Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments

Reviews Human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments Maher K Gandhi and Rajiv Khanna After initial infection, human A B cytomegalovirus remains in a persistent state with the host. Immunity against the virus controls Membrane replication, although intermitent viral Nucleocapsid shedding can still take place in Tegument the seropositive immunocompetent gB person. Replication of cytomegaloGenome virus in the absence of an effective immune response is central to the gH pathogenesis of disease. Therefore, complications are primarily seen in individuals whose immune system is –8 immature, or is suppressed by drug Figure 1. Human cytomegalovirus. (A) Shaded surface representation of 18 x 10 m resolution threedimensional reconstruction of icosahedrally ordered portion of intact human cytomegalovirus treatment or coinfection with other particle as viewed along a three-fold symmetry axis (adapted with permission from reference 2). (B) pathogens. Although our increasing Virtual three-dimensional model, showing various components of the cytomegalovirus (reproduced knowledge of the host-virus relation- with permission from Marko Reschke, http://www.biografix.de/). ship has lead to the development of new pharmacological strategies for cytomegalovirus- myeloid lineage constitute an important reservoir. associated infections, these strategies all have limitations— Productive (lytic) infection results in a co-ordinated eg, drug toxicities, development of resistance, poor oral sequence of events that leads to the synthesis of immediatebioavailability, and low potency. Immune-based therapies to early, early, and late viral proteins.5 Presence of the virus in a complement pharmacological strategies for the successful subset of CD34+ myeloid progenitor cells in bone marrow treatment of virus-associated complications should be has been established, with a small proportion of these cells prospectively investigated. containing cytomegalovirus genomic DNA without Lancet Infect Dis 2004; 4: 725–38

Human cytomegalovirus is a
human herpesvirus— HHV5—characterised by its restricted host range, production of nuclear as well as cytoplasmic inclusions, and its long life cycle. It is the largest known human herpesvirus, with a genome of about 230 kb.1 The virus has doublestranded linear DNA enveloped by a proteinaceous matrix (the tegument), which is surrounded by a lipid bilayer that contains viral glycoproteins (figure 1).2 Human cytomegalovirus can be transmitted via saliva, sexual contact, placental transfer, breastfeeding, blood transfusion, or solid-organ transplantation or haematopoietic stem-cell transplantation. Acquisition of the virus arises progressively from an early age, and in developed countries the overall seroprevalence is 30–70%.3 Homosexual men, poor socioeconomic groups, and residents of developing countries, however, have seroprevalence rates that can exceed 90%.4

Biology After primary infection, cytomegalovirus establishes lifelong latency or persistence within the person, in which cells of the

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detectable viral immediate-early gene expression, termed latent infection.5 In healthy carriers, viral DNA is also present in a small proportion of CD14+ monocytes and in dendritic cells and megakaryocytes.6–8 Permissiveness for cytomegalovirus replication is cell-type specific—ie, the virus can enter a cell, but because of transcriptional repression of the major immediate-early promoter there is no production of new virions. The permissiveness of myeloid cells for viral replication is related to their state of differentiation; monocytes are non-permissive, whereas differentiated macrophages and immature dendritic cells are permissive for productive infection.9,10

Congenital and neonatal infection One of the most important clinical manifestations of primary human cytomegalovirus infection is seen in MKG is Senior Research Officer and RK is Head of the Tumour Immunology Laboratory at the Queensland Institute of Medical Research, Brisbane, Australia. Correspondence: Dr Rajiv Khanna, Tumour Immunology Laboratory, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Queensland 4006, Australia. Tel +61 733620385; fax +61 7 38453510; email [email protected]

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Review newborn babies infected during pregnancy.11 In a seronegative mother, the risk of primary infection is 0·7–4·1%, which carries a 40% risk of congenital infection.12 The risk to the infant is greatest if infection occurs in the first trimester, since maternal cytomegalovirus antibodies have a protective role against intrauterine transmission (but not against development of symptoms once infection has arisen).13,14 Congenital cytomegalovirus-associated disease is difficult to identify and is frequently not recognised. Results of studies on infants with asymptomatic infection, however, indicate that 10–17% develop hearing defects or neurodevelopmental sequelae. More importantly, 5–10% of congenitally infected neonates have symptoms of irreversible CNS involvement in the form of microcephaly, encephalitis, seizures, deafness (a solitary finding in 10% of cases), upper motor neuron disorders, psychomotor retardation, and, rarely, myopathy and choroidoretinitis.15 Microcephaly alone does not confer as poor an outlook as hard neurological signs. These newborn babies also display other clinical features, including intrauterine growth retardation, jaundice, hepatosplenomegaly, thrombocytopenia, petechiae, and hepatitis, which tend to be self-limiting and resolve without treatment. Findings of long-term follow-up studies indicate that most affected infants (up to 80%) display serious life-long neurological abnormalities with severe life-threatening organ dysfunction and death in 10–20% of patients, not to mention the vast socioeconomic implications of life-long illness. A review panel from the Institute of Medicine concluded that a vaccine against human cytomegalovirus was secondary in need only to a vaccine for HIV.16 Cytomegalovirus infection can also be acquired perinatally, which is generally asymptomatic. However, up to 30% of perinatally infected infants display short-term, self-limiting symptoms of hepatosplenomegaly, lymphadenopathy, hepatitis, or pneumonia; neurological sequelae or hearing impairment does not typically arise with this mode of infection. Other than counselling, there is no specific treatment for mothers who acquire infection during pregnancy, or for neonates with congenital or perinatal infection. Supportive treatment and follow-up of infants for detection of potential sensorineural deafness is essential.

Infection of immunocompetent adults Primary cytomegalovirus infection in the immunocompetent host rarely causes serious illness.17 Uncommonly, it can result in a mononucleosis syndrome, which is indistinguishable from primary Epstein-Barr virus (EBV) infection, with persistent fever (generally for 2–3 weeks), myalgia, and cervical adenopathy. Unlike EBV-associated infectious mononucleosis, cytomegalovirus rarely causes tonsillopharyngitis or great splenomegaly. In developed countries, delayed exposure is causing an increasing incidence of infections in middle-aged adults (such as the seronegative child carer). Less frequent complications of primary infections include arthralgia and arthritis, ulcerative colitis, pneumonitis, hepatitis, aseptic meningitis, and myocarditis. 5–10% of patients with Guillain-Barré syndrome have serological evidence of primary infection, and antibodies to the ganglioside GM2 that are cross-

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reactive with cytomegalovirus-infected fibroblasts have been identified.18 Cytomegalovirus has also been indirectly implicated as a contributor to atherosclerosis; previous infection with the virus has been suggested as a strong independent risk factor for smooth-muscle proliferation, leading to restenosis after coronary angioplasty.19 However, subsequent reports20 have not confirmed this association.

