Christian Gilbert

Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms

Nucleoside analogues such as acyclovir and ganciclovir have been the mainstay of therapy for alphaherpesviruses (herpes simplex virus (HSV) and varicella-zoster virus (VZV)) and cytomegalovirus (CMV) infections, respectively. Drug-resistant herpesviruses are found relatively frequently in the clinic, almost exclusively among severely immunocompromised patients receiving prolonged antiviral therapy. For instance, close to 10% of patients with AIDS receiving intravenous ganciclovir for 3 months excrete a drug-resistant CMV isolate in their blood or urine and this percentage increases with cumulative drug exposure. Many studies have reported that at least some of the drug-resistant herpesviruses retain their pathogenicity and can be associated with progressive or relapsing disease. Viral mutations conferring resistance to nucleoside analogues have been found in either the drug activating/phosphorylating genes (HSV or VZV thymidine kinase, CMV UL97 kinase) and/or in conserved regions of the viral DNA polymerase. Currently available second line agents for the treatment of herpesvirus infections--the pyrophosphate analogue foscarnet and the acyclic nucleoside phosphonate derivative cidofovir--also inhibit the viral DNA polymerase but are not dependent on prior viral-specific activation. Hence, viral DNA polymerase mutations may lead to a variety of drug resistance patterns which are not totally predictable at the moment due to insufficient information on specific drug binding sites on the polymerase. Although some CMV and HSV DNA polymerase mutants have been found to replicate less efficiently in cell cultures, further research is needed to correlate viral fitness and clinical outcome.

Absence of Cytomegalovirus-Resistance Mutations after Valganciclovir Prophylaxis, in a Prospective Multicenter Study of Solid-Organ Transplant Recipients

We investigated the emergence of cytomegalovirus (CMV) ganciclovir-resistance mutations in 301 high-risk solid-organ transplant (SOT) recipients after oral prophylaxis, for 100 days, with either valganciclovir or ganciclovir. For patients treated with ganciclovir, the incidence of CMV UL97 mutations was 1.9% (2/103) at the end of prophylaxis and 6.1% (2/33) for patients with suspected CMV disease up to 1 year after transplantation. No resistance mutations were detected in samples from valganciclovir-treated patients. Dual polymerase (UL54) and UL97 resistance mutations were not seen. Valganciclovir was associated with negligible risk of resistance and thus constitutes a useful alternative to ganciclovir prophylaxis for CMV in high-risk SOT recipients.

Rate of Emergence of Cytomegalovirus (CMV) Mutations in Leukocytes of Patients with Acquired Immunodeficiency Syndrome Who Are Receiving Valganciclovir as Induction and Maintenance Therapy for CMV Retinitis

The emergence of mutations conferring ganciclovir resistance was evaluated in an open-label randomized clinical trial that compared oral valganciclovir with intravenous ganciclovir as induction therapy, followed by maintenance with valganciclovir, for newly diagnosed cytomegalovirus (CMV) retinitis in 148 patients with acquired immunodeficiency syndrome. The presence of CMV mutations was directly assessed in patient leukocytes by polymerase chain reaction, followed by restriction fragment-length polymorphism (RFLP) for detection of the most common UL97 mutations associated with ganciclovir resistance and by sequencing of the viral UL97 gene. The cumulative percentages of patients with UL97-mutant viruses at 3, 6, 12, and 18 months (based on the number of patients on treatment at each time point) was 2.2%, 6.5%, 12.8%, and 15.3%, respectively. Of the 20 relevant UL97 mutations found by sequencing in 14 patients, 14 (70%) were detected by RFLP analysis. The rate of emergence of ganciclovir-resistant viruses with use of oral valganciclovir is no greater than that reported with use of intravenous ganciclovir.

Clinical impact of ganciclovir-resistant cytomegalovirus infections in solid organ transplant patients

Abstract: Clinical consequences of ganciclovir resistant cytomegalovirus (CMV) infections were studied during 2 large prophylactic trials consisting of 100 days of valganciclovir or ganciclovir prophylaxis in solid organ transplant (SOT) recipients. The first one involved 301 high‐risk (CMV donor seropositive/recipient seronegative) SOT recipients excluding lung transplants followed for 12 months, whereas the second one involved 80 lung transplant patients evaluated over 6 months. Among the 7 patients (4 non‐lung and 3 lung transplant patients) carrying viruses with known ganciclovir resistance mutations in blood, adverse clinical outcome was only observed in the lung transplant recipients. Additionally, no CMV resistance mutations were observed in non‐lung transplant patients receiving valcanciclovir.

