Anthrax and botulism are dangerous infectious diseases that can be fatal unless detected and treated quickly. Fatalities from these diseases are primarily due to endopeptidase toxins secreted by the pathogens. Rapid and sensitive detection of the presence of active toxins is the key element for protection from natural outbreaks of anthrax and botulism, as well as from the threat of bioterrorism. We describe an ultrasensitive polymerase chain reaction (PCR)-based assay for detecting proteolytic activity of anthrax and botulinum toxins using composite probes consisting of covalent peptide-DNA conjugate for the detection of anthrax, and noncovalent protein-aptamer assembly to assay botulinum toxin activity. Probes immobilized on the solid-phase support are cleaved by toxins to release DNA, which is detected by real-time PCR. Both assays can detect subpicogram quantities of active toxins isolated from composite matrices. Special procedures were developed to isolate intact toxins from the matrices under mild conditions. The assay is rapid, uses proven technologies, and can be modified to detect other proteolytic and biopolymer-degrading enzymes.

Live anthrax vaccine containing spores from attenuated strains STI-1 of Bacillus anthracis is used in Russia and former CIS (Commonwealth of Independent States) to prevent anthrax. In this paper we studied the duration of circulation of antibodies specific to spore antigens, the protective antigen (PA), the lethal factor (LF) and their domains (D) in donors’ blood at different times after their immunization with live anthrax vaccine. The relationship between the toxin neutralization activity level and the level of antibodies to PA, LF and their domains was tested. The effect of age, gender and number of vaccinations on the level of adaptive post-vaccination immune response has been studied. It was shown that antibodies against PA-D1 circulate in the blood of donors for 1 year or more after immunization with live anthrax vaccine. Antibodies against all domains of LF and PA-D4 were detected in 11 months after vaccination. Antibodies against the spores were detected in 8 months after vaccination. A moderate positive correlation was found between the titers of antibodies to PA, LF, or their domains, and the TNA of the samples of blood serum from the donors.

Neutralization of the lethal toxin of Bacillus anthracis is an important topic of both fundamental medicine and practical health care, regarding the fight against highly dangerous infections. We have generated a neutralizing monoclonal antibody 1E10 against the lethal toxin of Bacillus anthracis and described the stages of receptor interaction between the protective antigen (PA) and the surface of eukaryotic cells, the formation of PA oligomers, assembly of the lethal toxin (LT), and its translocation by endocytosis into the eukaryotic cell, followed by the formation of a true pore and the release of LT into the cell cytosol. The antibody was shown to act selectively at the stage of interaction between Bacillus anthracis and the eukaryotic cell, and the mechanism of toxin-neutralizing activity of the 1E10 antibody was revealed. The interaction between the 1E10 monoclonal antibody and PA was found to lead to inhibition of the enzymatic activity of the lethal factor (LF), most likely due to a disruption of true pore formation by PA, which blocks the release of LF into the cytosol.

Botulism is one of the most serious food intoxications, manifesting as prolonged paralytic conditions. This disease is usually the result of the consumption of poor quality canned or smoked foods, so the inhabitants of many countries of the world are exposed to the risk of this kind of poisoning every year. In view of the severity of poisonings caused by botulinum neurotoxins, monoclonal antibodies (mAbs) show great promise because of their targeting action, lack of allergic reactions and serum sickness. The use of a cocktail of mAbs increases the “functional specificity” of their mixture, allowing them to bind to the active domains of different toxin chains and block their action. In this work, we obtained 14 murine mAbs to the catalytic and receptor-binding domain of botulinum toxin type A. The Sp2/0-Ag14 murine myeloma cell line and splenocytes from immunized mice of the BALB/c line were used as fusion partners. We have shown that the selected cocktail of three antibodies neutralizes native toxin more effectively than antibodies separately—complete neutralization is achieved at a toxin dose of 3LD50 and partial neutralization at 5LD50. We presume that this cocktail may be promising as a prototype for the creation of a therapeutic drug capable of neutralizing the toxin in the blood of patients.

Bacillus anthracis has three main virulence factors: an extracellular capsule and two binary toxins (lethal toxin—consists of a lethal factor and a protective antigen, and edema toxin—consists of an edema factor and a protective antigen). In the Russian Federation, the epidemiological situation regarding anthrax infection remains unfavorable. In the late stages of an anthrax infection, antibiotic therapy becomes ineffective and the patient dies within 24 h as a large amount of lethal toxin accumulates in the patient’s blood. Antibodies capable of neutralising lethal toxin (LT) can be an effective treatment for these patients. The objective of the study was to construct a chimeric monoclonal antibody targeting the protective antigen of the LT and to elucidate its mechanism of toxin neutralization. In this work, a chimeric monoclonal antibody (xi1E10) directed against the protective antigen was successfully produced. Both in vitro and in vivo experiments demonstrated the capacity of xi1E10 to neutralize lethal toxin. Confocal microscopy revealed that xi1E10 effectively suppresses the formation of a functional pore, thereby blocking the translocation of the lethal factor into the cytosol. These findings indicate that the monoclonal antibody xi1E10 represents a promising candidate for the development of a therapeutic drug.