New Locus of Drug Resistance in the Human Cytomegalovirus UL56 Gene Revealed by In Vitro Exposure to Letermovir and Ganciclovir

Abstract

Letermovir is a human cytomegalovirus (CMV) terminase inhibitor recently approved as prophylaxis in stem cell transplant recipients. In further studies of emerging drug resistance, a baseline laboratory CMV strain was serially propagated in cell culture under a combination of letermovir and ganciclovir. In eight experiments, UL56 terminase gene mutations were detected beginning at 10 passages and included novel amino acid substitutions V236A, L328V, and A365S in a region previously associated with letermovir resistance. Outside this region, the UL56 substitution C25F was detected at moderate drug concentrations in two experiments as either the first detected mutation or an addition to a preexisting V231L substitution. In all cases, mutation at UL56 codon 325 conferring absolute letermovir resistance eventually developed at a median of 20 passages. No UL97 kinase or UL54 DNA polymerase mutations relevant to ganciclovir resistance were detected until many passages after the first detection of the UL56 mutations. UL56 substitutions V236A, L328V, and A365S were shown to confer borderline or low-grade letermovir resistance, while C25F conferred a 5.4-fold increase in letermovir resistance (50% effective concentration [EC]) by itself and a 46-fold increase in combination with V231L. The evolution of resistance mutations sooner in UL56 than in UL54 or UL97 is consistent with prior observations, and UL56 codon 25 is a genetic locus for letermovir resistance distinct from loci previously described.

Emergence of letermovir resistance in a lung transplant recipient with ganciclovir-resistant cytomegalovirus infection

“…The viral DNA rebound began before high‐dose steroid therapy was given at week 94. At week 100, viral genotypic analysis revealed a CMV UL56 mutation C325Y, which is known to confer absolute resistance to letermovir (50% inhibitory concentration increased by >8000‐fold over wild‐type) . The previously detected UL97 mutation M460V was again detected, but the UL54 mutation L516P was no longer detected.…”

“…In the literature, the median duration of ganciclovir therapy prior to detection of resistance is about 22 weeks, although 3 of 17 lung recipients were reported to develop ganciclovir resistance much sooner . Extensive in vitro studies have reported the rapid evolution of letermovir resistance after drug exposure, much sooner than in parallel experiments with ganciclovir and foscarnet . Unlike ganciclovir and foscarnet, letermovir is affected by a single step mutation at UL56 codon 325 (C325) that confers absolute resistance with minimal to low impact on viral growth fitness .…”

“…Extensive in vitro studies have reported the rapid evolution of letermovir resistance after drug exposure, much sooner than in parallel experiments with ganciclovir and foscarnet . Unlike ganciclovir and foscarnet, letermovir is affected by a single step mutation at UL56 codon 325 (C325) that confers absolute resistance with minimal to low impact on viral growth fitness . In vitro, multiple independent selections of C325 mutations have been observed in the approximate order of frequency of C325F > C325R ~ C325Y > C325W, probably reflecting the relative growth fitness of the mutants.…”

“…The L516P mutant was resistant to ganciclovir and cidofovir but remained susceptible to foscarnet. This phenotype is qualitatively similar to the D515Y mutant involving the adjacent amino acid of the UL54 DNA polymerase gene product, though the L516P mutant conferred a slightly higher level of resistance to ganciclovir and cidofovir. The UL54 mutant with codons 981‐982 deleted was included as a known multidrug‐resistant control and showed the expected degree of resistance to all 3 polymerase inhibitors …”

Use of Letermovir as Salvage Therapy for Drug-Resistant Cytomegalovirus Retinitis

“…Genotyping confirmed treatment-emergent UL56 mutations in two patients, while a third patient had clinical evidence of resistance. Serial viral passage under letermovir selective pressure has been associated with a relatively rapid selection of UL56 mutations, particularly within codons 231 to 369 (16,17). The possibility exists that the observed cases of letermovir resistance resulted from a selection of resistant subpopulations of CMV rather than as a consequence of a low barrier to resistance.…”