Objectives: Cytomegalovirus infection and disease remain an issue in solid-organ transplant. Universal prophylaxis is more cost-effective than a preemptive strategy and is associated with significantly less Cytomegalovirus resistance after kidney transplant, especially in Cytomegalovirus-seropositive donors and Cytomegalovirus-seronegative recipients.

Materials and Methods: Registry data and meta-analyses have shown that mammalian target of rapamycin inhibitors (sirolimus- and everolimus-based immunosuppression) are associated with significantly less Cytomegalovirus events in de novo kidney transplant patients than in patients who are treated with calcineurin inhibitors plus mycophenolate-based immunosuppression.

Results: Recent pooled analyses of 3 randomized controlled trials in de novo kidney transplant patients, where immunosuppression was based on cyclosporine with either mycophenolate or everolimus, showed that patients who received everolimus had significantly less Cytomegalovirus events (Cytomegalovirus viremia, Cytomegalovirus infection/disease) than those who received mycophenolate, with or without cyto­megalovirus as prophylaxis. An even more recent prospective randomized controlled study on de novo kidney transplant patients with no anticyto­megalovirus prophylaxis demonstrated that everolimus-based immunosuppression plus low-dose tacrolimus was associated with significantly less Cytomegalovirus infection than standard-dose tacrolimus plus mycophenolate.

Conclusions: The potential benefits are not fully known of such a therapeutic strategy to limit the long-term indirect effects mediated by Cytomegalovirus infections.

Key words : Cytomegalovirus disease; Mammalian target of rapamycin inhibitors

Introduction

Cytomegalovirus (CMV), also referred to as Human herpesvirus 5, is a double-stranded DNA virus. As a virus, it replicates within cells using the host’s cellular machinery. Its genome size is about 230 kb, encoding over 200 proteins.¹ Cytomegalovirus replication occurs through a sequence that includes immediate-early, early, and late steps. This sequence also requires considerable time, thus explaining the slow growth of CMV in culture.

Cytomegalovirus is the most common viral pathogen to occur after kidney transplant.² Cytomegalovirus infection is usually acquired early in life, and approximately 40% to 70% of the world’s population is seropositive and harboring this latent virus. Although primary CMV infection is generally self-limiting in immunocompetent individuals, primary infection or reactivation of the latent virus in immunocompromised patients can have debilitating and even life-threatening consequences.^3,4

The use of CMV prophylaxis has resulted in a significant decrease in the incidence of CMV infection.^5,6 However, an issue is the occurrence of CMV infection/disease that can arise after ceasing CMV prophylaxis (ie, late CMV infection/disease).^7,8 Hence, CMV infection can be associated with allograft rejection and loss, mortality, interstitial fibrosis and tubular atrophy as shown in protocol biopsies, and increased posttransplant costs.^9-11 Thus, it is important to circumvent and decrease CMV infection after kidney transplant.

To accomplish this, we have 2 strategies: universal prophylaxis or preemptive therapy. Universal prophylaxis is given to every patient perceived at risk for CMV infection (ie, to CMV-seronegative recipients whose donor is CMV seropositive or when a recipient is CMV seropositive). Prophylaxis is given for either 6 months (seropositive donor-seronegative recipient combination) or 3 months (seropositive recipient).¹² However, this treatment is not fully effective and can be associated with adverse bone marrow events, which may lead to dose decrease or dose discontinuation, to late-onset CMV infection, and in some cases to drug resistance.^13-16 Preemptive therapy does not prevent viral replication, an event that has been associated with inferior transplant outcomes, and also requires intensive logistic coordination.^15,17-20

A third strategy has also emerged, which is to use a mammalian target of rapamycin (mTOR) inhibitor in the immunosuppressive regimen (ie, either sirolimus or everolimus), as suggested by recent data.^21-25

Cytomegalovirus and Mammalian Target of Rapamycin Pathway Interactions
Cytomegalovirus DNA translation occurs through a cap structure-dependent mechanism. Cap-dependent translation follows after activation of the phosphatidylinositol 3-kinase-Akt-mTOR pathway. In this pathway, phosphatidylinositol 3-kinase increases the cellular content of inositol triphosphate, which activates Akt; Akt itself activates mTOR. Interestingly, Akt is the cellular homolog of a viral protein (found in the Akt⁸ retrovirus).

Mammalian target of rapamycin kinase exists as 2 different complexes: mTORC126 and mTORC2.²⁷ These 2 complexes are distinguished by their substrate and specificity factors (raptor and rictor). The mTORC1 complex activates cap structure-dependent translation. The mTORC2 complex is less understood but is particularly implicated in cytoskeleton processes.

