An in vitro and in vivo evaluation of the efficacy of recombinant human liver prolidase as a catalytic bioscavenger of chemical warfare nerve agents

Abstract

In this study, we determined the ability of recombinant human liver prolidase to hydrolyze nerve agents in vitro and its ability to afford protection in vivo in mice. Using adenovirus containing the human liver prolidase gene, the enzyme was over expressed by 200- to 300-fold in mouse liver and purified to homogeneity by affinity and gel filtration chromatography. The purified enzyme hydrolyzed sarin, cyclosarin and soman with varying rates of hydrolysis. The most efficient hydrolysis was with sarin, followed by soman and by cyclosarin {apparent k_cat/K_m [(1.9 ± 0.3), (1.7 ± 0.2), and (0.45 ± 0.04)] × 10⁵M⁻¹min⁻¹, respectively}; VX and tabun were not hydrolyzed by the recombinant enzyme. The enzyme hydrolyzed P (+) isomers faster than the P (−) isomers. The ability of recombinant human liver prolidase to afford 24 hour survival against a cumulative dose of 2 × LD₅₀ of each nerve agent was investigated in mice. Compared to mice injected with a control virus, mice injected with the prolidase expressing virus contained (29 ± 7)-fold higher levels of the enzyme in their blood on day 5. Challenging these mice with two consecutive 1 × LD₅₀ doses of sarin, cyclosarin, and soman resulted in the death of all animals within 5 to 8 min from nerve agent toxicity. In contrast, mice injected with the adenovirus expressing mouse butyrylcholinesterase, an enzyme which is known to afford protection in vivo, survived multiple 1 × LD₅₀ challenges of these nerve agents and displayed no signs of toxicity. These results suggest that, while prolidase can hydrolyze certain G-type nerve agents in vitro, the enzyme does not offer 24 hour protection against a cumulative dose of 2 × LD₅₀ of G-agents in mice in vivo.

• Adenovirus
• butyrylcholinesterase
• catalytic bioscavenger
• chemical warfare nerve agents
• human liver prolidase
• in vivo delivery

Declaration of interest

This research was supported by the Defense Threat Reduction Agency (Project ID: D0003_09_RC_C)-Joint Science and Technology Office, Medical S&T Division. The views expressed in this manuscript are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. The experimental protocol was approved by the Animal Care and Use Committee of the US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council), and the Animal Welfare Act of 1966 (P.L. 89-544), as amended. The authors are grateful to Dr. John McDonough, USAMRICD, for help with the animal experiments and suggestions with the manuscript.