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An update on the management and prevention of cytomegalovirus infection following allogeneic hematopoietic stem cell transplantation Pilar Pérez Romero1, Pilar Blanco1, Estela Giménez2, Carlos Solano3 & David Navarro*,2,4 ABSTRACT A significant progress has been made in deciphering critical aspects of the biology and immunology of CMV infection in the allogeneic stem cell transplantation setting. Genetic traits predisposing to active CMV infection and CMV end-organ disease have begun to be delineated. Reliable molecular assays for CMV DNA load quantitation in body fluids have been developed. Elucidation of immune mechanisms affording control of CMV infection will help to improve the management of active CMV infection. Finally, the advent of new CMV-specific antivirals and promising vaccine prototypes as well as the development of fine procedures for large-scale ex vivo generation of functional CMV-specific T cells for adoptive T-cell transfer therapies will certainly minimize the negative impact of CMV on survival in these patients. Clinical significance of CMV infection in allogeneic stem cell transplant patients CMV causes significant morbidity and mortality in allogeneic stem cell transplant (allo-SCT) patients [1] . CMV end-organ disease including interstitial pneumonia, gastrointestinal disease, hepatitis, retinitis, encephalitis or systemic disease occur either early (100 days) after transplant and are related to viral cytopathogenicity (‘direct effect’) [1,2] . The incidence of late CMV end-organ disease has increased in recent years (15% CMV-seropositive allo-SCT recipients) and has been linked to prior active CMV infection or CMV end-organ disease, treatments involving ex vivo or in vivo with T-cell depletion, and the occurrence of acute or chronic graft versus host disease (GvHD). Likewise, low CD4 + T-cell counts, and lack of detectable CMV-specific immunity after 3 months of transplant have been recognized as biological factors significantly associated with the development of late CMV disease [3,4] . CMV encodes several proteins that display pro-inflammatory and immunosuppressive properties in vivo. This is the reason why CMV has also been associated with the occurrence of acute and chronic GvHD, with an increased risk of bacterial and fungal superinfection (‘indirect effects’) [5–9] . CMV-triggered ‘indirect effects’ have been well documented in the solid organ transplantation setting and have been associated with persistent virus replication in the transplanted allograft [10] ; The existence of CMV-related indirect effects in an allo-SCT setting remains to be definitively proven [11] . CMV-related morbidity in allo-SCT recipients is commonly linked to viral reactivation episodes in donor (D)+/recipient (R)+ or D-/R+, although it may also result from a primary infection

KEYWORDS

• allogeneic stem cell transplantation • CMV therapy • CMV vaccines • cytomegalovirus (CMV) • immunological monitoring • real-time PCR

Infectious Diseases, Microbiology & Preventive Medicine Unit, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain 2 Microbiology Service, Hospital Clínico Universitario, Fundación INCLIVA, Valencia, Spain 3 Hematology & Medical Oncology Service, Hospital Clínico Universitario, Fundación INCLIVA, Valencia, Spain 4 Department of Microbiology, School of Medicine, University of Valencia, Valencia, Spain *Author for correspondence: Tel.: +34 96 386 4657; Fax: +34 96 386 4173; [email protected] 1

10.2217/FVL.14.102 © 2015 Future Medicine Ltd

Future Virol. (2015) 10(2), 113–134

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Review Pérez Romero, Blanco, Giménez, Solano & Navarro (D+/R-), or from reinfection by a heterotypic strain carried by the allograft [1] . Active CMV infection, as diagnosed by the presence of CMV DNA either in plasma or whole blood (CMV DNAemia), develops in more than two-thirds of allo-SCT recipients in the first 100 days following transplantation [12,13] . In the absence of antiviral treatment, 25–30% of these patients will progress to CMV end-organ disease [2] . Clinical & genetic risk factors for active CMV infection & CMV end-organ disease CMV infection and CMV end-organ disease are particularly frequent in certain clinical settings: patients receiving a T-cell-depleted graft, a graft from an unrelated and/or an HLA-mismatched donor, the inclusion of antithymocyte globulin or alemtuzumab in the conditioning regimen, the use of mycophenolate mofetil in the GvHD prophylaxis regimen, and the administration of high dose of corticosteroids (>1 mg/kg) for severe (grades III-IV) GvHD therapy [14] . Receipt of a graft from a CMV-seronegative donor appears to have a negative impact on survival in the context of unrelated and myeloablative allo-SCT, in part at the expense of an increase in the rate of active CMV infection and CMV disease [13,15–17] . This effect has been associated with delayed reconstitution of functional CMV-specific T-cell responses [18] . In addition, both the overall hazard of CMV infection at any level and CMV endorgan disease occurring within the first 100 days in CMV-seropositive recipients appear to be similar in patients undergoing nonmyeloablative or myeloablative conditioning [19,20] . Nevertheless, a higher risk of late CMV disease was reported in the nonmyeloablative group [19] . Single nucleotide polymorphisms (SNPs) in several genes related to immune function may determine the susceptibility to CMV infection in the allo-SCT setting. In this context, certain SNPs in TLR-9, DC-SIGN, CCR5, IL-10 and MCP-1 genes appear to promote the development of either active CMV infection, CMV end-organ disease or both [21,22] . Other SNPs (in the chemokine receptor 5 gene and a single SNP upstream of the IL28B gene, encoding IFNλ3, previously associated with spontaneous and interferon-alpha treatment-induced clearance of HCV genotypes 1 and 4) [23] , have been shown to have an impact on the kinetics of CMV DNAemia clearance in this clinical setting [24,25] .

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CMV monitoring in the context of pre-emptive antiviral therapy strategies Pre-emptive antiviral therapy, consisting of the administration of antiviral therapy upon detection of CMV in blood to a certain level, is the first-choice strategy for the prevention of CMV end-organ disease over the universal antiviral prophylaxis strategy in the allo-SCT setting [26,27] . Currently, quantitative real-time PCR methods (QRT-PCR) are used at most centers over the pp65 antigenemia assay (AG) for guiding the inception of antiviral therapy. CMV DNA load triggering the initiation of preemptive antiviral therapy widely varies among centers. Likewise, no consensus does exist as to when antiviral therapy should be interrupted. Common practice is to initiate antiviral treatment when the CMV DNAemia level reaches a pre-established cut-off (from 1000 to 10,000 copies/ml in whole blood, or from 100 to 10,000 copies/ml of plasma), which is locally validated at each institution [26,27] . At some centers, the CMV DNA-load cut-off triggering the initiation of antiviral therapy depends upon the individual risk of patients for developing CMV disease, and the time at which CMV DNAemia occurs [27] . Alternatively, pre-emptive therapy may be initiated upon a significant increase in CMV DNAemia level (above the interassay coefficient of variation of the assay-around 0.5 log10) documented between two consecutive determinations performed 1 week apart [27] . In our experience, the percentage of patients receiving pre-emptive antiviral therapy for first or recurrent episodes of active CMV infection guided by QRT-PCR are comparable to patients guided by the AG assay [12] , although no consensus does exist on this issue [28] . Nevertheless, the duration of antiviral treatment for first episodes of active CMV infection appears to be significantly longer in the former group of patients when therapy is interrupted upon the second consecutive negative (undetectable) QRT-PCR result [12] . Pre-emptive antiviral therapy regimens have been shown to be highly effective in preventing the occurrence of CMV end-organ disease. In a recent study [29] , the cumulative incidence of CMV disease by day 100 in patients guided by PCR was comparable to that in patients guided by the AG assay (1.2 cells/μl, respectively [90,118–119] . Ohnishi et al. [124] reported that levels of T cells over 1 cell/μl, as quantified by an ELISPOT assay using individual pp65 peptides as antigens,

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were associated with the lack of subsequent CMV antigenemia. In turn, levels of IFN-γ CD8 + and CD4 + T cells above 3 and 1 cell/μl, respectively, as enumerated by ICS using CMV VR1814-infected DCs as stimulating antigen, have been shown to protect against CMV disease [95] . Likewise, Hebart et al. [125] found that the presence of more than five pp65-peptidespecific IFN-γ CD8 + T cells/μl was associated with protection from CMV DNAemia. Among the IFN-γ releasing assays, the QuantiFERONCMV assay (QFA; Cellestis Ltd, Melbourne, Australia) [128] is the only commercially available test that has been extensively evaluated. The QFA quantifies the level of IFN-γ, produced mostly by CMV-specific CD8 + T cells, upon the stimulation of whole blood with a 23 immunogenic peptides mapped within IE-1, IE-2, pp65, pp50 and gB and restricted by several widespread HLA-I alleles that cover >95% of the general population. The QFA is Conformité Européenne-labeled for in vitro diagnostic use in Europe. Its potential application in the allo-SCT setting has been evaluated [129–131] . These studies demonstrated the feasibility of the procedure for estimating the degree of CMV-specific T-cell reconstitution after transplant. MHC multimer-based assays are based on the direct staining CMV-specific CD8 + T cells with peptide-conjugated MHC class I tetramers, pentamers or more recently dextramers. This can be performed on low volume (0.5–1 ml) of freshly isolated or fixed PBMC or on whole blood, and it measures the frequency of T-cell phenotypes [132–134] . Although some studies have shown the clinical utility of tetramer-based immune monitoring in the setting of allo-SCT, it does not give information regarding whether the detected cells are functional, which is usually overcome by combining the assay with intracellular cytokine staining [135] . For these reasons, while multimers are powerful research tools, their value for diagnostic purposes may be limited. In addition multimer-peptide assays are limited to a few HLA haplotypes and thus may not be applicable for all patients. An FDA-approved, adenosine triphosphate release assay (Immuknow, Cylex™ Inc., USA) is used in several centers as a global measure of immunosuppression; however, it is not pathogen specific. A number of studies suggested that this assay may be useful for identifying patients at risk for post-transplantation viral infections [136–141] . Immunological monitoring may modulate the

future science group

Type of sample

Stimulating antigen


Standardized and simple to perform. Can be conducted at any center. Does not require knowledge of the HLA genotype High sensitivity, knowledge of HLA genotype not required. Reproducibility

Highly sensitivity, precise enumeration of CMV-specific CD8+ and CD4+ T cells. Cut-offs predicting protection from CMV DNAemia and CMV disease tentatively proposed Fast assay with single epitope-specific clone staining. Low blood volume. Cut-offs predicting protection from CMV DNAemia and CMV disease temptatively proposed

Intracellular cytokines secreted by CMV-specific T cells upon antigen stimulation

Allows for a comprehensive functional and phenotypic analysis of CMV-specific CD8+ and CD4+ T cells Peptide-specific T cells

Major advantages

IFN-γ secretion by CMV-stimulated CD8+ T cells

Immunological information

DC: Dendritic cell; ICS: Intracellular cytokine staining; PBMC: Peripheral blood mononuclear cell.