Infection of immunocompromised patients Initial infection with cytomegalovirus induces a primary immune response and subsequent establishment of longterm immunity, which restrains viral replication after reactivation from latency. Long-term immunosuppression can lead to uncontrolled replication and serious disease.21,22 Solid-organ transplantation

Cytomegalovirus replication can be detected in healthy cytomegalovirus-seropositive individuals affected by surgery-related stress, sepsis, and catecholamine release.23,24 Such conditions are typical soon after allogeneic solid-organ transplantation and, in the context of immunosuppression to prevent organ-rejection, result in a heightened risk of cytomegalovirus replication and ensuing disease. Despite improved treatment and surveillance, cytomegalovirus continues to be a great cause of morbidity. Furthermore, virus-associated disease and virus-associated post-transplant complications remain an important economic drain on individual transplantation programmes.25 Owing to the absence of a host-derived cytomegalovirusspecific immune response, the serologically negative patient in receipt of an organ from a serologically positive donor (R–/D+) is at greatest risk for cytomegalovirus-associated disease.26 Although matching of seronegative recipients to seronegative donors is best, in practice limited organ availability means R–/D– transplants are not generally possible. At time of cytomegalovirus disease onset, the viral load is higher in R–/D+ recipients than in other R/D combinations.27 Additional risk factors include use of T-cellspecific antibodies,28 and recipient seroconversion to HHV6.29 Although definitive proof is lacking, the degree of viral load within the transplanted organ is probably proportional to risk of subsequent disease. show that Results of histological studies30 cytomegalovirus-associated disease initially localises in the transplanted organ—for example, hepatitis arises generally in liver-transplant recipients and pancreatitis in pancreastransplant recipients—but can subsequently spread systemically, causing pneumonitis, enteritis, hepatitis, and (less frequently) retinitis and CNS involvement. Findings of studies31–36 investigating cytomegalovirus in graft rejection due to renal artery stenosis (kidney transplants), coronary artery stenosis (heart transplants), bronchiolitis obliterans (lung transplants), and vanishing bile duct syndrome (liver transplants) are conflicting, and the role of the virus remains to be established. Haematopoietic stem-cell transplantation

If a cytomegalovirus-seronegative donor is identified, the risk for cytomegalovirus transmission in stem-cell

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transplantation is mainly through blood products. Two comparable options exist for reducing this risk of transmission—use of blood products from seronegative donors, or use of leucocyte-depleted blood products.37 Cytomegalovirus infections observed in patients after allogeneic stem-cell transplants are due to reactivation of latent virus present in the seropositive recipient.21,22 Among (R–/D+) allogeneic stem-cell transplant recipients who receive only seronegative blood products, the incidence of cytomegalovirus infection is 17%.38 Pneumonitis is the most serious manifestation of cytomegalovirus infection after allogeneic stem-cell transplants, with a mortality of 60–80% without ganciclovir and 50% when treated with antiviral chemotherapy plus cytomegalovirus hyperimmunoglobulin.21,39 Cytomegalovirus-associated pneumonitis is characterised by increasing hypoxia and progression to respiratory failure, and seems to derive from a combination of direct cytopathic effects of viral replication and hostdependent immunopathological mechanisms. Initial reports were of cytomegalovirus-associated disease within 3 months after allogeneic stem-cell transplant. However, in two studies40,41 late-onset cytomegalovirus-associated disease was linked with chronic graft-versus-host disease (GvHD) and long-term ganciclovir administration, suggesting that recovery of the cytomegalovirus-specific T-cell responses necessary for protective immunity can be delayed in these patients. Clinical manifestations of infection that had only rarely been observed early post-transplant—eg, retinitis— have been described in patients who develop late-onset cytomegalovirus-associated disease.41–43 Risk factors for disease include seropositivity of the recipient, GvHD, and T-cell depletion of the allograft.44 Reports suggest that cytomegalovirus seropositivity of either donor or recipient could be associated with an increased risk of immunemediated complications such as GvHD.45 Conversely, donor seropositivity may reduce leukaemia relapse owing to an enhanced graft versus leukaemia effect.46,47 In HLA-A2 recipients, donor-derived cytomegalovirus-specific T cells can cross-react with HLA-A2-restricted recipient minor histocompatibility antigens,43 raising the possibility that donor cytomegalovirus-specific T-cells might be alloreactive and directly participating in the GvHD, or that the inflammatory cytokines evoked by severe GvHD contributed to its maintenance, or both.43 The increasing trend towards new methods of allogeneic stem-cell transplantation (including non-myeloablative transplants that need intense immunosuppressive regimens, such as fludarabine, antithymocyte immunoglobulin, or Campath-1H,48,49 or the use of T-cell depleted stem cells) could increase the risk of cytomegalovirus-associated disease. HIV infection

The range of disease in patients with AIDS is strikingly different, with retinitis (characterised by haemorrhagic retinal necrosis) the most frequent manifestation. Before the widespread availability of highly active antiretroviral therapy (HAART) in developed countries, retinitis was seen in up to 25% of patients.50 More recently, immune recovery vitritis associated with posterior segment inflammation has been

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observed, since the CD4+ T-cell count recovers after initiation of HAART.50,51 The inflammation resolves with steroid therapy. Other manifestations of cytomegalovirusassociated disease include encephalopathy and peripheral polyradiculopathy due to replication of the virus in the CNS. Pneumonitis is rarely observed. In patients with HIV, cytomegalovirus can exert indirect effects that manifest as acceleration of the time to AIDS and time to death. Whether infection and disease are simply markers of the immune dysfunction that follows HIV replication or whether cytomegalovirus infection itself promotes HIV progression, is unknown. Reduction of HIV viral load after the introduction of HAART has greatly reduced the cytomegalovirus load and disease.52,53 Diagnosis

The most widely used assays for cytomegalovirus in the immunocompromised patient test for viraemia, antigenaemia, DNA, and mRNA. There is no single best assay method for monitoring infection, but molecular assays are fast and automated, promise earlier treatment, and have reliable results. Panel 154–70 summarises the different diagnostic strategies and panel 271–73 outlines ways to monitor Panel 1. Diagnosis of cytomegalovirus infection and disease History/examination Presence of cytomegalovirus in tissue might simply be a bystander effect and does not necessarily imply causality. Cytomegalovirus-associated disease should be diagnosed on clinical grounds in combination with detection of virus. Viral cultures Requires at least 21 days to be reported negative. Not routinely done, but can be useful in resistance testing. Detection of early antigen fluorescent foci 24 h turn-around. Infected fibroblasts stained with fluorescent antibody specific for the antigen MIE p72.54 Insufficiently sensitive to be routinely recommended after allogeneic stem-cell transplantation.55,56 Antigenaemia assay Quantitates leucocytes positive for pp65. Since uninfected cells can harbour cytomegalovirus protein, provides an indirect assessment of infection.57 Reliable, rapid, and sensitive. In common use.58 Qualitiative PCR Can be used on whole blood, leucocytes, and plasma.59 Rapid and sensitive, easily automated. Threshold needs careful calibrating to over detection of cytomegalovirus infection. Quantitative PCR Allows response to treatment to be monitored. Useful as a surrogate marker of clinical or viral resistance. Correlation between high degrees of viral DNA and presence of clinical symptoms in transplant recipients and patients with AIDS and the congenital infection of pregnancy has been shown.60–65 Hybrid capture assay RNA probes used to detect viral DNA in an ELISA-type format. Can be used on whole blood stored for up to 48 h. Limited experience but results to date promising.66,67 Nucleic assay sequence-based amplification Allows the specific nucleic assay sequence-based amplification of unspliced viral mRNAs in a background of DNA. Experience in transplant and AIDS patients encouraging.68–70

