A new preclinical study by scientists at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) and the U.S. Naval Research Laboratory highlights a novel therapeutic approach against anthrax using an engineered enzyme that dismantles the bacterium’s protective capsule. Conducted in high-containment biosafety level (BSL) facilities, the research demonstrates that two forms of pegylated CapD enzymes can significantly protect mice from lethal Bacillus anthracis infection.
Published in ACS Omega, this work builds on prior efforts to harness CapD as a treatment strategy, particularly for combating antibiotic-resistant anthrax strains. The study tested new, clinically optimized formulations of CapD that lack research-use tags and compared the effects of attaching either a linear or a branched polyethylene glycol (PEG) molecule to improve therapeutic performance.
What Are Pegylated CapD Enzymes and Why Are They Being Studied?
CapD is a naturally occurring enzyme produced by Bacillus anthracis that plays a role in anchoring the bacterium’s protective capsule, which is made of polymers of d-glutamic acid (PDGA). This capsule inhibits immune recognition, allowing the pathogen to evade phagocytosis. When used therapeutically, CapD can degrade the capsule from the outside, stripping the bacteria of its defense and making it vulnerable to immune clearance. However, unmodified enzymes like CapD are typically unstable in the body, prone to rapid degradation by proteases and elimination by the immune system. To overcome these limitations, scientists attach polyethylene glycol (PEG) chains to CapD in a process known as pegylation. Pegylation enhances solubility, improves stability, extends circulation time, and reduces immunogenicity. By strengthening these pharmacological properties, pegylated CapD enzymes are being explored as a promising countermeasure against anthrax infections—particularly in cases involving antibiotic-resistant strains or scenarios where conventional therapies may fail.
Comparing Two PEG Strategies: Branched vs. Linear
The study evaluated two versions of tag-free CapD modified at a specific site with either:
- 1-prong PEG-CapD (linear PEG)
- 3-prong PEG-CapD (branched PEG)
Both versions:
- Maintained comparable enzyme activity
- Showed improved thermostability with melting temperatures above 50°C
- Retained partial structure even under acidic conditions, favoring robustness in vivo
Pharmacokinetic data revealed that the linear (1-prong) PEG version peaked faster in serum, while the branched (3-prong) PEG version remained in circulation longer, suggesting a trade-off between speed and persistence.
Effective Protection at High Doses of Virulent B. anthracis
Mice challenged with 10 or 100 LD50 doses of B. anthracis spores were treated with 40 mg/kg of either enzyme variant every 8 hours for 7 days. Results showed:
- 10 LD50 challenge:
- 1-prong PEG-CapD: 90% survival (both trials)
- 3-prong PEG-CapD: 70% and 100% survival
- 100 LD50 challenge:
- 1-prong PEG-CapD: up to 70% survival
- 3-prong PEG-CapD: up to 30% survival
In all experiments, survival was significantly higher than untreated controls, and no statistically significant difference was observed between the two PEG variants.
Relevance for Biosecurity and Antibiotic Resistance
Though ciprofloxacin remains effective against anthrax, resistant strains have been reported, and the CDC has highlighted the risk of drug-resistant B. anthracis. Enzyme-based therapies like pegylated CapD offer a critical alternative, especially in scenarios involving deliberate release or naturally resistant strains.
While immune responses to pegylated proteins could impact long-term use, the study’s success in high-containment animal models provides a strong rationale for advancing to nonhuman primate testing, the next step toward clinical translation.
Conducted under high-containment biosafety conditions, this study demonstrates the potential of both linear and branched pegylated CapD enzymes to protect against experimental anthrax. These findings support further development of enzyme therapeutics as countermeasures in a biodefense context and as tools to address future threats from multidrug-resistant bacterial pathogens.
Chua J, Mathur D, Lankford H, Meinig JM, Chabot DJ, Legler PM, Friedlander AM. In Vivo Comparison of Branched vs Linear Pegylation of a Capsule-Degrading Enzyme for Treatment of Anthrax. ACS Omega, 5 June 2025.