3. DNA Polymerase Inhibitors
Until recently, the prevention and treatment of CMV infection relied on the use of inhibitors of the viral DNA polymerase that is essential for viral replication
[6]. The first-line drugs include the nucleoside analog, GCV and its prodrug VGCV whereas the second-line drugs consist in the pyrophosphate analog, FOS and the nucleotide analogue, CDV (
Figure 1)
[29].
Figure 1. Chemical structures of the different DNA polymerase inhibitors, letermovir and maribavir. Concentrations of antivirals that reduce cytomegalovirus growth by 50% (EC50) are also indicated.
Upon entry into infected cells, GCV is phosphorylated by the viral pUL97 kinase
[30] and then converted into its triphosphorylated form by cellular kinases. The active form is a competitive inhibitor of the activity of the viral pUL54 DNA polymerase
[31]. GCV-triphosphate also blocks chain elongation following its incorporation into viral DNA
[32]. CDV requires only two phosphorylations by cellular kinases to be converted into its active form
[33]. CDV-diphosphate is incorporated into viral DNA and prevents chain elongation
[34]. FOS does not require any phosphorylation to be active. It directly binds to the pyrophosphate site on the viral DNA polymerase and prevents the incorporation of incoming nucleotides into viral DNA
[35].
4. Indications for DNA Polymerase Inhibitors
Oral VGCV (900 mg once daily for prophylaxis and twice daily for treatment) and intravenous GCV (5 mg/kg once daily for prophylaxis and twice daily for treatment, dose adjusted for renal function) are indicated in the prevention and in the treatment of active CMV infections. The intravenous formulation of FOS (60 mg/kg every 8 h or 90 mg/kg every 12 h, with a reduction in dose for renal dysfunction) is used for the treatment of CMV retinitis in individuals with acquired immunodeficiency syndrome (AIDS) and infections caused by GCV-resistant CMV in immunocompromised patients. The intravenous formulation of CDV (5 mg/kg once a week for 2 weeks then every 2 weeks) is used for the treatment of CMV retinitis in AIDS patients and is occasionally administered in transplant recipients with drug-resistant CMV infections.
5. Prevention and Treatment of CMV Infection
The prevention of active CMV infection is based on two main approaches, universal prophylaxis and pre-emptive therapy (
Figure 2)
[5]. Universal prophylaxis consists of administering an antiviral agent after the transplantation for a period of 3 or 6 months in the high-risk groups and up to 12 months in lung transplants
[19]. The aim of this approach is to maintain viral suppression during the period of the greatest risk for CMV infection or reactivation. Antiviral prophylaxis is effective for the prevention of CMV disease as well as to reduce CMV-associated effects. However, this strategy is associated with a relatively high rate of late-onset CMV diseases following cessation of antiviral administration
[4][36][4,36] and substantial toxicity. Universal prophylaxis is the main CMV prevention strategy in high-risk SOT recipients. The pre-emptive therapy approach is based on the determination of the viral DNA load every week for 3 or 6 months
[5]. The antiviral agent is administered only when the viral DNA load is higher than a defined threshold. Pre-emptive therapy reduces drug exposure and drug-associated toxicity. In the DNA polymerase inhibitors era, pre-emptive therapy was the preferred CMV prevention strategy in HSC recipients to avoid the myelotoxicity of GCV. In order to reduce the risk of delayed-onset CMV diseases after antiviral prophylaxis, a hybrid approach based on the use of prophylaxis during the high-risk periods, i.e., 3 to 6 months after transplantation, followed by a shift to pre-emptive therapy has been also evaluated
[37][38][37,38].
Figure 2. Strategies used for the prevention of CMV infection in solid organ transplant (SOT) and hematopoietic stem cell (HSC) recipients. Universal prophylaxis is based on the administration of antivirals (blue line) to all at-risk patients for 3 or 6 months after transplantation (Tx). During pre-emptive therapy (PET), the antiviral (blue triangle) is administered when the viral load (determined in blood every week for 3 or 6 months) is higher than a defined threshold (red circle) and stopped when the viral is below the threshold (white circle). D
+/R
−, donor positive/recipient negative for CMV; R
+, recipient positive for CMV. Adapted from Limaye et al.
[5].
Treatment of initial and recurrent episodes of CMV syndrome and tissue-invasive CMV diseases have been based on the administration of oral VGCV or intravenous GCV
[39]. Oral VGCV is preferred for mild to moderate CMV disease and intravenous GCV for life-threatening disease
[19]. The viral DNA load should be monitored every week
[19]. Antiviral therapy can be stopped at resolution of clinical symptoms and viral clearance in two consecutive samples one week apart.
6. When to Suspect CMV Resistance to Antiviral Drugs?
When the viremia increases or reaches high levels or when clinical symptoms do not resolve despite antiviral therapy, the emergence of drug viral resistance should be suspected
[19]. In SOT recipients, exposure to GCV is usually longer than 6 weeks with a median at 5 to 6 months before the emergence of resistance but it can be shorter than 6 weeks in lung transplant recipients.
Prolonged antiviral therapy with inadequate GCV levels is typically associated with the emergence of drug resistance
[40]. In SOT recipients, risk factors include the intensity of immunosuppression, a donor positive/recipient negative (D
+/R
−) status and lung transplantation
[41][42][43][41,42,43]. In HSC recipients, the risk of developing viral drug resistance is increased by a D
−/R
+ status, the depletion of T cells, a delayed immune reconstitution and active graft-versus-host disease
[44]. The emergence of drug resistance is usually associated with increased morbidity and mortality in transplant recipients.
