Correction: Novel decay dynamics revealed for virus-mediated drug activation in cytomegalovirus infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006299.].

Novel decay dynamics revealed for virus-mediated drug activation in cytomegalovirus infection.

Human cytomegalovirus (CMV) infection is a substantial cause of morbidity and mortality in immunocompromised hosts and globally is one of the most important congenital infections. The nucleoside analogue ganciclovir (GCV), which requires initial phosphorylation by the viral UL97 kinase, is the mainstay for treatment. To date, CMV decay kinetics during GCV therapy have not been extensively investigated and its clinical implications not fully appreciated. We measured CMV DNA levels in the blood of 92 solid organ transplant recipients with CMV disease over the initial 21 days of ganciclovir therapy and identified four distinct decay patterns, including a new pattern exhibiting a transient viral rebound (Hump) following initial decline. Since current viral dynamics models were unable to account for this Hump profile, we developed a novel multi-level model, which includes the intracellular role of UL97 in the continued activation of ganciclovir, that successfully described all the decline patterns observed. Fitting the data allowed us to estimate ganciclovir effectiveness in vivo (mean 92%), infected cell half-life (mean 0.7 days), and other viral dynamics parameters that determine which of the four kinetic patterns will ensue. An important clinical implication of our results is that the virological efficacy of GCV operates over a broad dose range. The model also raises the possibility that GCV can drive replication to a new lower steady state but ultimately cannot fully eradicate it. This model is likely to be generalizable to other anti-CMV nucleoside analogs that require activation by viral enzymes such as UL97 or its homologues.

Vertical Cytomegalovirus Transmission From HIV-Infected Women Randomized to Formula-Feed or Breastfeed Their Infants.

BACKGROUND: Cytomegalovirus (CMV) is associated with morbidity and mortality in human immunodeficiency virus (HIV)-exposed infants. We assessed the effect of and relative contribution of breastfeeding to CMV acquisition among infants delivered by HIV-infected mothers. METHODS: Between 1993 and 1998 pregnant, HIV-infected women in Nairobi, Kenya, were randomly assigned to breastfeed or formula-feed their infants in an HIV transmission study. Women were allocated equally between treatment arms, and the study was not blinded. The primary endpoint of this nested study was time to infant CMV infection. RESULTS: CMV infection was assessed in 138 breastfed and 134 formula-fed infants. Baseline characteristics were similar between arms. Breastfed infants acquired CMV earlier than formula-fed infants (median age of acquisition, 4.26 vs 9.87 months; P < .001) and had a higher 1-year probability of CMV infection (0.89 vs 0.69; P < .001). Breastfeeding was associated with a 1.6-fold increased risk of infant CMV acquisition independent of infant HIV status (multivariable hazard ratio, 1.61; 95% confidence interval, 1.20-2.16; P = .002). Approximately one third of CMV infections occurred during the peripartum period, with 40% acquired through breastfeeding and the remainder acquired through modes other than breast milk. CONCLUSIONS: Preventing CMV acquisition may be a priority for HIV-exposed infants, but there is a narrow window of opportunity for intervention. Approaches that reduce maternal cervical and breast milk CMV reactivation may help delay infant infection.

Glucocorticosteroids trigger reactivation of human cytomegalovirus from latently infected myeloid cells and increase the risk for HCMV infection in D+R+ liver transplant patients.

Graft rejection in transplant patients is managed clinically by suppressing T-cell function with immunosuppressive drugs such as prednisolone and methylprednisolone. In such immunocompromised hosts, human cytomegalovirus (HCMV) is an important opportunistic pathogen and can cause severe morbidity and mortality. Currently, the effect of glucocorticosteroids (GCSs) on the HCMV life cycle remains unclear. Previous reports showed enhanced lytic replication of HCMV in vitro in the presence of GCSs. In the present study, we explored the implications of steroid exposure on latency and reactivation. We observed a direct effect of several GCSs used in the clinic on the activation of a quiescent viral major immediate-early promoter in stably transfected THP-1 monocytic cells. This activation was prevented by the glucocorticoid receptor (GR) antagonist Ru486 and by shRNA-mediated knockdown of the GR. Consistent with this observation, prednisolone treatment of latently infected primary monocytes resulted in HCMV reactivation. Analysis of the phenotype of these cells showed that treatment with GCSs was correlated with differentiation to an anti-inflammatory macrophage-like cell type. On the basis that these observations may be pertinent to HCMV reactivation in post-transplant settings, we retrospectively evaluated the incidence, viral kinetics and viral load of HCMV in liver transplant patients in the presence or absence of GCS treatment. We observed that combination therapy of baseline prednisolone and augmented methylprednisolone, upon organ rejection, significantly increased the incidence of HCMV infection in the intermediate risk group where donor and recipient are both HCMV seropositive (D+R+) to levels comparable with the high risk D+R- group.

