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.

The adverse reactions of the current live smallpox vaccine, and potential use of smallpox as a bioterrorism weapon, have highlighted the need to develop a new effective vaccine for this infectious disease. In the present study, a DNA vaccine vector was produced, which was optimized for expression of the vaccinia virus L1 antigen in a mouse model. Plasmid-encoded IgM-tL1R, which contains a truncated L1R gene fused to an IgM signal sequence, was constructed and expressed under the regulation of an SV40 enhancer. The expressed recombinant tL1 proteins were successfully secreted into the culture media. The DNA vaccine was administered to mice by electroporation, and animals were subsequently challenged with the lethal dose of vaccinia virus. We observed that immunization with IgM-tL1R induced robust neutralizing antibody responses and provided complete protection against a vaccinia virus infection. Isotyping studies revealed a lower IgG1/IgG2a ratio following vaccination with IgM-tL1R, suggesting the stimulation of Th1 immune responses. Our results propose that an optimized DNA vaccine, IgM-tL1R, can be effective in eliciting an anti-vaccinia virus immune response and provide protection against lethal orthopoxvirus challenge.