Characterization of human influenza virus variants selected in vitro in the presence of the neuraminidase inhibitor GS 4071

Abstract

An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

FIG. 1

The 12-S3 variant is compromised for growth in culture. (A) MDCK cells were infected with the wild-type A/Victoria/3/75 virus, the 12-B1 variant, or the 12-S3 variant in a standard plaque assay. After 3 days at 37°C, the cell monolayers were stained to reveal the size of the plaques. (B) MDCK cells were infected with the wild-type virus or the 12-S3 variant at a multiplicity of infection of 0.001 PFU per cell. At the indicated times after infection, the titer of virus in the culture supernatant was determined. The data points are means and standard deviations obtained from duplicate wells in a representative experiment.

FIG. 2

Kinetics of inhibition of wild-type and mutant neuraminidase by GS 4071. Progress plots depicting the hydrolysis of MUNANA by the wild-type (A) and mutant (B) neuraminidase in the presence of varying concentrations of GS 4071. The concentration of GS 4071 present in each of the reactions is indicated.