Development of an in vitro potency assay for anti-anthrax lethal toxin neutralizing antibodies

Area of Science:

• Immunology
• Cell Biology
• Microbiology

Background:

• Bacillus anthracis lethal toxin (LT) down-regulates host inflammatory responses by reducing inflammatory mediators.
• Interleukin-8 (IL-8) reduction is a sensitive marker for LT intoxication in human neutrophil-like NB-4 cells.
• Therapeutic monoclonal antibodies (mAbs) with toxin-neutralizing (TN) activity can restore normal IL-8 levels.

Purpose of the Study:

• To develop and validate a human cell-based in vitro assay for measuring the TN activity of therapeutic anti-LT mAbs.
• To utilize the human umbilical vein endothelial cell line (HUVEC jr2) for high-throughput analysis of anti-LT mAb efficacy.
• To establish a biologically relevant alternative to existing animal models for evaluating anti-LT therapies.

Main Methods:

• An in vitro assay was developed using the adherent human endothelial cell line HUVEC jr2.
• The assay measures TN activity of therapeutic anti-LT mAbs by monitoring IL-8 levels as a marker of LT intoxication.
• HUVEC jr2 cells offer advantages over NB-4 cells, including adherence and no requirement for differentiation, facilitating microtitre plate format and high-throughput analysis.

Main Results:

• The HUVEC jr2 cell-based assay effectively measures the TN activity of therapeutic anti-LT mAbs.
• IL-8 serves as a reliable marker for LT intoxication in this assay system.
• The assay facilitates high-throughput analysis due to the properties of HUVEC jr2 cells.

Conclusions:

• A novel, human cell-based assay using HUVEC jr2 cells provides a valid method for evaluating therapeutic anti-LT mAbs.
• This assay is a more biologically relevant alternative to the mouse macrophage assay for assessing therapies against LT intoxication in humans.
• The assay enables efficient, high-throughput screening of potential treatments for Bacillus anthracis infections.