Toxin neutralizing activity of monoclonal antibodies against Bacillus anthracis lethal toxin

Abstract

Introduction. Anthrax is an infection caused by a Gram-positive, spore-forming bacteria Bacillus anthracis. It is difficult to diagnose the early stages of disease at the gastrointestinal or pulmonary forms, and in the later stages antibiotic therapy becomes ineffective since the pathogenesis and outcome of the disease are determined by anthrax toxins. The lethal toxin plays the leading role in the death of the patients. One way to neutralize anthrax toxin is to use monoclonal antibodies specific to the lethal toxin (LT) subunits: protective antigen (PA) or lethal factor (LF).

Aim of the study - to characterize and evaluate the toxin neutralizing activity of monoclonal antibodies (MAbs) against the lethal toxin of B. anthracis in vitro and in vivo.

Material and methods. MAbs 1D6F10, 4F6F8, 9D5C9 and 1E10 specific to PA were obtained and were generated in vivo in ascitic fluid. The immunoglobulin fraction from ascitic fluid was isolated by affinity chromatography on a column with Protein G Sepharose sorbent. The domain specificity and subclass of MAbs were determined. To identify the most promising toxin neutralizing MAbs we performed in vivo and in vitro experiments: on laboratory BALb/c mice and on murine macrophage cell line J774A.1, respectively. Cell viability in vitro was assessed using the MTT-test.

Results. As a result of the made experiments the most promising MAb 1E10 was chosen from all panel of obtained MAbs. MAb 1E10 interacts with IV domain of PA. It was shown that MAb 1E10 in concentration from 1 pg/ml and above in an experiment in vitro with murine macrophage cell line J774A.1 neutralizes LT of B. anthracis. It was found that MAb 1Е10 at a dose of 100 pg/mouse protects 100 % of animal from death upon subsequent administration of 4LD50 LT in an experiments in vivo with BALB/c mice. MAb 1E10 is perspective for receiving on its basis of chimeric MAbs with reduced immunogenicity for human.

Conclusion. It can be assumed that MAb 1Е10 inhibits the 1st stage of LT assembly -the interaction of PA with surface receptors of eukaryotic cells, and thus can have a toxin neutralizing effect.

Keywords:Bacillus anthracis, monoclonal antibodies; lethal toxin; protective antigen; lethal factor; toxin neutralizing activity

For citation: Romanenko Ya.O., Marin M.A., Riabko A.K., Kartseva A.S., Silkina M.V., Zeninskaya N.A., Shemyakin I.G., Dyatlov I.A., Firstova V.V. Toxin neutralizing activity of monoclonal antibodies against Bacillus anthracis lethal toxin. Immunologiya. 2021; 42 (3): 232-42. DOI: https://doi.org/10.33029/0206-4952-2021-42-3-232-242 (in Russian)

Funding. The work was supported by the Sectoral Scientific Program of the Russian Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (Registration number 116030310014).

Conflict of interests. The authors declare no conflict of interests.

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