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Ardila CM, Yadalam PK, Ramírez-Arbelaez J. Efficacy of antimicrobials in preventing resistance in solid organ transplant recipients: A systematic review of clinical trials. World J Transplant 2025; 15:98003. [PMID: 40104188 PMCID: PMC11612891 DOI: 10.5500/wjt.v15.i1.98003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/14/2024] [Revised: 09/26/2024] [Accepted: 10/28/2024] [Indexed: 11/26/2024] Open
Abstract
BACKGROUND In the absence of effective antimicrobials, transplant surgery is not viable, and antirejection immunosuppressants cannot be administered, as resistant infections compromise the life-saving goal of organ transplantation. AIM To evaluate the efficacy of antimicrobials in preventing resistance in solid organ transplant recipients. METHODS A systematic review was conducted using a search methodology consistent with the preferred reporting items for systematic reviews and meta-analyses. This review included randomized clinical trials that evaluated the efficacy of antimicrobial agents (prophylactic or therapeutic) aimed at preventing antimicrobial resistance. The search strategy involved analyzing multiple databases, including PubMed/MEDLINE, Web of Science, Embase, Scopus, and SciELO, as well as examining gray literature sources on Google Scholar. A comprehensive electronic database search was conducted from the databases' inception until May 2024, with no language restrictions. RESULTS After the final phase of the eligibility assessment, this systematic review ultimately included 7 articles. A total of 2318 patients were studied. The most studied microorganisms were cytomegalovirus, although vancomycin-resistant enterococci, Clostridioides difficile, and multidrug-resistant Enterobacterales were also analyzed. The antimicrobials used in the interventions were mainly maribavir, valganciclovir, ganciclovir, and colistin-neomycin. Of concern, all clinical trials showed significant proportions of resistant microorganisms after the interventions, with no statistically significant differences between the groups (mean resistance 13.47% vs 14.39%), except for two studies that demonstrated greater efficacy of maribavir and valganciclovir (mean resistance 22.2% vs 41.1% in the control group; P < 0.05). The total reported deaths in three clinical trials were 75, and there were 24 graft rejections in two studies. CONCLUSION All clinical trials reported significant proportions of antimicrobial-resistant microorganisms following interventions. More high-quality randomized clinical trials are needed to corroborate these results.
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Affiliation(s)
- Carlos M Ardila
- Department of Basic Sciences Faculty of Dentistry, Biomedical Stomatology Research Group, Universidad de Antioquia U de A, Medellín 0057, Colombia
- Department of Postdoctoral Program, CIFE University Center, Cuernavaca 62330, Morelos, Mexico
| | - Pradeep K Yadalam
- Department of Periodontics, Saveetha Dental College, Saveetha Institute of Medical and Health, SIMATS, Saveetha University, Chennai 600077, Tamil Nadu, India
| | - Jaime Ramírez-Arbelaez
- Department of Transplantation, Hospital San Vicente Fundación, Rionegro 054047, Colombia
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Zhu VZ, Horton MB, Haeusler GM, Yong MK. The emergence of letermovir and maribavir drug-resistant mutations: from clinical trials to real-world studies. Curr Opin Infect Dis 2024; 37:536-546. [PMID: 39331647 DOI: 10.1097/qco.0000000000001065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/29/2024]
Abstract
PURPOSE OF REVIEW Cytomegalovirus (CMV) infection is associated with severe clinical disease and high morbidity in immunocompromised hosts. Letermovir and maribavir, are two recently developed antiviral drugs used in the prevention and treatment of resistant and refractory CMV. Following the publication of landmark randomized trials and increased use, both clinical trial data and real-world experience has reported the development of antiviral drug resistance. The aim of this review was to comprehensively review the published literature on letermovir and maribavir drug resistance and to describe the clinical scenarios in which they may emerge. RECENT FINDINGS For letermovir, the most frequently detected resistance mutations occur in the UL56 gene (C325Y/W/F) and confer total resistance. Maribavir resistance mutations most often occur in the UL97 gene and resistance-associated variants (RAVs) T409M, H411Y, C480F have all been detected. The clinical context in which letermovir and maribavir resistance occurs include high viral loads at initiation, intensified immunosuppression, subtherapeutic drug exposure because of poor adherence, drug interactions, and inadequate central nervous system (CNS) penetration. Emergence of resistance mutations generally occurs within the first 3 months of initiation. SUMMARY The detection of letermovir and maribavir resistance mutations highlights an ongoing clinical challenge in the management of CMV.
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Affiliation(s)
- Violet Z Zhu
- National Centre for Infections in Cancer, Peter MacCallum Cancer Centre, Melbourne
- Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville
| | - Miles B Horton
- Immunology Division, Walter & Eliza Hall Institute of Medical Research, Melbourne
- Department of Medical Biology, The University of Melbourne
| | - Gabrielle M Haeusler
- National Centre for Infections in Cancer, Peter MacCallum Cancer Centre, Melbourne
- Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville
- Department of Infectious Diseases, The Royal Children's Hospital Melbourne
- Clinical Infections, Murdoch Childrens Research Institute
| | - Michelle K Yong
- National Centre for Infections in Cancer, Peter MacCallum Cancer Centre, Melbourne
- Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville
- Victorian Infectious Diseases Service, Royal Melbourne Hospital, Parkville
- Department of Infectious Diseases, Peter MacCallum Cancer Centre, Melbourne, Australia
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Marschall M, Schütz M, Wild M, Socher E, Wangen C, Dhotre K, Rawlinson WD, Sticht H. Understanding the Cytomegalovirus Cyclin-Dependent Kinase Ortholog pUL97 as a Multifaceted Regulator and an Antiviral Drug Target. Cells 2024; 13:1338. [PMID: 39195228 PMCID: PMC11352327 DOI: 10.3390/cells13161338] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2024] [Revised: 07/31/2024] [Accepted: 08/06/2024] [Indexed: 08/29/2024] Open
Abstract
Herpesviral protein kinases, such as the therapy-relevant pUL97 of human cytomegalovirus (HCMV), are important for viral replication efficiency as well as pathogenesis, and represent key antiviral drug targets. HCMV pUL97 is a viral cyclin-dependent kinase (CDK) ortholog, as it shares functional and structural properties with human CDKs. Recently, the formation of vCDK/pUL97-cyclin complexes and the phosphorylation of a variety of viral and cellular substrate proteins has been demonstrated. Genetic mapping and structural modeling approaches helped to define two pUL97 interfaces, IF1 and IF2, responsible for cyclin binding. In particular, the regulatory importance of interactions between vCDK/pUL97 and host cyclins as well as CDKs has been highlighted, both as determinants of virus replication and as a novel drug-targeting option. This aspect was substantiated by the finding that virus replication was impaired upon cyclin type H knock-down, and that such host-directed interference also affected viruses resistant to existing therapies. Beyond the formation of binary interactive complexes, a ternary pUL97-cyclin H-CDK7 complex has also been described, and in light of this, an experimental trans-stimulation of CDK7 activity by pUL97 appeared crucial for virus-host coregulation. In accordance with this understanding, several novel antiviral targeting options have emerged. These include kinase inhibitors directed to pUL97, to host CDKs, and to the pUL97-cyclin H interactive complexes. Importantly, a statistically significant drug synergy has recently been reported for antiviral treatment schemes using combinations of pharmacologically relevant CDK7 and vCDK/pUL97 inhibitors, including maribavir. Combined, such findings provide increased options for anti-HCMV control. This review focuses on regulatory interactions of vCDK/pUL97 with the host cyclin-CDK apparatus, and it addresses the functional relevance of these key effector complexes for viral replication and pathogenesis. On this basis, novel strategies of antiviral drug targeting are defined.
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Affiliation(s)
- Manfred Marschall
- Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (M.S.); (M.W.); (C.W.); (K.D.)
| | - Martin Schütz
- Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (M.S.); (M.W.); (C.W.); (K.D.)
| | - Markus Wild
- Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (M.S.); (M.W.); (C.W.); (K.D.)
| | - Eileen Socher
- Institute of Anatomy, Functional and Clinical Anatomy, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany;
| | - Christina Wangen
- Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (M.S.); (M.W.); (C.W.); (K.D.)
| | - Kishore Dhotre
- Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (M.S.); (M.W.); (C.W.); (K.D.)
| | - William D. Rawlinson
- Serology and Virology Division, NSW Health Pathology Microbiology, Prince of Wales Hospital, and Schools of Biomedical Sciences, Women’s and Children’s Health, Medicine and Biotechnology and Biomolecular Sciences, University of New South Wales, High Street, Sydney 2050, Australia;
| | - Heinrich Sticht
- Division of Bioinformatics, Institute of Biochemistry, FAU, 91054 Erlangen, Germany;
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Cochran WV, Dioverti MV, Langlee J, Barker LN, Shedeck A, Toman LP, Avery RK. Approaches and Challenges in the Current Management of Cytomegalovirus in Transplant Recipients: Highlighting the Role of Advanced Practice Providers (Nurse Practitioners and Physician Assistants). Ann Transplant 2024; 29:e941185. [PMID: 38650316 PMCID: PMC11055468 DOI: 10.12659/aot.941185] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Accepted: 01/22/2024] [Indexed: 04/25/2024] Open
Abstract
Cytomegalovirus (CMV) infection is associated with increased morbidity and mortality in hematopoietic cell transplant (HCT) and solid organ transplant (SOT) recipients, with traditional anti-CMV therapies limited by their associated toxicities and the development of resistance. Clinical providers are often faced with challenging and complicated CMV infections that require multiple courses of antiviral therapies. Increasingly, advanced practice providers (APPs) are playing an important role in the day-to-day management of transplant recipients with CMV infection, including resistant/refractory CMV and other complex CMV syndromes. Here, we provide an overview of current preventative and treatment strategies for CMV infection in HCT and SOT recipients, highlighting the challenging aspects of current management and the potential utility of newer antiviral agents. This article also focuses on how a multidisciplinary team, orchestrated by APPs, can improve CMV-associated patient outcomes. Protocols using antiviral agents for the prevention or treatment of CMV infections require carefully designed and meticulously implemented strategies to ensure the best clinical outcomes for patients. APPs, who have increasingly become the frontline providers of outpatient care for transplant recipients, are ideally positioned to design and carry out these protocols.