QuantiFERON-CMV Whole blood 21 peptides mapping within IE-1, IE-2, pp65, pp50 and gB and restricted by several widespread HLA-I variants (HLA-A1, A2, A3 and A24 and HLA-B7, B8, B27, B35, B44 and B52) ELISPOT PBMCs Single peptide, peptide pools, overlapping peptide libraries, infected cell lysates, recombinant proteins, purified viral proteins, DC loaded with peptides or infected with CMV panning the entire sequences of pp65 and IE-1 Flow cytometry Whole blood Single peptide, peptide pools, (ICS) or PBMCs overlapping peptide libraries, infected cell lysates, recombinant proteins, purified viral proteins, DC loaded with peptides or infected with CMV panning the entire sequences of pp65 and IE-1 MHC-multimerWhole blood Single immunogenic peptides peptides or PBMCs

Assay

Table 3. Methods for assessing CMV-specific immune responses in allogeneic stem cell transplant recipients.

Knowledge of HLA genotype required. Lack of reliable reagents for evaluation of CD4+ T-cell immunity. Lack of standardization

Lack of standardization. Expert personnel required. Need for access to a flow cytometer

High-sample volume needed. Does not differentiate between CD4+ and CD8+ T cells

Suboptimal sensitivity, with high percentage of indeterminate results. Does not evaluate CMV-specific CD4+ T-cell immunity

Major disadvantages

[132–135]

[79,89,95,116–123]

[16,124–125]

[128–131]

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An update on the management of CMV infection following allogeneic hematopoietic stem cell transplantation

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Review Pérez Romero, Blanco, Giménez, Solano & Navarro strategy of pre-emptive antiviral therapy in several manners. Antiviral therapy may be withheld or deferred in patients with evidence of CMVspecific immunity at the time of detection of CMV viremia, as these may be able to spontaneously clear the episode of CMV viremia without risk of developing CMV end-organ disease. A pilot study by Avetisyan et al. proved the feasibility of this approach [142] allowing for sparing antiviral treatment in 25% of patients. Likewise, shorter courses of antiviral drugs may be given to patients rapidly expanding functional CMVspecific T cells. This was proven in a proof-ofconcept study published by our group [143] . In turn, deficient immune reconstitution or expansion of CMV-specific T cells may allow the selection of patients that may benefit from antiviral prophylaxis or adoptive T-cell transfer therapy. To date, assessment of CMV-specific T-cell immunity is not routinely performed by most centers. Two main reasons account for this fact: these methods have not been standardized, hence, direct comparison of results obtained at different centers is not feasible; and although several surrogate marker for protection against active CMV infection and disease have been proposed (Table 3) , none of them has been clinically validated in large and controlled studies. Despite this, the potential contribution of immunological monitoring to the clinical and therapeutic management of CMV infection in the allo-SCT setting is being increasingly appreciated.

with second-line drugs can be problematic due to limited oral bioavailability, modest potency and significant toxicity [147,148] . To overcome these problems the development of new therapies, especially those with different molecular targets, is of paramount interest. Novel antivirals that are currently being developed or evaluated in clinical trials for CMV infection and their mechanisms of action are presented briefly in Table 4.

New antivirals: toward a prophylactic approach Ganciclovir and valganciclovir are the firstchoice drugs for treatment of active CMV infection and CMV end-organ disease in alloSCT recipients, with foscarnet and cidofovir as second-line alternatives [144] . When resistance to first-line therapies arises [145,146] , treatment

●●CMX-001

●●Maribavir

Maribavir is one of the most promising antiCMV drugs. It blocks the nuclear egress of newly formed HCMV virions by inhibiting the viral kinase UL97, and has been shown to display in vitro activity against ganciclovir-resistant strains [149] . Maribavir was successfully tested in allo-SCT patients in a Phase II trial [150] ; Nevertheless, in Phase III trials maribavir was no more effective than placebo or ganciclovir for preventing CMV viremia in SCT patients or liver transplant recipients, respectively [151,152] , which could have been related to the dosage employed [151,153] . To further define the optimal dosage for reducing viral replication, an open Phase II trial is currently underway in allo-SCT and solid organ transplant recipients refractory to treatment with ganciclovir/valganciclovir or foscarnet (NCT01611974). The future of this drug is uncertain, particularly since resistance to maribavir has already been reported [154] . CMX001 is an oral derivative of cidofovir with minimal toxicity [155] , and 300–400-fold more potency compared with intravenous cidofovir and high activity against CMV resistant to ganciclovir, foscarnet and cidofovir [156] . In a Phase II trial in seropositive allo-SCT patients, oral treatment with CMX001 at doses of 100 mg

Table 4. New antivirals for treatment of CMV infection. Antiviral

Mechanism of action

Maribavir CMX-001 Artesunate Letermovir (AIC246) Cyclopropavir Leflunomide Nucleic acid therapies

Inhibition of nuclear egress of newly formed CMV virions by inhibiting the viral kinase UL97 An orally bioavailable derivative of cidofovir that selectively inhibits the viral DNA polymerase Inhibition of the viral DNA polymerase (proposed mechanism) Inhibition of the viral pUL56 subunit blocking the cleaving and packaging of HCMV genomic DNA

[149–154]

Guanoside nucleoside analog that results in inhibition of viral DNA synthesis Inhibition of tegument acquisition by viral nucleocapsids Small interfering RNA molecules that prevent gene expression due to RNA silencing

[166–169]

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Ref. [155–157] [158–161] [162–165]

[170–174] [175–180]



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Table 5. CMV vaccines evaluated in allogeneic stem cell transplant recipients. Vaccine

Formulation and composition Phase Trial characteristics

ASP0113 (TransVAx/VCL-CB01) Plasmid (bivalent, pp65 Vical (USA)/Astellas (Japan) and gB) formulated with the poloxamer CRLI005 and benzalkonium chloride ASP0113 (TransVAx/VCL-CB01) Plasmid (bivalent, pp65 Astellas and gB) formulated with the poloxamer CRLI005 and benzalkonium chloride vCP260 Sanofi (Aventis; Attenuated canarypox-based France) Pasteur MSD (pp65)

weekly reduced the incidence of CMV replication, with no evidence of myelosuppression or nephrotoxicity [157] . A Phase III trial is currently recruiting CMV-seropositive allo-SCT patients to study efficacy and safety of CMX001 for the prevention of CMV infection (NCT01769170). ●●Artesunate

Artesunate is a widely used drug for treating cases of severe malaria that has recently been proposed as an effective treatment for mild CMV infection. In spite of showing an in vitro antiviral activity against multidrug-resistant CMV, the use of artesunate treatment in immunocompromised patients has been controversial [158] . Some results have shown inefficacy in preventing retinitis and associated CMV-resistant colitis in solid organ transplant recipients [159,160] . In addition, pre-emptive therapy with artesunate in allo-SCT patients failed to decrease CMV load in four out of six patients [161] . Further studies are needed to examine the role of artesunate in the treatment of CMV infection in allo-SCT recipients. ●●Letermovir (AIC246)

Letermovir has a novel mechanism of action which consists of interfering with the viral pUL56 subunit and blocking the cleaving and packaging of CMV genomic DNA [162,163] . Preclinical and Phase I trials showed good oral bioavailability, a favorable pharmacokinetic profile and a remarkable lack of toxicity [163,164] . A Phase II trial enrolling 131 CMV-seropositive allo-SCT patients showed that letermovir was effective in reducing the incidence of CMV infections compared with placebo when it was administrated at different doses for 12 weeks after engraftment [165] . The highest dose (240 mg per day) showed the greatest anti-CMV activity, with an acceptable safety profile. Thus, a Phase III trial is currently recruiting participants


II

III

II

Results

Ref.

Randomized/double-blind/placebo Occurrence and duration [111] of CMV viremia reduced in CMV-seropositive recipients Nonrandomized/open-label/no Recruiting [197] placebo

Nonrandomized/open-label/no placebo

Data not available

[198]

to test the efficacy of letermovir compared with placebo in the prevention of CMV infection in seropositive allo-SCT patients [181] . ●●Cyclopropavir

Cyclopropavir is a guanoside nucleoside analog tenfold more active against CMV than ganciclovir [166,167] . Pre-clinical studies have proven it to be effective in preventing mortality of MCMV infected mice and inhibiting replication of the virus in organs [168] . In addition, cyclopropavir can achieve in vivo therapeutic concentrations without prodrug modification [166] . Although resistance to cyclopropavir has been reported, these mutations are uncommon and most gancyclovir-resistant clinical isolates remain susceptible to cyclopropavir [169] . ●●Leflunomide

The in vitro anti-CMV properties of leflunomide were described in 1999, which involve blocking tegument acquisition by viral nucleocapsids [170] . The efficacy of leflunomide against CMV was first reported in kidney transplant patients [171] and later leflunomide was used in combination with foscarnet in allo-SCT patients infected with ganciclovir and foscarnet-resistant strains in a trial that showed a decrease in viral loads to undetectable levels [172] . In later studies leflunomide failed to control recurrent CMV infection [173,174] . ●●Nucleic acid therapies

Over the past two decades, the use of gene-based therapy in clinical practice against a wide range of diseases has been investigated. Small interfering RNA molecules are especially promising in silencing viral replication with reduced toxicity and high stability. The ability of RNA interference to target multiple regions of the viral genome could overcome drug resistance,


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Universal prophylaxis with new antivirals

Day 0

Day 30

Targeted prophylaxis with classic or new antivirals

Day 30

Day 0 Assessment of clinical and genetic factors increasing the risk of CMV end-organ disease

Assessment of CMV-specific T-cell immunity reconstitution

Pre-emptive antiviral therapy based on combined virological and immunological monitoring

Virological monitoring (once a week) DNAemia above cut-off

Spare treatment if detectable CMV-specific T-cell response

Immunological monitoring (once/2 weeks)

Interrupt antiviral treatment if expansion of CMV-specific T cells above a pre-established cut-off and significant decrease in viral

Adoptive T-cell transfer therapy if no expansion of CMV-specific T cells and lack of response to antiviral therapy

Figure 1. New approaches in the management of CMV infection in the allogeneic stem cell transplantation setting. (A) The advent of new antivirals that have high activity against CMV and cause less side effects than classic anti-CMV drugs may permit universal prophylaxis to be installed either at the time of transplant or engraftment. This strategy would prevent the occurrence of CMV end-organ disease and simultaneously reduce the risk of CMV-replication-derived ‘indirect effects’. Nevertheless, it might blunt the reconstitution of CMV-specific T-cell immunity. (B) In this strategy, antivirals (either classic or newly developed) would be selectively administered to patients with an increased risk of developing active CMV infection and CMV end-organ disease either at the time of transplant or at the time of engraftment.