Cap-dependent translation is initiated by the eukaryotic initiation factor 4F (eIF4F) complex. The eIF4F complex requires interaction between the different subunits, eIF4E and eIF4G, as a first step in cap-dependent translation. This interaction is inhibited by the eIF4E binding protein, a target of the mTORC1 complex. The mTORC1 complex phosphorylates eIF4E binding protein, preventing eIF4E binding protein from binding to eIF4G, thereby maintaining the activity of eIF4F. In addition, mTORC1 phosphorylates p70S6 kinase, a process that also promotes translation.^28-30 However, cap-dependent translation is tightly regulated in human cells. Several stress signals (eg, hypoxia, energy depletion) down-regulate cap-dependent translation (thereby CMV replication) through the mTOR pathway. These stress signals are generated by the CMV infection itself, by increasing metabolism to form new virions.

As a consequence, CMV (as well as other double-stranded DNA viruses) has evolved mechanisms to maintain favorable cellular conditions to replicate. This is especially important as CMV replication is a relatively slow, multistep process. These mechanisms are mediated by Akt activation plus maintenance of the activity of the phosphatidylinositol 3-kinase-Akt-mTOR pathway, even during cellular stress. These mechanisms intervene above the mTOR step in the pathway. Mammalian target of rapamycin inhibitors therefore retain the ability to down-regulate cap-dependent translation. In other words, the use of mTOR inhibitors is a way of restoring the naturally occurring stress-induced down-regulation of the mTOR pathway, thereby providing an antiviral effect.

It should be noted that the efficacy of mTOR inhibition during CMV infection seems to depend on the time after infection, with mTOR inhibitors (such as Torin1) being much more efficient in earlier steps than at later time points.³¹ Therefore, the exact role of mTOR inhibitors in viral replication and the time frame when mTOR inhibitors have antiviral activity deserve to be investigated.

Cytomegalovirus Incidence With Everolimus Versus Mycophenolate in De Novo Kidney Transplant Patients
In a meta-analysis performed by Webster and associates in 2006, which examined the outcomes of kidney transplant patients whose primary immuno­suppression was based on mTOR inhibitors compared with calcineurin inhibitor-based immuno­suppression, patients treated with mTOR inhibitors had a 51% reduction in CMV infection versus those treated with an antimetabolite plus calcineurin inhibitor (relative risk of 0.49; 95% confidence interval, 0.37-0.65). This result varied with CMV prophylaxis.²¹

Brennan and associates evaluated freedom from and incidence of CMV events (including viremia, infection, and syndrome, as well as CMV with organ involvement) in de novo kidney transplant patients whose immunosuppression included cyclosporine (standard-dose cyclosporine or reduced-dose cyclos­porine) plus either everolimus or mycophenolate (mycophenolate mofetil or enteric-coated myco­phenolate sodium [EC-MPS]).²⁴ Three multicenter, multicountry studies were discussed,²⁴ including the B201 (588 patients) and B251 (583 patients) studies, which were double-blind, randomized, parallel group studies in adult de novo kidney transplant patients. In these 2 studies, patients were randomized to receive standard-dose cyclosporine plus either everolimus at 1.5 or 3 mg/day or mycophenolate mofetil at 2 g/day, in addition to steroids and basiliximab as induction therapy. The 2 studies reported CMV events as part of the data collected on infections and adverse or serious adverse events, classified by the investigator as CMV viremia (infection with no clinical symptoms), CMV infection or syndrome (viremia plus clinical symptoms), or invasive CMV tissue disease.

The third study (A2309; 833 patients) was a phase IIIb, multicenter, open-label, noninferiority trial on adult de novo kidney transplant patients randomized to receive reduced-dose cyclosporine and everolimus at 1.5 or 3 mg/day or standard-dose cyclosporine plus EC-MPS at 1440 mg/day in addition to steroids and basiliximab as induction therapy. In that study, investigators specifically reported individual events (laboratory-defined CMV-positive antigenemia, polymerase chain reaction positive), CMV syndrome (fever that lasted 2 days, leukopenia, neutropenia, viral syndrome), and CMV disease (organ involvement) on a form designed to capture such events. A total of 2004 patients were included in these 3 studies (1.5 mg everolimus: 664 patients; 3 mg everolimus: 671 patient; mycophenolate: 669 patients). These 3 groups were similar in age, race, underlying cause of end-stage renal disease, cold ischemia time, donor age and type, and human leukocyte antigen mismatches. Donor and recipient CMV serostatus combinations were similar among the 3 groups, with the most frequent combination being seropositive donor-seropositive recipient; approximatively 17% of patients in each group were seropositive donor-seronegative recipient. The incidence of biopsy-proven acute rejection and percentage of patients receiving CMV prophylaxis after a rejection episode were similar in the 3 groups. In addition, in the B201 and B251 studies, CMV prophylaxis with ganciclovir, CMV hyperimmune globulins, or acyclovir was mandatory for all CMV-seronegative recipients with a CMV-seropositive donation and was recommended after antibody treatment for an acute rejection episode.

