Abstract
The aim of this research is to develop a human/murine chimeric Fab antibody which neutralizes the anthrax toxin, protective antigen (PA). The chimeric Fab was constructed using variable regions of murine anti-PA monoclonal antibody in combination with constant regions of human IgG. The chimeric PA6-Fab was expressed in E. coli. BL21 and evaluated by ELISA and co-immunoprecipitation- mass spectra. The potency of PA6-Fab to neutralize LeTx was examined in J774A.1 cell viability in vitro and in Fisher 344 rats in vivo. The PA6-Fab did not have domain similarity corresponding to the current anti PA mAbs, but specifically bound to anthrax PA at an affinity of 1.76 nM, and was able to neutralize LeTx in vitro and protected 56.9% cells at 20 μg/mL against anthrax LeTx. One hundred μg PA6-Fab could neutralize 300 μg LeTx in vivo. The PA6-Fab has potential as a therapeutic mAb for treatment of anthrax.
Highlights
Bacillus anthracis is a Gram-positive spore-forming bacterium that causes the clinical spectrum of anthrax
lethal factor (LF) or edema factor (EF) confers toxicity only after binding to the haptamer to form lethal toxin (LeTx) or edema toxin (EdTx); protective antigen (PA) plays a central role in the virulence of the pathogen [4]
Total RNAs were extracted from the PA6 hybridoma cells, and PA-6 cDNA was derived (Table 1)
Summary
Bacillus anthracis is a Gram-positive spore-forming bacterium that causes the clinical spectrum of anthrax. Aerosolised distribution of spores can rapidly spread among a population of people and cause lethal inhalational anthrax due to the toxins produced during the replication of infectious bacilli [1]. Anthrax toxins comprise protective antigen (PA), lethal factor (LF) and edema factor (EF) [3]. LF or EF confers toxicity only after binding to the haptamer to form lethal toxin (LeTx) or edema toxin (EdTx); PA plays a central role in the virulence of the pathogen [4]. There are currently six clinically useful anti-PA mAbs, only Raxibacumab [11], a human mAb, has been approved by USDA as a therapeutic anthrax mAb, in 2012. Raxibacumab binds poorly to PA with an affinity at
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