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Affiliation(s)
- Willa V. Cochran
- Department of Surgery, Johns Hopkins Hospital, Baltimore, MD, USA
- Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA
| | | | - Julie Langlee
- Department of Surgery, Johns Hopkins Hospital, Baltimore, MD, USA
| | | | - Audra Shedeck
- Sidney Kimmel Cancer Center, Johns Hopkins Hospital, Baltimore, MD, USA
| | - Lindsey P. Toman
- Department of Pharmacy, Johns Hopkins Hospital, Baltimore, MD, USA
| | - Robin K. Avery
- Department of Medicine, Johns Hopkins Hospital, Baltimore, MD, USA
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Monday LM, Keri V, Chandrasekar PH. Advances in pharmacotherapies for cytomegalovirus infection: what is the current state of play? Expert Opin Pharmacother 2024; 25:685-694. [PMID: 38717943 DOI: 10.1080/14656566.2024.2353627] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Accepted: 05/07/2024] [Indexed: 06/12/2024]
Abstract
INTRODUCTION Cytomegalovirus (CMV) remains a serious opportunistic infection in hematopoietic cell transplant (HCT) and solid-organ transplant (SOT) recipients. Traditional anti-CMV drugs are limited by toxicities and the development of resistance. Letermovir and maribavir are newly approved antivirals for the prevention and treatment of CMV. AREAS COVERED Prior reviews have discussed use of letermovir for prevention of CMV after HCT and maribavir for resistant or refractory (R/R) CMV post HCT or SOT. Subsequent data have expanded their use including letermovir for primary CMV prophylaxis in high-risk renal transplant recipients and new recommendations for extending prophylaxis through day + 200 in certain HCT patients. Data on the use of maribavir for first asymptomatic CMV infection post-HCT has also been published. This review compares the pharmacology of anti-CMV agents and discusses the updated literature of these new drugs in the prevention and treatment of CMV. EXPERT OPINION Letermovir and maribavir are much needed tools that spare toxicities of ganciclovir, foscarnet, and cidofovir. High cost is a challenge preventing their integration into clinical practice in resource-limited countries. Transplant centers need to exercise restraint in overuse to avoid resistance, particularly in the setting of high viral loads.
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Affiliation(s)
- Lea M Monday
- Division of Infectious Diseases, Department of Medicine, Wayne State University, Detroit, MI, USA
| | - Vishakh Keri
- Division of Infectious Diseases, Department of Medicine, Wayne State University, Detroit, MI, USA
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Chou S, Alain S, Cervera C, Chemaly RF, Kotton CN, Lundgren J, Papanicolaou GA, Pereira MR, Wu JJ, Murray RA, Buss NE, Fournier M. Drug Resistance Assessed in a Phase 3 Clinical Trial of Maribavir Therapy for Refractory or Resistant Cytomegalovirus Infection in Transplant Recipients. J Infect Dis 2024; 229:413-421. [PMID: 37506264 PMCID: PMC10873177 DOI: 10.1093/infdis/jiad293] [Citation(s) in RCA: 41] [Impact Index Per Article: 41.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 07/18/2023] [Accepted: 07/27/2023] [Indexed: 07/30/2023] Open
Abstract
BACKGROUND This drug resistance analysis of a randomized trial includes 234 patients receiving maribavir and 116 receiving investigator-assigned standard therapy (IAT), where 56% and 24%, respectively, cleared cytomegalovirus DNA at week 8 (treatment responders). METHODS Baseline and posttreatment plasma samples were tested for mutations conferring drug resistance in viral genes UL97, UL54, and UL27. RESULTS At baseline, genotypic testing revealed resistance to ganciclovir, foscarnet, or cidofovir in 56% of patients receiving maribavir and 68% receiving IAT, including 9 newly phenotyped mutations. Among them, 63% (maribavir) and 21% (IAT) were treatment responders. Detected baseline maribavir resistance mutations were UL27 L193F (n = 1) and UL97 F342Y (n = 3). Posttreatment, emergent maribavir resistance mutations were detected in 60 (26%) of those randomized to maribavir, including 49 (48%) of 103 nonresponders and 25 (86%) of the 29 nonresponders where viral DNA initially cleared then rebounded while on maribavir. The most common maribavir resistance mutations were UL97 T409M (n = 34), H411Y (n = 26), and C480F (n = 21), first detected 26 to 130 (median 56) days after starting maribavir. CONCLUSIONS Baseline maribavir resistance was rare. Drug resistance to standard cytomegalovirus antivirals did not preclude treatment response to maribavir. Rebound in plasma cytomegalovirus DNA while on maribavir strongly suggests emerging drug resistance. CLINICAL TRIALS REGISTRATION NCT02931539.
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Affiliation(s)
- Sunwen Chou
- Division of Infectious Diseases, Oregon Health and Science University, Portland, Oregon, USA
- Research and Development Service, Veterans Affairs Portland Health Care System, Portland, Oregon, USA
| | - Sophie Alain
- Department of Virology and National Reference Center for Herpesviruses, Limoges University Hospital, UMR Inserm 1092, University of Limoges, Limoges, France
| | - Carlos Cervera
- Division of Infectious Diseases, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
| | - Roy F Chemaly
- Department of Infectious Diseases, Infection Control, and Employee Health, University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Camille N Kotton
- Transplant and Immunocompromised Host Infectious Diseases, Infectious Diseases Division, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
| | - Jens Lundgren
- Centre for Health and Infectious Disease Research, Department of Infectious Diseases, Rigshospitalitet, University of Copenhagen, Copenhagen, Denmark
| | - Genovefa A Papanicolaou
- Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA
- Department of Medicine, Weill Cornell Medicine, New York, New York, USA
| | - Marcus R Pereira
- Department of Medicine, Columbia University Medical Center, New York, New York, USA
| | - Jingyang J Wu
- Takeda Development Center Americas, Inc., Lexington, Massachusetts, USA
| | - Rose Ann Murray
- Takeda Development Center Americas, Inc., Lexington, Massachusetts, USA
| | - Neil E Buss
- Medical Expressions, Büren, Solothurn, Switzerland
| | - Martha Fournier
- Takeda Development Center Americas, Inc., Lexington, Massachusetts, USA
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Chou S, Watanabe J. Ganciclovir and maribavir cross-resistance revisited: Relative drug susceptibilities of canonical cytomegalovirus mutants. Antiviral Res 2024; 222:105792. [PMID: 38163624 PMCID: PMC10922325 DOI: 10.1016/j.antiviral.2023.105792] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2023] [Revised: 12/26/2023] [Accepted: 12/27/2023] [Indexed: 01/03/2024]
Abstract
Therapeutic use of maribavir for human cytomegalovirus infection has renewed attention to the extent of cross-resistance with ganciclovir as the existing standard therapy. Each drug selects in vivo for a characteristic set of resistance mutations in the viral UL97 kinase gene. To improve the calibration of relative susceptibilities to each drug, genetic variants at relevant UL97 codons were extensively phenotyped using the same baseline viral clone, cell culture conditions and growth readout. Ganciclovir-selected mutations at codons 460, 520, 592, 594, 595 and 603 conferred 2.8-fold (C603Y) to 12-fold (M460I) increases in ganciclovir 50% inhibitory concentrations (EC50) over wild type baseline, while conferring maribavir EC50 fold changes ranging from 0.21-fold (M460I) to 1.9-fold (A594V). Maribavir-selected mutations at codons 409, 411 and 480 conferred maribavir EC50 fold changes ranging from 17 (H411Y) to 210 (C480F), while conferring ganciclovir EC50 fold changes ranging from 0.7 (H411Y) to 2.3 (C480F). The P-loop substitution F342Y, selected by either drug, is confirmed to confer 4.7-fold and 6-fold increases in maribavir and ganciclovir EC50s respectively, and suggests this part of the ATP-binding domain of UL97 to be involved in moderate resistance to both drugs. The maribavir hypersensitivity of M460I and M460V may be advantageous.
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Affiliation(s)
- Sunwen Chou
- Department of Veterans Affairs Medical Center, Portland, OR, USA; Division of Infectious Diseases, Oregon Health and Science University, USA.
| | - Justin Watanabe
- Department of Veterans Affairs Medical Center, Portland, OR, USA
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Piret J, Boivin G. Management of Cytomegalovirus Infections in the Era of the Novel Antiviral Players, Letermovir and Maribavir. Infect Dis Rep 2024; 16:65-82. [PMID: 38247977 PMCID: PMC10801527 DOI: 10.3390/idr16010005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/17/2023] [Revised: 01/08/2024] [Accepted: 01/12/2024] [Indexed: 01/23/2024] Open
Abstract
Cytomegalovirus (CMV) infections may increase morbidity and mortality in immunocompromised patients. Until recently, standard antiviral drugs against CMV were limited to viral DNA polymerase inhibitors (val)ganciclovir, foscarnet and cidofovir with a risk for cross-resistance. These drugs may also cause serious side effects. This narrative review provides an update on new antiviral agents that were approved for the prevention and treatment of CMV infections in transplant recipients. Letermovir was approved in 2017 for CMV prophylaxis in CMV-seropositive adults who received an allogeneic hematopoietic stem cell transplant. Maribavir followed four years later, with an indication in the treatment of adult and pediatric transplant patients with refractory/resistant CMV disease. The target of letermovir is the CMV terminase complex (constituted of pUL56, pUL89 and pUL51 subunits). Letermovir prevents the cleavage of viral DNA and its packaging into capsids. Maribavir is a pUL97 kinase inhibitor, which interferes with the assembly of capsids and the egress of virions from the nucleus. Both drugs have activity against most CMV strains resistant to standard drugs and exhibit favorable safety profiles. However, high-level resistance mutations may arise more rapidly in the UL56 gene under letermovir than low-grade resistance mutations. Some mutations emerging in the UL97 gene under maribavir can be cross-resistant with ganciclovir. Thus, letermovir and maribavir now extend the drug arsenal available for the management of CMV infections and their respective niches are currently defined.