In the A2309 study, CMV prophylaxis (≥ 30 d; ganciclovir, CMV hyperimmune globulins, acyclovir, or valganciclovir, according to local practice) was mandatory for all CMV-seronegative recipients who received a kidney from a CMV-seropositive donor; the patients received CMV prophylaxis according to local practices. Slightly more than half of the patients in each of the groups received CMV prophylaxis and most (79%-86%) were seronegative recipients with seropositive donations. The specific CMV prophylaxis agent used did not differ significantly among the 3 groups, and the most common agents used were ganciclovir (53%-59%) followed by valganciclovir (29%-37%).²⁴

The mycophenolate doses given were similar across the studies; mean cyclosporine trough levels were consistently higher for the mycophenolate groups compared with the everolimus groups. Everolimus trough levels ranged from 2.2 to 5.9 ng/mL for the 1.5-mg everolimus group and 4.5 ng/mL for the 3-mg everolimus group.

The authors found that, among recipients who did not receive CMV prophylaxis, a significantly greater proportion of kidney transplant patients who received 1.5 mg everolimus (93.5%) or 3 mg everolimus (95.5%) were free from CMV events (viremia, infections/syndrome, or disease) compared with those who received mycophenolate (85.2%; P < .001). In a Cox proportional hazards model, the adjusted hazard ratio showed that mycophenolate was associated with a significantly higher risk of CMV events (adjusted hazard ratio of 2.81; 95% confidence interval, 1.77-4.5; P < .001). Among patients who received CMV as prophylaxis, a significantly greater proportion of recipients who were given 1.5 mg everolimus (93.9%) or 3 mg everolimus (93.2%) were free from CMV events (viremia, infections/syndromes, or disease) compared with patients treated with mycophenolate. The adjusted hazard ratio from the Cox proportional hazards model for mycophenolate versus everolimus showed that mycophenolate was associated with a significantly increased risk of CMV events (adjusted hazard ratio of 1.82; 95% confidence interval, 1.18-2.8; P = .006). Table 1 shows the proportions within the 1.5-mg everolimus, 3-mg everolimus, and myco­phenolate groups presenting with CMV viremia, CMV infection/syndrome, or CMV with organ involvement according to having or not having CMV prophylaxis.

Regarding the group at highest risk for CMV (seropositive donor-seronegative recipient), there were numerically lower incidences of CMV infection and syndrome with everolimus (16% with 1.5 mg everolimus, 18% with 3 mg everolimus, and 25% with mycophenolate; P = not significant).

Finally, among patients who experienced a CMV event, both 1.5 and 3 mg everolimus were associated with a significantly (P < .001) longer mean time until a first CMV event versus those who received mycophenolate (mean number of days to a first CMV event: 194 d with 1.5 mg everolimus, 190 d with 3 mg everolimus, 124 d with mycophenolate).

Cytomegalovirus Events and Everolimus in Pediatric Patients
Pape and associates reported on 20 children (median age of 12 y; range, 1-17 y) who were initially treated with basiliximab, cyclosporine (aiming at trough levels of 200-250 ng/mL), and steroids.³² At 2 weeks after transplant, cyclosporine doses were reduced by 50% (to trough levels of 75-100 ng/mL) and to 50 to 75 ng/mL after 6 months, with everolimus started at 1.6 mg/m²/day and targeted trough levels of 3 to 6 ng/mL. At 6 months after kidney transplant, prednisone was given on alternate days and then stopped 3 months later. Eighty percent of the recipients were CMV seronegative; CMV prophylaxis was not given to these patients. Although 50% of patients were at risk for CMV events (seropositive donor-seronegative recipient), the authors observed only 2 primary CMV infections (CMV DNAemia detection). In addition, 1 incident of CMV reinfection occurred.

Ettenger and associates reported on a series of 19 pediatric patients (≤ 16 y old) who received steroid immunosuppression, everolimus (0.8 mg/m² twice daily; trough levels ≥ 3 ng/mL), and cyclosporine (trough levels of 200-350 ng/mL until day 30, then 75-150 ng/mL until day 60, and then 50-100 ng/mL).³³ Sixteen of the 19 patients also received basiliximab as an induction therapy. Ten patients were CMV seropositive, and 4 were seronegative recipients with seropositive donations; CMV prophylaxis was mandatory only in the latter category. The authors observed only 1 occurrence of CMV infection.

Everolimus-Based Immunosuppression as a Means to Prevent Cytomegalovirus Infection
Tedesco-Silva and associates recently conducted a randomized clinical trial with de novo kidney transplant patients in which the primary endpoint was the incidence of CMV infection/disease with respect to type of immunosuppression (ie, tacrolimus-based immunosuppression with either everolimus or mycophenolate in the absence of CMV prophylaxis).²⁵ This was a single-center, prospective, randomized, 12-month, open-label controlled trial that aimed to compare the incidence of CMV infection or disease in de novo kidney transplant patients without CMV prophylaxis.

