Abstract
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.
Highlights
Anthrax, a disease caused by the gram-positive, spore-forming, rod-shaped bacterium Bacillus anthracis, is highly lethal and an appealing biological weapon owing to its great durability and longevity [1,2,3]
The pcDNA 3.1 was digested with EcoRI and XhoI and the protective antigen (PA)-D4 gene containing the signal sequence was inserted into the corresponding sites to obtain PA-D4 expression vector immunoglobulin M (IgM)-D4 and immunoglobulin kappa (IgK)-D4, respectively. (iii) Plasmids carrying the enhancer from Simian Virus 40 (SV40) were cloned
We designed a codon-optimized synthetic gene encoding PA-D4, that with most of the rare codons replaced by codons that are most frequently used in human cells
Summary
A disease caused by the gram-positive, spore-forming, rod-shaped bacterium Bacillus anthracis, is highly lethal and an appealing biological weapon owing to its great durability and longevity [1,2,3]. Since the intentional contamination of mail with B. anthracis spores in the United States in 2001, there is increasing concern about its use in terrorist attacks. There are three forms of anthrax characterized by the route of infection, namely, cutaneous, gastrointestinal, and inhalational. The most dangerous form, inhalational anthrax, results from inhalation of spores aerosolized in a particle size small enough to reach the alveoli. The spores are ingested by alveolar macrophages, and surviving spores are transported to the mediastinal lymph nodes. Rapid germination occurs resulting in a fatality rate approaching 100% if left untreated [4,5]
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