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Figure 1. New approaches in the management of CMV infection in the allogeneic stem cell transplantation setting (cont.). (B) In this sense, the development of a risk score taking into consideration pretransplant or post-transplant clinical factors as well as host genetic factors predisposing to CMV end-organ disease is of critical relevance. Alternatively, antiviral prophylaxis may be administered exclusively to patients with unreconstituted CMV-specific T-cell responses at the time of engraftment provided that active CMV infection had not developed. (C) We show a potential intervention strategy based on combined virological (plasma or whole blood realtime PCR) an immunological monitoring of functional CMV-specific T-cell responses. Virological monitoring is routinely performed on a weekly basis within the first 100 days after transplant. Immunological monitoring may be conducted every 2 weeks in the absence of CMV DNAemia and weekly within episodes of active CMV infection. Antiviral treatment may be spared in patients exhibiting DNAemia values above the cutoff level for the inception of antiviral therapy provided that a measurable CMV-specific T-cell response is detected at the time the therapeutic decision has to be made. In addition, early interruption of pre-emptive therapy might be attempted if sufficient expansion of functional CMV-specific T cells is achieved concomitant with a significant decrease in viral load. In addition adoptive T-cell transfer therapy may be considered in cases of persistent CMV DNAemia in the absence of a significant expansion of functional CMV-specific T cells.

reducing the possibility for the virus to escape from the siRNA therapy [175] . Efficacy of interfering RNA molecules targeting CMV genes has been reported in animal models of retinitis [176–178] however, success in clinical trials has been limited [179,180] . It would be desirable for future design of siRNA therapeutics including both, essential and nonessential CMV gene targets such as UL54, UL97 and UL122/123 that have shown antiviral activity in cell culture. In addition, before using siRNA as an alternative therapy against viruses some issues such as protecting siRNA from nuclease digestion should be solved [140] . Antisense technology uses short chains of modified nucleotides to inhibit messenger ribonucleic acid translation. Fomivirsen is the only USA-approved antisense oligonucleotide for the treatment of CMV-induced retinitis in AIDS patients. This antisense RNA inhibits IE2 expression, however because it produces adverse effects it has had limited clinical use [182–185] . New therapeutic approaches: adoptive T-cell transfer therapies Pioneer studies conducted in the early 1990s demonstrated the efficacy of adoptive transfer of donor-derived CMV-specific cytotoxic T lymphocytes to control episodes of CMV replication refractory to conventional antiviral therapy [86,87] . Since then, several pilot studies have yielded data supporting the clinical utility of this approach [186–190] and currently two Phase II clinical trials are underway [191,192] . Improved methods in selection of virus-specific T cells on the basis of magnetic-activated cell


sorting technology have been developed. In this sense, direct ex vivo isolation of virus-specific T cells using reversible MHC multimers or shorttime stimulated cytokine-secreting T cells has been successfully applied in pilot. CMV vaccines The development of a vaccine for CMV has been identified as a high-priority goal in biomedical research [193] . Although numerous preclinical studies and a handful of Phase I and II clinical trials have been conducted with several CMV vaccine candidates, no vaccine has yet been licensed. Preclinical and clinical studies have been complicated by the paucity of animal models that accurately mimic human infection [194] , and lack of a well-established clinical end point for evaluating the efficacy of CMV vaccines [195] . Despite these obstacles, multiple strategies and candidate CMV vaccines have been developed during the past four decades [196] . A few of these (Table 5) have been evaluated in CMV-seropositive allo-SCT recipients. Live attenuated vaccines based on the Towne and AD169 strains were developed in the 1960s with the idea that they would express all or almost all relevant antigens to the immune system [199,200] . Both vaccines were safe and well-tolerated in seronegative adults, and while a live-attenuated Towne vaccine prevented severe disease in renal transplant recipients, it did not prevent infection with natural strains. In an attempt to improve the immunogenicity of Towne vaccine, a formulation based on four different chimeras of Towne/Toledo strains was developed by replacing regions of the Towne


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Review Pérez Romero, Blanco, Giménez, Solano & Navarro genome with sequences from Toledo strain. These vaccines were safe in CMV-seropositive subjects [201] and a Phase I trial of the four chimeric vaccines in CMV-seronegative adults is currently underway (NCT01195571). Advances in molecular biology and recombinant DNA technology over the past three decades have prompted the use of recombinant proteins, DNA-based vaccine and virus-vectored vaccines for development of CMV vaccines. A recombinant gB vaccine formulated with the MF59 adjuvant was reported to be safe and immunogenic [202,203] , and was able to boost antibodies and the T-cell response in CMVseropositive women [204] . This success warranted a Phase II trial in seronegative healthy young women of child-bearing age [205] , in which the vaccine demonstrated an efficacy of 50% against primary infection. A separate Phase II clinical trial enrolling patients awaiting kidney or liver transplantation (NCT00299260) showed that the gB/MF59 vaccine significantly induced gB-antibody titers in both seronegative and seropositve patients versus placebo [206] . Despite these results it was unclear whether gB + MF59 prototype would continue further development for use in these populations. Recently, another gB-based vaccine formulated with the AS01 adjuvant has been shown to be safe and immunogenic in a Phase I trial (NCT01357915). Plasmid DNA vaccines represent an attractive strategy for vaccination because they can induce both cellular and humoral immune responses against target antigens [207] . A bivalent DNA vaccine encoding the pp65 and gB antigens (ASP0113) was designed to prevent CMV viremia in high-risk patients undergoing hematopoietic cell transplantation and solid organ transplant [208] . This DNA-vaccine was tested in a Phase I trial (NCT02103426) in which it demonstrated a favorable safety profile in healthy CMV-seropositive and seronegative adults [209] . The vaccine has also been tested in a Phase II trial enrolling CMV-seropositive bone marrow transplant recipients in which the vaccine was well tolerated and reduced the occurrence, recurrence, viremia and duration of episodes of replication. Although the number of CMV-specific T cells expressing IFN-γ and antigB antibody titers were higher in the vaccine group than in placebo group at all time points after transplantation, these differences were not statistically significant and they did not find difference in the rates of viremia requiring antiviral

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therapy between the studied groups [111] . A study to further evaluate safety and tolerability of this vaccine prototype in subjects undergoing hematopoietic cell transplant nevertheless is currently underway [197] . Two candidate CMV peptide vaccines composed of the HLA A*0201 pp65495-503 cytotoxic CD81 T-cell epitope fused to two different universal T-helper epitopes (either the synthetic Pan DR epitope [PADRE] or a natural Tetanus sequence) were evaluated in a Phase Ib trial (NCT00722839) for safety and ability to elicit pp65 T cells in HLA A*0201 in healthy volunteers expressing the HLA A*0201 MHC class I allele sequence [210] . Results in healthy volunteers pave the way for future trials in HCT donors for the purpose of transferring CMV immunity to their recipients. AVX601, a replication-defective Venezuelan equine encephalitis virus expressing gB and an in-frame pp65/IE1 fusion protein, was recently evaluated in a Phase I trial (NCT00439803) [211] . CMV-seronegative individuals received the vaccine or the placebo at 0, 8 and 24 weeks after enrollment [212] . The vaccine was well tolerated and 4 weeks after the third dose of vaccination most of the CMV-seronegative individuals developed neutralizing antibodies detected by microneutralization assay. Additionally, the CMV-specific T-cell response measured by ELISPOT assay was detected in almost 97% of vaccinated recipients after the second dose [212] . Further studies are required to demonstrate the ability of this vaccine to prevent CMV infection. Conclusion & future perspective Despite significant progress in our understanding of CMV immunobiology and major advancements in the management of CMV infection in the allo-SCT setting, CMV remains a relavant cause of morbidity and mortality in the allo-SCT recipient. The advent of highly effective and less-toxic anti-CMV drugs, the standardization of biological assays for immune monitoring, the simplification of methods for large-scale ex vivo generation of functional CMV-specific T cells for adoptive T-cell transfer therapies, the optimization of certain vaccine formulations and elucidation of genetic traits and biological factors increasing the risk of CMV viremia and ultimately of end-organ disease will certainly have a major impact in the management of CMV infection in the allo-SCT setting in the near future, in



Review

EXECUTIVE SUMMARY Clinical relevance ofCMV infection in allogeneic stem cell transplant patients ●●

CMV continues to be a relevant cause of morbidity and mortality in allogeneic stem cell transplant (allo-SCT) recipients.

●●

CMV causes end-organ disease by virtue of its cytopathogenicity. CMV may also be related to the development of graft versus host disease and increase the risk of bacterial and fungal superinfection.

●●

Currently, CMV end-organ disease occurs most frequently at late times (after day 100) following transplantation.

●●

CMV DNAemia occurs in more than two-thirds of allo-SCT recipients in the first 100 days following transplantation.

Risk factors for active CMV infection & CMV end-organ disease ●●

Patients receiving a T-cell-depleted graft, or an unrelated and/or an HLA-mismatched allograft are at high risk of developing CMV-infection related events.

●●

The use of antithymocyte globulin or alemtuzumab in the conditioning regimen and mycophenolate mofetil in the graft versus host disease prophylaxis regimen predispose to CMV infection and end-organ disease.

●●

The use of high dose of corticosteroids (>1 mg/kg) for graft versus host disease therapy impairs CMV-specific T-cell immunity increasing the risk of CMV-related morbidity and mortality.

●●

For CMV-seropositive patients, the receipt of an allograft from an unrelated CMV-seronegative donor increases the risk of active CMV infection and disease.

●●

Single nucleotide polymorphisms in Toll-like receptor 9, dendritic cell-specific ICAM3-grabbing nonintegrin, chemokine

receptor 5, IL-10, and monocyte chemoattractant protein 1 and IL28B genes are associated with either a higher risk of developing either active CMV infection, CMV end-organ disease or both and higher CMV replicative levels within episodes. Preventative strategies of CMV end-organ disease: CMV surveillance ●●

The pre-emptive antiviral strategy is used at most centers for prevention of CMV end-organ disease. Universal prophylaxis at engraftment may be recommended in patients at highest risk for CMV disease.

●●

Quantitation of CMV DNAemia in either whole blood or plasma are equally suitable for CMV surveillance after allo-SCT. Real-time PCR assays are currently recommended for CMV DNA load monitoring.

●●

CMV DNAemia thresholds for initiation or interruption of pre-emptive therapy vary widely across centers. No

consensus has been reached on this matter. The advent of the 1st WHO standards will allow for the harmonization of these cut-offs among centers. Clinical value of the analysis of the kinetics of CMV DNA load in blood ●●

Analysis of the kinetics of CMV DNAemia may help to optimize pre-emptive antiviral therapy strategies.

Clinical value of monitoring of CMV-specific immune responses ●●

There are several methods for quantitation of functional CMV-specific T-cell responses. Only one of them, the Quantiferon CMV assay has been sufficiently standardized.

●●

Assessment of CMV-specific T-cell immunity may be ancillary to routine virological monitoring to treat patients with active CMV infection on an individual basis.

●●

Several immune surrogate markers for protection against active CMV infection and CMV end-organ disease have been proposed. Its potential use in clinical practice awaits extensive evaluation.

New antivirals: towarda prophylactic approach ●●

New drugs with high activity against CMV and better toxicity profile in comparison with ganciclovir and foscarnet have been developed. Of these, Letermovir, which interferes with CMV DNA replication in a novel manner, looks particularly promising.

CMV vaccines ●●

Several recombinant and plasmid-based vaccine candidates have been shown to be effective in the prevention of CMV viremia or CMV end-organ disease in the allo-SCT setting.



125

Review Pérez Romero, Blanco, Giménez, Solano & Navarro that it will allow the implementation of preventative strategies for CMV-related morbidity on an individual basis (Figure 1) . Until then, preemptive antiviral strategies based upon CMV DNA load monitoring in blood will continue to be the first-line choice for prevention of CMV disease over prophylaxis, even in high-risk patients. In this context the recent development of the 1st WHO standard will allow for widespread clinical validation of CMV DNAemia thresholds for initiation and interruption of antiviral therapy. Acknowledgement The authors thank M McConnell for critical reading of the manuscript.

