New "Checkmate" Method Provides Powerful Tool for Preventing Spread of Future Epidemics

By Eric Sauter and Mika Ono

Scientists from The Scripps Research Institute have developed a breakthrough methodology that can be used to rapidly predict how viruses such as avian influenza H5N1, a dangerous strain of "bird flu," will mutate in response to attacks by the immune system. The new approach, dubbed "checkmate analysis," may also predict which antibodies or small molecule therapeutics will best neutralize these viral mutations before they can develop into global epidemics.

In a high-powered collaboration, Richard A. Lerner, president of The Scripps Research Institute, Sydney Brenner, recipient of the 2002 Nobel Prize in Medicine and a faculty member of The Salk Institute for Biological Studies, Tobin J. Dickerson, assistant professor of chemistry at Scripps Research and several Scripps Research colleagues developed the methodology. Because of its simplicity and low cost, this innovative approach will be accessible to scientists around the world.

The new study was published in an advanced online edition of journal Proceedings of the National Academy of Sciences during the week of July 16 to 20, 2007.

"Our new 'checkmate analysis' allows scientists to explore all the possible routes that a virus might take to escape an immune response or a small molecule therapeutic," Lerner said. "The result is a detailed chemical map of the trajectories of viral escape and antibody response."

During the course of an infection, new viruses and new neutralizing antibodies are selected and discarded, as the microbe and the host struggle for dominance. During this process, the immune system operates largely in a reactive mode instead of anticipating the next viral mutation. To date, new vaccines and other treatments have also been designed against what the virus has done rather than what it might do—but the new methodology devised by Lerner, Brenner, Dickerson, and colleagues could change that.

The new method starts with large libraries of mutant viral proteins and antibodies that are expressed on a phage surface. (Phages, also called "bacteriophages," are single-stranded DNA viruses, which infect only bacteria.) These two factions are then used to challenge each other.

Because this approach is both simple and inexpensive, the new methodology is within reach of almost any biomedical laboratory on the planet.

"Currently, high-throughput screening is limited to those who have access to expensive equipment," Lerner said. "This work will put high-throughput screening into the hands of the worldwide scientific community."

That was the idea from the start, according to Dickerson. "We envisioned that this could be utilized in almost any environment—in a field laboratory in the Arctic or a research hospital in Africa," he said. "That's the real power of these findings, that we've reduced everything to a simple bacteriophage assay."

Scripps is already noted for its pioneering work in combinatorial antibody libraries. But, before this study, no one had succeeded in expressing functional viral proteins on phages. While methods for generating combinatorial small molecules and antibody libraries were well established, the expression of functional viral proteins on phages posed additional challenges. These proteins are frequently assembled in cell membranes and often composed of various subunits, as is the case with hemagglutinin (HA), a glycoprotein on the influenza virus surface that binds the virus to infected cells.

Lerner, Brenner, Dickerson, and colleagues bridged this technological gap using some elegant chemistry to design a new system, which they then successfully applied to expressing hemagglutinin on the phage surface.

A sample protocol, the study suggested, might involve starting with a population of hemagglutinin-containing phages plus a similar population of antibodies or small molecules that prevent binding. The hemagglutinin would be mutated and the viral escape variants (which preserve binding capacity), selected. These new viral mutations could then be used to screen for new antibodies or small molecules that can bind and hold the escaped mutants.

As a result, the threat to the virus escalates as the population of new antibodies and small molecule antagonists grows and are added to each new analytical cycle. In the case of an

immunological checkmate analysis, the sequence analysis of successful viral mutants provides a map of escape routes the virus can use, while the antibody sequences provide information about the chemical basis of a successful immune response.

"This really is a new way to think about the study of viral evolution," Dickerson said. "The ultimate scenario is that, in a single series of experiments, you can look forward and backward in evolutionary time to see where the avian flu virus has been and where it's going."

The new methodology, the study emphasizes, isn't limited to viruses but could be applied to any protein-ligand interaction that was open to disruption. For instance, the checkmate analysis of escape mutants could be useful in profiling trajectories of enzymes important to shifting cancer phenotypes or changes leading to antibiotic resistance in bacteria. The methodology also has potential for evaluating organic compounds or small peptides rather than antibodies that disrupt the interaction between protein and ligand. In that case, the new methodology would allow analysis of direct binding at the level of single molecules without special reagents or complicated equipment.

This is not the first time Lerner and Brenner have worked together. In 1992, they collaborated on a combinatorial chemistry technique to tag each polymer in a library with an individually coded DNA molecule for more effective identification in drug screening. This technique was termed "encoded combinatorial chemistry" and is now a subject of intense study.

In addition to Lerner, Brenner, and Dickerson, authors of the study, "Phage Escape Libraries for Checkmate Analysis," include Kathleen M. McKenzie, Amanda S. Hoyt, Malcolm R. Wood of The Scripps Research Institute, and Kim D. Janda of The Scripps Research Institute, The Skaggs Institute for Chemical Biology, and the Worm Institute for Research and Medicine (WIRM). See PNAS at http://www.pnas.org/cgi/content/abstract/0705362104v1.

The study was supported by the Skaggs Institute for Chemical Biology and the National Institutes of Health.

Send comments to: mikaono[at]scripps.edu

"This work will put high-throughput screening into the hands of the worldwide scientific community."

—Richard Lerner