Novel UL97 drug resistance mutations identified at baseline in a clinical trial of maribavir for resistant or refractory cytomegalovirus infection

Abstract

In a Phase 2 clinical trial, 120 subjects with cytomegalovirus (CMV) infection refractory or resistant to standard therapy were randomized equally to 3 doses of oral maribavir treatment, and 70% achieved undetectable plasma CMV DNA within 12 weeks. At study entry, standard diagnostic UL97 genotyping was available for 71 subjects, with 60 (85%) revealing well-characterized ganciclovir resistance mutations that did not preclude a therapeutic response to maribavir. Central laboratory testing of a range of UL97 codons (288-468) not fully covered by standard genotyping was done on 93 subjects at baseline. This detected no previously known maribavir resistance mutations, but identified atypical mutations in 3 subjects, including a P-loop substitution F342Y, and ATP-binding region substitutions K359E/Q. By recombinant phenotyping, K359E and K359Q each conferred a nearly 4-fold increased ganciclovir 50% inhibitory concentration (EC50) without maribavir resistance, whereas F342Y conferred a 6-fold increased ganciclovir EC50 and a 4.5-fold increased maribavir EC50. The subject with F342Y detected at baseline did not achieve plasma CMV DNA clearance after 12 weeks of maribavir therapy and later developed an additional UL97 substitution H411Y known to confer 12- to 20-fold increased MBV EC50 by itself. The combination of F342Y and H411Y was shown to increase the maribavir EC50 by 56-fold. Diagnostic genotyping of UL97 should be expanded to cover the ATP-binding region beginning at codon 335 to enable the detection of atypical resistance mutations and further correlation of their clinical significance.

Keywords: Antiviral drug resistance; Cross-resistance; Cytomegalovirus; Ganciclovir; Maribavir.

Figure 1.. Comparative growth curves of viral strains

Wild type control virus and UL97 mutants were inoculated at equal multiplicity of infection of 0.02 as calibrated by culture supernatant SEAP activity at 24 hours. At each of 4 to 8 days post-inoculation, culture supernatants were assayed for SEAP activity (relative light units, RLU) as a measure of viral growth. Data points are the mean and standard deviation of 4 replicates set up simultaneously.

Figure 2.. UL97 kinase structure model

A published structure model of the UL97 kinase ATP-binding region based on yeast GCN2 kinase (Chou and Marousek, 2008) is updated to show residue 342, a locus of cross-resistance, in the central portion of the P-loop (residues 335–346), and residue 359 located downstream of the functionally critical K355 residue. The ATP-competitive site of MBV binding is shown. Mutations conferring MBV resistance (substitutions V353A, T409M and H411Y) are expected to disrupt this binding. The common ganciclovir resistance mutations at residue 460 are in a separate catalytic loop. Original figure copyright © American Society for Microbiology, Journal of Virology, 82, 2008, 246–253, DOI: 10.1128/JVI.01787-07