In vitro and in vivo activities of recombinant anthrax protective antigen co-expressed with thioredoxin inEscherichia coli

Abstract

Because of the central role it plays in the formation of lethal toxin and edema toxin, protective antigen (PA) is the principal target for the development of vaccines against anthrax. In the present study, we explored and compared the in vitro and in vivo activities of recombinant anthrax protective antigen (rPA) and receptor binding domain of protective antigen (PA4). As a result, the fully soluble rPA and PA4 proteins were successfully expressed in Escherichia coli by co-expression with thioredoxin (Trx), and the rPA was active in forming cytotoxic lethal toxins, indicating that the rPA protein retains a functionally biological activity. Furthermore, immunization with rPA protein induced stronger PA-specific immune responses in mice than PA4 protein. The protection elicited by immunization with PA4 suggests the presence of common neutralizing epitopes between rPA and PA4, but the immunization with rPA protein induced stronger neutralizing antibodies and protective levels against challenge with the B. anthracis strain A16R than the PA4 protein. The sera neutralizing antibodies titers correlated well with anti-PA group ELISA antibodies titers and the in vivo protective potency. Based on the results of cell cytotoxicity assays and the observed immune responses and protective potency, we concluded that the soluble rPA protein retains the in vitro and in vivo functionally biological activity and can be developed into a highly effective human subunit vaccine candidate against anthrax.

Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors

“…1G, H) encoding the PA4 were constructed in previous studies [7,14]. In this study, a series of recombinant DNA plasmids, including the conventional non-viral DNA vaccines and SFV replicon DNA vaccines encoding the PA4 or PA [23] with a signal sequence or not, were constructed and used for vaccination ( Fig. 1A-C, E, and F).…”

“…Mice from all groups were challenged intraperitoneally (i.p.) with the indicated dose of B. anthracis A16R at 4 weeks after the last vaccination [23]. The mice were observed for 1 week after challenge, and survival was determined for each vaccination group.…”

“…The in vitro expression of PA or PA4 in DNA-transfected cells was detected by immunoblots as previously described [20,23]. Briefly, BHK-21 cells were seeded into six-well tissue culture plates at 2 × 10 5 cells/well and grown to 70-80% confluence, then were transfected with complexes of each plasmid DNA and Lipofectamine 2000 (Invitrogen, CA) following the manufacturer's instructions and harvested with lysis buffer after 24 h posttransfection.…”

“…Sera from mice in the different groups were screened for anti-PA antibodies by ELISA as previously described [23]. Briefly, ELISA plates (Corning Inc., Corning, NY) were coated overnight at 4 • C with 100 l recombinant PA (2 g/ml), which was expressed and purified in Escherichia coli [23].…”

Expression of Aβ-Fc Fusion Protein in Transgenic Potato

“…Biotechnology and genetic engineering techniques have been used to produce subunit vaccines that stimulate a strong immune response. To create the subunit vaccine, the genes encoding the appropriate subunits are isolated from the genome of the infectious agent and expressed in bacteria (Ma et al, 2013), yeast (Wang et al, 2013), insects (Kanai et al, 2013), and plant host cells (Chen and Lai, 2013), which then produce large quantities of the subunit molecules. Subunit vaccines are safe for use in immunocompromised patients because they do not cause disease (Emini et al, 1986).…”