Combined use of pritelivir with acyclovir or foscarnet suppresses evolution of HSV-1 drug resistance

Abstract

The widespread use of antivirals in immunocompromised individuals has led to frequent occurrences of drug-resistant herpes simplex virus 1 (HSV-1) infections. Current antivirals target the viral DNA polymerase (DP), resulting in cross-resistance patterns that emphasize the need for novel treatment strategies. In this study, we assessed whether combining antivirals with different targets affects drug resistance emergence by passaging wild-type HSV-1 under increasing concentrations of acyclovir (ACV), foscarnet (phosphonoformic acid, PFA), or the helicase-primase inhibitor pritelivir (PTV), individually or in combination (ACV + PTV or PFA + PTV). The resistance selection procedure was initiated from two different drug concentrations for each condition. Deep sequencing and subsequent phenotyping showed the rapid acquisition of resistance mutations under monotherapy pressure, whereas combination therapy resulted in either no mutations or mutations conferring ACV and/or PFA resistance. Notably, mutations associated with PTV resistance were not detected after five passages under combination pressure. Strains resistant to both ACV and PTV were eventually obtained upon further passaging under ACV + PTV pressure initiated from lower drug concentrations. PFA + PTV dual treatment induced PFA resistance mutations in the DP, but PTV resistance mutations were not acquired, even after 15 passages. Our data suggest that combining the helicase-primase inhibitor PTV with a DP inhibitor may be an effective strategy to prevent drug resistance evolution in HSV-1.

Keywords: antiviral resistance; combination therapy; herpes simplex virus 1.

Figure 1.

Anti-HSV-1 activity of PTV in combination with ACV or PFA. HEL fibroblasts infected with HSV-1 wild-type strain KOS were exposed to various concentrations of one or two antivirals (n = 2). Inhibitory effects of the antivirals were assessed by determining the reduction in HSV-1 virus titers. Interaction plots for (a) ACV and PTV and (b) PFA and PTV were generated using SynergyFinder 2.0 using the ZIP model. Synergy (δ) scores were calculated to quantify the deviation from the expected response for the given dose pair, with a δ score < −10 implying antagonism, a δ score between −10 and 10 implying additivity, and a δ score >10 implying synergism. Overall synergy scores, based on all measured drug combinations, are shown above the plots, and the black squares indicate the most synergistic 3-by-3 dose region in the dose–response matrix.

Figure 2.

Chronological detection of drug resistance mutations under PTV (combination) pressure using plaque-purified wild-type virus. The frequency (%) of mutations in the virus cultures was defined before and following 2, 5, 10, and 15 passages by deep sequencing of the viral TK (UL23 gene), DP (UL30 gene), helicase (UL5 gene), and primase (UL52 gene). PTVr^L and PTVr^H were cultured for five passages (until the dotted line). Bold: previously undescribed change with an unknown effect on drug susceptibility; italics: known drug resistance mutations (Chibo et al. 2004, Saijo et al. 2005, Collot et al. 2016).

Figure 3.

Mutation map of the HSV-1 TK (UL23), DP (UL30), helicase (UL5), and primase (UL52) mutations identified in this study. Conserved sites are indicated by black boxes. Alterations that were not detected in a viral clone or with a frequency of >2% at Passages 5, 10, and/or 15 were excluded. Bold: previously undescribed changes. * Pre-existing as minor variant in the parental KOS strain.