Anthrax has long been considered the most probable bioweapon-induced disease. The protective
antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of
anthrax. In the current study, we evaluated the efficiency of a genetic vaccination with the fourth domain (D4) of PA, which is responsible for initial binding of the
anthrax toxin to the cellular receptor. The eukaryotic expression vector was designed with the
immunoglobulin M (
IgM)
signal sequence encoding for PA-D4, which contains
codon-optimized genes. The expression and secretion of
recombinant protein was confirmed in vitro in 293T cells transfected with plasmid and detected by western blotting, confocal microscopy, and
enzyme-linked
immunosorbent assay (ELISA). The results revealed that PA-D4
protein can be efficiently expressed and secreted at high levels into the culture medium. When plasmid
DNA was given intramuscularly to mice, a significant PA-D4-specific antibody response was induced. Importantly, high titers of
antibodies were maintained for nearly 1 year. Furthermore, incorporation of the SV40 enhancer in the plasmid
DNA resulted in approximately a 15-fold increase in serum antibody levels in comparison with the plasmid without enhancer. The
antibodies produced were predominantly the
immunoglobulin G2 (
IgG2) type, indicating the predominance of the Th1 response. In addition, splenocytes collected from immunized mice produced PA-D4-specific
interferon gamma (IFN-γ). The biodistribution study showed that plasmid
DNA was detected in most organs and it rapidly cleared from the injection site. Finally,
DNA vaccination with electroporation induced a significant increase in immunogenicity and successfully protected the mice against
anthrax spore challenge. Our approach to enhancing the immune response contributes to the development of
DNA vaccines against
anthrax and other biothreats.