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Review cytomegalovirus-specific immunity. The diagnosis of invasive disease is not straightforward, since the presence of cytomegalovirus in a diseased tissue does not necessarily imply a causal relation. Diagnosis should be based on appropriate clinical signs and symptoms of disease in conjunction with detection of virus from the appropriate involved tissue (with the exception of retinitis). For the purpose of consistent reporting of cytomegalovirus in clinical trials, definitions of infection and disease have been developed.74 Pharmacological strategies

Aciclovir and valaciclovir (inhibitors of thymidine kinase, which intriguingly is not expressed in cytomegalovirus) have only modest effect in vitro on replication. However, results of large studies of prophylaxis show a reduction of cytomegalovirus episodes and, for aciclovir, an improvement in survival.75 In these studies, the frequency of cytomegalovirus-associated disease was not reduced and, therefore, if either drug is to be used as prophylaxis after stem-cell therapy, a strategy of pre-emptive therapy should also be given. By contrast, valaciclovir does provide effective prophylaxis in renal transplant patients and is associated with a reduced rate of graft rejection.76 The table40,58,77–90 outlines drugs used in the prophylaxis, pre-emptive therapy, and treatment of cytomegalovirus infection. Pre-emptive therapy is the administration of chemotherapy against a virus at a time of particularly high risk for disease but before symptomatic disease. Ganciclovir is both myelosuppressive and might induce viral resistance, and the purpose of a pre-emptive strategy for cytomegalovirus is to restrict ganciclovir exposure to those patients who have detectable cytomegalovirus reactivation. The two risk factors that are most important in identifying the population for pre-emptive treatment are presence of cytomegalovirus-associated viraemia and presence of GvHD. Treatment is often started at the time of the first positive cytomegalovirus monitoring test,55,58 but treatment Panel 2. Monitoring of cytomegalovirus-specific immunity Serology Highly specific and sensitive in immunocompetent individuals. IgM titres rise 2–6 weeks after infection and can persist for 2 years and be detectable during episodes of reactivation. IgG seropositivity is usually lifelong although antibody concentrations can decline with age. Unreliable and not used in monitoring of cytomegalovirus reactivation in immunocompromised patients. IgG antibody avidity assays (low avidity suggests infection within 3 months) can be useful in identifying pregnant women at risk of transmitting intrauterine infection.71 Tetramer assays Remains a research method to detect peptide-specific CD8+ T-cells. Data in allogeneic stem-cell transplant setting suggest a protective threshold below which patients at high risk of reactivation can be identified.72,73 Cytokine assays Research method to enable high throughput detection of virus-specific T-cells. Being assessed in transplant patients. As with tetramer assays, when combined with sensitive, rapid viral detection techniques, may be of use in monitoring post-transplant patients and to assist decisionmaking in pre-emptive or prophylaxis strategies.

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at first steroid use for GvHD has also been shown to be effective.91 In general, pre-emptive ganciclovir use requires that patients be monitored once or twice weekly during the period of maximum risk (until day +100 after allogeneic stem-cell transplantation). Longer monitoring is recommended in patients with GvHD, unrelated or HLA haploidentical allogeneic stem-cell transplant, and in patients with previous cytomegalovirus reactivation. There continues to be much debate over the relative merits of preemptive versus prophylaxis therapy,92,93 with studies in solidorgan transplant recipients providing conflicting results as to which is better for the prevention of cytomegalovirusassociated disease.76,94

Immune regulation Humoral immunity

The virion envelope carries a range of glycoproteins with glycoprotein B recognised as the predominant target for neutralising antibodies. The importance of the cytomegalovirus-specific humoral response remains unclear. Clinical studies indicate that the humoral response plays a part in preventing the blood-borne spread of the virus. Indeed, results of studies in animals show that previous immunisation with glycoprotein B induces a neutralising antibody response, which protects these animals from a lethal challenge,95 and immunisation of pregnant guineapigs against guineapig cytomegalovirus glycoprotein B protects unborn babies from infection.96 This notion is supported by studies in people, the results of which show that the probability of transmission of infection from mother to fetus greatly increases if the antibody response to cytomegalovirus is of low avidity and poor neutralising activity.80 The importance of the humoral response could also be supported by the evidence that primary infection is often more frequent and severe in seronegative rather than seropositive solid-organ transplant recipients. Indeed, previous immunisation with attenuated cytomegalovirus Towne strain or passive transfer of high-titre immunoglobulins against cytomegalovirus reduces the severity of disease in transplant patients.97 Furthermore, after allogeneic stem-cell transplantation, the generation of high titres of glycoprotein-specific neutralising antibodies in response to viral replication seems to be associated with improved survival.98 Innate immunity: natural killer cells

Natural killer (NK) cells play a part in clearance of experimental murine cytomegalovirus infection.99 Neonatal mice are rendered susceptible to lethal infection by ablation of their NK cells. Certain adult strains of mice are resistant to murine cytomegalovirus infection in vivo, but become susceptible when NK cells are depleted—their resistance maps to a single autosomal dominant locus, termed Cmv1, contained in the NK gene complex (NKC) on mouse chromosome 6. Results of research suggest this resistance is mediated by the murine NK-cell activation receptor Ly49H.100 In people there are few data. One case-report101 described a patient prone to recurring severe herpesvirus infections (including an episode of cytomegalovirus-

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Drugs used in prophylaxis, pre-emptive therapy, and treatment of cytomegalovirus infection Drug Intravenous immunoglobulin

Mechanism of action Provides passive humoral immunity against cytomegalovirus

Intravenous ganciclovir

Nucleoside analogue monophosphorylated in infected cells by gene product of UL97, and is a competitive inhibitor of DNA polymerase UL54. Mutations in UL97 gene can confer resistance.

Oral ganciclovir

As above

Intraocular ganciclovir

As above

Oral valganciclovir Intravenous foscarnet

Ganciclovir prodrug; mechanism as above Pyrophosphate analogue. Inhibits HCMV replication

Intravenous cidofovir

Once-weekly acyclic nucleoside analogue. Inhibits HCMV replication

Intraocular fomiversen

Antisense oligonucleotide inhibits translation of the cytomegalovirus major immediate early proteins Benximidazole A promising new class of compound with excellent in-vitro data and good bioavailability.90 ribonucleosides. Inhibits HCMV DNA synthesis and nuclear egress. Mutations in UL27may confer resistance.

