Triple combination antiviral drug (TCAD) composed of amantadine, oseltamivir, and ribavirin impedes the selection of drug-resistant influenza A virus

Abstract

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.

Figure 1. Percent of Resistant Virus Variants Generated at Passage 5 as a Function of Drug Concentration.

Wild type influenza A/Hawaii/31/2007 (H1N1) virus was passaged five times in MDCK cells, with the concentrations of drugs in each regimen kept fixed in between passages. The drug concentrations used are given in Table 2 and are discussed in the Materials and Methods. Concentrations 1 to 4 correspond to 1/9, 1/3, 1, and 3 times the clinically relevant concentration for each drug, respectively. The percent of virus variants bearing resistance-associated substitutions in M2 channel (V27A, A30T, or S31N) are presented as the mean of triplicate qASPCR reactions with 95% confidence intervals from a single well of MDCK cells. Concentration 3 represents the clinically relevant concentrations of all three drugs (AMT, OSC, and RBV, see Materials and Methods).

Figure 2. Percent of AMT-Resistant Virus Variants Generated as a Function of Passage Number.

Wild type influenza A/Hawaii/31/2007 (H1N1) virus was passaged five times in MDCK cells, with the concentrations of drugs in each regimen kept fixed in between passages. The percent of virus variants bearing resistance-associated substitutions in M2 (V27A, A30T, or S31N) at each passage are presented as the mean of triplicate qASPCR reactions with 95% confidence intervals from a single well of MDCK cells. Each panel represents an increase in the concentration of each drug in the various regimens, with Concentration 3 representing the clinically relevant concentrations of all three drugs (see Materials and Methods). Drug concentrations are provided in units of µg/mL.

Figure 3. Passage History of Wild Type Influenza A/Hawaii/31/07 under Escalating Drug Concentrations.

Wild type influenza A/Hawaii/31/2007 (H1N1) virus was passaged in the presence of escalating concentrations of each drug regimen for a total of ≥25 cumulative days in culture, or until the drug concentration reached the 50% of the cytotoxic concentration (TC₅₀) of the drug as a single agent.

Figure 4. Each Drug in TCAD Contributes to the Suppression of Virus Breakthrough.

MDCK cells in 96-well plates were infected with influenza A/Hawaii/31/2007 (H1N1) virus in the presence of a combination of two drugs at fixed concentrations with varying concentrations of the third drug, using 12 replicates for each condition. The fixed concentrations of the double combinations were 0.30 µg/mL OSC and 0.60 µg/mL RBV, 0.6 µg/mL AMT and 0.6 µg/mL RBV, or 0.6 µg/mL AMT and 0.3 mg/mL OSC. Following 5 serial passages, the number of wells for each condition having virus breakthrough, defined as >50% cytopathic effect, was determined by neutral red uptake.