Cytomegalovirus (CMV) Glycoprotein B Genotypes and Response to Antiviral Therapy, in Solid-Organ–Transplant Recipients with CMV Disease

Cytomegalovirus (CMV) can be classified into 4 glycoprotein B (gB) genotypes, on the basis of sequence variation in the UL55 gene. We assessed the effect that CMV gB genotype has on virologic and clinical response to therapy, in 50 solid-organ-transplant recipients with CMV disease. CMV loads were determined at regular intervals after the start of therapy. Genotype results were correlated with CMV-load kinetics in response to therapy with ganciclovir. At the onset of treatment, the distribution of CMV gB genotypes was as follows: gB1, 19/50 (38%); gB2, 9/50 (18%); gB3, 12/50 (24%); gB4, 2/50 (4%); and mixed-genotype infection, 8/50 (16%). Between viral genotype groups, time to clearance of CMV, failure to clear CMV, and calculated CMV-load half-life after the start of therapy were not significantly different. The CMV gB genotype did not affect the rate of disease recurrence or occurrence of tissue-invasive disease. It appears that the gB genotype, which causes CMV disease, does not significantly influence CMV-load kinetics or clinical response to therapy.

Emergence and prevalence of cytomegalovirus UL97 mutations associated with ganciclovir resistance in AIDS patients

Objectives: To evaluate the prevalence of the most common cytomegalovirus (CMV) UL97 mutations associated with ganciclovir resistance directly in polymorphonuclear leukocytes (PMNL) of patients with AIDS and CMV retinitis. Also to correlate the presence (or absence) of these mutations with the systemic CMV viral load and the ophthalmologic outcome of these subjects. Methods: Monthly blood samples were obtained from 19 patients with AIDS and CMV retinitis who had been treated with systemic ganciclovir for ≥ 2 months. Detection of CMV UL97 mutations was done using nested PCR amplification followed by restriction enzyme analysis. The viral load was assessed with a polymerase chain reaction-based assay and non-isotopic hybridization detection. Results: CMV UL97 mutations were detected in PMNL of four of 13 (30.8%) patients who had been treated with ganciclovir for ≥ 3 months but in none of six patients who had been treated for < 3 months. All four patients with detectable UL97 mutations were presenting evidence of retinitis progression at the time those mutations were first detected (mean, 145.7 days of ganciclovir) and three of four patients had a viral DNA load > 10 000 copies per 105 PMNL contrasting with the copy numbers in the 15 subjects without mutations (mean, 492.9 copies per 105 PMNL after a mean of 146.8 days of ganciclovir). Conclusions: The prevalence of the most common CMV UL97 mutations associated with ganciclovir resistance in PMNL of patients with AIDS treated for ≥ 3 months (30.8%) appears to be higher than the rate of emergence of ganciclovir-resistant CMV isolates as previously reported using phenotypic assays (about 8%). Moreover, the detection of these mutations is associated with a considerable increase in the CMV DNA load in the blood as well as with progression of CMV retinitis during ganciclovir therapy.

Human cytomegalovirus glycoprotein B genotypes in blood of AIDS patients: Lack of association with either the viral DNA load in leukocytes or presence of retinitis

It has been suggested that human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes could be used as a marker for viral virulence in patients with AIDS. The present study was designed to evaluate a possible association between specific gB genotypes, the presence of HCMV retinitis, and the HCMV viral load. Fifty‐four blood samples were obtained from 54 HIV‐ and HCMV‐infected patients. Twenty‐seven of these patients were asymptomatic for HCMV, whereas the other 27 patients had been diagnosed recently with HCMV retinitis. HCMV gB genotyping was carried out by using restriction enzyme analysis of PCR‐amplified PMNL extracts. Determination of the HCMV viral load in the same specimens was carried out using a quantitative‐PCR. HCMV gB genotype 2 was found more frequently than other genotypes in PCR‐amplified polymorphonuclear leukocytes (PMNL) of patients with AIDS (P < 0.05) but not more frequently in samples from patients with HCMV retinitis. No significant association was found between any HCMV gB genotypes and the viral load in blood. In conclusion, the actual HCMV gB genotyping system using PMNL provides no additional benefit over the viral load in blood for identification of HIV‐infected subjects at risk of HCMV disease. J. Med. Virol. 59:98–103, 1999.