Maribavir

Comments Results of randomised trials of prophylaxis in allogeneic stem-cell transplant recipients inconsistent. Results of a randomised study of prophylaxis in renal-transplant patients showed a reduction in cytomegalovirus-related syndromes in immunoglobulin recipients.77 Combination of ganciclovir plus immunoglobulin for cytomegalovirus-associated pneumonitis is gold standard, but no randomised trial has been done. Results of uncontrolled studies conflicting.78,79 Results of 2 randomised trials of prophylaxis after allogeneic stem-cell transplantation40,80 showed reduced incidence of viraemia, and incidence of cytomegalovirus-associated disease was reduced in one but no improvement in mortality. Studies done before widespread use of granulocyte colony stimulating factor (G-CSF). Associated with delayed immune reconstitution and late cytomegalovirus-associated disease. After stem-cell transplantation, findings of a randomised study of pre-emptive therapy versus non-selective ganciclovir prophylaxis showed prophylaxis was more effective in preventing early disease; but pre-emption reduced risks of late disease, neutropenia, and fungal disease, equalising the risk for cytomegalovirus-associated disease and survival.58 Resistance reported in AIDS and solid-organtransplant patients with lung transplant recipients being disproportionately affected.81 Recommended as retinitis therapy, where higher maintenance doses might be needed for retinopathy inproximity to the macular. In AIDS patients continued prophylaxis should be maintained until CD4+ T-cell increase achieved. For cytomegalovirus reactivation in blood, ganciclovir administered at 5 mg/kg twice daily for at least 2 weeks. If neutropenia is a problem, G-CSF can be administered or foscarnet substituted. Bioavailability about 5%. Results of small prophylactic study in 21 allogeneic stem-cell transplant recipients show poor tolerability and high rate of cytomegalovirus infection.82 Might have role in maintenance therapy. Implantable intraocular devices, providing sustained release of ganciclovir, have been used.83 Delay retinitis progression (versus intravenous ganciclovir), but association with retinal detachment and endopthalmitis. Has better bioavailability than ganciclovir and dependent on ongoing assessement is likely to become established as prophylaxis of choice in transplant setting. Inhibits cytomegalovirus replication and its major side-effect, renal toxicity and electrolyte imbalances, can be reduced with careful monitoring and hydration.84,85 Results of randomised study in allogeneic stemcell-transplant recipients and liver and renal transplant patients of combined foscarnet and ganciclovir (both at half dose) versus full-dose ganciclovir monotherapy did not support a synergistic effect, with increased toxicity seen with two-drug therapy.86 Findings of small retrospective survey show that cidofovir could salvage allogeneic stem-cell transplant patients with cytomegalovirus-associated pneumonitis, failing primary therapy.87 Only studied in uncontrolled trials and is nephrotoxic (should be co-administered with probenecid to prevent renal tubule damage). Should be reserved for second-line treatment. A potent and specific agent for first-line and second-line treatment of cytomegalovirus-associated retinitis in AIDS patients.88,89

associated pneumonitis) who was specifically deficient in NK cells. In a study of 43 patients102 with cytomegalovirus reactivation after allogeneic stem-cell transplantation in which 12 cases were fatal, the capacity to develop nonspecific NK-cell cytotoxicity correlated with the patient’s ability to recover from infection.

rapidly and disease is rare,103 but cases have been reported in T-cell depleted autografts. By contrast, after allogeneic transplantation, recipients who lack cytomegalovirusspecific CD4+ and CD8+ T cells are at an increased risk of developing disease.41 Role of CD8+ T cells

Adaptive immunity: cell-mediated immune responses

Cytomegalovirus-specific T-cell responses are important in the control of virus replication, and hence in protection against disease. In seropositive recipients who undergo either autologous or allogeneic stem-cell transplantation, the incidence of cytomegalovirus reactivation is similar, but the incidence of virus-associated disease is different. After unmanipulated autologous stem-cell transplantation, cytomegalovirus-specific T-cell responses reconstitute fairly

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An analysis of virus-specific T-cell responses during primary infection suggests that cytomegalovirus-specific CD8+ T cells play an important part in limiting virus-infected cells in vivo.104 The presence of
interferon-producing CD8+ T cells in patients with AIDS seems to be protective against cytomegalovirus-associated retinitis.105 In a pivotal study of 14 allogeneic stem-cell recipients, Walter and colleagues and Riddell and co-workers106,107 showed that infusions of donorderived cytomegalovirus-specific CD8+ T cells restored pp65

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specific cytotoxic T lymphocyte immunity. Neither cytomegalovirus viraemia nor cytomegalovirus-associated disease developed in any of the 14 patients (although nine had an R–/D+ transplant and were thus at low risk for infection). Until recently, much of our understanding of the CD8+ cytotoxic T lymphocyte response to cytomegalovirus has been based on the pp65 and IE-1 antigens, and little was known about T-cell control over other antigens expressed during the different stages of infection.108–110 However, the development of ex-vivo T-cell assays—eg, enzyme-linked immunospot assay (ELISPOT) and MHC-peptide tetramers—in combination with predictive bioinformatics has allowed rapid profiling of T-cell responses to a large panel of cytomegalovirus antigens expressed during different phases of replication.111 These studies show that CD8+ T-cell responses to the virus often contain multiple antigen-specific reactivities, which are not constrained to pp65 or IE-1 antigens. These observations have been confirmed by Manley and colleagues112 who noted that a large fraction of CD8+ cytomegalovirus-specific cytotoxic T lymphocytes in healthy seropositive donors recognise viral antigens other than pp65 and IE-1, which were perceived to be immunodominant targets for the cytotoxic T lymphocyte response. Various important conclusions can be drawn based on these studies: (1) T-cell reactivity against pp65, pp50, IE-1, glycoprotein B, and IE-2 seems to form a substantial part of cytomegalovirus-specific cytotoxic T lymphocyte responses in a high proportion of healthy virus carriers (figure 2);

(2) subdominant or occasional T-cell reactivity is directed towards pp28, pp150, pp71, and US proteins (figure 2); (3) pp65 and IE-1 constitute only 40% of the total T-cell responses, while 60% of the CD8+ T-cell response is directed towards other antigens; (4) immunodominant CD8+ T-cell reactivity is restricted through a limited number of HLA class I alleles such as HLA A1, HLA A2, HLA A23/A24, HLA B7, HLA B8, HLA B35, HLA B40, and HLA B57/B58 (a comprehensive list of the T-cell epitopes mapped108–111,113–123 is shown in figure 3); and (5), these studies indicate that successful cytomegalovirus-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broader repertoire of antigens than previously thought. Role of CD4+ T cells

Although much emphasis has been placed on the role of MHC class I-restricted CD8+ T cells in the recognition of infected cells, there is increasing evidence that CD4+ T cells also play an important part in the control of infection. The CD4+ T-cell response against individual cytomegalovirus proteins has been analysed in healthy virus carriers, with strong proliferative responses to pp65 in all patients. Furthermore, responses to glycoproteins B and H, IE72, IE86, and UL69 were noted in some individuals.124 Several pp65-specific
-interferon producing CD4+ T-cell epitopes have also been identified.125 In otherwise healthy children who have acquired cytomegalovirus, prolonged viral

HCMV genome TRL1-14

UL1-132

Proteins studied

IRL region

US1-34

TRS1

Strength of reactivities detected

pp65 (UL83) pp50 (UL44) Strong reactivity

IE-1 (UL122) gB (UL55) IE-2 (UL123) gH (UL75) pp28 (UL99)

Subdominant reactivities

pp150 (UL32) pp71 (UL82) US2 US3 US6

Occasional weak reactivities

US11 UL16 UL18

Figure 2. Schematic representation of strength of CD8+ and CD4+ T-cell responses against cytomegalovirus antigens in healthy virus carriers. Relative reactivity for each protein is indicated on basis of strength of T-cell responses detected by ex-vivo (ELISPOT and/or MHC-peptide tetramers) and/or invitro (T-cell expansion and cytotoxicity) assays.