The patients’ immunosuppression regimens were as follows. Group 1 received rabbit antithymocyte globulin (rATG) as a single 3-mg/kg bolus within the first 24 hours after graft reperfusion, tacrolimus 0.05 mg/kg twice daily (trough levels of 3-5 ng/mL), 1.5 mg everolimus twice daily (whole blood trough levels of 4-8 ng/mL), and steroids. Group 2 received intravenous basiliximab at 20 mg on days 0 and 4, tacrolimus 0.1 mg twice daily (trough levels of 3-8 ng/mL) for the first 3 months, which was then reduced to 3 to 5 ng/mL, 1.5 mg everolimus twice daily (trough levels of 3-8 ng/mL), and steroids. Group 3 patients received intravenous basiliximab at 20 mg on days 0 and 4, tacrolimus 0.1 mg/kg twice daily (trough levels of 6-8 ng/mL), steroids, and EC-MPS (720 mg twice daily). Because no CMV prophylaxis was given, the authors administered a preemptive strategy consisting of weekly monitoring of CMV viral replication (pp65-CMV antigenemia test) over a 6-month period.

Regarding the primary endpoint, the authors found that groups that received everolimus-based immunosuppression had significantly less CMV infection/disease (4.7% in group 1, 10.8% in group 2, 37.6% in group 3; P < .001). Similar results were shown for incidence of CMV infection (1.2% for group 1, 3.9% for group 2, 25.7% for group 3; P = .001) and CMV disease (3.5% for group 1, 6.9% for group 2, 11.9% for group 3; P = .037). Moreover, the difference regarding incidence of a CMV event was only statistically significant for seropositive recipients with seropositive donation (2.8% for group 1, 7.5% for group 2, 32% for group 3; P = .001). For seronegative recipients with seropositive donations, although there were numerically less CMV events with everolimus treatment, this difference did not reach significance (60% in group 1, 60% in group 2, 83.3% in group 3).²⁵

Regarding the primary endpoint (CMV event) at 1 year after transplant, patients treated with rATG and everolimus showed a 90% proportional reduction, whereas those treated with basiliximab and everolimus had a 75% proportional reduction in the incidence of CMV infection/disease versus those who received basiliximab and EC-MPS. Moreover, there were no differences in the mean time until the first incidence of CMV infection/disease or the duration of treatment between the 3 groups.When the authors examined the recurrence of CMV infection/disease, they found recurrence in 4.7% of patients treated with rATG and everolimus, 17.6% of patients treated with basiliximab and everolimus, and 57.4% of patients treated with basiliximab and EC-MPS (P < .001). The number of first CMV events after treatment for acute rejection was higher in patients who received basiliximab-everolimus and basiliximab-EC-MPS than in patients who received rATG-everolimus. However, some patients in the latter group developed tissue-invasive CMV. In the basiliximab and EC-MPS group, 12 patients who had recurrent CMV infection/disease were converted from EC-MPS to everolimus.

When the authors performed a multivariate analysis to identify the risk factors for CMV infection/disease, rATG induction versus basiliximab and tacrolimus whole blood trough levels were not risk factors. Conversely, being seronegative with a seropositive donation versus other types (odds ratio of 14.6; 95% confidence interval, 4.16-51.49; P = .001) and treatment for acute rejection (odds ratio of 2.309; 95% confidence interval, 1.063-5.014; P = .03) were independent risk factors for CMV events. In contrast, the use of everolimus (vs EC-MPS) was an independent predictive factor for protection against CMV events (odds ratio of 0.163; 95% confidence interval, 0.072-0.368; P = .001).

Finally, there were no significant statistical differences in the 3 groups regarding acute rejection rates, patient and graft survival rates, proteinuria, and de novo cancer. Of note, anemia and thrombocytopenia occurred at similar rates across the 3 groups, whereas leucopenia was more frequently reported as an adverse event in the EC-MPS group than in the everolimus groups (99% vs 4.7% for rATG-everolimus and 1% for basiliximab-everolimus; P < .05).

In conclusion, until very recently, there has only been indirect evidence that mTOR inhibitors, particularly everolimus, have an anti-CMV effect. However, recent data from a randomized controlled trial have shown for the first time that, in de novo kidney transplant patients, everolimus-based immunosuppression with low tacrolimus and no CMV prophylaxis significantly decreased CMV infection/disease compared with a regimen based on standard tacrolimus plus mycophenolate. Other randomized controlled trials investigating this issue are ongoing. Clinicians should be aware that, in cases of CMV infection after kidney transplant, mycophenolate could be replaced by everolimus to limit CMV-related adverse effects.

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