Geno- and Phenotypic Characterization of Human Cytomegalovirus Mutants SelectedIn Vitroafter Letermovir (AIC246) Exposure

Abstract

Letermovir is a novel antiviral compound currently in clinical development for the prevention of human cytomegalovirus (HCMV) infections. In contrast to all currently approved anti-HCMV drugs that target the viral DNA polymerase, letermovir acts via a distinct mode of action involving the viral terminase subunit pUL56. To extend our understanding of potential letermovir resistance mechanisms, we used marker transfer to characterize mutations identified in letermovir-resistant HCMV variants that were selected in cell culture. Human cytomegalovirus (HCMV) disease continues to be a serious and life-threatening condition in immunocompromised patients such as transplant recipients. Currently approved anti-HCMV drugs, including (val)ganciclovir, cidofovir, and foscarnet, are associated with profound toxicity as well as drug resistance that restricts their long-term clinical benefit. All these substances ultimately target the viral DNA polymerase (pUL54), though ganciclovir requires prior activation by the viral pUL97 kinase. Accordingly, mutations in open reading frame (ORF) UL97 are associated with (val)ganciclovir resistance whereas mutations in ORF UL54 can confer cross-resistance to all approved anti-HCMV drugs, an increasingly frequent scenario representing a serious threat for specific patient populations (1-3).Letermovir (AIC246) is a new anti-HCMV drug with a distinct but well-characterized mechanism of action targeting the viral terminase subunit pUL56 (4, 5). The drug has proven to be well tolerated in numerous (pre)clinical studies and has demonstrated clinical efficacy in a recent phase IIb trial, meeting all primary endpoints as a prophylactic drug (6).In line with its distinct mode of action, in vitro studies have shown that letermovir is active against HCMV strains resistant to current anti-HCMV drugs (4, 5, 7). In vivo confirmation of resistance-breaking potential was demonstrated in a patient infected with a multidrug-resistant HCMV strain (8). So far, no letermovir-resistant HCMV strains have been reported from clinical trials. Nonetheless, as letermovir is a direct-acting antiviral, the emergence of resistance is ultimately to be expected.HCMV resistance testing previously required virus cultivation and phenotypic characterization of virus isolates; this is slow, labor-intensive, and nonstandardized and often has a poor success rate (3, 9). Current standard clinical practice uses genotyping and virtual phenotyping based, e.g., on mutation databases (10). Once a database is established, this genetic approach is faster, more sensitive, and more cost-effective and provides detailed and quantitative resistance information to the physician (3). However, genetic monitoring of drug resistance requires detailed knowledge of potential resistance mutations in order to develop virtual phenotyping profiles for screening databases (6).To date, 2 letermovir resistance mutations leading to amino acid substitutions have been identified in vitro, both within the coding region for the pUL56 subunit of the viral termi...

RapidIn VitroEvolution of Human Cytomegalovirus UL56 Mutations That Confer Letermovir Resistance

“…Any difference in fitness of the mutant viruses compared to baseline strains is not discernible among the growth curves. This is consistent with a previous report (7). wild type (wt) and UL56 mutant virus stocks were inoculated onto human foreskin fibroblast monolayers at an MOI of ϳ0.01.…”

“…LMV EC 50 s are shown in Table 3 and were determined using a standardized reporterbased yield reduction assay. Published data derived using a different phenotyping assay reported a comparable baseline LMV EC 50 of 3 to 5 nM and EC 50 ratios of 5, 160 to 218, and Ͼ8,000 for V231L, L241P, and C325Y, respectively (7,23,24). EC 50 ratios of Ͼ3,000 can be interpreted as absolute LMV resistance, because viral yield reduction occurs at visibly cytotoxic LMV concentrations.…”

“…More recently, letermovir (LMV; formerly AIC246) advanced to phase III clinical trials for prevention of CMV infection in stem cell transplant recipients after a successful dose-ranging phase II study (6). Published in vitro studies of LMV resistance after exposure to drug concentrations 10-fold higher than the baseline 50% effective concentration (EC 50 ) identified mutations at UL56 codons 231, 236, 241, 325, and 369, distinct from those reported for the older terminase inhibitors (7). These UL56 mutations were found to confer diverse levels of LMV resistance, ranging from a 5-fold increase in EC 50 for V231L to Ͼ5,000-fold for C325Y.…”

Treatment of CMV infection after allogeneic hematopoietic stem cell transplantation

“…The study is expected to complete the accrual in January 2017 for primary outcome (NCT02137772). Overall, in vitro LMV resistant CMV strains have been reported [79,80,81].…”

Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation

“…One of the most promising new drug candidates, Letermovir (MK-8228; AIC246), which demonstrated excellent efficacy and safety in phase 2 clinical trials in kidney transplant patients and in hematopoietic stem cell recipients (28,29) and recently met the primary endpoint in a phase III clinical study (30) (ClinicalTrials registration no. NCT02137772) prevents cleavage of the viral DNA concatemers and formation of mature virions, apparently by targeting the terminase subunit pUL56 (31)(32)(33)(34). Importantly, Letermovir is able to inhibit HCMV variants that are resistant to other antiviral agents (35)(36)(37), and compared to the approved HCMV drugs, its toxicity is negligible.…”