The molecular adjuvant mC3d enhances the immunogenicity of FimA from type I fimbriae of Salmonella enterica serovar Enteritidis

Abstract

The fimbriae of Salmonella enterica serovar Enteritidis are used for colonization and invasion into host cells, and have drawn considerable interest because fimbriae can serve as potential immunogens against many pathogenic bacteria that colonize on epithelial surfaces. The purpose of the study is to use a molecular adjuvant, C3d, to enhance the immunogenicity of FimA proteins against Salmonella enterica serovar Enteritidis. FimA of type I fimbriae from Salmonella enteritidis and FimA with one copy of mC3d, two copies of mC3d2 and three copies of mC3d3 were cloned into the expression vector pCold-TF. Soluble fusion proteins of FimA with different copy of mC3d were induced by IPTG and expressed into Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant proteins from pCold-TF-fimA, TF-fimA-mC3d, TF-fimA-mC3d2, TF-fimA-mC3d3 were 70 kDa, 100 kDa, 130 kDa and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visualized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract interaction. The recombinant proteins were purified by Ni-TED (tris-carboxymethyl ethylene diamine), immobilized metal ion affinity chromatography (IMAC). Balb/c mice were subcutaneously immunized with the purified proteins and the immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for FimA-specific antibody. The immunized mice were challenged with a 10-fold LD50 dose (i.e., 100 CFU) of Salmonella enterica serovar Enteritidis standard strain (SD-2) 1 week after the second immunization. The immunized mice with the fusion proteins FimA-mC3d2 and FimA-mC3d3 had increased levels of ELISA titer of antibody that were 2 and 4 logs, respectively, more immunogenic than the TF-FimA protein alone. The challenge results showed that immune protection rate in the mice immunized with 10 μg of FimA, FimA-mC3d2, and FimA-mC3d3 were 50%, 75% and 100%, respectively. We conclude that mC3d can be expressed in a prokaryotic vector and enhance the immune response of the recombinant protein. FimA-mC3d3 is potentially a subunit vaccine against S. enterica serovar Enteritidis infection.