References Papers of special note have been highlighted as: • of interest; •• of considerable interest 1

•

2

3

4

Boeckh M, Nichols WG, Papanicolaou G, Rubin R, Wingard JR, Zaia J. Cytomegalo virus in hematopoietic stem cell transplant recipients: current status, known challenges, and future strategies. Biol. Blood Marrow Transplant. 9(9), 543–558 (2003). A comprehensive review dealing with several critical issues of CMV infection in the allogeneic stem cell transplant (allo-SCT) setting. Ljungman P. CMV infections after hematopoietic stem cell transplantation. Bone Marrow Transplant. 42(Suppl. 1), S70–S72 (2008). Ozdemir E, Saliba RM, Champlin RE et al. Risk factors associated with late cytomegalo virus reactivation after allogeneic stem cell transplantation for hematological malignancies. Bone Marrow Transplant. 40(2), 125–136 (2007). Boeckh M, Leisenring W, Riddell SR et al. Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of viral load and T-cell immunity. Blood 101(2), 407–414 (2003).

•• A seminal study defining risk factors for late CMV disease. 5

126

Nichols WG, Corey L, Gooley T, Davis C, Boeckh M. High risk of death due to bacterial and fungal infection among cytomegalovirus (CMV)-seronegative recipients of stem cell transplants from seropositive donors: evidence for indirect effects of primary CMV

Financial & competing interests disclosure This research was supported by grants (09/1117, 12/1992, 11/01357, 14/01291) from FIS (Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain). This work was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III - cofinanced by European Development Regional Fund ‘A way to achieve Europe’ ERDF, Spanish Network for the Research in Infectious Diseases [REIPI RD12/0015]. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

infection. J. Infect. Dis. 185(3), 273–282 (2002). 6

Marr KA, Carter RA, Boeckh M, Martin P, Corey L. Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors. Blood 100(13), 4358–4366 (2002).

•• A large study linking CMV disease with the development of invasive aspergillosis. 7

8

9

Wang LR, Dong LJ, Zhang MJ, Lu DP. Correlations of human herpesvirus 6B and CMV infection with acute GVHD in recipients of allogeneic haematopoietic stem cell transplantation. Bone Marrow Transplant. 42(10), 673–677 (2008). Garcia-Vidal C, Upton A, Kirby KA, Marr KA. Epidemiology of invasive mold infections in allogeneic stem cell transplant recipients: biological risk factors for infection according to time after transplantation. Clin. Infect. Dis. 47(8), 1041–1050 (2008). Cantoni N, Hirsch HH, Khanna N et al. Evidence for a bidirectional relationship between cytomegalovirus replication and acute graft-versus-host disease. Biol. Blood Marrow Transplant. 16(9), 1309–1314 (2010).

10 Freeman RB. The ‘indirect’ effects of

cytomegalovirus infection. Am. J. Transplant. 9(11), 2453–2458 (2009). •

An interesting review dealing with the so-called ‘indirect effects’ induced by CMV, especially in the solid organ transplantation setting.

11 Gimenez E, Solano C, Nieto J et al. An

investigation on the relationship between the occurrence of CMV DNAemia and the development of invasive aspergillosis in the


allogeneic stem cell transplantation setting. J. Med. Virol. 86(4), 568–575 (2014). 12 Solano C, Munoz-Cobo B, Gimenez E et al.

Pre-emptive antiviral therapy for active CMV infection in adult allo-SCT patients guided by plasma CMV DNAemia quantitation using a real-time PCR assay: clinical experience at a single center. Bone Marrow Transplant. 48(7), 1010–1012 (2013). 13 Boeckh M, Nichols WG. The impact of

cytomegalovirus serostatus of donor and recipient before hematopoietic stem cell transplantation in the era of antiviral prophylaxis and preemptive therapy. Blood 103(6), 2003–2008 (2004). 14 Ljungman P, Perez-Bercoff L, Jonsson J et al.

Risk factors for the development of cytomegalovirus disease after allogeneic stem cell transplantation. Haematologica 91(1), 78–83 (2006). •• Large study defining risk factors for CMV disease. 15 Ljungman P, Brand R, Einsele H, Frassoni F,

Niederwieser D, Cordonnier C. Donor CMV serologic status and outcome of CMVseropositive recipients after unrelated donor stem cell transplantation: an EBMT megafile analysis. Blood 102(13), 4255–4260 (2003). 16 Ganepola S, Gentilini C, Hilbers U et al.

Patients at high risk for CMV infection and disease show delayed CD8+ T-cell immune recovery after allogeneic stem cell transplantation. Bone Marrow Transplant. 39(5), 293–299 (2007). 17 Ljungman P, Brand R, Hoek J et al. Donor

cytomegalovirus status influences the outcome of allogeneic stem cell transplant: a study by the European group for blood and


An update on the management of CMV infection following allogeneic hematopoietic stem cell transplantation marrow transplantation. Clin. Infect Dis. 59(4), 473–481 (2014). •

A large multicenter study addressing the effect of CMV serostatus on survival following allo-SCT.

recipient CCR5, MCP-1, IL-10, and TLR9 genes. J. Med. Virol. 87(29), 248–255 (2015).

18 Zhou W, Longmate J, Lacey SF et al. Impact

of donor CMV status on viral infection and reconstitution of multifunction CMV-specific T cells in CMV-positive transplant recipients. Blood 113(25), 6465–6476 (2009). •

A pioneer work proving differences in the degree of CMV-specific immune reconstitution in CMV-seropositive allo-SCT recipients depending upon the CMV serostatus of donors.

•

20 Nakamae H, Kirby KA, Sandmaier BM

et al. Effect of conditioning regimen intensity on CMV infection in allogeneic hematopoietic cell transplantation. Biol. Blood Marrow Transplant. 15(6), 694–703 (2009). 21 Loeffler J, Steffens M, Arlt EM et al.

Polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation. J. Clin. Microbiol. 44(5), 1847–1850 (2006). •

A comprehensive analysis of the effect of several single nucleotide polymorphisms (SNPs) in several genes related to innate and adaptive immunity on the incidence of active CMV infection and CMV disease in the allo-SCT setting.

22 Mezger M, Steffens M, Semmler C et al.

Investigation of promoter variations in dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN) (CD209) and their relevance for human cytomegalovirus reactivation and disease after allogeneic stem-cell transplantation. Clin. Microbiol. Infect. 14(3), 228–234 (2008). 23 Ge D, Fellay J, Thompson AJ et al. Genetic

variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461(7262), 399–401 (2009). 24 Corrales I, Gimenez E, Solano C et al.

Incidence and dynamics of active cyto megalovirus infection in allogeneic stem cell transplant patients according to single nucleotide polymorphisms in donor and


The effect of quantification standards used in real-time CMV PCR assays on guidelines for initiation of therapy in allogeneic stem cell transplant patients. Bone Marrow Transplant. 39(4), 237–238 (2007).

Pioneer study linking an SNP upstream of the IL28B gene with the dynamics of active CMV infection in the allo-SCT setting.

34 Herrmann B, Larsson VC, Rubin CJ et al.

Comparison of a duplex quantitative real-time PCR assay and the COBAS Amplicor CMV Monitor test for detection of cytomegalovirus. J. Clin. Microbiol. 42(5), 1909–1914 (2004). 35 Hong KM, Najjar H, Hawley M, Press RD.

Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients. Clin. Chem. 50(5), 846–856 (2004).

26 Ljungman P, De La Camara R, Cordonnier C

et al. Management of CMV, HHV-6, HHV-7 and Kaposi-sarcoma herpesvirus (HHV-8) infections in patients with hematological malignancies and after SCT. Bone Marrow Transplant. 42(4), 227–240 (2008).

19 Junghanss C, Boeckh M, Carter RA et al.

Incidence and outcome of cytomegalovirus infections following nonmyeloablative compared with myeloablative allogeneic stem cell transplantation, a matched control study. Blood 99(6), 1978–1985 (2002).

33 Harrington SM, Buller RS, Storch GA et al.

25 Bravo D, Solano C, Gimenez E et al. Effect of

the IL28B Rs12979860 C/T polymorphism on the incidence and features of active cytomegalovirus infection in allogeneic stem cell transplant patients. J. Med. Virol. 86(5), 838–844 (2014).

•

36 Kalpoe JS, Kroes AC, De Jong MD et al.

Validation of clinical application of cytomegalovirus plasma DNA load measurement and definition of treatment criteria by analysis of correlation to antigen detection. J. Clin. Microbiol. 42(4), 1498–1504 (2004).

Consensus document for the management of CMV infection in allo-SCT recipients.

27 Boeckh M, Ljungman P. How we treat

cytomegalovirus in hematopoietic cell transplant recipients. Blood 113(23), 5711–5719 (2009).

37 Leruez-Ville M, Ouachee M, Delarue R et al.

•• A practical approach to the management of CMV infection in the allo-SCT setting.

Monitoring cytomegalovirus infection in adult and pediatric bone marrow transplant recipients by a real-time PCR assay performed with blood plasma. J. Clin. Microbiol. 41(5), 2040–2046 (2003).

28 Gerna G, Lilleri D, Caldera D, Furione M,

Zenone Bragotti L, Alessandrino EP. Validation of a DNAemia cutoff for preemptive therapy of cytomegalovirus infection in adult hematopoietic stem cell transplant recipients. Bone Marrow Transplant. 41(10), 873–879 (2008).

38 Limaye AP, Huang ML, Leisenring W,

Stensland L, Corey L, Boeckh M. Cytomegalovirus (CMV) DNA load in plasma for the diagnosis of CMV disease before engraftment in hematopoietic stem-cell transplant recipients. J. Infect. Dis. 183(3), 377–382 (2001).

29 Green ML, Leisenring W, Stachel D et al.

Efficacy of a viral load-based, risk-adapted, preemptive treatment strategy for prevention of cytomegalovirus disease after hemato poietic cell transplantation. Biol. Blood Marrow Transplant. 18(11), 1687–99 (2012). 30 Gimeno C, Solano C, Latorre JC et al.

Quantification of DNA in plasma by an automated real-time PCR assay (cytomegalovirus PCR kit) for surveillance of active cytomegalovirus infection and guidance of preemptive therapy for allogeneic hematopoietic stem cell transplant recipients. J. Clin. Microbiol. 46(10), 3311–3318 (2008). 31 Boeckh M, Huang M, Ferrenberg J et al.

Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR. J. Clin. Microbiol. 42(3), 1142–1148 (2004). 32 Griscelli F, Barrois M, Chauvin S, Lastere S,

Bellet D, Bourhis JH. Quantification of human cytomegalovirus DNA in bone marrow transplant recipients by real-time PCR. J. Clin. Microbiol. 39(12), 4362–4369 (2001).

Review

•

A seminal study demonstrating the clinical utility of CMV DNA monitoring in the management of CMV infection in the allo-SCT setting.

39 Machida U, Kami M, Fukui T et al.

Real-time automated PCR for early diagnosis and monitoring of cytomegalovirus infection after bone marrow transplantation. J. Clin. Microbiol. 38(7), 2536–2542 (2000). 40 Mori T, Okamoto S, Watanabe R et al.

