V-4142. Modeling the Kinetics of Influenza A Resistance Under Drug Pressure
Session: Poster Session: Respiratory Viruses I: Influenza and RSV
Tuesday, October 28, 2008: 12:00 AM
Room: Hall C
Background: One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. For instance, it is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the M2 gene. Previously, we have used mathematical models to demonstrate that resistance to amantadine occurs within 48-72 hours of drug treatment. Methods: We used an in vitro kinetic model to examine the effects of amantadine drug pressure on the kinetics and genotypic profile of resistance in influenza A. MDCK cells were infected with a low multiplicity of virus in the presence of amantadine and the infection was monitored over seven days, allowing multiple rounds of replication. Viral load at each time points was determined by quantitative PCR, and the genotype and percentage of mutant species were determined by Sanger sequencing and Taq-MAMA. Results: Influenza A replicated to high titers in the in vitro kinetic system, with the peak viral load occurring at 48 hours post infection. Amantadine inhibited virus replication by ~2-logs at early time points. However, by 72 hours, all amantadine treated arms had similar viral load levels as the untreated arm. Sequencing of samples revealed that mutations in the M2 gene appeared as early as 48 hours under drug pressure, and Taq-MAMA assays showed that resistant mutants were the predominant species by 72 hours. The resistance mutations isolated from the in vitro system were identical to those identified in the clinic for amantadine resistant viruses (V27A, A30T, and S31N). Conclusions: We have optimized an in vitro system to mimic the course of influenza A infection in humans, and our data suggest that the emergence of resistance can be accurately modeled in vitro. This system provides a foundation to model resistance to other drugs with different mechanisms of action or to combinations of drugs.
Alan Perelson, PhD1, David Engelthaler, MS2, Elizabeth Driebe, MS3, Gregory Went, PhD4, Jack Nguyen, PhD5, Paul Keim, PhD2 and  J. T. Nguyen,
Adamas Pharmaceuticals Role(s): Employee, Received: Salary., (1)Los Alamos National Laboratory, Los Alamos, NM, (2)The Translational Genomics Research Institute, (3)Translational Genomics Research Institute, Flagstaff, AZ, (4)Adamas Pharmaceuticals, (5)Adamas Pharmaceuticals, Emeryville, CA