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Affiliation(s)
| | - Guy Boivin
- Centre de Recherche en Infectiologie, CHU de Québec-Université Laval, Quebec City, QC G1V 4G2, Canada;
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Gourin C, Alain S, Hantz S. Anti-CMV therapy, what next? A systematic review. Front Microbiol 2023; 14:1321116. [PMID: 38053548 PMCID: PMC10694278 DOI: 10.3389/fmicb.2023.1321116] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2023] [Accepted: 11/06/2023] [Indexed: 12/07/2023] Open
Abstract
Human cytomegalovirus (HCMV) is one of the main causes of serious complications in immunocompromised patients and after congenital infection. There are currently drugs available to treat HCMV infection, targeting viral polymerase, whose use is complicated by toxicity and the emergence of resistance. Maribavir and letermovir are the latest antivirals to have been developed with other targets. The approval of letermovir represents an important innovation for CMV prevention in hematopoietic stem cell transplant recipients, whereas maribavir allowed improving the management of refractory or resistant infections in transplant recipients. However, in case of multidrug resistance or for the prevention and treatment of congenital CMV infection, finding new antivirals or molecules able to inhibit CMV replication with the lowest toxicity remains a critical need. This review presents a range of molecules known to be effective against HCMV. Molecules with a direct action against HCMV include brincidofovir, cyclopropavir and anti-terminase benzimidazole analogs. Artemisinin derivatives, quercetin and baicalein, and anti-cyclooxygenase-2 are derived from natural molecules and are generally used for different indications. Although they have demonstrated indirect anti-CMV activity, few clinical studies were performed with these compounds. Immunomodulating molecules such as leflunomide and everolimus have also demonstrated indirect antiviral activity against HCMV and could be an interesting complement to antiviral therapy. The efficacy of anti-CMV immunoglobulins are discussed in CMV congenital infection and in association with direct antiviral therapy in heart transplanted patients. All molecules are described, with their mode of action against HCMV, preclinical tests, clinical studies and possible resistance. All these molecules have shown anti-HCMV potential as monotherapy or in combination with others. These new approaches could be interesting to validate in clinical trials.
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Affiliation(s)
- Claire Gourin
- INSERM, CHU Limoges, University of Limoges, RESINFIT, Limoges, France
| | - Sophie Alain
- INSERM, CHU Limoges, University of Limoges, RESINFIT, Limoges, France
- CHU Limoges, Laboratoire de Bactériologie-Virologie-Hygiène, National Reference Center for Herpesviruses, Limoges, France
| | - Sébastien Hantz
- INSERM, CHU Limoges, University of Limoges, RESINFIT, Limoges, France
- CHU Limoges, Laboratoire de Bactériologie-Virologie-Hygiène, National Reference Center for Herpesviruses, Limoges, France
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Hume J, Lowry K, Whiley DM, Irwin AD, Bletchly C, Sweeney EL. Application of the ViroKey® SQ FLEX assay for detection of cytomegalovirus antiviral resistance. J Clin Virol 2023; 167:105556. [PMID: 37566984 DOI: 10.1016/j.jcv.2023.105556] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2023] [Revised: 07/13/2023] [Accepted: 08/03/2023] [Indexed: 08/13/2023]
Abstract
BACKGROUND Cytomegalovirus (CMV) is a viral infection which establishes lifelong latency, often reactivating and causing disease in immunosuppressed individuals, including haematopoietic stem cell transplant (HSCT) recipients. Treatment can be problematic due to antiviral resistance which substantially increases the risk of patient mortality. Diagnostic testing capabilities for CMV antiviral resistance in Australia and elsewhere have traditionally relied on gene-specific Sanger sequencing approaches, however, are now being superseded by next generation sequencing protocols. OBJECTIVE Provide a snapshot of local mutations and explore the feasibility of the ViroKeyࣨ® SQ FLEX Genotyping Assay (Vela Diagnostics Pty Ltd) by examining sequencing success. METHOD Performed sequencing on adult (n = 38) and paediatric (n = 81) plasma samples, over a large range of viral loads (above and below the assay recommended threshold of ≥1,000 International Units (IU)/mL; noting most of our paediatric samples have loads <1,000 IU/mL). RESULTS Eleven test runs (including three repeat runs; 14 to 15 samples per run) were conducted, and four runs were deemed valid. The overall individual sample success rate for the four evaluable test runs was 71.2% (42/59 samples); 80.4% (37/46) samples ≥1,000 IU/mL were valid. Ten clinically important antiviral resistance mutations were detected, the most common being A594V in the UL97 gene, found in 6 (5%) samples. CONCLUSIONS A range of technical issues were experienced, however with improvement this platform could be a useful addition to routine pathology workflows, providing timely antiviral resistance results for patients undergoing HSCT.
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Affiliation(s)
- Jocelyn Hume
- Faculty of Medicine, The University of Queensland Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland, Australia; Pathology Queensland Central Laboratory, Brisbane, Queensland, Australia
| | - Kym Lowry
- Faculty of Medicine, The University of Queensland Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland, Australia; Queensland Paediatric Infectious Diseases (QPID) Sakzewski Laboratory, Centre for Children's Health Research, Queensland Children's Hospital, Brisbane, Queensland, Australia
| | - David M Whiley
- Faculty of Medicine, The University of Queensland Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland, Australia; Pathology Queensland Central Laboratory, Brisbane, Queensland, Australia
| | - Adam D Irwin
- Faculty of Medicine, The University of Queensland Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland, Australia; Infection Management and Prevention Service, Queensland Children's Hospital, Brisbane, Queensland, Australia
| | - Cheryl Bletchly
- Pathology Queensland Central Laboratory, Brisbane, Queensland, Australia
| | - Emma L Sweeney
- Faculty of Medicine, The University of Queensland Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland, Australia.
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11
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Bassel M, Romanus D, Bo T, Sundberg AK, Okala S, Hirji I. Retrospective chart review of transplant recipients with cytomegalovirus infection who received maribavir in the Phase 3 SOLSTICE trial: Data at 52 weeks post-maribavir treatment initiation. Antivir Ther 2023; 28:13596535231195431. [PMID: 37657421 DOI: 10.1177/13596535231195431] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/03/2023]
Abstract
BACKGROUND Cytomegalovirus (CMV) infection is a frequent complication in haematopoietic cell/solid organ transplant (HCT/SOT) recipients. Previous studies report all-cause mortality rates of 31% and 50% in HCT/SOT recipients post-treatment initiation with conventional anti-CMV therapies for refractory or resistant CMV. METHODS This was a multi-country, retrospective medical chart review study of HCT/SOT recipients with refractory CMV infection with or without resistance (R/R) who were randomized to the maribavir arm in the open-label Phase 3 SOLSTICE trial. Patients came from 21 SOLSTICE sites across 6 countries; each site randomized ≥3 patients to the maribavir arm. Patients were followed for 52 weeks (SOLSTICE trial period: 20 weeks; follow-up chart review period: 32 weeks). The primary outcomes were mortality and graft status. RESULTS Of 234 patients who were randomized and received maribavir in SOLSTICE, chart abstraction was completed for all 109 patients enrolled across 21 trial sites (SOT, 68/142; HCT, 41/92). At 52 weeks, overall mortality was 15.6% (17/109) and survival probability was 0.84. Among SOT recipients, survival probability was 0.96, and 3 (4.4%) deaths occurred during the chart review period. For the HCT recipients, survival probability was 0.65 with 14 (34.1%) deaths; 8 occurred during SOLSTICE and 6 during the chart review period. No new graft loss or re-transplantation occurred during the chart review period. CONCLUSIONS Overall mortality at 52 weeks post-maribavir treatment initiation in this sub-cohort of patients from the SOLSTICE trial was lower than that previously reported for similar populations treated with conventional therapies for R/R cytomegalovirus infection.
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Affiliation(s)
| | - Dorothy Romanus
- Takeda Development Center Americas, Inc., Lexington, MA, USA
| | - Tien Bo
- Takeda Development Center Americas, Inc., Lexington, MA, USA
| | | | | | - Ishan Hirji
- Takeda Development Center Americas, Inc., Lexington, MA, USA
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12
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Demin MV, Tikhomirov DS, Tupoleva TA, Filatov FP. [Resistance to antiviral drugs in human viruses from the subfamily Betaherpesvirinae]. Vopr Virusol 2022; 67:285-294. [PMID: 36515284 DOI: 10.36233/0507-4088-136] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Indexed: 12/07/2022]
Abstract
The review provides information on the mechanisms of the emergence of resistance to antiviral drugs in human viruses from the subfamily Betaherpesvirinae. Data on the principles of action of antiviral drugs and their characteristics are given. The occurrence rates of viral resistance in various groups of patients is described and information about the possible consequences of the emergence of resistance to antiviral drugs is given. Information is provided regarding the virus genes in which mutations occur that lead to viral resistance, and a list of such mutations that have described so far is given. The significance of the study of mutations leading to the resistance of the virus to antiviral drugs for medical practice is discussed.
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Affiliation(s)
- M V Demin
- National Medical Research Center of Hematology of the Ministry of Health of Russia
| | - D S Tikhomirov
- National Medical Research Center of Hematology of the Ministry of Health of Russia
| | - T A Tupoleva
- National Medical Research Center of Hematology of the Ministry of Health of Russia
| | - F P Filatov
- I.I. Mechnikov Research Institute of Vaccines and Serums of the Ministry of Education and Science of Russia.,National Research Center of Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of Russia
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13
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Abstract
Maribavir was approved by the U.S. Food and Drug Administration in November 2021 for the treatment of adult and pediatric patients with post-transplant cytomegalovirus (CMV) infection/disease that is refractory to treatment (with or without genotypic resistance) with ganciclovir, valganciclovir, cidofovir, or foscarnet. Maribavir is an oral benzimidazole riboside with potent and selective multimodal anti-CMV activity. It utilizes a novel mechanism of action which confers activity against CMV strains that are resistant to traditional anti-CMV agents, and also offers a more favorable safety profile relative to the dose-limiting side effects of previously available therapies. Maribavir was initially studied as an agent for CMV prophylaxis in solid organ and hematopoietic stem cell recipients, but initial phase III trials failed to meet clinical efficacy endpoints. It has been more recently studied as a therapeutic agent at higher doses for refractory-resistant (R-R) CMV infections with favorable outcomes. After an overview of maribavir's chemistry and clinical pharmacology, this review will summarize clinical efficacy, safety, tolerability, and resistance data associated with maribavir therapy.
