Development of an in Vitro Potency Assay for Anti-anthrax Lethal Toxin Neutralizing Antibodies

Gail Whiting,Sjoerd Rijpkema,Michael Baker

doi:10.3390/toxins4010028

Gail Whiting, Sjoerd Rijpkema + Show 1 more

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https://doi.org/10.3390/toxins4010028

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Journal: Toxins	Publication Date: Jan 19, 2012
Citations: 36	License type: CC BY 4.0

Affiliation: National Institute for Biological Standards and Control

Abstract

Lethal toxin (LT) of Bacillus anthracis reduces the production of a number of inflammatory mediators, including transcription factors, chemokines and cytokines in various human cell lines, leading to down-regulation of the host inflammatory response. Previously we showed that the reduction of interleukin-8 (IL-8) is a sensitive marker of LT-mediated intoxication in human neutrophil-like NB-4 cells and that IL-8 levels are restored to normality when therapeutic monoclonal antibodies (mAb) with toxin-neutralising (TN) activity are added. We used this information to develop cell-based assays that examine the effects of TN therapeutic mAbs designed to treat LT intoxication and here we extend these findings. We present an in vitro assay based on human endothelial cell line HUVEC jr2, which measures the TN activity of therapeutic anti-LT mAbs using IL-8 as a marker for intoxication. HUVEC jr2 cells have the advantage over NB-4 cells that they are adherent, do not require a differentiation step and can be used in a microtitre plate format and therefore can facilitate high throughput analysis. This human cell-based assay provides a valid alternative to the mouse macrophage assay as it is a more biologically relevant model of the effects of toxin-neutralising antibodies in human infection.

Highlights

Bacillus anthracis is a spore-forming bacterium, which occurs naturally in soils throughout the world and causes the disease anthrax
At 4 h, significant decreases in mRNA were seen for IL-6, IL-8 and CCL20; this pattern concurred with previous findings in NB-4 cells by Barson et al [15]
The lowest concentration of 0.2 nM protective antigen (PA) and 0.02 nM lethal factor (LF) reduced IL-8 by a significant degree for the unstimulated cells only (Figure 2A). Both the unstimulated HUVEC jr2 cells and those stimulated with LPS gave a clearly defined dose response with the concentrations of lethal toxin (LT) used for both IL-6 and IL-8 and statistical analysis revealed that the decline in IL-6 and IL-8 was significant

Summary

Introduction

Bacillus anthracis is a spore-forming bacterium, which occurs naturally in soils throughout the world and causes the disease anthrax. B. anthracis produces two binary toxins; edema toxin (ET) and lethal toxin (LT). ET is composed of protective antigen (PA) and edema factor (EF) whereas LT comprises PA and lethal factor (LF) [1,2]. PA binds to cell surface receptors and following cleavage by furin, polymerises into a heptameric structure that can bind EF and LF and promote their entry into the cell. LF is a zinc metalloprotease that cleaves the amino terminus of the mitogen-activated protein kinase (MAPK) kinases, preventing binding to downstream mitogen activated protein kinases such as extracellular regulated kinase (ERK) or p38, leading to the complete inhibition of the MAP kinase signalling pathway and, cell cycle arrest and cell death [4,5,6]

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