The Gram-positive bacterium Bacillus anthracis is a very strong candidate for potential bio- weaponization, and believed to have actually been weaponized by the former Soviet Union. Anthrax spores are readily found in nature or produced in the laboratory, are resistant to harsh conditions, and can survive for a long time in the environment. The microscopic spores could be formulated in powder form, sprays, food, or water. Two key toxins generated by combination of the protective antigen (PA) with either lethal factor (LF) or edema factor (EF) play a critical role in B. anthracis virulence. Current CDC recommendations following potential exposure to aerosolized B. anthracis spores consist of a combination of oral antibiotics and PA-based anthrax vaccine. However, in practice, these treatments cannot adequately address the adverse effects of bacterial toxins released post exposure. In this R41 proposal we intend to develop a novel approach to target neutralizing anti- toxin antibodies specifically to the site of infection. The approach exploits the cell wall targeting domains (CWT) of well characterized phage endolysins: PlyG, PlyL and PlyB which bind with species-specificity and high affinity to cell wall components of B. anthracis. Theses CWTs will be fused to specific antitoxin neutralizing monoclonal antibodies to generate Infection Site Targeted Antitoxin antibodies (ISTAbs). ISTAbs are expected to accumulate at the site of infection where they are needed most, and capture and sequester the toxins, thus immediately neutralizing the effects of the toxins and preventing their release into circulation. Bacterium-toxin complex is then expected to be cleared by phagocytes. In this proposal, we will use three anthrax-PA neutralizing monoclonal antibodies fused to high affinity phage endolysin CWTs to generate ISTAbs.
In Aim 1 we will screen for best binding CWTs from ten phage endolysins, including those from PlyG, PlyL, and PlyB. We will characterize them based on in vivo and in vitro binding.
In Aim 2, based on Aim 1 results, we will select 3 CWTs 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 activity.
In Aim 3 we will further characterize the selected ISTAbs based on stability; bacterial cell binding specificity and affinity, and performance in opsonophagocytic killing assays.
In Aim 4, we will perform efficacy testing in pre-challenge and post challenge treatment mouse models and also explore potential immunogenicity of the ISTAbs. Since ISTAb technology provides two therapeutic advantages: immediate toxin neutralization at the site of infection and opsonophagocytic killing by phagocyte, there is a high probability that these molecules will synergize with existing antibiotics. The combination of immediate toxin clearance, phagocytic killing, and concurrent use of antibiotics is expected to create synergy and yield a treatment that is far superior to the current standard of vaccine plus antibiotics. Furthermore, this technology can be applied to a variety of other bacterial pathogens where toxins play a key role in pathogenesis. Overall, this approach has board application as a platform technology across multiple pathogens.
Bacillus anthracis, a spore-forming bacterium, causes disease in animals and humans by three routes of infection: inhalational, gastrointestinal, and skin. Bacillus anthracis is a Tier 1 agent that poses the greatest risk of misuse, with significant potential for mass casualties and threat to public health and safety. For instance, in 2001, powdered anthrax spores were deliberately placed inside letter envelopes that were mailed to two U.S. Senators and several news media offices. Twenty-two people, including 12 mail handlers, contracted anthrax and five died as a result (http://www.cdc.gov/anthrax/bioterrorism/threat.html). Three toxins are involved in anthrax disease: protective antigen (PA), lethal factor (LF) and edema factor (EF). In this proposal we seek to use a novel technology called Infection site targeted antitoxin antibodies (ISTAbs) to target antitoxins exactly to the site fo infection. This technology can provide a novel approach for treatment of Bacillus anthracis infections.