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14
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Piret J, Goyette N, Boivin G. In vitro activity of letermovir against human cytomegalovirus isolates with different drug susceptibility phenotypes. Antiviral Res 2022; 202:105328. [PMID: 35490740 DOI: 10.1016/j.antiviral.2022.105328] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Revised: 04/21/2022] [Accepted: 04/25/2022] [Indexed: 11/16/2022]
Abstract
Letermovir (LTV) is approved for the prophylaxis of human cytomegalovirus (HCMV) infection in adult seropositive recipients of an allogeneic hematopoietic stem cell transplant. Here, we report on the in vitro activity of LTV against a large panel of clinical HCMV isolates and recombinant viruses with different drug susceptibility phenotypes to currently-approved DNA polymerase inhibitors or maribavir. No pre-existing mutations conferring resistance to LTV were detected by Sanger sequencing in clinical HCMV isolates susceptible or resistant to DNA polymerases inhibitors. The susceptibility of LTV against the different recombinant HCMV mutants with amino acid substitutions in the UL97 kinase or in the UL54 DNA polymerase was similar to that of the wild type virus. LTV was also effective against recombinant HCMV harboring UL97 mutations conferring resistance to maribavir.
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Affiliation(s)
- Jocelyne Piret
- CHU de Québec-Université Laval, Quebec City, Quebec, Canada
| | | | - Guy Boivin
- CHU de Québec-Université Laval, Quebec City, Quebec, Canada.
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15
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Gandhi RG, Kotton CN. Evaluating the Safety of Maribavir for the Treatment of Cytomegalovirus. Ther Clin Risk Manag 2022; 18:223-232. [PMID: 35308097 PMCID: PMC8926008 DOI: 10.2147/tcrm.s303052] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2021] [Accepted: 02/28/2022] [Indexed: 12/18/2022] Open
Abstract
Purpose of Review Cytomegalovirus (CMV) infections are a common complication in solid organ (SOT) and hematopoietic stem cell transplant (HSCT) recipients, leading to increased morbidity and mortality. Currently available treatment options have reduced the burden of infection, but utilization of these agents can be limited by toxicities such as nephrotoxicity and/or myelosuppression as well as emergence of resistance. The expansion of our current armamentarium towards CMV infection is crucial. Here, we review an emerging therapy, maribavir, and the safety and efficacy of this potential new agent for the prophylaxis and treatment of CMV infections including resistant/refractory disease. Recent Findings Maribavir is a novel agent with CMV activity approved by Federal Food and Drug Administration (FDA) in December 2021 for resistant/refractory disease. Compared to currently available treatment for CMV infection, maribavir has a unique mechanism of action, retains activity against most (val)ganciclovir resistant strains, provides a more predictable pharmacokinetic profile, and fewer severe toxicities. Maribavir has been studied in phase 2 and 3 studies with ongoing phase 3 studies. While maribavir failed to meet the primary endpoints in the initial phase 3 study for prophylaxis therapy in allogeneic-HSCT and liver transplant recipients, results from the phase 2 study when used for pre-emptive therapy after HSCT show similar efficacy to valganciclovir, and results from the phase 3 study examining resistant/refractory disease demonstrate superiority to investigator-initiated therapy of (val)ganciclovir, foscarnet, or cidofovir. Summary Maribavir provides a new agent for the management of resistant/refractory CMV infection. Results of the recently published phase 3 study provide further insight into the role of this novel therapy.
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Affiliation(s)
- Ronak G Gandhi
- Department of Pharmacy, Massachusetts General Hospital, Boston, MA, USA
- Correspondence: Ronak G Gandhi, Senior Attending Pharmacist – Infectious Diseases, Department of Pharmacy, Massachusetts General Hospital, 55 Fruit Street, GRB 005, Boston, MA02114, USA, Tel +1 617-643-6570, Fax +1 617-726-9232, Email
| | - Camille N Kotton
- Infectious Diseases Division, Massachusetts General Hospital, Boston, MA, USA
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16
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Avery RK, Alain S, Alexander BD, Blumberg EA, Chemaly RF, Cordonnier C, Duarte RF, Florescu DF, Kamar N, Kumar D, Maertens J, Marty FM, Papanicolaou GA, Silveira FP, Witzke O, Wu J, Sundberg AK, Fournier M, SOLSTICE Trial Investigators. Maribavir for Refractory Cytomegalovirus Infections With or Without Resistance Post-Transplant: Results From a Phase 3 Randomized Clinical Trial. Clin Infect Dis 2021; 75:690-701. [PMID: 34864943 PMCID: PMC9464078 DOI: 10.1093/cid/ciab988] [Citation(s) in RCA: 161] [Impact Index Per Article: 40.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2021] [Indexed: 01/11/2023] Open
Abstract
BACKGROUND Therapies for refractory cytomegalovirus infections (with or without resistance [R/R]) in transplant recipients are limited by toxicities. Maribavir has multimodal anti-cytomegalovirus activity through the inhibition of UL97 protein kinase. METHODS In this phase 3, open-label study, hematopoietic-cell and solid-organ transplant recipients with R/R cytomegalovirus were randomized 2:1 to maribavir 400 mg twice daily or investigator-assigned therapy (IAT; valganciclovir/ganciclovir, foscarnet, or cidofovir) for 8 weeks, with 12 weeks of follow-up. The primary endpoint was confirmed cytomegalovirus clearance at end of week 8. The key secondary endpoint was achievement of cytomegalovirus clearance and symptom control at end of week 8, maintained through week 16. RESULTS 352 patients were randomized (235 maribavir; 117 IAT). Significantly more patients in the maribavir versus IAT group achieved the primary endpoint (55.7% vs 23.9%; adjusted difference [95% confidence interval (CI)]: 32.8% [22.80-42.74]; P < .001) and key secondary endpoint (18.7% vs 10.3%; adjusted difference [95% CI]: 9.5% [2.02-16.88]; P = .01). Rates of treatment-emergent adverse events (TEAEs) were similar between groups (maribavir, 97.4%; IAT, 91.4%). Maribavir was associated with less acute kidney injury versus foscarnet (8.5% vs 21.3%) and neutropenia versus valganciclovir/ganciclovir (9.4% vs 33.9%). Fewer patients discontinued treatment due to TEAEs with maribavir (13.2%) than IAT (31.9%). One patient per group had fatal treatment-related TEAEs. CONCLUSIONS Maribavir was superior to IAT for cytomegalovirus viremia clearance and viremia clearance plus symptom control maintained post-therapy in transplant recipients with R/R cytomegalovirus. Maribavir had fewer treatment discontinuations due to TEAEs than IAT. Clinical Trials Registration. NCT02931539 (SOLSTICE).
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Affiliation(s)
- Robin K Avery
- Division of Infectious Diseases, Johns Hopkins University, Baltimore, Maryland, USA
| | - Sophie Alain
- Department of Virology and National Reference Center for Herpesviruses, Limoges University Hospital, UMR Inserm 1092, University of Limoges, Limoges, France
| | - Barbara D Alexander
- Division of Infectious Diseases and International Health, Duke University, Durham, North Carolina, USA
| | - Emily A Blumberg
- Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Roy F Chemaly
- Department of Infectious Diseases, Infection Control, and Employee Health, University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | - Catherine Cordonnier
- Haematology Department, Henri Mondor Hospital and University Paris-Est-Créteil, Créteil, France
| | - Rafael F Duarte
- Department of Haematology, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain
| | - Diana F Florescu
- Infectious Diseases Division, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Nassim Kamar
- Department of Nephrology and Organ Transplantation, Toulouse Rangueil University Hospital, INFINITY-Inserm U1291-CNRS U5051, University Paul Sabatier, Toulouse, France
| | - Deepali Kumar
- Transplant Centre, University Health Network, Toronto, Ontario, Canada
| | - Johan Maertens
- Haematology Department, University Hospitals Leuven, KU Leuven, Leuven, Belgium
| | | | - Genovefa A Papanicolaou
- Infectious Disease Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA,Department of Medicine, Weill Cornell Medicine, New York, New York, USA
| | - Fernanda P Silveira
- Department of Medicine, Division of Infectious Diseases, University of Pittsburgh and University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA
| | - Oliver Witzke
- Department of Infectious Diseases, West German Centre of Infectious Diseases, University Medicine Essen, University Duisburg-Essen, Essen, Germany
| | - Jingyang Wu
- Biostatistics, Takeda Development Center Americas, Inc, Lexington, Massachusetts, USA
| | - Aimee K Sundberg
- Clinical Sciences, Takeda Development Center Americas, Inc, Lexington, Massachusetts, USA
| | - Martha Fournier
- Correspondence: M. Fournier, Takeda Development Center Americas, Inc, 300 Shire Way, Lexington, MA 02421 ()
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17
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Imlay HN, Kaul DR. Letermovir and Maribavir for the Treatment and Prevention of Cytomegalovirus Infection in Solid Organ and Stem Cell Transplant Recipients. Clin Infect Dis 2021; 73:156-160. [PMID: 33197929 DOI: 10.1093/cid/ciaa1713] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2020] [Accepted: 11/12/2020] [Indexed: 02/06/2023] Open
Abstract
Until recently, available drugs for cytomegalovirus (CMV) prevention and treatment in transplant patients included (val)ganciclovir, foscarnet, and cidofovir. Use of these drugs is limited by toxicity and the development of resistance. The 2017 approval of letermovir for prevention of CMV after stem cell transplant marked the first approval of an anti-CMV agent since 2003. The role of letermovir in treatment of established CMV infection or disease remains largely unstudied, although early reports suggest that a low barrier to resistance will likely limit efficacy as primary therapy for patients with refractory or resistant disease. The investigational agent maribavir has shown promise as preemptive treatment; in patients with refractory or resistant disease the emergence of resistance while on treatment has been observed and ongoing studies will define efficacy in this population. Both agents have unique mechanisms of action limiting cross resistance, and neither exhibit myelotoxicity or nephrotoxicity.