The incidence of GCV resistance is less than 5–12% in most SOTs but may be as high as 18% in lung transplant recipients
[41][45][46][41,45,46] and 31% in intestinal and multi-visceral organ transplants
[47][48][47,48]. In HSC recipients, the incidence of GCV resistance is usually less than 5% in recipients of an allogeneic graft
[49][50][49,50] but can be as high as 15% in recipients of a haploidentical graft
[51].
As FOS and CDV are less frequently used in the clinic, the temporal emergence of CMV strains resistant to these drugs has only been reported in human immunodeficiency virus (HIV)-infected individuals. One small study found an incidence of phenotypic resistance to FOS of 9, 26, 37 and 37% after 3, 6, 9 and 12 months of therapy using an EC
50 cutoff value of 400 μM (i.e., the concentration of antiviral that reduces CMV growth by 50%)
[52]. Another study reported rates of 13, 24 and 37% after 6, 9 and 12 months using an EC
50 cutoff value of 600 μM
[53]. The data on CDV resistance (EC
50 value ≥ 2–4 μM) seem to indicate a resistance rate similar to those observed with GCV and FOS
[52].
7. CMV Mutations Conferring Resistance to DNA Polymerase Inhibitors
Mutations conferring resistance to GCV initially arise in the pUL97 kinase and impair drug phosphorylation
[54]. Mutations conferring resistance to GCV usually emerge at codons 460 and between codons 590 and 607 of the pUL97 kinase (
Figure 3A)
[55]. Subsequent mutations emerge in the pUL54 DNA polymerase and can confer a high level of resistance and cross-resistance between two or three antiviral drugs
[56]. In pUL54 DNA polymerase, drug resistance mutations are widely distributed in the conserved regions of the enzyme (
Figure 3B)
[55]. GCV and CDV cross-resistant mutations are located in the exonuclease domain and in conserved region V of the polymerase domain. Mutations conferring resistance to FOS or both FOS and GCV are located in conserved regions II, VI and III of the polymerase domain. Mutations in both the pUL97 kinase and pUL54 DNA polymerase result in high levels of resistance to GCV
[57][58][59][57,58,59].
Figure 3. Confirmed cytomegalovirus resistance mutations to DNA polymerase inhibitors. Panel (A) shows a representation of the pUL97 kinase with its conserved regions (grey boxes) and the localization of amino acid substitutions conferring resistance to ganciclovir (vertical bars). The ATP-binding site, the phosphate transfer (P-transfer) domain, the nucleoside-binding site (NBS) and some regions conserved among the protein kinase family (i.e., I, II, III, VIB, VII, VIII and IX) are indicated above the boxes. The shaded area corresponds to the codon 590–603 region where different amino acid deletions were identified (i.e., deletions 591–594; 591–607; 595; 595–603; 600 and 601–603). Panel (B) shows a representation of pUL54 DNA polymerase with its conserved regions (grey boxes) and the localization of amino acids associated with resistance to ganciclovir (GCVR), foscarnet (FOSR) and/or cidofovir (CDVR) (colored bars). The Roman numbers (I to VII) and δ-region C correspond to conserved regions in the polymerase domain. Exo I, Exo II and Exo III are conserved motifs in the exonuclease domain.
8. Management of Refractory/Resistant CMV Disease in the DNA Polymerase Inhibitors Era
Based on the relative increase in their EC
50 values, UL97 mutations result in insignificant (<2×, low-grade (2–5×) or moderate (5–15×) levels of resistance to GCV (
Table 1)
[19]. Infection with insignificant or low-grade-resistant UL97 mutants can preferentially be treated with a high dose of intravenous GCV (10 mg/kg twice daily, adjusted for renal function)
[19]. Infection with UL97 mutants that are moderately resistant to GCV and UL54 mutants that are susceptible to FOS can be treated with a full dose of FOS (60 mg/kg every 8 h or 90 mg/kg every 12 h, with reduction in dose for renal dysfunction). Infection with UL54 mutants that are resistant to FOS can be treated with CDV (5 mg/kg once a week for 2 weeks and then every 2 weeks) whereas a combination of GCV and FOS at reduced doses
[60][61][60,61] could be administered in case of resistance to CDV.
Table 1. Relative levels of ganciclovir resistance of CMV UL97 mutants.
Relative levels of ganciclovir resistance of CMV UL97 mutants.
Genotype Frequency |
Relative Increase in EC50 Value Compared to Wild Type |
<2× (Insignificant) |
2–5× (Low-Grade) |
5–15× (Moderate) |
Most common |
|
C592G |
M460I/V, H520Q, A594V, L595S, C603W |
Less common at codons 460, 590–607 |
E596D, N597D, K599E/R, L600I, T601M, C603S, D605E, C607F |
A591V, A594E/T/S, E596G/Q, C603S, E596G, 600del2, C607F |
M460T, A594G/P, 595del, L595F/W/del, E596Y, 597del2, 599del, K599T, 600del, 601del, 601del2, C603R, C607Y, del(≥3) |
Atypical loci |
M615V, Y617H, A619V, L634Q, E655K, A674T |
K359E/N/Q, E362D, L405P, I610T, A613V |
F342S/Y, K355M, V356G, V466G, C480R, C518Y, P521L |