Lack of resistance development during brincidofovir therapy in animal models of orthopoxvirus infection.

Abstract

Brincidofovir (BCV, commercially known as Tembexa®) is an antiviral drug with broad-spectrum activity against double-stranded DNA viruses, including orthopoxviruses. BCV was developed and approved for treating human smallpox in adults and children, including neonates, under the FDA Animal Rule. The efficacy data for licensure were obtained using two surrogate models of smallpox: the rabbitpox virus (RPXV) in New Zealand White rabbits and ectromelia virus (ECTV) in Balb/c mice. The potential for BCV resistance development was evaluated in RPXV- and ECTV-infected animals treated with BCV from pivotal nonclinical studies. Next-generation sequencing (NGS) was employed to identify potential resistance-associated substitutions (RAS) in the DNA polymerase gene and the two largest RNA polymerase subunit genes in samples from RPXV- or ECTV-infected animals. NGS-identified potential RAS mutants were then constructed in vaccinia virus (VACV) using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and virus reactivation methods. Plaque reduction assays evaluated the sensitivity of VACV mutants to BCV. No previously identified viral DNA polymerase BCV or cidofovir RAS were detected at or above 1% frequency in the RPXV or ECTV samples by NGS. Positive controls containing known BCV resistance-associated mutations (A314T and A684T) in the viral DNA polymerase at frequencies of 1% or 5% were routinely detected. Several novel mutations were identified in mouse ECTV samples; however, none, when introduced into VACV, conferred BCV resistance in a plaque reduction assay. In conclusion, no BCV resistance-associated mutations were detected in either animal model under the conditions of this treatment regimen.