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Affiliation(s)
- Hannah N Imlay
- University of Utah, Department of Internal Medicine, Division of Infectious Diseases, Salt Lake City, Utah, USA
| | - Daniel R Kaul
- University of Michigan, Department of Internal Medicine, Division of Infectious Diseases, Ann Arbor, Michigan, USA
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18
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Trompet E, Temblador A, Gillemot S, Topalis D, Snoeck R, Andrei G. An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness. Viruses 2021; 13:v13060985. [PMID: 34073189 PMCID: PMC8227558 DOI: 10.3390/v13060985] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2021] [Revised: 05/11/2021] [Accepted: 05/24/2021] [Indexed: 12/04/2022] Open
Abstract
Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.
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19
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Piret J, Boivin G. Antiviral Drugs Against Herpesviruses. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1322:1-30. [PMID: 34258735 DOI: 10.1007/978-981-16-0267-2_1] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The discovery of the nucleoside analogue, acyclovir, represented a milestone in the management of infections caused by herpes simplex virus and varicella-zoster virus. Ganciclovir, another nucleoside analogue, was then used for the management of systemic and organ-specific human cytomegalovirus diseases. The pyrophosphate analogue, foscarnet, and the nucleotide analogue, cidofovir, have been approved subsequently and constitute the second-line antiviral drugs. However, the viral DNA polymerase is the ultimate target of all these antiviral agents with a possible emergence of cross-resistance between these drugs. Recently, letermovir that targets the viral terminase complex was approved for the prophylaxis of human cytomegalovirus infections in hematopoietic stem cell transplant recipients. Other viral targets such as the protein kinase and the helicase-primase complex are also evaluated for the development of novel potent inhibitors against herpesviruses.
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Affiliation(s)
| | - Guy Boivin
- CHU de Québec-Laval University, Quebec City, QC, Canada.
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20
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Li X, Akinyemi IA, You JK, Rezaei MA, Li C, McIntosh MT, Del Poeta M, Bhaduri-McIntosh S. A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents. J Virol 2020; 94:e01179-20. [PMID: 32796077 PMCID: PMC7565614 DOI: 10.1128/jvi.01179-20] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Accepted: 08/06/2020] [Indexed: 12/23/2022] Open
Abstract
Epstein-Barr virus (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers, including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that through the activities of the viral protein kinase conserved across herpesviruses and two cellular proteins, ATM and KAP1, a lytic cycle amplification loop is established, and disruption of this loop disables the EBV lytic cascade. We therefore devised a high-throughput screening assay, screened a small-molecule-compound library, and identified 17 candidates that impair the release of lytically replicated EBV. The identified compounds will (i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses, and (ii) be developed into anticancer agents, as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for antiherpesviral and anticancer drugs.IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small-molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even antiherpesviral potential, as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both antiherpesviral and anticancer drugs.
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Affiliation(s)
- Xiaofan Li
- Division of Infectious Diseases, Department of Pediatrics, University of Florida, Gainesville, Florida, USA
| | - Ibukun A Akinyemi
- Child Health Research Institute, Department of Pediatrics, University of Florida, Gainesville, Florida, USA
| | - Jeehyun Karen You
- Department of Microbiology and Immunology, Stony Brook University, Stony Brook, New York, USA
| | - Mohammad Ali Rezaei
- Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA
| | - Chenglong Li
- Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, Florida, USA
| | - Michael T McIntosh
- Child Health Research Institute, Department of Pediatrics, University of Florida, Gainesville, Florida, USA
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA
| | - Maurizio Del Poeta
- Department of Microbiology and Immunology, Stony Brook University, Stony Brook, New York, USA
- Division of Infectious Diseases, Stony Brook University, Stony Brook, New York, USA
- Veterans Administration Medical Center, Northport, New York, USA
| | - Sumita Bhaduri-McIntosh
- Division of Infectious Diseases, Department of Pediatrics, University of Florida, Gainesville, Florida, USA
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA
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21
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Chou S, Song K, Wu J, Bo T, Crumpacker C. Drug resistance mutations and associated phenotypes detected in clinical trials of maribavir for treatment of cytomegalovirus infection. J Infect Dis 2020; 226:576-584. [PMID: 32726419 PMCID: PMC9441206 DOI: 10.1093/infdis/jiaa462] [Citation(s) in RCA: 55] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2020] [Accepted: 07/24/2020] [Indexed: 01/26/2023] Open
Abstract
Background In separate phase 2 trials, 120 patients received maribavir for cytomegalovirus (CMV) infection failing conventional therapy (trial 202) and 119 received maribavir for asymptomatic infection (trial 203). Overall, 172 cleared their CMV infection (CMV DNA <200 copies/mL) within 6 weeks. Methods Baseline and posttreatment plasma samples were tested for mutations in viral genes UL97, UL54, and/or UL27. Selected viral mutants were phenotyped for drug susceptibility. Results Baseline samples revealed UL54 mutations newly phenotyped as conferring resistance to standard DNA polymerase inhibitor(s), including K493N, P497S, K513T, L565V, V823A, A987V, and E989D. Of 29 patients (including 25 from trial 202) who cleared but later experienced recurrent CMV infection while on maribavir, 23 had available UL97 genotyping data; 17 had known resistance mutations (T409M or H411Y) and 5 additional had UL97 C480F alone. The newly phenotyped mutation C480F conferred high-grade maribavir resistance and low-grade ganciclovir resistance. Among 25 who did not respond to >14 days of therapy, 9 showed T409M or H411Y and 4 others showed C480F alone. Conclusions After maribavir therapy (400–1200 mg twice daily), UL97 mutations T409M, H411Y, or C480F emerge to confer maribavir resistance in patients with recurrent CMV infection while on therapy or no response to therapy. Clinical Trials Registration NCT01611974 and EudraCT 2010-024247-32.
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Affiliation(s)
- Sunwen Chou
- Oregon Health & Science University and VA Health Care System, Portland OR
| | - Kening Song
- Shire Human Genetic Therapies Inc., Lexington MA, a Takeda Company
| | - Jingyang Wu
- Shire Human Genetic Therapies Inc., Lexington MA, a Takeda Company
| | - Tien Bo
- Shire Human Genetic Therapies Inc., Lexington MA, a Takeda Company
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22
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Abstract
PURPOSE OF REVIEW CMV DNA polymerase inhibitors such as ganciclovir and foscarnet have dramatically reduced the burden of CMV infection in the HCT recipient. However, their use is often limited by toxicities and resistance. Agents with novel mechanisms and favorable toxicity profiles are critically needed. We review recent developments in CMV antivirals and immune-based approaches to mitigating CMV infection. RECENT FINDINGS Letermovir, an inhibitor of the CMV terminase complex, was approved in 2017 for primary CMV prophylaxis in adult seropositive allogeneic HCT recipients. Maribavir, an inhibitor of the CMV UL97 kinase, is currently in two phase 3 treatment studies. Adoptive immunotherapy using third-party T cells has proven safe and effective in preliminary studies. Vaccine development continues, with several promising candidates currently under study. No longer limited to DNA polymerase inhibitors, the prevention and treatment of CMV infections in the HCT recipient is a rapidly evolving field which should translate into improvements in CMV-related outcomes.
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Affiliation(s)
- Morgan Hakki
- Division of Infectious Diseases, Department of Medicine, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Mail code L457, Portland, OR, 97239, USA.
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23
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Advances in the genotypic diagnosis of cytomegalovirus antiviral drug resistance. Antiviral Res 2020; 176:104711. [PMID: 31940472 DOI: 10.1016/j.antiviral.2020.104711] [Citation(s) in RCA: 72] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Revised: 12/22/2019] [Accepted: 01/09/2020] [Indexed: 12/20/2022]
Abstract
Cytomegalovirus (CMV) drug resistance mutation maps are updated with recent information for polymerase inhibitors, the terminase inhibitor letermovir and the UL97 kinase inhibitor maribavir. Newly mapped mutations and their phenotypes provide more detail on cross-resistance properties and suggest the need to expand the CMV gene regions covered in diagnostic testing. Next-generation deep sequencing technology offers a more sensitive, higher resolution view of emerging antiviral resistance and is recommended for use in clinical trials. Issues of standardization and diagnostic utility in comparison with traditional Sanger sequencing remain unresolved. Quality control is important for the accurate and reproducible detection of mutant viral populations in clinical specimens.
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24
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Trimpert J, Osterrieder N. Herpesvirus DNA Polymerase Mutants—How Important Is Faithful Genome Replication? CURRENT CLINICAL MICROBIOLOGY REPORTS 2019. [DOI: 10.1007/s40588-019-00135-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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25
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Chou S, Wu J, Song K, Bo T. Novel UL97 drug resistance mutations identified at baseline in a clinical trial of maribavir for resistant or refractory cytomegalovirus infection. Antiviral Res 2019; 172:104616. [PMID: 31568799 DOI: 10.1016/j.antiviral.2019.104616] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2019] [Revised: 09/26/2019] [Accepted: 09/26/2019] [Indexed: 12/27/2022]
Abstract
In a Phase 2 clinical trial, 120 subjects with cytomegalovirus (CMV) infection refractory or resistant to standard therapy were randomized equally to 3 doses of oral maribavir treatment, and 70% achieved undetectable plasma CMV DNA within 12 weeks. At study entry, standard diagnostic UL97 genotyping was available for 71 subjects, with 60 (85%) revealing well-characterized ganciclovir resistance mutations that did not preclude a therapeutic response to maribavir. Central laboratory testing of a range of UL97 codons (288-468) not fully covered by standard genotyping was done on 93 subjects at baseline. This detected no previously known maribavir resistance mutations, but identified atypical mutations in 3 subjects, including a P-loop substitution F342Y, and ATP-binding region substitutions K359E/Q. By recombinant phenotyping, K359E and K359Q each conferred a nearly 4-fold increased ganciclovir 50% inhibitory concentration (EC50) without maribavir resistance, whereas F342Y conferred a 6-fold increased ganciclovir EC50 and a 4.5-fold increased maribavir EC50. The subject with F342Y detected at baseline did not achieve plasma CMV DNA clearance after 12 weeks of maribavir therapy and later developed an additional UL97 substitution H411Y known to confer 12- to 20-fold increased MBV EC50 by itself. The combination of F342Y and H411Y was shown to increase the maribavir EC50 by 56-fold. Diagnostic genotyping of UL97 should be expanded to cover the ATP-binding region beginning at codon 335 to enable the detection of atypical resistance mutations and further correlation of their clinical significance.
