Infection Site Targeted Antitoxin Antibody (ISTAb) against Bacillus anthracis

Project Summary The Gram positive bacterium Bacillus anthracis is a very strong candidate for potential bioweaponizationand believed to have actually been weaponized by the former Soviet UnionAnthrax spores are readily found in nature or produced in the laboratoryare resistant to harsh conditionsand can survive for a long time in the environmentThe microscopic spores could be formulated in powder formspraysfoodor waterTwo key toxins generated by combination of the protective antigenPAwith either lethal factorLFor edema factorEFplay a critical role in Banthracis virulenceCurrent CDC recommendations following potential exposure to aerosolized Banthracis spores consist of a combination of oral antibiotics and PA based anthrax vaccineHoweverin practicethese treatments cannot adequately address the adverse effects of bacterial toxins released post exposureIn this Rproposal we intend to develop a novel approach to target neutralizing antitoxin antibodies specifically to the site of infectionThe approach exploits the cell wall targeting domainsCWTof well characterized phage endolysinsPlyGPlyL and PlyB which bind with species specificity and high affinity to cell wall components of BanthracisTheses CWTs will be fused to specific antitoxin neutralizing monoclonal antibodies to generate Infection Site Targeted Antitoxin antibodiesISTAbsISTAbs are expected to accumulate at the site of infection where they are needed mostand capture and sequester the toxinsthus immediately neutralizing the effects of the toxins and preventing their release into circulationBacterium toxin complex is then expected to be cleared by phagocytesIn this proposalwe will use three anthrax PA neutralizing monoclonal antibodies fused to high affinity phage endolysin CWTs to generate ISTAbsIn Aimwe will screen for best binding CWTs from ten phage endolysinsincluding those from PlyGPlyLand PlyBWe will characterize them based on in vivo and in vitro bindingIn Aimbased on Aimresultswe will selectCWTs for generating up to nine ISTAbs by fusing the CWTs with three highly neutralizing anti Anthrax monoclonal antibody as scaffold and characterize them for in vitro binding and toxin neutralizing activityIn Aimwe will further characterize the selected ISTAbs based on stabilitybacterial cell binding specificity and affinityand performance in opsonophagocytic killing assaysIn Aimwe will perform efficacy testing in pre challenge and post challenge treatment mouse models and also explore potential immunogenicity of the ISTAbsSince ISTAb technology provides two therapeutic advantagesimmediate toxin neutralization at the site of infection and opsonophagocytic killing by phagocytethere is a high probability that these molecules will synergize with existing antibioticsThe combination of immediate toxin clearancephagocytic killingand concurrent use of antibiotics is expected to create synergy and yield a treatment that is far superior to the current standard of vaccine plus antibioticsFurthermorethis technology can be applied to a variety of other bacterial pathogens where toxins play a key role in pathogenesisOverallthis approach has board application as a platform technology across multiple pathogens