Dose-adjusted preemptive therapy for cytomegalovirus disease based on real-time polymerase chain reaction after allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 29(9), 777–782 (2002). 41 Nitsche A, Steuer N, Schmidt CA et al.

Detection of human cytomegalovirus DNA by real-time quantitative PCR. J. Clin. Microbiol. 38(7), 2734–2737 (2000). 42 Pumannova M, Roubalova K, Vitek A,

Sajdova J. Comparison of quantitative


127

Review Pérez Romero, Blanco, Giménez, Solano & Navarro competitive polymerase chain reactionenzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation. Diagn. Microbiol. Infect. Dis. 54(2), 115–120 (2006). 43 Ruell J, Barnes C, Mutton K et al. Active

52 Abbate I, Finnstrom N, Zaniratti S et al.

Evaluation of an automated extraction system in combination with Affigene CMV Trender for CMV DNA quantitative determination: comparison with nested PCR and pp65 antigen test. J. Virol. Meth. 151(1), 61–65 (2008). Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation. J. Clin. Microbiol. 46(4), 1241–1245 (2008).

44 Schaade L, Kockelkorn P, Ritter K,

Pan J, Hayden RT. Detection of cyto megalovirus in whole blood using three different real-time PCR chemistries. J. Mol. Diagn. 11(1), 54–59 (2009). Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients. J. Clin. Microbiol. 49(8), 2899–2904 (2011).

Quantitative analysis of cytomegalovirus load using a real-time PCR assay. J. Med. Virol. 60(4), 455–462 (2000). 47 Caliendo AM, Ingersoll J, Fox-Canale AM

•

48 Gouarin S, Vabret A, Scieux C et al.

Multicentric evaluation of a new commercial cytomegalovirus real-time PCR quantitation assay. J. Virol. Meth. 146(1–2), 147–154 (2007).

Baldanti F. Kinetics of human cytomegalovirus (HCMV) DNAemia in transplanted patients expressed in international units as determined with the Abbott RealTime CMV assay and an in-house assay. J. Clin. Virol. 55(4), 317–322 (2012). 57 Clari MA, Bravo D, Costa E et al.

Comparison of the new Abbott Real Time CMV assay and the Abbott CMV PCR Kit for the quantitation of plasma cyto megalovirus DNAemia. Diagn. Microbiol. Infect. Dis. 75(2), 207–209 (2013). 58 Hirsch HH, Lautenschlager I, Pinsky BA

50 Yerly S, Perrin L, Van Delden C et al.

Cytomegalovirus quantification in plasma by an automated real-time PCR assay. J. Clin. Virol. 38(4), 298–303 (2007). 51 Allice T, Cerutti F, Pittaluga F et al.

Evaluation of a novel real-time PCR system for cytomegalovirus DNA quantitation on whole blood and correlation with pp65antigen test in guiding pre-emptive antiviral treatment. J. Virol. Meth. 148(1–2), 9–16 (2008).

128

Demonstrates the critical effect of the DNA extraction method on the CMV DNA loads produced by different commercially available quantitative real-time PCR (QRT-PCR) assays.

56 Furione M, Rognoni V, Cabano E,

49 Hanson KE, Reller LB, Kurtzberg J,

Horwitz M, Long G, Alexander BD. Comparison of the Digene Hybrid Capture System Cytomegalovirus (CMV) DNA (version 2.0), Roche CMV UL54 analytespecific reagent, and QIAGEN RealArt CMV LightCycler PCR reagent tests using AcroMetrix OptiQuant CMV DNA quantification panels and specimens from allogeneic-stem-cell transplant recipients. J. Clin. Microbiol. 45(6), 1972–1973 (2007).

61 Gracia-Ahufinger I, Tormo N, Espigado I

et al. Differences in cytomegalovirus plasma viral loads measured in allogeneic hemato poietic stem cell transplant recipients using two commercial real-time PCR assays. J. Clin. Virol. 48(2), 142–146 (2010). 62 Fryer JF, Heath AB, Anderson R, Minor PD,

the Collaborative Study Group. Collaborative Study to Evaluate the Proposed 1st WHO International Standard for Human Cytomegalovirus (HCMV) for Nucleic Acid Amplification (NAT)-Based Assays (WHO/BS/10.2138) (2010). www.who.int/iris/handle/10665/70521

55 Bravo D, Clari MA, Costa E et al.

46 Tanaka N, Kimura H, Iida K et al.

et al. Evaluation of real-time PCR laboratory-developed tests using analytespecific reagents for cytomegalovirus quantification. J. Clin. Microbiol. 45(6), 1723–1727 (2007).

Stellrecht KA, Press RD. Multi-Site PCR-based CMV viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology. J. Mol. Diagn. 11(2), 87–92 (2009).

54 Vincent E, Gu Z, Morgenstern M, Gibson C,

45 Tanaka Y, Kanda Y, Kami M et al.

Monitoring cytomegalovirus infection by antigenemia assay and two distinct plasma real-time PCR methods after hematopoietic stem cell transplantation. Bone Marrow Transplant. 30(5), 315–319 (2002).

60 Wolff DJ, Heaney DL, Neuwald PD,

53 Michelin BD, Hadzisejdic I, Bozic M et al.

CMV disease does not always correlate with viral load detection. Bone Marrow Transplant. 40(1), 55–61 (2007). Kleines M. Detection of cytomegalovirus DNA in human specimens by LightCycler PCR. J. Clin. Microbiol. 38(11), 4006–4009 (2000).

•• A comprehensive multicenter study stressing the interassay (intercenter) variability of QRT-PCR assays.

et al. An international multicenter performance analysis of cytomegalovirus load tests. Clin. infect. Dis. 56(3), 367–373 (2013). •

A large multicenter study evaluating the first US FDA-approved QRT-PCR assay.

59 Pang XL, Fox JD, Fenton JM, Miller GG,

Caliendo AM, Preiksaitis JK. Interlaboratory comparison of cytomegalovirus viral load assays. Am. J. Transplant. 9(2), 258–268 (2009).


•

Description and evaluation of the 1st CMV WHO standard.

63 Clari MA, Bravo D, Costa E et al.

Comparison of the new Abbott Real Time CMV assay and the Abbott CMV PCR Kit for the quantitation of plasma cyto megalovirus DNAemia. Diagn. Microbiol. Infect. Dis. 75(2), 207–209 (2013). 64 Hayden RT, Yan X, Wick MT et al. Factors

contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis. J. Clin. Microbiol. 50(2), 337–345 (2012). 65 Caliendo AM. The long road toward

standardization of viral load testing for cytomegalovirus. Clin. Iinfect. Dis. 56(3), 374–375 (2013). •

A thoughtful reflection on the benefits and shortcomings of QRT-PCR assays in the management of CMV infection in transplant recipients.

66 Kraft CS, Armstrong WS, Caliendo AM.

Interpreting quantitative cytomegalovirus DNA testing: understanding the laboratory perspective. Clin. Infect. Dis. 54(12), 1793–1797 (2012). •• A practical review highlighting the difficulties in interpreting CMV DNA load measurements produced by QRT-PCR assays. 67 Hayden RT, Shahbazian MD, Valsamakis A

et al. Multicenter evaluation of a commercial cytomegalovirus quantitative standard: effects of commutability on interlaboratory


An update on the management of CMV infection following allogeneic hematopoietic stem cell transplantation concordance. J. Clin. Microbiol. 51(11), 3811–3817 (2013). 68 Solano C, Navarro D. Clinical virology of

cytomegalovirus infection following hematopoietic transplantation. Future Virol. 5(1), 111–124 (2010). 69 Garrigue I, Doussau A, Asselineau J et al.

Prediction of cytomegalovirus (CMV) plasma load from evaluation of CMV whole-blood load in samples from renal transplant recipients. J. Clin. Microbiol. 46(2), 493–498 (2008). 70 Gurtler V, Mayall BC, Wang J,

Ghaly-Derias S. The increased sensitivity of human cytomegalovirus (HCMV) PCR quantitation in whole blood affects reproductive rate (Ro) measurement. J. Virol. Meth. 196, 179–184 (2014). 71 Xie L, Liang XN, Deng Y et al. Effects of

storage time on Cytomegalovirus DNA stability in plasma determined by quantitative real-time PCR. J. Virol. Meth. 207, 196–199 (2014). 72 Atkinson C, Emery VC. Cytomegalovirus

quantification: where to next in optimising patient management? J. Clin. Virol. 51(4), 223–228 (2011). 73 Andrews PA, Emery VC, Newstead C.

Summary of the British Transplantation Society Guidelines for the Prevention and Management of CMV disease after solid organ transplantation. Transplantation 92(11), 1181–1187 (2011). •

An up-to-date guide for the management of active CMV infection in allo-SCT recipients.

74 Emery VC, Sabin CA, Cope AV, Gor D,

Hassan-Walker AF, Griffiths PD. Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation. Lancet 355(9220), 2032–2036 (2000). •• A Pioneer study on the clinical utility of the analyses of CMV DNA kinetics in transplant recipients. 75 Stachel D, Kirby K, Corey L, Boeckh M. CMV

Viral load as predictor for transplant-related mortality in the era of pre-emptive therapy. Bone Marrow Transplant. 41(Suppl.), S46 (2008). 76 Gimenez E, Munoz-Cobo B, Solano C,

Amat P, Navarro D. Early kinetics of plasma cytomegalovirus DNA load in allogeneic stem cell transplant recipients in the era of highly sensitive real-time PCR assays: does it have any clinical value? J. Clin. Microbiol. 52(2), 654–656 (2014). •

A new approach for triggering the inception of pre-emptive antiviral therapy in allo-SCT recipients.


77 Nichols WG, Corey L, Gooley T et al. Rising

pp65 antigenemia during preemptive anticytomegalovirus therapy after allogeneic hematopoietic stem cell transplantation: risk factors, correlation with DNA load, and outcomes. Blood 97(4), 867–874 (2001). 78 Gerna G, Lilleri D, Zecca M et al. Rising

antigenemia levels may be misleading in pre-emptive therapy of human cyto megalovirus infection in allogeneic hematopoietic stem cell transplant recipients. Haematologica 90(4), 526–533 (2005).

85 Riddell SR, Watanabe KS, Goodrich JM,

Li CR, Agha ME, Greenberg PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T-cell clones. Science 257(5067), 238–241 (1992). •• Proof of concept study that set the basis for the use of adoptive T-cell transfer therapies for active CMV infection or disease. 86 Hakki M, Riddell SR, Storek J et al. Immune

reconstitution to cytomegalovirus after allogeneic hematopoietic stem cell transplantation: impact of host factors, drug therapy, and subclinical reactivation. Blood 102(8), 3060–3067 (2003).

79 Tormo N, Solano C, Benet I et al. Lack of

prompt expansion of cytomegalovirus pp65 and IE-1-specific IFNgamma CD8 + and CD4 + T cells is associated with rising levels of pp65 antigenemia and DNAemia during pre-emptive therapy in allogeneic hemato poietic stem cell transplant recipients. Bone Marrow Transplant. 45(3), 543–549 (2010). 80 Buyck HC, Griffiths PD, Emery VC. Human

cytomegalovirus (HCMV) replication kinetics in stem cell transplant recipients following anti-HCMV therapy. J. Clin. Virol. 49(1), 32–36 (2010). 81 Munoz-Cobo B, Solano C, Costa E et al.