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Affiliation(s)
- Sunwen Chou
- Division of Infectious Diseases, Oregon Health and Science University, Department of Veterans Affairs Medical Center, Portland, Oregon, USA.
| | | | | | - Tien Bo
- Shire, a Takeda Company, Lexington, MA, USA
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26
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Piret J, Boivin G. Clinical development of letermovir and maribavir: Overview of human cytomegalovirus drug resistance. Antiviral Res 2019; 163:91-105. [PMID: 30690043 DOI: 10.1016/j.antiviral.2019.01.011] [Citation(s) in RCA: 63] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2018] [Revised: 01/17/2019] [Accepted: 01/18/2019] [Indexed: 01/28/2023]
Abstract
The prevention and treatment of human cytomegalovirus (HCMV) infections is based on the use of antiviral agents that currently target the viral DNA polymerase and that may cause serious side effects. The search for novel inhibitors against HCMV infection led to the discovery of new molecular targets, the viral terminase complex and the viral pUL97 kinase. The most advanced compounds consist of letermovir (LMV) and maribavir (MBV). LMV inhibits the cleavage of viral DNA and its packaging into capsids by targeting the HCMV terminase complex. LMV is safe and well tolerated and exhibits pharmacokinetic properties that allow once daily dosing. LMV showed efficacy in a phase III prophylaxis study in hematopoietic stem cell transplant (HSCT) recipients seropositive for HCMV. LMV was recently approved under the trade name Prevymis™ for prophylaxis of HCMV infection in adult seropositive recipients of an allogeneic HSCT. Amino acid substitutions conferring resistance to LMV selected in vitro map primarily to the pUL56 and rarely to the pUL89 and pUL51 subunits of the HCMV terminase complex. MBV is an inhibitor of the viral pUL97 kinase activity and interferes with the morphogenesis and nuclear egress of nascent viral particles. MBV is safe and well tolerated and has an excellent oral bioavailability. MBV was effective for the treatment of HCMV infections (including those that are refractory or drug-resistant) in transplant recipients in two phase II studies and is further evaluated in two phase III trials. Mutations conferring resistance to MBV map to the UL97 gene and can cause cross-resistance to ganciclovir. MBV-resistant mutations also emerged in the UL27 gene in vitro and could compensate for the inhibition of pUL97 kinase activity by MBV. Thus, LMV and probably MBV will broaden the armamentarium of antiviral drugs available for the prevention and treatment of HCMV infections.
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Affiliation(s)
- Jocelyne Piret
- Research Center in Infectious Diseases, CHU of Quebec and Laval University, Quebec City, QC, Canada
| | - Guy Boivin
- Research Center in Infectious Diseases, CHU of Quebec and Laval University, Quebec City, QC, Canada.
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27
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Chou S, Ercolani RJ, Derakhchan K. Antiviral activity of maribavir in combination with other drugs active against human cytomegalovirus. Antiviral Res 2018; 157:128-133. [PMID: 30040968 PMCID: PMC6097806 DOI: 10.1016/j.antiviral.2018.07.013] [Citation(s) in RCA: 44] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2018] [Revised: 07/15/2018] [Accepted: 07/19/2018] [Indexed: 12/11/2022]
Abstract
The human cytomegalovirus (CMV) UL97 kinase inhibitor maribavir is in Phase III clinical trials as antiviral therapy, including use for infections refractory or resistant to standard therapy. To assess its activity in combination with approved and experimental CMV antivirals, and with the mTor inhibitor rapamycin (sirolimus), drug effects were tested by in vitro checkerboard assays and the data were analyzed using a three dimensional model based on an independent effects definition of additive interactions. Baseline virus and representative drug-resistant mutants were tested. According to the volume of synergy at 95% confidence, maribavir showed additive interactions with foscarnet, cidofovir, letermovir and GW275175X when tested against wild type and mutant viruses, strong antagonism with ganciclovir, and strong synergy with rapamycin, the latter suggesting a potentially useful therapeutic combination.
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Affiliation(s)
- Sunwen Chou
- Division of Infectious Diseases, Oregon Health & Science University, USA; Portland Veterans Affairs Health Care System, OR USA.
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28
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Topalis D, Gillemot S, Snoeck R, Andrei G. Thymidine kinase and protein kinase in drug-resistant herpesviruses: Heads of a Lernaean Hydra. Drug Resist Updat 2018; 37:1-16. [PMID: 29548479 DOI: 10.1016/j.drup.2018.01.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Herpesviruses thymidine kinase (TK) and protein kinase (PK) allow the activation of nucleoside analogues used in anti-herpesvirus treatments. Mutations emerging in these two genes often lead to emergence of drug-resistant strains responsible for life-threatening diseases in immunocompromised populations. In this review, we analyze the binding of different nucleoside analogues to the TK active site of the three α-herpesviruses [Herpes Simplex Virus 1 and 2 (HSV-1 and HSV-2) and Varicella-Zoster Virus (VZV)] and present the impact of known mutations on the structure of the viral TKs. Furthermore, models of β-herpesviruses [Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6)] PKs allow to link amino acid changes with resistance to ganciclovir and/or maribavir, an investigational chemotherapeutic used in patients with multidrug-resistant HCMV. Finally, we set the basis for the understanding of drug-resistance in γ-herpesviruses [Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus (KSHV)] TK and PK through the use of animal surrogate models.
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Affiliation(s)
- Dimitri Topalis
- Rega Institute for Medical Research, KU Leuven, Herestraat 49-box 1043, 3000 Leuven, Belgium.
| | - Sarah Gillemot
- Rega Institute for Medical Research, KU Leuven, Herestraat 49-box 1043, 3000 Leuven, Belgium.
| | - Robert Snoeck
- Rega Institute for Medical Research, KU Leuven, Herestraat 49-box 1043, 3000 Leuven, Belgium.
| | - Graciela Andrei
- Rega Institute for Medical Research, KU Leuven, Herestraat 49-box 1043, 3000 Leuven, Belgium.
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29
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How I treat resistant cytomegalovirus infection in hematopoietic cell transplantation recipients. Blood 2016; 128:2624-2636. [PMID: 27760756 DOI: 10.1182/blood-2016-06-688432] [Citation(s) in RCA: 134] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2016] [Accepted: 10/17/2016] [Indexed: 12/20/2022] Open
Abstract
Cytomegalovirus (CMV) infection is a significant complication in hematopoietic cell transplantation (HCT) recipients. Four antiviral drugs are used for preventing or treating CMV: ganciclovir, valganciclovir, foscarnet, and cidofovir. With prolonged and repeated use of these drugs, CMV can become resistant to standard therapy, resulting in increased morbidity and mortality, especially in HCT recipients. Antiviral drug resistance should be suspected when CMV viremia (DNAemia or antigenemia) fails to improve or continue to increase after 2 weeks of appropriately dosed and delivered antiviral therapy. CMV resistance is diagnosed by detecting specific genetic mutations. UL97 mutations confer resistance to ganciclovir and valganciclovir, and a UL54 mutation confers multidrug resistance. Risk factors for resistance include prolonged or previous anti-CMV drug exposure or inadequate dosing, absorption, or bioavailability. Host risk factors include type of HCT and degree of immunosuppression. Depending on the genotyping results, multiple strategies can be adopted to treat resistant CMV infections, albeit no randomized clinical trials exist so far, after reducing immunosuppression (if possible): ganciclovir dose escalation, ganciclovir and foscarnet combination, and adjunct therapy such as CMV-specific cytotoxic T-lymphocyte infusions. Novel therapies such as maribavir, brincidofovir, and letermovir should be further studied for treatment of resistant CMV.
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30
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Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance. Antimicrob Agents Chemother 2016; 60:3845-8. [PMID: 27044553 DOI: 10.1128/aac.00214-16] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2016] [Accepted: 04/01/2016] [Indexed: 01/11/2023] Open
Abstract
Eight in vitro selection experiments under brincidofovir pressure elicited the known cytomegalovirus DNA polymerase amino acid substitutions N408K and V812L and the novel exonuclease domain substitutions D413Y, E303D, and E303G, which conferred ganciclovir and cidofovir resistance with 6- to 11-fold resistance to brincidofovir or 17-fold when E303G was combined with V812L. The new exonuclease domain I resistance mutations selected under brincidofovir pressure add to the single instance previously reported and show the expected patterns of cross-resistance.
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Rapid In Vitro Evolution of Human Cytomegalovirus UL56 Mutations That Confer Letermovir Resistance. Antimicrob Agents Chemother 2015; 59:6588-93. [PMID: 26259791 DOI: 10.1128/aac.01623-15] [Citation(s) in RCA: 146] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2015] [Accepted: 08/03/2015] [Indexed: 11/20/2022] Open
Abstract
Letermovir (LMV) is an experimental cytomegalovirus terminase inhibitor undergoing phase 3 clinical trials. Viral mutations have been described at UL56 codons 231 to 369 that confer widely variable levels of LMV resistance. In this study, 15 independent experiments propagating an exonuclease mutant viral strain in escalating LMV concentrations replicated 6 of the 7 published UL56 mutations and commonly elicited additional resistance-conferring mutations at UL56 codons 231, 236, 237, 244, 257, 261, 325, and 329. Mutations were first detected earlier in LMV (median, 3 passages) than in 8 parallel experiments with foscarnet (median, 15 passages). As LMV concentrations increased, the typical initial UL56 change F261L, which confers low-grade resistance, combined or was replaced with mutations conferring higher-grade resistance, eventually enabling normal viral growth in 30 μM LMV (>5,000-fold the 50% effective concentration [EC50] for the wild type). At high LMV concentrations, the UL56 changes C325F/R were commonly detected, as well as a combination of changes at codons 236, 257, 329, and/or 369. Recombinant viruses containing individual UL56 mutations and combinations were constructed to confirm their resistance phenotypes and normal growth in cell culture. Several double and triple mutants showed much higher LMV resistance than the respective single mutants, particularly those including changes at both codons 236 and 257. The multiplicity of pathways to high-grade LMV resistance with minimal viral growth impact suggests a low viral genetic barrier and the need for close monitoring during treatment of active infection.