The "silent" global burden of congenital cytomegalovirus.

Human cytomegalovirus (CMV) is a leading cause of congenital infections worldwide. In the developed world, following the virtual elimination of circulating rubella, it is the commonest nongenetic cause of childhood hearing loss and an important cause of neurodevelopmental delay. The seroprevalence of CMV in adults and the incidence of congenital CMV infection are highest in developing countries (1 to 5% of births) and are most likely driven by nonprimary maternal infections. However, reliable estimates of prevalence and outcome from developing countries are not available. This is largely due to the dogma that maternal preexisting seroimmunity virtually eliminates the risk for sequelae. However, recent data demonstrating similar rates of sequelae, especially hearing loss, following primary and nonprimary maternal infection have underscored the importance of congenital CMV infection in resource-poor settings. Although a significant proportion of congenital CMV infections are attributable to maternal primary infection in well-resourced settings, the absence of specific interventions for seronegative mothers and uncertainty about fetal prognosis have discouraged routine maternal antibody screening. Despite these challenges, encouraging results from prototype vaccines have been reported, and the first randomized phase III trials of prenatal interventions and prolonged postnatal antiviral therapy are under way. Successful implementation of strategies to prevent or reduce the burden of congenital CMV infection will require heightened global awareness among clinicians and the general population. In this review, we highlight the global epidemiology of congenital CMV and the implications of growing knowledge in areas of prevention, diagnosis, prognosis, and management for both low (50 to 70%)- and high (>70%)-seroprevalence settings.

Compartmentalized cytomegalovirus replication and transmission in the setting of maternal HIV-1 infection.

BACKGROUND: Cytomegalovirus (CMV) infection is associated with adverse outcomes in human immunodeficiency virus (HIV)-exposed infants. Determinants of vertical CMV transmission in the setting of maternal HIV-1 infection are not well-defined. METHODS: CMV and HIV-1 levels were measured in plasma, cervical secretions, and breast milk of 147 HIV-1-infected women to define correlates of maternal CMV replication and infant CMV acquisition. RESULTS: Although few women had detectable CMV in plasma (4.8%), the majority had detectable CMV DNA in cervical secretions (66%) and breast milk (99%). There was a strong association between cervical CMV detection during pregnancy and later breast milk levels (ß = 0.47; P = .005). Plasma HIV-1 level and CD4 counts were associated with CMV in the cervix and breast milk. However HIV-1 levels within the cervix and breast milk were not associated with CMV within these compartments. Maternal breast milk CMV levels (hazard ratio [HR], 1.4; P = .003) and maternal CD4 < 450 cells/mm(3) (HR, 1.8; P = .008) were independently associated with infant CMV acquisition; each log10 increase in breast milk CMV was associated with a 40% increase in infant infection. The breast milk CMV level required to attain a 50% probability of CMV transmission increased with higher maternal CD4 counts, increasing from 3.55 log10 CMV DNA copies/mL at a CD4 count of 350 cells/mm(3) to 5.50 log10 CMV DNA copies/mL at a CD4 count of 1000 cells/mm(3). CONCLUSIONS: Breast milk CMV levels and maternal CD4 count are major determinants of CMV transmission in the setting of maternal HIV-1. Maternal immune reconstitution or lowering breast milk CMV levels may reduce vertical CMV transmission.

CMV infected or not CMV infected: that is the question.

Cytomegalovirus (CMV) infection is widespread in the human population. Normally, in adolescents and adults, prior exposure to CMV can readily be determined by IgG assays. However, in individuals where antibody production is impaired, such as patients with common variable immunodeficiency disease or in the very young where maternal antibodies are present, diagnosis of CMV infection is problematic using such assays. In this issue of the European Journal of Immunology, a study by Sester and colleagues [Eur J Immunol 2013. 43: 1099-1108] using CD4(+) T-cell immunity as a marker of infection clearly differentiates young children with prior exposure to CMV from those who only have passive maternal antibody. This information will quickly find application in the pretransplant screening of young children for CMV infection and help with the stratification of these children to identify those who are truly CMV negative and are therefore at risk for future CMV infection and disease if receiving an organ from a CMV-positive donor as discussed in this Commentary.

Effect of maintenance immunosuppressive drugs on virus pathobiology: evidence and potential mechanisms.