A case of ganciclovir-resistant cytomegalovirus (CMV) retinitis in a patient with AIDS: longitudinal molecular analysis of the CMV viral load and viral mutations in blood compartments.

Objectives: To study the temporal relationships between cytomegalovirus (CMV) viral load and specific UL97 mutations in polymorphonuclear leukocytes (PMNL) and plasma samples from a patient with AIDS who developed ganciclovir‐resistant CMV retinitis. Methods: Sequential PMNL and plasma samples were analysed for determination of the CMV viral load using non‐molecular methods and a quantitative polymerase chain reaction (PCR) assay. Screening of the same samples for the most common mutations conferring ganciclovir resistance was performed using nested PCR and restriction enzyme analysis. Results: At the time of progression of CMV retinitis (after 6 months of ganciclovir), a rapid increase in the CMV DNA load was found in both PMNL and plasma samples. This increase paralleled the emergence of a specific mutation (V594) in the same samples and recovery of ganciclovir‐resistant blood isolates. In this patient, however, the only tests that substantially predicted the progression of CMV disease were the quantitative PCR assay using PMNL and to a lesser extent the pp65 antigenemia assay. Conclusions: Quantitative evaluation of the CMV viral load in PMNL using sensitive assays such as PCR appears to be a promising approach for monitoring antiviral therapy in subjects with AIDS. In addition, common mutations conferring ganciclovir resistance can be detected directly in PMNL and plasma samples.

Characterization of Human Cytomegalovirus (HCMV) UL97 Mutations Found in a Valganciclovir/Oral Ganciclovir Prophylactic Trial by Use of a Bacterial Artificial Chromosome Containing the HCMV Genome

A method based on manipulation of the human cytomegalovirus genome in a bacterial artificial chromosome was developed to determine the role played by 6 UL97 mutations of unknown significance. These mutations were found in blood samples from solid-organ transplant recipients in a trial comparing valganciclovir and oral ganciclovir prophylaxis. Recombinant viruses containing UL97 mutations P405L, A427V, M550I, A582V, Y617H, and A674T were generated in a bacterial system. Viral stocks were subsequently reconstituted in human fibroblasts, and ganciclovir susceptibilities were tested using a plaque-reduction assay. All recombinant viruses containing these unknown mutations were found to be susceptible to ganciclovir.

Role of Helix P of the Human Cytomegalovirus DNA Polymerase in Resistance and Hypersusceptibility to the Antiviral Drug Foscarnet

ABSTRACT Mutations in the human cytomegalovirus DNA polymerase (UL54) can not only decrease but also increase susceptibility to the pyrophosphate (PPi) analogue foscarnet. The proximity of L802M, which confers resistance, and K805Q, which confers hypersusceptibility, suggests a possible unifying mechanism that affects drug susceptibility in one direction or the other. We found that the polymerase activities of L802M- and K805Q-containing mutant enzymes were literally indistinguishable from that of wild-type UL54; however, susceptibility to foscarnet was decreased or increased, respectively. A comparison with the crystal structure model of the related RB69 polymerase suggests that L802 and K805 are located in the conserved α-helix P that is implicated in nucleotide binding. Although L802 and K805 do not appear to make direct contacts with the incoming nucleotide, it is conceivable that changes at these residues could exert their effects through the adjacent, highly conserved amino acids Q807 and/or K811. Our data show that a K811A substitution in UL54 causes reductions in rates of nucleotide incorporation. The activity of the Q807A mutant is only marginally affected, while this enzyme shows relatively high levels of resistance to foscarnet. Based on these data, we suggest that L802M exerts its effects through subtle structural changes in α-helix P that affect the precise positioning of Q807 and, in turn, its presumptive involvement in binding of foscarnet. In contrast, the removal of a positive charge associated with the K805Q change may facilitate access or increase affinity to the adjacent Q807.