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HCMV genome

FEQPTETPP (HLA B41)* IIYTRNHEVK (HLA A2)*

TENGSFVAGY (HLA A24)

UL16 pp150 (UL32) pp50 (UL44) gB (UL55)

gH (UL75) pp65 (UL83)

UL1-132

RLLQTGIHV (HLA A2) NLVPMVATV (HLA A2) LMNGQQIFL (HLA A2) MLNIPSINV (HLA A2) RIFAELEGV (HLA A2) ILARNLVPM (HLA A2) HVRVSQPSL (HLA B7) QARLTVSGL (HLA B7) IGDQYVKVY (HLA A1) TVQGQNLKY (HLA A1) YRIQGKLEY (HLA A1/B27) FPTKDVAL (HLA B35) YSEHPTFTSQY (HLA A1) RIFAELEGV (HLA A2) ATVQGQNLK (HLA A11) KMQVIGDQY (HLA B15) CEDVPSGKL (HLA B40) HERNGFTVL (HLA B40) AELEGVWQPA (HLA B40) SEHPTFTSQY (HLA B44) DALPGPCI (HLA B51) RCPEMISVL (HLA Cw1) QYDPVAALF (HLA Cw4) VVCAHELVC (HLA Cw8) VAFTSHEHF (HLA Cw12) VVCAHELVC (HLA Cw15) VLGPISGHV (HLA A2) GPISGHVLK (HLA A11) QYDPVAALF (HLA A24) FTSQYRIQGKL (HLA A24) VYALPLKML (HLA A24) FVFPTKDVALR (HLA A68) RPHERNGFTV (HLA B7) TPRVTGGGAM (HLA B7)I PSINVHHY (HLA B35) VFPTKDVAL (HLA B35) PTFTSQYRIQGKL (HLA B38) EFFWDANDIY (HLA B44) QMWQARLTV (HLA B52) CPSQEPMSIYVY (HLA B35)*

UL18

pp28 (UL99)

IE-1 (UL123)

IE-2 (UL123)

US2

US3

RIWCLVVCV (HLA A2) AVGGAVASV (HLA A2) LDEGIMVVY (HLA A1) ATSTGDVVY (HLA A1) NTDFRVLEL (HLA A1) YAYIYTTYL (HLA B41)* VFETSGGLVV (HLA A29)* DYSNTHSTRYV (HLA DR7)

LLIDPTSGL (HLA A2) ARVYEIKCR (HLA B27) SLLSEFCRV (HLA A2) VLAELVKQI (HLA A2) VLEETSVML (HLA A2) YILEETSVM (HLA A2) CLQNALDIL (HLA A2) KARAKKDEL (HLA B7) QIKVRVDMV (HLA B8) ELRRKMMYM (HLA B8) ELKRKMMYM (HLA B8) IKEHMLKKY (HLA A1) DEEEAIVAY (HLA A1) CVETMCNEY (HLA A1) RRKMMYMCY (HLA B27) AYAQKIFKIL (HLA A23/A24) YIGADPLRV (HLA A2) SMMWMRFFV (HLA A2) TLLVLFIVYV (HLA A2) LLVLFIVYV (HLA A2) VYVTVDCNL (HLA A24) YYVECEPRCL (HLA A24)

US11

TRS1

US1-34

YLFSLVVLV (HLA A2) LLFRTLLVYL (HLA A2) TLLVYLFSL (HLA A2) VYLFSLVVL (HLA A24)

ALVNAVNKL (HLA A2) ALVNFLRHL (HLA A2) LIEDFDIYV (HLA A2) NVRRSWEEL (HLA B7) KARDHLAVL (HLA B7) SPWAPTAPL (HLA B7) RPSTPRAAV (HLA B7) WPRERAWAL (HLA B7) GQTEPIAFV (HLA B7) TTVYPPSSTAK (HLA A3)

LIFGHLPRV (HLA A2) LMLLKNGTV (HLA A2) HELLVLVKKAQL (HLA DR11)

IRL region

LLNCAVTKL (HLA A2) VTEHDTLLY (HLA A1) YEQHKITSY (HLA B44) RGDPFDKNY (HLA A1) GLDRNSGNY (HLA A1) TLLNCAVTK (HLA A3) TVRSHCVSK (HLA A3)

TRL1-14

LYPRPPGSGL (HLA A24) LYTSRMVTNL (HLA A24)

Figure 3. MHC class I-restricted and class II-restricted T lymphocyte epitopes from cytomegalovirus proteins defined at peptide level.108–111,113–123 *Unpublished epitopes.

shedding is associated with a persistent and selective deficiency of virus-specific CD4+ T-cell immunity126 and not a deficient virus-specific CD8+ T-cell response.127 The loss of CD4+ T-cell-mediated immunity might be particularly relevant immediately after transplantation when high doses of immunosuppressive drugs could disrupt the balance

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between viral replication and cellular immune responses. Sester and colleagues128 showed that the clinical symptoms of cytomegalovirus-associated disease in renal transplant patients were preceded by a great decrease in cytomegalovirus-specific CD4+ T-cell frequencies and an increase in viral load. In renal transplant patients, functional

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Review CD8+ T-cell and antibody responses can be insufficient to control primary infection, and formation of effectormemory CD4+ T cells is necessary for recovery of infection.129 Further evidence for a crucial role for cytomegalovirusspecific CD4+ T cells was documented by Einsele and colleagues,130 who showed that reconstitution of virusspecific CD4+ T-cell responses after adoptive transfer of CD4+ and CD8+ cytomegalovirus-specific T-cell lines resulted in a great fall in viral load in seven patients who had undergone allogeneic stem-cell transplants. Transfer of virus-specific CD4+ T cells was also coincident with the expansion of cytomegalovirus-specific CD8+ T cells from precursors that, without T-cell help, might not have been activated. Conventionally, CD4+ T-cell responses during latent infection were thought to play an indirect part by providing T-cell help in maintaining virus-specific CD8+ memory response and in generation of virus-specific antibody responses.131 Cytomegalovirus-specific CD4+ T cells might also play a direct part in controlling infection by killing virus-infected cells. Results of studies on normal asymptomatic seropositive individuals and women throughout pregnancy showed that CD4+ cytotoxic T lymphocyte responses were mainly directed towards the glycoprotein B antigen.132 Findings of a subsequent analysis show that much of the CD4+ cytotoxic T lymphocyte response to glycoprotein B and glycoprotein H antigens is directed towards highly conserved regions present in both clinical isolates of human cytomegalovirus and isolates of virus from non-human primates.133 Perturbation of innate and adaptive immune response