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32
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Iwahori S, Hakki M, Chou S, Kalejta RF. Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97. J Biol Chem 2015; 290:19666-80. [PMID: 26100623 DOI: 10.1074/jbc.m115.660043] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2015] [Indexed: 01/10/2023] Open
Abstract
The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.
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Affiliation(s)
- Satoko Iwahori
- From the Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin 53706 and
| | - Morgan Hakki
- the Division of Infectious Diseases, Oregon Health and Science University and
| | - Sunwen Chou
- the Division of Infectious Diseases, Oregon Health and Science University and Veterans Affairs Portland Health Care System, Portland, Oregon 97239
| | - Robert F Kalejta
- From the Institute for Molecular Virology and McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin 53706 and
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33
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Gable J, Acker TM, Craik CS. Current and potential treatments for ubiquitous but neglected herpesvirus infections. Chem Rev 2014; 114:11382-412. [PMID: 25275644 PMCID: PMC4254030 DOI: 10.1021/cr500255e] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2014] [Indexed: 02/07/2023]
Affiliation(s)
- Jonathan
E. Gable
- Department
of Pharmaceutical Chemistry, University
of California, San Francisco, 600 16th Street, San Francisco, California 94158-2280, United States
- Graduate
Group in Biophysics, University of California,
San Francisco, 600 16th
Street, San Francisco, California 94158-2280, United States
| | - Timothy M. Acker
- Department
of Pharmaceutical Chemistry, University
of California, San Francisco, 600 16th Street, San Francisco, California 94158-2280, United States
| | - Charles S. Craik
- Department
of Pharmaceutical Chemistry, University
of California, San Francisco, 600 16th Street, San Francisco, California 94158-2280, United States
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34
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Human cytomegalovirus intrahost evolution-a new avenue for understanding and controlling herpesvirus infections. Curr Opin Virol 2014; 8:109-15. [PMID: 25154343 DOI: 10.1016/j.coviro.2014.08.001] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2014] [Revised: 07/31/2014] [Accepted: 08/02/2014] [Indexed: 01/31/2023]
Abstract
Human cytomegalovirus (HCMV) is exquisitely adapted to the human host, and much research has focused on its evolution over long timescales spanning millennia. Here, we review recent data exploring the evolution of the virus on much shorter timescales, on the order of days or months. We describe the intrahost genetic diversity of the virus isolated from humans, and how this diversity contributes to HCMV spatiotemporal evolution. We propose mechanisms to explain the high levels of intrahost diversity and discuss how this new information may shed light on HCMV infection and pathogenesis.
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35
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Improved detection of emerging drug-resistant mutant cytomegalovirus subpopulations by deep sequencing. Antimicrob Agents Chemother 2014; 58:4697-702. [PMID: 24890586 DOI: 10.1128/aac.03214-14] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.
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36
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Differential properties of cytomegalovirus pUL97 kinase isoforms affect viral replication and maribavir susceptibility. J Virol 2014; 88:4776-85. [PMID: 24522923 DOI: 10.1128/jvi.00192-14] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo.
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37
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Komatsu TE, Pikis A, Naeger LK, Harrington PR. Resistance of human cytomegalovirus to ganciclovir/valganciclovir: A comprehensive review of putative resistance pathways. Antiviral Res 2014; 101:12-25. [DOI: 10.1016/j.antiviral.2013.10.011] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Revised: 10/20/2013] [Accepted: 10/21/2013] [Indexed: 11/26/2022]
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Cytomegalovirus mutants resistant to ganciclovir and cidofovir differ in susceptibilities to synguanol and its 6-ether and 6-thioether derivatives. Antimicrob Agents Chemother 2013; 58:1809-12. [PMID: 24379208 DOI: 10.1128/aac.02544-13] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The methylenecyclopropane nucleoside (MCPN) analogs synguanol and its 6-alkoxy (MBX2168) and 6-alkylthio (MBX1616) derivatives retained good in vitro activities against several common ganciclovir-resistant UL97 kinase variants of human cytomegalovirus. Foscarnet-MCPN cross-resistance was observed among UL54 polymerase variants. UL54 exonuclease domain ganciclovir-cidofovir dual-resistant variants were remarkably more hypersensitive to these MCPNs than to cyclopropavir, with some 50% effective concentration ratios that were <0.1× the wild type. Different categories of MCPNs may have therapeutically exploitable mechanistic differences in viral DNA polymerase inhibition.
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Human cytomegalovirus pUL97 regulates the viral major immediate early promoter by phosphorylation-mediated disruption of histone deacetylase 1 binding. J Virol 2013; 87:7393-408. [PMID: 23616659 DOI: 10.1128/jvi.02825-12] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human cytomegalovirus (HCMV) is a common agent of congenital infection and causes severe disease in immunocompromised patients. Current approved therapies focus on inhibiting viral DNA replication. The HCMV kinase pUL97 contributes to multiple stages of viral infection including DNA replication, controlling the cell cycle, and virion maturation. Our studies demonstrate that pUL97 also functions by influencing immediate early (IE) gene expression during the initial stages of infection. Inhibition of kinase activity using the antiviral compound maribavir or deletion of the UL97 gene resulted in decreased expression of viral immediate early genes during infection. Expression of pUL97 was sufficient to transactivate IE1 gene expression from the viral genome, which was dependent on viral kinase activity. We observed that pUL97 associates with histone deacetylase 1 (HDAC1). HDAC1 is a transcriptional corepressor that acts to silence expression of viral genes. We observed that inhibition or deletion of pUL97 kinase resulted in increased HDAC1 and decreased histone H3 lysine 9 acetylation associating with the viral major immediate early (MIE) promoter. IE expression during pUL97 inhibition or deletion was rescued following inhibition of deacetylase activity. HDAC1 associates with chromatin by protein-protein interactions. Expression of active but not inactive pUL97 kinase decreased HDAC1 interaction with the transcriptional repressor protein DAXX. Finally, using mass spectrometry, we found that HDAC1 is uniquely phosphorylated upon expression of pUL97. Our results support the conclusion that HCMV pUL97 kinase regulates viral immediate early gene expression by phosphorylation-mediated disruption of HDAC1 binding to the MIE promoter.
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40
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Abstract
Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease.
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Affiliation(s)
- Rob C Hoeben
- Department of Molecular Cell Biology, Leiden University Medical Centre, 2333 ZC Leiden, The Netherlands.
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41
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Chou S, Hakki M, Villano S. Effects on maribavir susceptibility of cytomegalovirus UL97 kinase ATP binding region mutations detected after drug exposure in vitro and in vivo. Antiviral Res 2012; 95:88-92. [PMID: 22664236 DOI: 10.1016/j.antiviral.2012.05.013] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2012] [Revised: 05/16/2012] [Accepted: 05/24/2012] [Indexed: 11/25/2022]
Abstract
Resistance to the experimental human cytomegalovirus (CMV) UL97 kinase inhibitor maribavir has been mapped to UL97 mutations at codons 353, 397, 409 and 411, in the kinase ATP-binding region, and to mutations in the UL27 gene. We studied the maribavir susceptibility phenotypes of additional UL97 mutations observed in vitro and in clinical trials, and the effect of simultaneous mutation in both UL97 and UL27. In vitro selection under maribavir identified a new locus of UL97 mutation within the conserved kinase p-loop (L337M), which conferred low grade maribavir resistance (3.5-fold increased EC50) without ganciclovir cross-resistance. During maribavir Phase III CMV prevention clinical trials, three previously unknown UL97 sequence variants were detected in plasma samples after 27-98 days of drug exposure (I324V, S334G and S386L). These variants did not confer any drug resistance despite proximity to mutations that confer maribavir resistance. The UL27 resistance mutation R233S, when added to strains containing UL97 mutations L337M or V353A, doubled their maribavir EC50s. These results expand the range of UL97 maribavir-resistance mutations into another part of the kinase ATP-binding region, but offer no genotypic evidence that development of drug resistance affected the outcomes of Phase III maribavir clinical trials after drug exposure of up to 14 weeks. There is a potential for increased maribavir resistance in UL27-UL97 double mutants.
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Affiliation(s)
- Sunwen Chou
- Division of Infectious Diseases, Oregon Health and Science University, Portland, OR 97239, USA.
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42
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Cyclopropavir susceptibility of cytomegalovirus DNA polymerase mutants selected after antiviral drug exposure. Antimicrob Agents Chemother 2011; 56:197-201. [PMID: 21968367 DOI: 10.1128/aac.05559-11] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human cytomegalovirus (CMV) UL54 DNA polymerase (pol) mutants with known patterns of resistance to current antivirals ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) were tested for cyclopropavir (CPV) susceptibility by a standardized reporter-based yield reduction assay. Exonuclease and A987G (region V) mutations at codons commonly associated with dual GCV-CDV resistance in clinical isolates paradoxically conferred increased CPV susceptibility. Various polymerase catalytic region mutations conferring FOS resistance with variable low-grade GCV and CDV cross-resistance also conferred CPV resistance, with 50% effective concentration (EC(50)) increases of 3- to 13-fold. CPV EC(50) values against several pol mutants were increased about 2-fold by adding UL97 mutation C592G. Propagation of a CMV exonuclease mutant under CPV selected for pol mutations less often than UL97 mutations. In 21 experiments, one instance each of mutations E756D and M844V, which were shown individually to confer 3- to 4-fold increases in CPV EC(50), was detected. Unlike GCV and CDV, exonuclease mutations are not a preferred mechanism of CPV resistance, but mutations in and near pol region III may confer CPV resistance by affecting its recognition as an incoming base for DNA polymerization.