Recent evidence suggesting a potential anti-CMV effect of mTORis is of great interest to the transplant community. However, the concept of an immunosuppressant with antiviral properties is not new, with many accounts of the antiviral properties of several agents over the years. Despite these reports, to date, there has been little effort to collate the evidence into a fuller picture. This manuscript was developed to gather the evidence of antiviral activity of the agents that comprise a typical immunosuppressive regimen against viruses that commonly reactivate following transplant (HHV1 and 2, VZV, EBV, CMV and HHV6, 7, and 8, HCV, HBV, BKV, HIV, HPV, and parvovirus). Appropriate immunosuppressive regimens posttransplant that avoid acute rejection while reducing risk of viral reactivation are also reviewed. The existing literature was disparate in nature, although indicating a possible stimulatory effect of tacrolimus on BKV, potentiation of viral reactivation by steroids, and a potential advantage of mammalian target of rapamycin (mTOR) inhibition in several viral infections, including BKV, HPV, and several herpesviruses.

Acute cytomegalovirus infection is associated with increased frequencies of activated and apoptosis-vulnerable T cells in HIV-1-infected infants.

Cytomegalovirus (CMV) coinfection is associated with infant HIV-1 disease progression and mortality. In a cohort of Kenyan HIV-infected infants, the frequencies of activated (CD38(+) HLA-DR(+)) and apoptosis-vulnerable (CD95(+) Bcl-2(-)) CD4(+) and CD8(+) T cells increased substantially during acute CMV infection. The frequency of activated CD4(+) T cells was strongly associated with both concurrent CMV coinfection (P = 0.001) and HIV-1 viral load (P = 0.05). The frequency of apoptosis-vulnerable cells was also associated with CMV coinfection in the CD4 (P = 0.02) and CD8 (P < 0.001) T cell subsets. Similar observations were made in HIV-exposed uninfected infants. CMV-induced increases in T cell activation and apoptosis may contribute to the rapid disease progression in coinfected infants.

Influence of cytomegalovirus infection on immune cell phenotypes in patients with common variable immunodeficiency.

BACKGROUND: A subset of patients with common variable immunodeficiency (CVID) have debilitating inflammatory complications strongly associated with cytomegalovirus (CMV) infection and a hyperproliferative CMV-specific T-cell response. OBJECTIVES: We studied the T-cell response to CMV and the global effect of this virus on immune effector cell populations in patients with CVID. METHODS: Antibody staining, peptide stimulation, and proliferation assays were used to profile CMV-specific T-cell function. RESULTS: CMV infection drives the CD4/CD8 ratio inversion that is characteristic of CVID. The late effector CD8(+) T-cell subset is expanded in CMV-infected patients with CVID. This expansion is largely attributable to CMV-specific cells and correlates with inflammatory disease; within the CMV-specific population, the frequency of late effector cells correlates inversely with the frequency of cells expressing programmed death 1. Supernatants from proliferating CMV-specific CD8(+) cells from patients with inflammatory disease can confer proliferative potential on cells from patients with noninflammatory CVID and healthy subjects. Blocking experiments showed that this proliferation is mediated in part by IFN-γ and TNF-α. CONCLUSIONS: These data strengthen the association of CMV with inflammatory pathology in patients with CVID, explain some of the well-known T-cell abnormalities associated with this condition, and provide a plausible mechanism for the documented therapeutic activity of anti-TNF-α and antiviral chemotherapy in managing CVID-associated inflammatory disease.

Cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant in transplant recipients: a phase 2 randomised placebo-controlled trial.