Dynamics of cytomegalovirus (CMV) plasma DNAemia in initial and recurrent episodes of active CMV infection in the allogeneic stem cell transplantation setting: implications for designing preemptive antiviral therapy strategies. Biol. Blood Marrow Transplant. 17(11), 1602–1611 (2011).

87 Ozdemir E, St John LS, Gillespie G et al.

Cytomegalovirus reactivation following allogeneic stem cell transplantation is associated with the presence of dysfunctional antigen-specific CD8 + T cells. Blood 100(10), 3690–3697 (2002). 88 Aubert G, Hassan-Walker AF, Madrigal JA

et al. Cytomegalovirus-specific cellular immune responses and viremia in recipients of allogeneic stem cell transplants. J. Infect. Dis. 184(8), 955–963 (2001). 89 Cwynarski K, Ainsworth J, Cobbold M et al.

Direct visualization of cytomegalovirusspecific T-cell reconstitution after allogeneic stem cell transplantation. Blood 97(5), 1232–1240 (2001). 90 Solano C, Benet I, Clari MA et al.

Enumeration of cytomegalovirus-specific interferongamma CD8 + and CD4 + T cells early after allogeneic stem cell transplantation may identify patients at risk of active cytomegalovirus infection. Haematologica 93(9), 1434–1436 (2008).

82 Li CR, Greenberg PD, Gilbert MJ,

Goodrich JM, Riddell SR. Recovery of HLA-restricted cytomegalovirus (CMV)specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis. Blood 83(7), 1971–1979 (1994). 83 Quinnan GV Jr, Kirmani N, Rook AH et al.

Cytotoxic t cells in cytomegalovirus infection: HLA-restricted T-lymphocyte and non-Tlymphocyte cytotoxic responses correlate with recovery from cytomegalovirus infection in bone-marrow-transplant recipients. N. Engl. J. Med. 307(1), 7–13 (1982). •• Pioneer work demonstrating the relevance of CMV-specific T cells in the control of CMV infection in the allo-SCT setting. 84 Reusser P, Riddell SR, Meyers JD, Greenberg

PD. Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease. Blood 78(5), 1373–1380 (1991).

Review

91 Sylwester AW, Mitchell BL, Edgar JB et al.

Broadly targeted human cytomegalovirusspecific CD4 + and CD8 + T cells dominate the memory compartments of exposed subjects. J. Exp. Med. 202(5), 673–685 (2005). 92 Einsele H, Roosnek E, Rufer N et al. Infusion

of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood 99(11), 3916–3922 (2002). •

Demonstrates successful adoptive transfer donor-derived CMV-specific T-cell lines into stem cell transplant recipients lacking CMV-specific T-cell proliferation.

93 Krol L, Stuchly J, Hubacek P et al. Signature

profiles of CMV-specific T-cells in patients with CMV reactivation after hematopoietic SCT. Bone Marrow Transplant. 46(8), 1089–1098 (2011). 94 Lacey SF, La Rosa C, Zhou W et al. Functional

comparison of T cells recognizing


129

Review Pérez Romero, Blanco, Giménez, Solano & Navarro cytomegalovirus pp65 and intermediate-early antigen polypeptides in hematopoietic stem-cell transplant and solid organ transplant recipients. J. Infect. Dis. 194(10), 1410–1421 (2006). 95 Lilleri D, Fornara C, Chiesa A, Caldera D,

Alessandrino EP, Gerna G. Human cytomegalovirus-specific CD4 + and CD8 + T-cell reconstitution in adult allogeneic hematopoietic stem cell transplant recipients and immune control of viral infection. Haematologica 93(2), 248–256 (2008). 96 Makedonas G, Hutnick N, Haney D et al.

Perforin and IL-2 upregulation define qualitative differences among highly functional virus-specific human CD8 T cells. PLoS Pathog. 6(3), e1000798 (2010). 97 Nebbia G, Mattes FM, Smith C et al.

Polyfunctional cytomegalovirus-specific CD4 + and pp65 CD8 + T cells protect against high-level replication after liver transplantation. Am. J. Transplant. 8(12), 2590–2599 (2008). 98 Lachmann R, Bajwa M, Vita S et al.

Polyfunctional T cells accumulate in large human cytomegalovirus-specific T cell responses. J. Virol. 86(2), 1001–1009 (2012). 99 Munoz-Cobo B, Solano C, Benet I et al.

Functional profile of cytomegalovirus (CMV)-specific CD8 + T cells and kinetics of NKG2C + NK cells associated with the resolution of CMV DNAemia in allogeneic stem cell transplant recipients. J. Med. Virol. 84(2), 259–267 (2012). 100 Espigado I, De La Cruz-Vicente F,

Benmarzouk-Hidalgo OJ et al. Timing of CMV-specific effector memory T-cells predicts viral replication and Survival after allogeneic hematopoietic stem cell transplantation. Transpl. Int. doi:10.1111/ tri.12406 (2014) (Epub ahead of print). •

Novel study demonstrating that early immune reconstitution after transplantation has an impact increase survival.

101 Barron MA, Gao D, Springer KL et al.

Relationship of reconstituted adaptive and innate cytomegalovirus (CMV)-specific immune responses with CMV viremia in hematopoietic stem cell transplant recipients. Clin. Iinfect. Dis. 49(12), 1777–1783 (2009). 102 Foley B, Cooley S, Verneris MR et al.

Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood 119(11), 2665–2674 (2012). 103 Kuijpers TW, Baars PA, Dantin C,

Van Den Burg M, Van Lier RA, Roosnek E.

130

Human NK cells can control CMV infection in the absence of T cells. Blood 112(3), 914–915 (2008). 104 Lopez-Verges S, Milush JM, Schwartz BS

et al. Expansion of a unique CD57(+) NKG2Chi natural killer cell subset during acute human cytomegalovirus infection. Proc. Natl Acad. Sci. USA 108(36), 14725–14732 (2011). 105 Kheav VD, Busson M, Scieux C et al.

Favorable impact of natural killer cell reconstitution on chronic graft-versus-host disease and cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation. Haematologica doi:10.3324/ haematol.2014.108407 (2014) (Epub ahead of print). 106 Fouts AE, Chan P, Stephan JP, Vandlen R,

Feierbach B. Antibodies against the gH/gL/UL128/UL130/UL131 complex comprise the majority of the anticytomegalovirus (anti-CMV) neutralizing antibody response in CMV hyperimmune globulin. J. Virol. 86(13), 7444–7447 (2012). 107 Genini E, Percivalle E, Sarasini A,

Revello MG, Baldanti F, Gerna G. Serum antibody response to the gH/gL/pUL128–131 five-protein complex of human cytomegalovirus (HCMV) in primary and reactivated HCMV infections. J. Clin. Virol. 52(2), 113–118 (2011). 108 Lilleri D, Kabanova A, Lanzavecchia A,

Gerna G. Antibodies against neutralization epitopes of human cytomegalovirus gH/gL/pUL128–130–131 complex and virus spreading may correlate with virus control in vivo. J. Clin. Immunol. 32(6), 1324–1331 (2012). 109 Munoz I, Gutierrez A, Gimeno C et al. Lack

of association between the kinetics of human cytomegalovirus (HCMV) glycoprotein B (gB)-specific and neutralizing serum antibodies and development or recovery from HCMV active infection in patients undergoing allogeneic stem cell transplant. J. Med. Virol. 65(1), 77–84 (2001). 110 Crough T, Khanna R. Immunobiology of

human cytomegalovirus: from bench to bedside. Clin. Microbiol. Rev. 22(1), 76–98 (2009). •• A comprehensive review of CMV biology. 111 Kharfan-Dabaja MA, Boeckh M, Wilck MB

et al. A novel therapeutic cytomegalovirus DNA vaccine in allogeneic haemopoietic stem-cell transplantation: a randomised, double-blind, placebo-controlled, Phase 2 trial. Lancet Infect. Dis. 12(4), 290–299 (2012).


•• A Phase II trial as proof of concept for TransVax for clinically significant viraemia in the allo-SCT setting. 112 Ljungman P. Would monitoring CMV

immune responses allow improved control of CMV in stem cell transplant patients. J. Clin. Virol. 35(4), 493–495 (2006). 113 Baldanti F, Lilleri D, Gerna G. Monitoring

human cytomegalovirus infection in transplant recipients. J. Clin. Virol. 41(3), 237–241 (2008). 114 Egli A, Humar A, Kumar D. State-of-the-art

monitoring of cytomegalovirus-specific cell-mediated immunity after organ transplant: a primer for the clinician. Clin. Infect. Dis. 55(12), 1678–1689 (2012). •• An up-to-date review discussing the clinical impact of measuring CMV-specific immune response on the management of CMV after organ transplantation. 115 Li Pira G, Kern F, Gratama J, Roederer M,

Manca F. Measurement of antigen specific immune responses: 2006 update. Cytometry B Clin. Cytom. 72(2), 77–85 (2007). 116 Lilleri D, Gerna G, Fornara C, Lozza L,

Maccario R, Locatelli F. Prospective simultaneous quantification of human cytomegalovirus-specific CD4 + and CD8 + T-cell reconstitution in young recipients of allogeneic hematopoietic stem cell transplants. Blood 108(4), 1406–1412 (2006). 117 Moins-Teisserenc H, Busson M, Scieux C

et al. Patterns of cytomegalovirus reactivation are associated with distinct evolutive profiles of immune reconstitution after allogeneic hematopoietic stem cell transplantation. J. Infect. Dis. 198(6), 818–826 (2008). 118 Tormo N, Solano C, Benet I et al. Kinetics of

cytomegalovirus (CMV) pp65 and IE-1specific IFNgamma CD8 + and CD4 + T cells during episodes of viral DNAemia in allogeneic stem cell transplant recipients: potential implications for the management of active CMV infection. J. Med. Virol. 82(7), 1208–1215 (2010). 119 Tormo N, Solano C, Benet I et al.

Reconstitution of CMV pp65 and IE-1specific IFN-gamma CD8(+) and CD4(+) T-cell responses affording protection from CMV DNAemia following allogeneic hematopoietic SCT. Bone Marrow Transplant. 46(11), 1437–1443 (2011). 120 Pourgheysari B, Piper KP, Mclarnon A et al.

Early reconstitution of effector memory CD4 + CMV-specific T cells protects against CMV reactivation following allogeneic SCT. Bone Marrow Transplant. 43(11), 853–861 (2009).


An update on the management of CMV infection following allogeneic hematopoietic stem cell transplantation 121 Foster AE, Gottlieb DJ, Sartor M,

129 Fleming T, Dunne J, Crowley B. Ex vivo

Hertzberg MS, Bradstock KF. Cytomegalovirus-specific CD4 + and CD8 + T-cells follow a similar reconstitution pattern after allogeneic stem cell transplantation. Biol. Blood Marrow Transplant. 8(9), 501–511 (2002). 122 Gallez-Hawkins G, Thao L, Lacey SF et al.

monitoring of human cytomegalovirusspecific CD8 (+) T-Cell responses using the QuantiFERON-CMV assay in allogeneic hematopoietic stem cell transplant recipients attending an Irish hospital. J. Med. Virol. 82(3), 433–440 (2010). Performance of the QuantiFERONcytomegalovirus (CMV) assay for detection and estimation of the magnitude and functionality of the CMV-specific gamma interferon-producing CD8 (+) T-cell response in allogeneic stem cell transplant recipients. Clin. Vaccine Immunol. 19(5), 791–796 (2012).