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43
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Chou S, Marousek G, Auerochs S, Stamminger T, Milbradt J, Marschall M. The unique antiviral activity of artesunate is broadly effective against human cytomegaloviruses including therapy-resistant mutants. Antiviral Res 2011; 92:364-8. [PMID: 21843554 DOI: 10.1016/j.antiviral.2011.07.018] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2011] [Revised: 07/13/2011] [Accepted: 07/21/2011] [Indexed: 12/26/2022]
Abstract
Current therapy options to treat infections with human cytomegalovirus face severe limitations leading to a continued search for novel drug candidates. Here, we describe novel characteristics of the strong antiviral potency of the drug artesunate. In vitro virus replication systems were applied to analyze a number of laboratory and clinically relevant strains of human cytomegalovirus. An inhibitory block at a very early stage of infection was demonstrated. Time-of-addition experiments indicated that the antiviral efficacy could be optimized when artesunate was applied as fractional doses consecutively added post-infection. Artesunate showed a clearly higher anti-cytomegaloviral activity than its parental drug artemisinin (approximately 10-fold) or other artesunate-related compounds. Mean IC(50) values of artesunate for a variety of standard therapy-resistant virus mutants were within a 2-fold range compared to wild-type virus. Furthermore, a synergistic effect was identified when artesunate was combined with the mechanistically distinct antiviral compound maribavir. These findings point to unique antiviral properties of artesunate which may offer an advantage over standard antiviral therapy particularly in cases of drug resistance.
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Affiliation(s)
- Sunwen Chou
- Division of Infectious Diseases, Oregon Health and Science University, Portland, Oregon, USA
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Recombinant phenotyping of cytomegalovirus UL54 mutations that emerged during cell passages in the presence of either ganciclovir or foscarnet. Antimicrob Agents Chemother 2011; 55:4019-27. [PMID: 21709106 DOI: 10.1128/aac.00334-11] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Selection of human cytomegalovirus variants in the presence of ganciclovir or foscarnet led to 18 DNA polymerase mutations, 14 of which had not been previously studied. Using bacterial artificial chromosome technology, each of these mutations was individually transferred into the genome of a reference strain. Following reconstitution of infectious viral stocks, each mutant was assessed for its drug susceptibility and growth kinetics in cell culture. Computer-assisted three-dimensional (3D) modeling of the polymerase was also used to position each of the mutations in one of four proposed structural domains and to predict their influence on structural stability of the protein. Among the 10 DNA polymerase mutations selected with ganciclovir, 7 (P488R, C539R, L545S, V787L, V812L, P829S, and L862F) were associated with ganciclovir resistance, whereas 2 (F595I and V946L) conferred only foscarnet resistance. Among the eight mutations selected with foscarnet, only two (T552N and S585A) conferred foscarnet resistance, whereas four (N408D, K500N, L802V, and L957F) had an impact on ganciclovir susceptibility. Surprisingly, the combination of mutations, some of which were not associated with resistance for a specific antiviral, resulted in increasing resistance effects. 3D modeling suggested that none of the mutated residues were directly involved in the polymerase catalytic site but rather had an influence on drug susceptibility by modifying the structural flexibility of the protein. Our study significantly adds to the number of DNA polymerase mutations conferring in vitro drug resistance and emphasizes the point that evaluation of individual mutations may not accurately reflect the phenotype conferred by multiple mutations.
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Boutolleau D, Burrel S, Agut H. Genotypic characterization of human cytomegalovirus UL97 phosphotransferase natural polymorphism in the era of ganciclovir and maribavir. Antiviral Res 2011; 91:32-5. [PMID: 21570426 DOI: 10.1016/j.antiviral.2011.04.015] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2011] [Revised: 04/12/2011] [Accepted: 04/27/2011] [Indexed: 01/17/2023]
Abstract
The molecular mechanisms of human cytomegalovirus (CMV) resistance to both ganciclovir and maribavir reported so far rely predominantly on the presence of mutations within UL97 phosphotransferase. The accurate interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objective of this work was to extend the catalog of CMV UL97 phosphotransferase natural polymorphisms. The full-length UL97 gene sequence analysis from 4 laboratory strains and 35 clinical samples from patients who had not received any previous anti-CMV treatment was performed. At the nucleotide level, the interstrain identity was >98%. At the amino acid level, ten natural polymorphisms never previously described were identified. Together with all previous results reported in the literature, a new map of UL97 phosphotransferase natural polymorphism could be settled in the era of ganciclovir and maribavir.
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Denison MR, Graham RL, Donaldson EF, Eckerle LD, Baric RS. Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity. RNA Biol 2011; 8:270-9. [PMID: 21593585 PMCID: PMC3127101 DOI: 10.4161/rna.8.2.15013] [Citation(s) in RCA: 377] [Impact Index Per Article: 26.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2010] [Revised: 01/27/2011] [Accepted: 01/28/2011] [Indexed: 12/18/2022] Open
Abstract
In order to survive and propagate, RNA viruses must achieve a balance between the capacity for adaptation to new environmental conditions or host cells with the need to maintain an intact and replication competent genome. Several virus families in the order Nidovirales, such as the coronaviruses (CoVs) must achieve these objectives with the largest and most complex replicating RNA genomes known, up to 32 kb of positive-sense RNA. The CoVs encode sixteen nonstructural proteins (nsp 1-16) with known or predicted RNA synthesis and modification activities, and it has been proposed that they are also responsible for the evolution of large genomes. The CoVs, including murine hepatitis virus (MHV) and SARS-CoV, encode a 3'-to-5' exoribonuclease activity (ExoN) in nsp14. Genetic inactivation of ExoN activity in engineered SARS-CoV and MHV genomes by alanine substitution at conserved DE-D-D active site residues results in viable mutants that demonstrate 15- to 20-fold increases in mutation rates, up to 18 times greater than those tolerated for fidelity mutants of other RNA viruses. Thus nsp14-ExoN is essential for replication fidelity, and likely serves either as a direct mediator or regulator of a more complex RNA proofreading machine, a process previously unprecedented in RNA virus biology. Elucidation of the mechanisms of nsp14-mediated proofreading will have major implications for our understanding of the evolution of RNA viruses, and also will provide a robust model to investigate the balance between fidelity, diversity and pathogenesis. The discovery of a protein distinct from a viral RdRp that regulates replication fidelity also raises the possibility that RNA genome replication fidelity may be adaptable to differing replication environments and selective pressures, rather than being a fixed determinant.
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Affiliation(s)
- Mark R Denison
- Department of Pediatrics and Microbiology & Immunology, Vanderbilt University Medical Center, Nashville, TN, USA
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Abstract
The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.
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Uil TG, Vellinga J, de Vrij J, van den Hengel SK, Rabelink MJWE, Cramer SJ, Eekels JJM, Ariyurek Y, van Galen M, Hoeben RC. Directed adenovirus evolution using engineered mutator viral polymerases. Nucleic Acids Res 2010; 39:e30. [PMID: 21138963 PMCID: PMC3061072 DOI: 10.1093/nar/gkq1258] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.
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Affiliation(s)
- Taco G Uil
- Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, 2300 RC, The Netherlands
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Shannon-Lowe CD, Emery VC. The effects of maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein. HERPESVIRIDAE 2010; 1:4. [PMID: 21429239 PMCID: PMC3050433 DOI: 10.1186/2042-4280-1-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/04/2010] [Accepted: 12/07/2010] [Indexed: 11/21/2022]
Abstract
Background The UL97 protein kinase of human cytomegalovirus phosphorylates the antiviral drug ganciclovir and is the target of maribavir action. A detailed enzyme kinetic analysis of maribavir on the various enzymatic functions of wild type and ganciclovir resistant forms of UL97 is required. Methods Wild type and site directed mutant forms of the human cytomegalovirus UL97 gene product were expressed using recombinant baculoviruses and the purified products used to assess the effects of maribavir on the ganciclovir (GCV) kinase and protein kinase (PK) activities. Results Maribavir was a potent inhibitor of the autophosporylation of the wild type and all the major GCV resistant UL97 mutants analysed (M460I, H520Q, A594V and L595F) with a mean IC50 of 35 nM. The M460I mutation resulted in hypersensitivity to maribavir with an IC50 of 4.8 nM. A maribavir resistant mutant of UL97 (L397R) was functionally compromised as both a GCV kinase and a protein kinase (~ 10% of wild type levels). Enzyme kinetic experiments demonstrated that maribavir was a competitive inhibitor of ATP with a Ki of 10 nM. Discussion Maribavir is a potent competitive inhibitor of the UL97 protein kinase function and shows increased activity against the M460I GCV-resistant mutant which may impact on the management of GCV drug resistance in patients.
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Affiliation(s)
- Claire D Shannon-Lowe
- Department of Infection, Centre for Virology, UCL (Royal Free Campus Campus), Rowland Hill Street, Hampstead, London NW3 2QG, UK.
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Prichard MN, Kern ER. The search for new therapies for human cytomegalovirus infections. Virus Res 2010; 157:212-21. [PMID: 21095209 DOI: 10.1016/j.virusres.2010.11.004] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2010] [Revised: 11/11/2010] [Accepted: 11/12/2010] [Indexed: 12/19/2022]
Abstract
Ganciclovir (GCV), the therapy of choice for human cytomegalovirus (CMV) infections and foscarnet, a drug used to treat GCV-resistant CMV infections was approved more than twenty years ago. Although cidofovir and a prodrug of GCV have since been added to the armamentarium, a highly effective drug without significant toxicities has yet to be approved. Such a therapeutic agent is required for treatment of immunocompromised hosts and infants, which bear the greatest burden of disease. The modest antiviral activity of existing drugs is insufficient to completely suppress viral replication, which results in the selection of drug-resistant variants that remain pathogenic, continue to replicate, and contribute to disease. Sustained efforts, largely in the biotech industry and academia, have identified highly active lead compounds that have progressed into clinical studies with varying levels of success. A few of these compounds inhibit new molecular targets, remain effective against isolates that have developed resistance to existing therapies, and promise to augment existing therapies. Some of the more promising drugs will be discussed with an emphasis on those progressing to clinical studies. Their antiviral activity both in vitro and in vivo, spectrum of antiviral activity, and mechanism of action will be reviewed to provide an update on the progress of potential new therapies for CMV infections.
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Affiliation(s)
- Mark N Prichard
- Department of Pediatrics, University of Alabama School of Medicine, Birmingham, AL 35233-1711, USA.
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