BACKGROUND: Cytomegalovirus end-organ disease can be prevented by giving ganciclovir when viraemia is detected in allograft recipients. Values of viral load correlate with development of end-organ disease and are moderated by pre-existing natural immunity. Our aim was to determine whether vaccine-induced immunity could do likewise. METHODS: We undertook a phase-2 randomised placebo controlled trial in adults awaiting kidney or liver transplantation at the Royal Free Hospital, London, UK. Exclusion criteria were pregnancy, receipt of blood products (except albumin) in the previous 3 months, and simultaneous multiorgan transplantation. 70 patients seronegative and 70 seropositive for cytomegalovirus were randomly assigned from a scratch-off randomisation code in a 1:1 ratio to receive either cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant or placebo, each given at baseline, 1 month and 6 months later. If a patient was transplanted, no further vaccinations were given and serial blood samples were tested for cytomegalovirus DNA by real-time quantitative PCR (rtqPCR). Any patient with one blood sample containing more than 3000 cytomegalovirus genomes per mL received ganciclovir until two consecutive undetectable cytomegalovirus DNA measurements. Safety and immunogenicity were coprimary endpoints and were assessed by intention to treat in patients who received at least one dose of vaccine or placebo. This trial is registered with ClinicalTrials.gov, NCT00299260. FINDINGS: 67 patients received vaccine and 73 placebo, all of whom were evaluable. Glycoprotein-B antibody titres were significantly increased in both seronegative (geometric mean titre 12,537 (95% CI 6593-23,840) versus 86 (63-118) in recipients of placebo recipients; p<0.0001) and seropositive (118,395; 64,503-217,272) versus 24,682 (17,909-34,017); p<0.0001) recipients of vaccine. In those who developed viraemia after transplantation, glycoprotein-B antibody titres correlated inversely with duration of viraemia (p=0.0022). In the seronegative patients with seropositive donors, the duration of viraemia (p=0.0480) and number of days of ganciclovir treatment (p=0.0287) were reduced in vaccine recipients. INTERPRETATION: Although cytomegalovirus disease occurs in the context of suppressed cell-mediated immunity post-transplantation, humoral immunity has a role in reduction of cytomegalovirus viraemia. Vaccines containing cytomegalovirus glycoprotein B merit further assessment in transplant recipients. FUNDING: National Institute of Allergy and Infectious Diseases, Grant R01AI051355 and Wellcome Trust, Grant 078332. SPONSOR: University College London (UCL).

Inflammation in common variable immunodeficiency is associated with a distinct CD8(+) response to cytomegalovirus.

BACKGROUND: Common variable immunodeficiency is the most common primary immunodeficiency. A subset of patients has debilitating inflammatory complications. OBJECTIVES: We investigated the role of cytomegalovirus (CMV), and the T-cell response targeted at this virus, in this inflammatory disease. METHODS: Phenotypic and functional assays were used to profile CMV-specific T cells in patients with common variable immunodeficiency with and without inflammatory complications. Highly sensitive immunohistochemistry was used to detect CMV antigens at sites of inflammation. RESULTS: Cytomegalovirus was significantly associated with inflammatory disease, which occurred in 31 of 43 (72%) virus-exposed patients and 8 of 31 (26%) naive patients (P = .0001). CMV pp65-NLVPMVATV epitope-specific CD8(+) T-cell frequencies were significantly elevated in inflammatory patients, but these cells did not show evidence of exhaustion, with low levels of programmed death-1 and high T-cell receptor avidity. Rather, they showed features consistent with high in vivo functionality and proliferative activity including reduced levels of the anti-inflammatory marker CD73 (1.67% of NLV(+) cells were CD73(+) vs 42.01% in noninflammatory patients; P = .004) and increased Ki-67 expression (37% vs 2% in noninflammatory patients; P < .0001). In vitro, the CMV-specific T cells showed high antigen-specific proliferative potential compared with cells from noninflammatory patients. By using sensitive immunohistochemistry, we detected for the first time viral antigen at the sites of inflammation, indicative of active viral replication. CONCLUSION: Our data strongly support a direct role for CMV and a hyperreactive CMV-specific immune response in the debilitating chronic inflammatory complications of common variable immunodeficiency.

Sequential mutations associated with adaptation of human cytomegalovirus to growth in cell culture.

Mutations that occurred during adaptation of human cytomegalovirus to cell culture were monitored by isolating four strains from clinical samples, passaging them in various cell types and sequencing ten complete virus genomes from the final passages. Mutational dynamics were assessed by targeted sequencing of intermediate passages and the original clinical samples. Gene RL13 and the UL128 locus (UL128L, consisting of genes UL128, UL130 and UL131A) mutated in all strains. Mutations in RL13 occurred in fibroblast, epithelial and endothelial cells, whereas those in UL128L were limited to fibroblasts and detected later than those in RL13. In addition, a region containing genes UL145, UL144, UL142, UL141 and UL140 mutated in three strains. All strains exhibited numerous mutations in other regions of the genome, with a preponderance in parts of the inverted repeats. An investigation was carried out on the kinetic growth yields of viruses derived from selected passages that were predominantly non-mutated in RL13 and UL128L (RL13+UL128L+), or that were largely mutated in RL13 (RL13-UL128L+) or both RL13 and UL128L (RL13-UL128L-). RL13-UL128L- viruses produced greater yields of infectious progeny than RL13-UL128L+ viruses, and RL13-UL128L+ viruses produced greater yields than RL13+UL128L+ viruses. These results suggest strongly that RL13 and UL128L exert at least partially independent suppressive effects on growth in fibroblasts. As all isolates proved genetically unstable in all cell types tested, caution is advised in choosing and monitoring strains for experimental studies of vulnerable functions, particularly those involved in cell tropism, immune evasion or growth temperance.

