid DNA Mediated by Electrotransfer

“…In the case of the HIV gp120, fusion to an IgG provided a more effective protection against a chimera virus challenge (Shimada et al, 2010), and MIPa chemokine helped for APC targeting (Ruffini et al, 2010). Fusion of the host-derived component of complement C3d to the extracellular region of murine VEGFR-2 led to a significantly increased survival in mice melanoma and colon cancer models by inducing a strong humoral response , with similar findings in a bladder cancer model (Liang et al, 2010).…”

Vaccination approach to anti-angiogenic treatment of cancer

“…Similarly to the group of Niethammer et al, Liang et al [69] also designed a DNA vaccine aimed at inducing a specific immune response against VEGFR2. In this case, however, the extracellular domain of Flk-1 was fused to C3d3, a complement fragment that enhances the immune response and is applied as adjuvant.…”