123 Lilleri D, Gerna G, Zelini P et al. Monitoring

of human cytomegalovirus and virus-specific T-cell response in young patients receiving allogeneic hematopoietic stem cell transplantation. PLoS ONE 7(7), e41648 (2012). 124 Ohnishi M, Sakurai T, Heike Y et al.

Evaluation of cytomegalovirus-specific T-cell reconstitution in patients after various allogeneic haematopoietic stem cell transplantation using interferon-gammaenzyme-linked immunospot and human leucocyte antigen tetramer assays with an immunodominant T-cell epitope. Br. J. Haematol. 131(4), 472–479 (2005). 125 Hebart H, Daginik S, Stevanovic S et al.

Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses by interferon-gamma-enzymelinked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation. Blood 99(10), 3830–3837 (2002). 126 Gimenez E, Solano C, Azanza JR, Amat P,

Navarro D. Monitoring of trough plasma ganciclovir levels and peripheral blood cytomegalovirus (CMV)-specific CD8 + T cells to predict CMV DNAemia clearance in preemptively treated allogeneic stem cell transplant recipients. Antimicrob. Agents Chemother. 58(9), 5602–5605 (2014). •

Demonstrates that measurement of plasma ganciclovir levels does not reliably predict response to therapy.

127 Borchers S, Bremm M, Lehrnbecher T et al.

Sequential anti-cytomegalovirus response monitoring may allow prediction of cytomegalovirus reactivation after allogeneic stem cell transplantation. PLoS ONE 7(12), e50248 (2012). 128 Walker S, Fazou C, Crough T et al. Ex vivo

monitoring of human cytomegalovirusspecific CD8 + T-cell responses using QuantiFERON-CMV. Transpl. Infect. Dis. 9(2), 165–170 (2007).


137 Ge S, Pao A, Vo A et al. Immunologic

parameters and viral infections in patients desensitized with intravenous immuno globulin and rituximab. Transpl. Immunol. 24(3), 142–148 (2011). 138 Husain S, Raza K, Pilewski JM et al.

Experience with immune monitoring in lung transplant recipients: correlation of low immune function with infection. Transplantation 87(12), 1852–1857 (2009).

130 Clari MA, Munoz-Cobo B, Solano C et al.

Cytomegalovirus immune reconstitution occurs in recipients of allogeneic hemato poietic cell transplants irrespective of detectable cytomegalovirus infection. Biol. Blood Marrow Transplant. 11(11), 890–902 (2005).

•

Compared QuantiFERON-CMV assay with flow cytometry intracellular cytokine staining (ICS) method for the detection of CMV-specific gamma interferon (IFN-γ)producing CD8 + T-cell responses in allo-SCT recipients.

139 Mizuno S, Nakatani K, Muraki Y et al.

Combination assays for evaluation of immune function and CYP3A5 genotype to identify the risk of infectious complications and mortality in living donor liver transplant patients. Ann. Transplant. 18, 349–357 (2013). 140 Nishikawa K, Mizuno S, Masui S et al.

Usefulness of monitoring cell-mediated immunity for predicting post-kidney transplantation viral infection. Transplant. Proc. 46(2), 552–555 (2014). 141 Zeevi A, Husain S, Spichty KJ et al. Recovery

of functional memory T cells in lung transplant recipients following induction therapy with alemtuzumab. Am. J. Transplant. 7(2), 471–475 (2007).

131 Tey SK, Kennedy GA, Cromer D et al.

Clinical assessment of anti-viral CD8+ T cell immune monitoring using QuantiFERONCMV(R) assay to identify high risk allogeneic hematopoietic stem cell transplant patients with CMV infection complications. PLoS ONE 8(10), e74744 (2013).

142 Avetisyan G, Aschan J, Hagglund H,

Ringden O, Ljungman P. Evaluation of intervention strategy based on CMV-specific immune responses after allogeneic SCT. Bone Marrow Transplant. 40(9), 865–869 (2007).

132 Borchers S, Bremm M, Lehrnbecher T et al.

Sequential anti-cytomegalovirus response monitoring may allow prediction of cytomegalovirus reactivation after allogeneic stem cell transplantation. PLoS ONE 7(12), e50248 (2012). 133 Gratama JW, Boeckh M, Nakamura R et al.

Immune monitoring with iTAg MHC Tetramers for prediction of recurrent or persistent cytomegalovirus infection or disease in allogeneic hematopoietic stem cell transplant recipients: a prospective multicenter study. Blood 116(10), 1655–1662 (2010). 134 Koehl U, Dirkwinkel E, Koenig M et al.

Reconstitution of cytomegalovirus specific T cells after pediatric allogeneic stem cell transplantation: results from a pilot study using a multi-allele CMV tetramer group. Klin. Padiatr. 220(6), 348–352 (2008). 135 Gratama JW, Cornelissen JJ. Clinical utility

of tetramer-based immune monitoring in allogeneic stem cell transplantation. BioDrugs 17(5), 325–338 (2003). 136 De Paolis P, Favaro A, Piola A et al.

“Immuknow” to measurement of cellmediated immunity in renal transplant recipients undergoing short-term evaluation. Transplant. Proc. 43(4), 1013–1016 (2011).

Review

•

A pioneer study proving the clinical value of CMV-specific T-cell immunity monitoring in the management of active CMV infection in the allo-SCT setting.

143 Solano C, Benet I, Remigia MJ et al.

Immunological monitoring for guidance of preemptive antiviral therapy for active cytomegalovirus infection in allogeneic stem-cell transplant recipients: a pilot experience. Transplantation 92(4), e17–e20 (2011). •

A proof of concept study showing the clinical value of monitoring CMV-specific T-cell responses.

144 Eid AJ, Razonable RR. New developments in

the management of cytomegalovirus infection after solid organ transplantation. Drugs 70(8), 965–981 (2010). 145 Emery VC, Griffiths PD. Prediction of

cytomegalovirus load and resistance patterns after antiviral chemotherapy. Proc. Natl Acad. Sci. USA 97(14), 8039–8044 (2000). •• Interesting paper describing mathematical models that provide a framework to predict the virologic course of patients at therapeutic initiation.


131

Review Pérez Romero, Blanco, Giménez, Solano & Navarro 146 Torres-Madriz G, Boucher HW.

156 Dropulic LK, Cohen JI. Update on new

Immunocompromised hosts: perspectives in the treatment and prophylaxis of cytomegalovirus disease in solid-organ transplant recipients. Clin. Infect. Dis. 47(5), 702–711 (2008). 147 Lurain NS, Chou S. Antiviral drug resistance

of human cytomegalovirus. Clin. Microbiol. Rev. 23(4), 689–712 (2010). 148 Strasfeld L, Chou S. Antiviral drug resistance:

mechanisms and clinical implications. Infect. Dis. Clin. North Am. 24(2), 413–437 (2010). 149 Drew WL, Miner RC, Marousek GI, Chou S.

Maribavir sensitivity of cytomegalovirus isolates resistant to ganciclovir, cidofovir or foscarnet. J. Clin. Virol. 37(2), 124–127 (2006). 150 Winston DJ, Young JA, Pullarkat V et al.

Maribavir prophylaxis for prevention of cytomegalovirus infection in allogeneic stem cell transplant recipients: a multicenter, randomized, double-blind, placebocontrolled, dose-ranging study. Blood 111(11), 5403–5410 (2008). 151 Marty FM, Ljungman P, Papanicolaou GA

et al. Maribavir prophylaxis for prevention of cytomegalovirus disease in recipients of allogeneic stem-cell transplants: a Phase 3, double-blind, placebo-controlled, randomised trial. Lancet Infect. Dis. 11(4), 284–292 (2011). •

Shows that maribavir prophylaxis does not prevent CMV disease when started after engraftment.

152 Winston DJ, Saliba F, Blumberg E et al.

Efficacy and safety of maribavir dosed at 100 mg orally twice daily for the prevention of cytomegalovirus disease in liver transplant recipients: a randomized, double-blind, multicenter controlled trial. Am. J. Transplant. 12(11), 3021–3030 (2012). 153 Webel R, Hakki M, Prichard MN,

154 Avery RK, Marty FM, Strasfeld L et al. Oral

maribavir for treatment of refractory or resistant cytomegalovirus infections in transplant recipients. Transpl. Infect. Dis. 12(6), 489–496 (2010). 155 Beadle JR, Hartline C, Aldern KA et al.

Alkoxyalkyl esters of cidofovir and cyclic cidofovir exhibit multiple-log enhancement of antiviral activity against cytomegalovirus and herpesvirus replication in vitro. Antimicrob. Agents Chemother. 46(8), 2381–2386 (2002).

132

157 Marty FM, Winston DJ, Rowley SD et al.

cyclopropavir and ganciclovir in human cytomegalovirus-infected cells. Antimicrob. Agents Chemother. 58(4), 2329–2333 (2014). 167 Gentry BG, Vollmer LE, Hall ED et al.

CMX001 to prevent cytomegalovirus disease in hematopoietic-cell transplantation. N. Engl. J. Med. 369(13), 1227–1236 (2013). •• Clinical trial with 230 patients demonstrating that treatment with oral CMX001 significantly reduces the incidence of CMV events in recipients of hematopoietic cell transplants.

Resistance of human cytomegalovirus to cyclopropavir maps to a base pair deletion in the open reading frame of UL97. Antimicrob. Agents Chemother. 57(9), 4343–4348 (2013). 168 Kern ER, Bidanset DJ, Hartline CB, Yan Z,

Zemlicka J, Quenelle DC. Oral activity of a methylenecyclopropane analog, cyclopropavir, in animal models for cytomegalovirus infections. Antimicrob. Agents Chemother. 48(12), 4745–4753 (2004).

158 He R, Park K, Cai H et al. Artemisinin-

derived dimer diphenyl phosphate is an irreversible inhibitor of human cyto megalovirus replication. Antimicrob. Agents Chemother. 56(7), 3508–3515 (2012).

169 Chou S, Bowlin TL. Cytomegalovirus UL97

mutations affecting cyclopropavir and ganciclovir susceptibility. Antimicrob. Agents Chemother. 55(1), 382–384 (2011).

159 Lau PK, Woods ML, Ratanjee SK, John GT.

Artesunate is ineffective in controlling valganciclovir-resistant cytomegalovirus infection. Clin. Infect. Dis. 52(2), 279 (2011).

170 Waldman WJ, Knight DA, Lurain NS et al.

Novel mechanism of inhibition of cyto megalovirus by the experimental immuno suppressive agent leflunomide. Transplantation 68(6), 814–825 (1999).