Most herpesviruses have evolved unique mechanisms to avoid recognition by the various effectors of the innate and adaptive immune system.134 The important role played by T cells and NK cells in the control of cytomegalovirus infection is implied by the evolution in the virus of multiple strategies that interfere with these immune responses. Indeed, cytomegalovirus is a paradigm for viral immune evasion. Mechanisms include downregulation of MHC class I molecules—eg, US2, US3, US6, and US11— and MHC class II molecules—US2—on antigen-presenting cells, expression of MHC class I homologues—UL18—and NK-cell evasion—eg, UL40 and UL16.135 Cytomegalovirus also encodes a homologue of the immunosuppressive cytokine interleukin 10, several chemokine receptors that might assist viral dissemination, and various antiapoptotic gene products.136 A comprehensive summary of various immunomodulatory genes encoded by cytomegalovirus and their potential effect on innate and adaptive immune responses is summarised in figure 4. It is noteworthy that, despite these immune evasive strategies, the inhibition of class I antigen presentation observed in cytomegalovirusinfected cells in vitro is not sufficient to prevent the induction of a broad repertoire of CD8+ cytotoxic T lymphocytes after natural infection in vivo.112 Furthermore, findings of studies by Holtappels and colleagues137 on

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murine cytomegalovirus show that although immune subversion is a reality, these mechanisms are apparently not as efficient as the term immune evasion implies. The most important consequence is that even the concerted action of all immune subversive proteins during the early and late phase of viral infection is not enough to prevent immune recognition in the epitopes from cytomegalovirus-encoded protein. Holtappels and coworkers claim that “there exists no immune evasion of mCMV in fibroblasts”. These observations are supported by studies by Elkington and colleagues111 who showed that the immune evasive genes themselves can be targeted by virus-specific CD8+ T cells.

Immune-based treatment Adoptive immunotherapy

The discovery that recovery of cytomegalovirus-specific Tcell immunity was associated with a decreased risk of developing disease after allogeneic stem-cell transplantation,41 coupled with data from murine models in which adoptive transfer of CD8+ T cells protected against viral challenge,137 led to attempts to passively restore cytomegalovirus cellular immunity in people. After the work of Walter and Riddell and colleagues,106,107 several laboratories developed novel strategies to select virusspecific CD8+ T cells for adoptive transfer, including the use of peptide-pulsed dendritic cells,139 coculture of dentritic cells with viral lysates,140 artificial antigen presenting cells,141 and positive selection of virus-specific cytokine-releasing T cells.142 Cytomegalovirus-specific CD8+ T cells can also be isolated with peptide-MHC class I tetramers either after invitro culture143 or direct from peripheral blood.144 In the experiments of Walter and colleagues,106 the frequency of adoptively transferred CD8+ T-cell clones declined progressively in patients who did not develop a concomitant virus-specific CD4+ T-helper response. This observation emphasises the importance of CD4+ T-cell responses in maintaining long-term virus-specific CD8+ Tcell immunity. A single pre-emptive infusion of polyclonal antigen-experienced cytomegalovirus-specific donor CD4+ and CD8+ T cells was both feasible and safe and resulted in a 3–5 log in-vivo expansion of virus-specific T-cell response after allogeneic stem-cell transplantation145 associated with a reduced incidence of cytomegalovirus reactivation. However, adoptive immunotherapy is confined to those recipients with virus-seropositive donors and to research centres that have the appropriate resources and expertise. Furthermore, there is a concern that selective pressure from an overly narrow anticytomegalovirus T-cell repertoire could lead to the emergence of viral escape mutates resistant to immune control. Prophylactic vaccination

In 1999, the US Institute of Medicine did a comprehensive assessment of potential targets for vaccine development,16 and assigned cytomegalovirus vaccine level 1 priority. The main objective for the cytomegalovirus vaccine, as identified by the Institute of Medicine, is to prevent symptomatic congenital disease. Every year more than 17 000 congenital

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cytomegalovirus-associated infections are recorded in the USA and Europe alone.146 There are three ways to reduce this number: (1) prevention of cytomegalovirus infection of pregnant women, (2) modification of the infection in such a way that the virus is not transmitted to the fetus, or (3) modification of the fetal infection so that it does not cause disease. Another important objective for the vaccine, again identified by the Institute of Medicine, is to prevent disease in immunocompromised individuals, particularly transplant patients. It is important to mention here that the immune correlates needed to prevent congenital cytomegalovirus disease and cytomegalovirus-associated pathogenesis in transplant patients might be quite distinct; both humoral and cell-mediated immune responses might be necessary to prevent congenital disease, whereas cellular immune response might alone be sufficient to prevent virusassociated complications in transplant patients. Over the past three decades several attempts have been made to design a prophylactic vaccine (figure 5). The first attempt was made in the mid-1970s and was based on an attenuated virus isolate referred to as Towne strain vaccine.147,148 To date this formulation has been tested in more than 800 individuals. Generally, the vaccine was found to be safe with few side-effects, and induced CD4+ and CD8+ T-cell immunity and antibody responses. Studies with Towne vaccine revealed that previous immunisation of seronegative renal transplant recipients reduced the severity

IgG Fc receptor (function unclear)

gpTRL11

Binds LIR1, ligand to HLAG (found on fetal trophoblasts). May have a role in utero

gpUL18

Chemokine receptor. postulated to modulate host type I/II interferon effects

gpUL27

of cytomegalovirus-associated disease by 85%.149,150 However, the vaccine did not prevent infection. Similarly, immunisation of seronegative women with Towne vaccine did not prevent infection, whereas immunisation of seropositive women protected them from reinfection.151 Although earlier trials with Towne vaccine induced low degrees of virus-specific antibody response, more recent studies with a different lot of this vaccine induced a strong neutralising antibody response comparable to natural viral infection.152 In an attempt to improve the immunogenicity of the Towne vaccine, a recombinant cytomegalovirus vaccine has been generated, which includes genome sequences from the Towne strain and Toledo strain.153 Toledo strain is a lowpassage isolate and was used for wild-type challenge studies of Towne vaccine.154 This recombinant vaccine formulation is in clinical trials in seropositive individuals.155 However, long-term safety of such a vaccine, especially in pregnant women, will remain a major concern for regulatory authorities. One of the most promising strategies, which will have reduced safety concerns, involves the use of subunit vaccines. This method allows the selection of the most relevant antigens for vaccination and directs the immune response specifically towards the protective antigens. Over the past decade or so there have been several attempts to create such a vaccine. Much of the interest has focused on

Orphan chemokine receptors. May be important in viral dissemination

gpUL33

TRL1-14

25% homology with human IL10. Reduces MHC class I/II expression. Regulates lymphocyte proliferation

gpUL78

gpUL111a

UL1-132 gpUL16

gpUL20

Chemokine receptor. Sequesters CC chemokines and fractaline. May assist viral dissemination

gpUS28 IRL region

gpUL40

UL83

Encodes a peptide loaded into HLA-E, which binds to inhibitory NK receptor CD94/NKG2A

Inhibits proteosomal processing of IE1

US1-34

TRS1

gpUL118

gpUS2 gpUS3 gpUS6 gpUS11 Binds to non-classic MHC proteins (MICB, ULBP1 andULBP2), preventing NKG2D receptor triggered NK cell activation

Inhibit T cell response

IgG Fc receptor (function unclear)

MHC class I/II down-regulation

•Blocks TAP and tapasindependent peptide loading •Ubiquitin-dependent retrograde dislocation of MHC from ER •Prevent egress of MHC class I from the ER to the Golgi

Figure 4. Comprehensive layout of cytomegalovirus-encoded immune evasion gene products. Each product is indicated with its location on the virus genome and potential function associated with these virus products.

