LCMV PCR In Vitro Assay Replaces Longstanding Animal Test

The alternative method developed and validated by Charles River could serve as a reference for the replacement of other animal-based assays to detect adventitious agents

For decades animal research facilities, including Charles River, used the Mouse Antibody Production (MAP) test to detect lymphocytic choriomeningitis virus (LCMV) and other inapparent viruses that can be difficult to recognize because they don't cause any noticeable symptoms in the host. LCMV originates from mice but can be transferred to humans through adventitious agents - viral contaminants that can be introduced to biological materials during production through contaminated materials - causing illness or even death. Due to the risks posed to humans, additional testing, like MAP, has historically been offered for this agent to confirm that customer test articles are free of LCMV.

In 2023, due to the ethical concerns of using live animals for this test, Charles River's In Vivo Biosafety (IVB) lab in Wilmington, MA., announced plans to discontinue this animal-based test and investigated alternatives to test for LCMV, aligning with the global company's 3Rs mission and Alternative Methods Advancement Project.

How does MAP work?

To perform the MAP test, mice are first inoculated with customer test articles. After 14 days, which is enough time for its immune system to start making antibodies, a subset of mice are injected with highly concentrated LCMV. If the mouse survives it means it is producing antibodies, signifying test article contamination with LCMV. On the other hand, if the mouse dies, then this indicates the absence of LCMV in client test articles since the mouse did not produce antibodies. IVB sought assistance from Charles River's Biologics site in Wayne, PA to help develop a GMP-validated PCR assay that could both successfully identify this pathogen and alleviate animal welfare concerns. In short, mortality should not be the endpoint of an in vivo test.

What do we mean by GMP and PCR?

Good Manufacturing Practices (GMP) are quality assurance regulations enforced by the US Food and Drug Administration, providing for systems that assure proper design, monitoring, and control of manufacturing processes and facilities. These practices are what protect consumers from purchasing an ineffective or sometimes dangerous product. Documentation is the key to success when it comes to making something GMP-compliant. Any company that manufactures drug materials must follow GMP to ensure drug safety, including Charles River.

PCR - an in vitro test that amplifies genetic matter specific to the pathogen in question - is an accepted alternative to in vivo testing as stated in regulatory guidelines. The Wayne team, led by Drs. Sung Ryeol Park and Michael Hantman, had an established system for developing and validating molecular biology assays in a GMP-compliant manner, with many years of experience. In collaboration with IVB and across multiple departments, the project team developed the LCMV PCR assay, providing a faster, animal-free, robust, and cost-effective alternative to the in vivo MAP test.

This PCR assay allows for the detection and quantification of LCMV in customer test articles. The introduction of this new screening approach is expected to replace 400 to 600 mice annually and could serve as a reference for the replacement of other animal-based assays to detect for additional adventitious agents.

Dr. Park, along with Hayden Hufnal, Byong Yoo, Carmen Li, Tegegn Jaleta, Ph.D., and Charles Michaelis received a Charles River 3Rs award this year for their work on the PCR assay. While the scope of this initial testing is complete, science continues to evolve, and viruses continue to mutate. A new form of LCMV could develop, and as is the nature of research, there are opportunities that may arise to improve and refine the assay even further in the future, says Dr. Park.

The LCMV RT-qPCR assay is only offered to Charles River clients, but Park says she did present a poster, shown on left, at this year at the 8th Annual Viral Safety and Viral Clearance Summit about the development and qualification of the assay. The event is sponsored by Charles River. "During my presentation I met with an FDA staff member and had an opportunity to share that we expect to save ~600 mice per year by replacing a single animal-based assay," she said. "The FDA staff seemed quite impressed with the potential impact."

Lindsey Cable joined Charles River Laboratories in 2023 as an Internal Communications Specialist. In this position, she provides communications support primarily for the Research Models & Services business. Prior to joining Charles River, she worked with government clients, such as the U.S. Census Bureau, the D.C. Department of Health, and the Department of Veteran's Affairs, Office of Mental Health and Suicide Prevention as project manager and writer for national campaigns. It was while working with these clients that Lindsey found her enjoyment of scientific writing. She received her bachelor's degree in journalism and public policy from the University of North Carolina at Chapel Hill in 2019.