Mechanisms of Antibody-Mediated Toxin Neutralization

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DESCRIPTION (provided by applicant): Antibodies have been used to protect against the effects of biological toxins for over a century. It is generally accepted that the mechanism whereby antibodies protect is by blocking the entry of toxins into cells. However, this ignores an important body of evidence demonstrating that antibodies to the A-chain of A-B toxins have equal or greater neutralizing activity than anti-B chain antibodies. We will present data demonstrating this with ricin toxin. Based upon these results we have formed the hypothesis that antibodies can protect by altering the intracellular processing and routing of the toxin and that the affinity of antibody binding may determine its ability to do so. To test this hypothesis we propose the following Specific Aims: Specific Aim 1: To produce and characterize a panel of high affinity anti-ricin A chain antibodies. The relationship between antibody affinity, in vitro neutralization and in vivo protection will be studied. Specific Aim 2: To study the intracellular routing of fluorescent-labeled ricin toxin in the presence of neutralizing and non-neutralizing antibodies. Confocal, deconvolution, and electron microscopy, and subcellular isolations will be used to study effects of antibody on subcellular localization of ricin. Specific Aim 3: To study the intracellular association between toxin and antibody. Double-label studies and fluorescence-energy transfer (FRET) will be used to determine how long antibody remains bound to the toxin during intracellular processing. Ricin is a prototype A-B toxin, a group that includes many bacterial and plant toxins. In addition to defining basic processes, the studies proposed in this application have utility for the development of vaccines and treatments for intoxications.