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Canarypox gB followed by gB/MF59 •Phase I trial in healthy seronegative adults •Vaccine found safe •Induced neutralising antibody and CD8+ and CD4+ T cell responses •Efficacy data not available

Recombinant gB + MF59/alum •Tested in seronegative and seropositive adults and young women •Vaccine found safe •Induced neutralising antibody and some CD8+ and CD4+ T-cell responses •Efficacy data not available

Canarypox gB •Animal testing showed vaccine was safe and induced CD8+ CTL and neutralising antibody •Human vaccination failed to induce neutralising antibody

Recombinant viral

DNA and subunit vaccines pp65 and gB bivalent DNA plasmid •Animal testing showed vaccine safe and induced CD8+ T-cell responses •Phase I testing in healthy seronegative individuals initiated •Testing of trivalent DNA vaccine based on pp65, gB, and IE-1 also initiated •Efficacy data not available

CTL epitope fused to helper epitope •Animal-testing showed vaccine was safe and induced CD8+CTL •Phase I testing in transplant patients initiated in 2004 •Efficacy data not available

Other vaccine approaches

HCMV dense bodies •Sub-viral particles produced by HCMV-infected cells •Animal testing showed vaccine safe and induced CD8+ T-cell response •Phase I testing in seronegative adults in progress •Efficacy data not available

Canarypox pp65 •Tested in people seronegative people •Vaccine found safe and induced pp65-specific CD8+ and CD4+ T-cell response •No neutralising antibody •Efficacy data not available Towne strain of HCMV •Tested in healthy people and renal transplant recipients •Vaccine found safe and induced CD4+ and CD8+ T-cell response but suboptimal antibody response •Modified severity of HCMV disease but failed to prevent infection Live attenuated Towne/Toledo recombinant •Four chimeras designed •Phase I trial in healthy seropositive people •Vaccine found safe •No information on immunogenicity available as yet •Efficacy data not available

HCMV polyepitope •Multiple CD8+ T-cell epitopes linked as string of beads •Animal testing showed vaccine was safe and induced CD8+CTL •Stimulation with HCMV polyepitope activates human memory CTL response in vitro.

Figure 5. Outline of cytomegalovirus vaccine development strategies. Live attenuated, subunit, and recombinant viral vector vaccine are the three primary areas of vaccine development. Recently, other approaches, such as cytotoxic T lymphocyte epitope fused to helper epitope, cytomegalovirus dense bodies, and polyepitope have been proposed as alternative strategies for vaccine development.

the cytomegalovirus-encoded glycoprotein B (also referred to as UL55), which is a major target for neutralising antibody response; during natural cytomegalovirus infection, glycoprotein B is a target for CD8+ and CD4+ T cells.111,133,156 The first attempt to use glycoprotein B in a subunit vaccine was reported by Chrion Corporation (Emeryville, CA, USA), which used a combination of recombinantly produced glycoprotein B protein and an oil-in-water-based adjuvant, MF59.157 The findings of the studies on this vaccine show that this formulation induced a strong neutralising antibody response, but its efficacy in terms of cytomegalovirus infection remains to be identified. More recently, a combination of glycoprotein B with the dominant cytotoxic T lymphocyte target for cytomegalovirus, pp65 (referred to as UL83), has been proposed as a way of improving the efficacy of this subunit vaccine.155,157 Although delivery of recombinant proteins would raise reduced safety concerns, the formulation of such vaccines might be greatly constrained when two or more proteins are needed to achieve higher degrees of protection. To overcome this difficulty, Plotkin and colleagues158 have proposed the exploitation of canarypox-based vectors to deliver multiple

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cytomegalovirus antigens. Although initial testing in seropositive individuals with recombinant canarypox encoding glycoprotein B did not induce great glycoprotein B-specific neutralising antibody responses,159 subsequent use of this vector in a prime-boost strategy after immunisation with Towne strain, greatly enhanced the immune response induced by the Towne vaccine. More recently, a canarypox vector with pp65 showed significant induction of CD8+ cytotoxic T lymphocyte and CD4+ helper T-cell responses, which were detectable up to 26 months after vaccination. Additionally, Wang and colleagues160 have inserted glycoprotein B, pp65, and pp150 in a single poxvirus-based vector, which would allow priming of the immune response to multiple antigens with a single immunisation. Other interesting formulations under consideration include DNA vaccines encoding pp65 or glycoprotein B, or both,156,161,162 and cytomegalovirus dense bodies (non-infectious particles rich in major antigenic virus proteins).163 A limitation of both glycoprotein B and pp65-based vaccines is the assumption that protection from cytomegalovirus can be achieved by induction of immunity against a single antigen derived from the virion. However,

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the results of studies in healthy virus carriers indicate that an ideal vaccine would need to include all those antigens that induce broad cellular and humoral protective immunity, and would need to be applicable to most of the population, irrespective of ethnicity.112,146 It is also noteworthy that vaccine formulations based on a combination of multiple full-length virus antigens are likely to present other limitations. Expression of virus proteins, such as pp65, can inhibit proteasomal processing of IE-1 through an associated kinase activity.164 Moreover, other genes associated with the early phase of infection are known to interfere at various steps of the MHC class I processing pathway and presentation.165 An alternative approach involves the delivery of the vaccine as minimum T-cell epitopes166 or as polyepitope technology designed to express minimum cytomegalovirus CD4+ and CD8+ cytotoxic T lymphocyte with neutralising antibody epitopes fused together as a synthetic protein rather than in their natural context within the viral protein.167,168 Such epitope-based vaccines might find application within a conventional vector suitable for people or injected directly as purified DNA. The development of a vaccine remains a challenge and depends on collaboration between research laboratories and the biotech industry. This challenge has been recognised by the US National Vaccine Advisory Committee, which has published various recommendations,169 asking for: (1) the National Institutes of Health to be prepared to fund phase I and II trials of candidate vaccines and to continue to fund studies of epidemiology of infection and disease, (2) the Centres for Disease Control and Prevention to expand its epidemiological surveillance to gather population-based data on congenital infection, and (3) vaccine trial sponsors to seek early advice from the Food and Drug Administration to develop better clinical trial designs. References 1