160 Shapira MY, Resnick IB, Chou S et al.

Artesunate as a potent antiviral agent in a patient with late drug-resistant cyto megalovirus infection after hematopoietic stem cell transplantation. Clin. Infect. Dis. 46(9), 1455–1457 (2008).

171 John GT, Manivannan J, Chandy S, Peter S,

Jacob CK. Leflunomide therapy for cytomegalovirus disease in renal allograft recepients. Transplantation 77(9), 1460–1461 (2004).

161 Wolf DG, Shimoni A, Resnick IB et al.

Human cytomegalovirus kinetics following institution of artesunate after hematopoietic stem cell transplantation. Antiviral. Res. 90(3), 183–186 (2011).

172 Avery RK, Bolwell BJ, Yen-Lieberman B et al.

Use of leflunomide in an allogeneic bone marrow transplant recipient with refractory cytomegalovirus infection. Bone Marrow Transplant. 34(12), 1071–1075 (2004).

162 Goldner T, Hewlett G, Ettischer N,

Ruebsamen-Schaeff H, Zimmermann H, Lischka P. The novel anticytomegalovirus compound AIC246 (Letermovir) inhibits human cytomegalovirus replication through a specific antiviral mechanism that involves the viral terminase. J. Virol. 85(20), 10884–10893 (2011). 163 Lischka P, Hewlett G, Wunberg T et al.

Rawlinson WD, Marschall M, Chou S. Differential properties of cytomegalovirus pUL97 kinase isoforms affect viral replication and maribavir susceptibility. J. Virol. 88(9), 4776–4785 (2014).

166 Gentry BG, Drach JC. Metabolism of

antivirals under development for the treatment of double-stranded DNA virus infections. Clin. Pharmacol. Ther. 88(5), 610–619 (2010).

173 Battiwalla M, Paplham P, Almyroudis NG

et al. Leflunomide failure to control recurrent cytomegalovirus infection in the setting of renal failure after allogeneic stem cell transplantation. Transpl. Infect. Dis. 9(1), 28–32 (2007). 174 Verkaik NJ, Hoek RA, Van Bergeijk H et al.

Leflunomide as part of the treatment for multidrug-resistant cytomegalovirus disease after lung transplantation: case report and review of the literature. Transpl. Infect. Dis. 15(6), E243–249 (2013).

In vitro and in vivo activities of the novel anticytomegalovirus compound AIC246. Antimicrob. Agents Chemother. 54(3), 1290–1297 (2010). 164 Verghese PS, Schleiss MR. Letermovir

treatment of human cytomegalovirus infection antiinfective agent. Drugs Future 38(5), 291–298 (2013). 165 Chemaly RF, Ullmann AJ, Stoelben S et al.

Letermovir for cytomegalovirus prophylaxis in hematopoietic-cell transplantation. N. Engl. J. Med. 370(19), 1781–1789 (2014). •• A recent Phase II clinical trial with 131 allo-SCT demostrating that letermovir was effective in reducing the incidence of CMV infection.


175 Stevenson M. Therapeutic potential of RNA

interference. N. Engl. J. Med. 351(17), 1772–1777 (2004). •

An interesting article reviewing the use of RNA interference as an attractive therapeutic approach.

176 Bai Z, Li L, Wang B et al. Inhibition of

human cytomegalovirus infection by IE86-specific short hairpin RNA-mediated RNA interference. Biosci. Biotechnol. Biochem. 74(7), 1368–1372 (2010).


An update on the management of CMV infection following allogeneic hematopoietic stem cell transplantation 177 Dittmer A, Bogner E. Specific short hairpin

188 Cobbold M, Khan N, Pourgheysari B et al.

RNA-mediated inhibition of viral DNA packaging of human cytomegalovirus. FEBS Lett. 580(26), 6132–6138 (2006). 178 Duan Q J, Tao R, Hu MF, Shang SQ.

Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J. Exp. Med. 202(3), 379–386 (2005). 189 Feuchtinger T, Opherk K, Bethge WA et al.

Efficient inhibition of human cyto megalovirus UL122 gene expression in cell by small interfering RNAs. J. Basic. Microbiol. 49(6), 531–537 (2009).

Adoptive transfer of pp65-specific T cells for the treatment of chemorefractory cyto megalovirus disease or reactivation after haploidentical and matched unrelated stem cell transplantation. Blood 116(20), 4360–4367 (2010).

179 Reich SJ, Fosnot J, Kuroki A et al. Small

interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol. Vis. 9, 210–216 (2003).

190 Stemberger C, Graef P, Odendahl M et al.

Lowest numbers of primary CD8(+) T cells can reconstitute protective immunity upon adoptive immunotherapy. Blood 124(4), 628–637 (2014).

180 De Fougerolles A, Vornlocher HP,

Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 6(6), 443–453 (2007).

191 Preventative/preemptive adoptive transfer of

peptide stimulated CMV/EBV specific T-cells in patients after allogeneic stem cell transplantation. https://clinicaltrials.gov/ct2/show

181 MK-8228 (Letermovir) versus placebo in the

prevention of clinically-significant cyto megalovirus (CMV) infection in adult, CMV-seropositive allogeneic hematopoietic stem cell transplant recipients (MK-8228-001). https://clinicaltrials.gov/ct2/show 182 Anderson KP, Fox MC, Brown-Driver V,

Martin MJ, Azad RF. Inhibition of human cytomegalovirus immediate-early gene expression by an antisense oligonucleotide complementary to immediate-early RNA. Antimicrob. Agents Chemother. 40(9), 2004–2011 (1996).

192 T-lymphocyte infusion or standard therapy in

treating patients at risk of Cytomegalovirus infection after a donor stem cell transplant. https://clinicaltrials.gov/ct2/show 193 Stratton K. Vaccines for the 21st Century:

a Tool for Decision Making. National Academy Press, Washington, DC, USA, (2000). •• An interesting document reviewing the development of vaccines for the 21st century. 194 Schleiss MR, Heineman TC. Progress toward

an elusive goal: current status of cyto megalovirus vaccines. Expert. Rev. Vaccines 4(3), 381–406 (2005).

183 Mulamba GB, Hu A, Azad RF, Anderson KP,

Coen DM. Human cytomegalovirus mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen (ISIS 2922). Antimicrob. Agents Chemother. 42(4), 971–973 (1998). 184 Devi GR. siRNA-based approaches in cancer

195 Adler SP. Human CMV vaccine trials: what if

CMV caused a rash? J. Clin. Virol. 41(3), 231–236 (2008). 196 Rieder F, Steininger C. Cytomegalovirus

vaccine: Phase II clinical trial results. Clin. Microbiol. Infect. 20(Suppl. 5), 95–102 (2013).

therapy. Cancer Gene Ther. 13(9), 819–829 (2006). 185 Hamilton ST, Milbradt J, Marschall M,

Rawlinson WD. Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122/123 gene transcripts. PLoS ONE 9(6), e97231 (2014). 186 Peggs KS, Thomson K, Samuel E et al.

Directly selected cytomegalovirus-reactive donor T cells confer rapid and safe systemic reconstitution of virus-specific immunity following stem cell transplantation. Clin. Infect. Dis. 52(1), 49–57 (2011). 187 Blyth E, Clancy L, Simms R et al. Donor-

derived CMV-specific T cells reduce the requirement for CMV-directed pharmaco therapy after allogeneic stem cell transplantation. Blood 121(18), 3745–3758 (2013).


•

A comprehensve review of CMV vaccine prototypes.

197 A study to evaluate safety and tolerability of

a therapeutic vaccine, ASP0113, in subjects undergoing allogeneic hematopoietic cell transplant. www.clinicaltrial.gov/ct2/show 198 Accelerated immunization to induce

Cytomegalovirus immunity in stem cell donors. https://clinicaltrials.gov/ct2/show 199 Elek SD, Stern H. Development of a vaccine

against mental retardation caused by cytomegalovirus infection in utero. Lancet 1(7845), 1–5 (1974).

Review

200 Plotkin SA, Farquhar J, Horberger E. Clinical

trials of immunization with the Towne 125 strain of human cytomegalovirus. J. Infect. Dis. 134(5), 470–475 (1976). 201 Heineman TC, Schleiss M, Bernstein DI et al.

A Phase 1 study of 4 live, recombinant human cytomegalovirus Towne/Toledo chimeric vaccines. J. Infect. Dis. 193(10), 1350–1360 (2006). 202 Mitchell DK, Holmes SJ, Burke RL,

Duliege AM, Adler SP. Immunogenicity of a recombinant human cytomegalovirus gB vaccine in seronegative toddlers. Pediatr. Infect. Dis. J. 21(2), 133–138 (2002). 203 Spaete RR, Saxena A, Scott PI et al. Sequence

requirements for proteolytic processing of glycoprotein B of human cytomegalovirus strain Towne. J. Virol. 64(6), 2922–2931 (1990). 204 Sabbaj S, Pass RF, Goepfert PA, Pichon S.

Glycoprotein B vaccine is capable of boosting both antibody and CD4 T-cell responses to cytomegalovirus in chronically infected women. J. Infect. Dis. 203(11), 1534–1541 (2011). 205 Pass RF, Zhang C, Evans A et al. Vaccine

prevention of maternal cytomegalovirus infection. N. Engl. J. Med. 360(12), 1191–1199 (2009). •• This Phase II clinical trial demonstrate in 234 subjects receiving ramdomly either placebo or CMV glycoprotein B vaccine demonstrate the potential of the vaccine to decrease incident cases of maternal and congenital CMV infection. 206 Griffiths PD, Stanton A, Mccarrell E et al.

Cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant in transplant recipients: a Phase 2 randomised placebocontrolled trial. Lancet 377(9773), 1256–1263 (2011). •• A Phase II clinical trial using glycoprotein-B vaccine with MF59 adjuvant demonstrating that humoral immunity has a role in reduction of cytomegalovirus viraemia. 207 Selinsky C, Luke C, Wloch M et al.

A DNA-based vaccine for the prevention of human cytomegalovirus-associated diseases. Hum. Vaccin. 1(1), 16–23 (2005). 208 Wloch MK, Smith LR, Boutsaboualoy S et al.

Safety and immunogenicity of a bivalent cytomegalovirus DNA vaccine in healthy adult subjects. J. Infect. Dis. 197(12), 1634–1642 (2008). 209 Liu MA, Ulmer JB. Human clinical trials of

plasmid DNA vaccines. Adv. Genet. 55, 25–40 (2005).


133

Review Pérez Romero, Blanco, Giménez, Solano & Navarro 210 La Rosa C, Longmate J, Lacey SF et al. Clinical

evaluation of safety and immunogenicity of PADRE-cytomegalovirus (CMV) and tetanus-CMV fusion peptide vaccines with or without PF03512676 adjuvant. J. Infect. Dis. 205(8), 1294–1304 (2012).

134

211 Reap EA, Morris J, Dryga SA et al.

Development and preclinical evaluation of an alphavirus replicon particle vaccine for cytomegalovirus. Vaccine 25(42), 7441–7449 (2007).


212 Bernstein DI, Reap EA, Katen K et al.

Randomized, double-blind, Phase 1 trial of an alphavirus replicon vaccine for cytomegalovirus in CMV seronegative adult volunteers. Vaccine 28(2), 484–493 (2009).
