Utilising animal models to evaluate oseltamivir efficacy against influenza A and B viruses with reduced in vitro susceptibility

Abstract

The neuraminidase (NA) inhibitor (NAI) oseltamivir (OST) is the most widely used influenza antiviral drug. Several NA amino acid substitutions are reported to reduce viral susceptibility to OST in in vitro assays. However, whether there is a correlation between the level of reduction in susceptibility in vitro and the efficacy of OST against these viruses in vivo is not well understood. In this study, a ferret model was utilised to evaluate OST efficacy against circulating influenza A and B viruses with a range of in vitro generated 50% inhibitory concentrations (IC50) values for OST. OST efficacy against an A(H1N1)pdm09 and an A(H1N1)pdm09 virus with the H275Y substitution in neuraminidase was also tested in the macaque model. The results from this study showed that OST had a significant impact on virological parameters compared to placebo treatment of ferrets infected with wild-type influenza A viruses with normal IC50 values (~1 nM). However, this efficacy was lower against wild-type influenza B and other viruses with higher IC50 values. Differing pathogenicity of the viruses made evaluation of clinical parameters difficult, although some effect of OST in reducing clinical signs was observed with influenza A(H1N1) and A(H1N1)pdm09 (H275Y) viruses. Viral titres in macaques were too low to draw conclusive results. Analysis of the ferret data revealed a correlation between IC50 and OST efficacy in reducing viral shedding but highlighted that the current WHO guidelines/criteria for defining normal, reduced or highly reduced inhibition in influenza B viruses based on in vitro data are not well aligned with the low in vivo OST efficacy observed for both wild-type influenza B viruses and those with reduced OST susceptibility.

Fig 1. Viral shedding data from ferrets exposed to different viruses and dosed with either OST or Placebo.

The data for all ferrets are shown after standardising to first day of TCID₅₀ positive, as this day varied between different ferrets, and each individual ferret within a group is identified by a separate colour. The variability in first day of viral shedding is summarised in S2 Fig. The bar graphs represent the mean viral titre for all ferrets within the group on each day. The B stands for baseline.

Fig 2. A scatter plot showing the relationship between %ΔAUC (difference between placebo and OST dosed animals) of viral shedding of ferrets and the OST IC₅₀.

This graph shows the significant correlation between in vitro OST IC₅₀ and in vivo effect on viral shedding. The regression model reveals the following relationship where y = e^4.35–0.03x. The shaded region of the graph is the 95% confidence interval of the line of best fit, calculated by bootstrapping over 2500 iterations.

Fig 3. Influenza virus titres from nasal washes and tracheal lavages from macaques infected with either H1N1pdm09 or H1N1pdm09 (H275Y) virus and treated with either OST or placebo.

Three macaques were allocated per group and artificially infected with 106 TICD₅₀/ml of viral inoculum. No significant differences were observed in viral titre between animals treated with OST compared to those treated with placebo. Panel a) shows viral titre data from nasal washes of animals and b) shows viral tire data from tracheal lavages.