Zanamivir Susceptibility Monitoring and Characterization of Influenza Virus Clinical Isolates Obtained during Phase II Clinical Efficacy Studies

Abstract

Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log 10 50% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (n ‫؍‬ 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.

Overexpression of the α-2,6-Sialyltransferase in MDCK Cells Increases Influenza Virus Sensitivity to Neuraminidase Inhibitors

“…However, there is no good correlation between virus sensitivity to NAI in vivo and in laboratory cell cultures. The sensitivity of clinical virus isolates to NA inhibitors can vary in cell culture assays dramatically (up to complete insensitivity) despite a uniform high sensitivity of the enzyme in NA-inhibition tests (1,3,37). This problem is likely due to a mismatch between virus receptors in humans and in available cell culture systems.…”

Evaluation of Neuraminidase Enzyme Assays Using Different Substrates To Measure Susceptibility of Influenza Virus Clinical Isolates to Neuraminidase Inhibitors: Report of the Neuraminidase Inhibitor Susceptibility Network

“…To date, relatively few viruses with altered susceptibility to NIs have been identified, as part of either preclinical or clinical development programs (2,5,7,8,9,12,16). Several NA mutants with reduced susceptibility have been selected by using zanamivir in vitro (E119G/A/D and R292K), but only one virus strain (R152K) with altered susceptibility has been isolated from an immunocompromised human treated with zanamivir (5).…”

Enhanced Expression of an α2,6-Linked Sialic Acid on MDCK Cells Improves Isolation of Human Influenza Viruses and Evaluation of Their Sensitivity to a Neuraminidase Inhibitor

“…A plaque reduction assay with MDCK cells was used to evaluate the susceptibility of laboratory strains of human influenza viruses to NA inhibitors (9). However, this MDCK cell assay cannot be used with clinical human isolates, especially recent strains, as the growth of many recent human influenza isolates is not inhibited by NA inhibitors in these cells, and the viruses are apparently judged to be resistant to the drugs even though their enzymatic activities are drug sensitive (1,7,27,30,32). In lieu of a reliable cell culture assay for determining the susceptibility of influenza viruses to NA inhibitors, a sialidase enzyme assay has been adapted for this purpose (22,27), but it is not suitable for large-scale screening.…”