Widespread resistance among circulating
influenza A strains to at least one of the anti-
influenza drugs is a major public health concern. A triple combination
antiviral drug (
TCAD) regimen comprised of
amantadine,
oseltamivir, and
ribavirin has been shown to have synergistic and broad spectrum activity against
influenza A strains, including
drug resistant strains. Here, we used mathematical modeling along with three different experimental approaches to understand the effects of single agents, double combinations, and the
TCAD regimen on resistance in
influenza in vitro, including: 1) serial passage at constant
drug concentrations, 2) serial passage at escalating
drug concentrations, and 3) evaluation of the contribution of each component of the
TCAD regimen to the suppression of resistance. Consistent with the modeling which demonstrated that three drugs were required to suppress the emergence of resistance in
influenza A, treatment with the
TCAD regimen resulted in the sustained suppression of
drug resistant viruses, whereas treatment with
amantadine alone or the
amantadine-
oseltamivir double combination led to the rapid selection of resistant variants which comprised ∼100% of the population. Furthermore, the
TCAD regimen imposed a high genetic barrier to resistance, requiring multiple mutations in order to escape the effects of all the drugs in the regimen. Finally, we demonstrate that each
drug in the
TCAD regimen made a significant contribution to the suppression of virus breakthrough and resistance at clinically achievable concentrations. Taken together, these data demonstrate that the
TCAD regimen was superior to double combinations and single agents at suppressing resistance, and that three drugs at a minimum were required to impede the selection of
drug resistant variants in influenza A virus. The use of mathematical modeling with multiple experimental designs and molecular readouts to evaluate and optimize
combination drug regimens for the suppression of resistance may be broadly applicable to other
infectious diseases.