Patients with B cell chronic lymphocytic leukaemia have an expanded population of CD4 perforin expressing T cells enriched for human cytomegalovirus specificity and an effector-memory phenotype.

We have previously shown an expansion of cytotoxic antigen-experienced CD4(+)T cells (CTLs) that express perforin (PF) in the peripheral blood of patients with B cell chronic lymphocytic leukaemia (B-CLL). Increased frequencies of CD4(+)CTLs have since been attributed to chronic viral infections, particularly, human cytomegalovirus (HCMV). The present study examined the involvement of CD4(+)CTLs in responses to HCMV in B-CLL, and characterized their differentiation. We studied 36 HCMV seropositive (SP) and seronegative B-CLL patients and 20 healthy age-matched individuals. The HCMV reactivity of CD4(+)PF(+) and CD4(+)PF(-) cells was determined by interferon-gamma expression, and expression of CD45RA and CCR7 was assessed by flow cytometry. Fluorescence in-situ hybridization was used to measure relative telomere lengths. CD4(+)PF(+)T cell expansion in B-CLL patients and controls was strongly associated with HCMV seropositivity. CD4(+)PF(+) compared to CD4(+)PF(-) cells from SP B-CLL patients elicited major histocompatibility complex (MHC) class II-restricted responses to HCMV. CD4(+)PF(+)T cells from patients and controls were enriched with highly differentiated T-effector/memory (CCR7(-)) and revertant (CCR7(-)CD45RA(+)) phenotype. CD4(+)PF(+)T cells from B-CLL patients had shorter telomeres than CD4(+)PF(-)T cells, indicating an extensive replicative history. We conclude that persistent exposure to HCMV antigens in SP B-CLL patients leads to an expansion of the circulating MHC class II-restricted CD4(+)PF(+)T cell population with effector/memory phenotype.

Diagnosis of post-transplant lymphoproliferative disorder in solid organ transplant recipients - BCSH and BTS Guidelines.

A joint working group established by the Haemato-oncology subgroup of the British Committee for Standards in Haematology (BCSH) and the British Transplantation Society (BTS) has reviewed the available literature and made recommendations for the diagnosis and management of post-transplant lymphoproliferative disorder (PTLD) in adult recipients of solid organ transplants. This review details the risk factors predisposing to development, initial features and diagnosis. It is important that the risk of developing PTLD is considered when using post transplant immunosuppression and that the appropriate investigations are carried out when there are suspicions of the diagnosis. These must include tissue for histology and computed tomography scan to assess the extent of disease. These recommendations have been made primarily for adult patients, there have been some comments made with regard to paediatric practice.

Management of post-transplant lymphoproliferative disorder in adult solid organ transplant recipients - BCSH and BTS Guidelines.

A joint working group established by the Haemato-oncology subgroup of the British Committee for Standards in Haematology (BCSH) and the British Transplantation Society (BTS) has reviewed the available literature and made recommendations for the diagnosis and management of post-transplant lymphoproliferative disorder in adult recipients of solid organ transplants. This review details the therapeutic options recommended including reduction in immunosuppression (RIS), transplant organ resection, radiotherapy and chemotherapy. Effective therapy should be instituted before progressive disease results in declining performance status and multi-organ dysfunction. The goal of treatment should be a durable complete remission with retention of transplanted organ function with minimal toxicity.

Sequence flexibility of the immunodominant HLA A*0201 restricted ppUL83 CD8 T-cell epitope of human cytomegalovirus.

The cytomegalovirus ppUL83 protein contains an immunodominant A*0201 restricted epitope between residues 495 and 503. We investigated the tolerance of this epitope to sequence variation in the context of peptide binding to HLA A*0201 and the ability to induce an Interferon gamma (IFNgamma) response through engagement with the T-cell receptor (TCR). The majority of mutations investigated resulted in a decrease in the production of IFNgamma indicating that if such variants occurred in vivo they would not be recognized by CD8 T-cell clones specific for the wild-type epitope. The mechanistic basis for the majority of the mutant peptides was their failure to bind and stabilize class I HLA cell surface expression. However, one peptide with a mutation at the P5 position (methionine to cysteine) resulted in a significant enhanced binding to HLA A*0201 and also an increase in cell surface expression over the wild-type peptide but was unable to engage with the CD8 TCR and trigger IFNgamma production. This peptide acted as a competitive inhibitor of the wild-type peptide but could not fully inhibit IFNgamma production by the latter. We subsequently investigated whether mutations of the HLA A*0201 epitope were evident in immunocompromized patients experiencing either rapid exponential or persistent cytomegalovirus replication.