2

3 4 5

6

7

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McGavran MH, Smith MG. Ultrastructural, cytochemical, and microchemical observations on cytomegalovirus (salivary gland virus) infection of human cells in tissue culture. Exp Mol Pathol 1965; 76: 1–10. Chen DH, Jiang H, Lee M, Liu F, Zhou ZH. Threedimensional visualization of tegument/capsid interactions in the intact human cytomegalovirus. Virology 1999; 260: 10–16. Pass RF. Epidemiology and transmission of cytomegalovirus. J Infect Dis 1985; 152: 243–48. Sohn YM, Oh MK, Balcarek KB, Cloud GA, Pass RF. Cytomegalovirus infection in sexually active adolescents. J Infect Dis 1991; 163: 460–63. Mocarski ES, Courcelle CT. Cytomegalovirus and their replication. In: Knippe DM, Howley PM, eds. Fields virology. Philedelphia: Lippincott Willams and Wilkins, 2001: 2629–73. Taylor-Wiedeman J, Sissons JG, Borysiewicz LK, Sinclair JH. Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol 1991; 72: 2059–64. Mendelson M, Monard S, Sissons P, Sinclair J. Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors. J Gen Virol 1996; 77: 3099–102. Crapnell K, Zanjani ED, Chaudhuri A, Ascensao JL, St Jeor S, Maciejewski JP. In vitro infection of megakaryocytes and their precursors by human cytomegalovirus. Blood 2000; 95: 487–93. Soderberg-Naucler C, Fish KN, Nelson JA. Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell 1997; 91: 119–26.

Search strategy and selection criteria Data for this Review were identified by searches of Medline and references from relevant articles; numerous articles were identified through searches of the extensive files of the authors. Search terms were “cytomegalovirus”, “congenital”, “renal transplant”, “lung transplant”, “heart transplant”, “liver transplant”, “bone marrow transplant”, “HIV”, and “immunity”. English language papers were reviewed. No time limits for these searches were applied.

Concluding remarks Despite major clinical and scientific advances, the aim of supplementing pharmacological strategies with immunebased treatments for cytomegalovirus-associated infection remains elusive. As scientific advances are made, so new challenges for laboratories developing novel treatments emerge. Key among these is the regulatory issue involved in immunotherapy. Regulatory authorities are applying restrictive criteria based on a model of product development, which is best suited to the drug industry—ie, a patented commercial product to be manufactured by a company or its licensed manufacturers. For the future benefit of patients with cytomegalovirus-associated disease, regulatory understanding is essential so there can be continued academic-led interventional research into virusspecific immunotherapies. Conflicts of interest

MKG has no conflicts of interest. RK has submitted an international patent application (PCT/AU02/00829), which includes a large panel of T-cell epitopes for vaccine development and immune monitoring. RK’s laboratory is funded by competitive grants from the National Health and Medical Research Council (NH&MRC, Australia), Cooperative Research Centre for Vaccine Technology, and National Institutes of Health, USA (1 R21 CA106172-01A1). RK is an NH&MRC Senior Research Fellow. MKG is supported by a Research Fellowship from the British Society for Haematology.

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Review 27 Sia IG, Wilson JA, Groettum CM, Espy MJ, Smith TF, Paya CV. Cytomegalovirus (CMV) DNA load predicts relapsing CMV infection after solid organ transplantation. J Infect Dis 2000; 181: 717–20. 28 Portela D, Patel R, Larson-Keller JJ, et al. OKT3 treatment for allograft rejection is a risk factor for cytomegalovirus disease in liver transplantation. J Infect Dis 1995; 171: 1014–18. 29 Dockrell DH, Prada J, Jones MF, et al. Seroconversion to human herpesvirus 6 following liver transplantation is a marker of cytomegalovirus disease. J Infect Dis 1997; 176: 1135–40. 30 Tolkoff-Rubin NE, Rubin RH. The interaction of immunosuppression with infection in the organ transplant recipient. Transplant Proc 1994; 26 (5 suppl 1): 16–19. 31 Evans PC, Soin A, Wreghitt TG, Taylor CJ, Wight DG, Alexander GJ. An association between cytomegalovirus infection and chronic rejection after liver transplantation. Transplantation 2000; 69: 30–35. 32 Luckraz H, Charman SC, Wreghitt T, Wallwork J, Parameshwar J, Large SR. Does cytomegalovirus status influence acute and chronic rejection in heart transplantation during the ganciclovir prophylaxis era? J Heart Lung Transplant 2003; 22: 1023–27. 33 Luckraz H, Sharples L, McNeil K, Wreghitt T, Wallwork J. Cytomegalovirus antibody status of donor/recipient does not influence the incidence of bronchiolitis obliterans syndrome in lung transplantation. J Heart Lung Transplant 2003; 22: 287–91. 34 van den Berg AP, Klompmaker IJ, Hepkema BG, et al. Cytomegalovirus infection does not increase the risk of vanishing bile duct syndrome after liver transplantation. Transplant Int 1996; 9 (suppl 1): S171–73. 35 Hosenpud JD. Coronary artery disease after heart transplantation and its relation to cytomegalovirus. Am Heart J 1999; 138: S469–72. 36 Brennan DC. Cytomegalovirus in renal transplantation. J Am Soc Nephrol 2001; 12: 848–55. 37 Bowden RA, Slichter SJ, Sayers M, et al. A comparison of filtered leukocyte-reduced and cytomegalovirus (CMV) seronegative blood products for the prevention of transfusionassociated CMV infection after marrow transplant. Blood 1995; 86: 3598–603. 38 Goodrich JM, Boeckh M, Bowden R. Strategies for the prevention of cytomegalovirus disease after marrow transplantation. Clin Infect Dis 1994; 19: 287–98. 39 Ljungman P. Immune reconstitution and viral infections after stem cell transplantation. Bone Marrow Transplant 1998; 21 (suppl 2): S72–74. 40 Goodrich JM, Bowden RA, Fisher L, Keller C, Schoch G, Meyers JD. Ganciclovir prophylaxis to prevent cytomegalovirus disease after allogeneic marrow transplant. Ann Intern Med 1993; 118: 173–78. 41 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 1994; 83: 1971–79. 42 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 2003; 101: 407–14. 43 Gandhi MK, Wills MR, Okecha G, et al. Late diversification in the clonal composition of human cytomegalovirus-specific CD8+ T cells following allogeneic hemopoietic stem cell transplantation. Blood 2003; 102: 3427–38. 44 Einsele H, Hebart H, Kauffmann-Schneider C, et al. Risk factors for treatment failures in patients receiving PCR-based preemptive therapy for CMV infection. Bone Marrow Transplant 2000; 25: 757–63. 45 Broers AE, van Der Holt R, van Esser JW, et al. Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-celldepleted stem cell transplantation. Blood 2000; 95: 2240–45. 46 Nachbaur D, Bonatti H, Oberaigner W, et al. Survival after bone marrow transplantation from cytomegalovirus seropositive sibling donors. Lancet 2001; 358: 1157–59. 47 Ljungman P, Brand R, Einsele H, Frassoni F, Niederwieser D, Cordonnier C. Donor CMV serologic status and outcome of CMV-seropositive recipients after unrelated donor stem cell transplantation: an EBMT megafile analysis. Blood 2003; 102: 4255–60.

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