Even with recent advances in antiviral therapy, cytomegalovirus (CMV) remains the most important pathogen affecting the immunocompromised host (1). CMV infection is usually acquired early in life, and approximately 40% to 70% of the world’s population is seropositive and harbors latent virus. Although primary CMV infection is generally self-limiting in immunocompetent individuals, primary infection or reactivation of latent virus in immunocompromised patients can have debilitating and even life-threatening consequences (1, 2).
CMV infection and disease are commonly observed after solid-organ transplantation, and are leading causes of clinical complications and recipient mortality (3, 4). Recipients who are seronegative for CMV, generally as a consequence of lack of previous virus exposure, are at greatest risk of disease after receiving allografts from infected, seropositive donors (donor positive/recipient negative combinations) (3). Without adequate preventive therapy to control viral replication, it is estimated that approximately 58% to 80% of solid-organ transplant recipients develop active CMV disease (5, 6).
Both invasive disease (“direct effects”) and immunologic phenomena (“indirect effects”) can arise after CMV infection in solid-organ transplant recipients (3). The direct effects can be divided into organ-specific manifestations of viral infection (including nephritis, hepatitis, and carditis) and more generalized symptoms termed “CMV syndrome” (fever, weakness, and myalgia). Indirect effects reflect altered immune responses associated with CMV infection, including allograft dysfunction, acute and chronic rejection, and opportunistic infections (1, 3, 7–9).
It is generally accepted that CMV is best dealt with by prophylactic or preemptive antiviral approaches (10). However, recent clinical trials involving immunosuppressants that target the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus indicate that the agents are not only effective in preventing rejection but also impact on the risk of CMV disease and, therefore, might enable a more novel clinical approach (11–17). Mechanistically, mTOR inhibitors act by blocking the serine-threonine kinase activity of the protein complex mTORC1 (18, 19) by binding to the intracellular mTOR receptor FKBP12 (18, 20). The mTOR inhibitor-FKBP12 complex subsequently binds to mTOR, thereby preventing its association with the essential mTORC1 component Raptor (18), leading to inhibition of translational processes dependent on mTORC1 activity (19). Hence, blocking the mTOR pathway prevents cell-cycle progression from G1 into S phase (21) in cytokine-stimulated T-cells, which largely accounts for the immunosuppressive potency of mTOR inhibitors. Emerging evidence also suggests that mTOR inhibitors may exhibit antiviral effects through interruption of certain mTORC pathways or by invoking immune deviation.
Retrospective analysis of CMV infection and immunosuppressive strategies is difficult because of potential bias and other obstacles such as lack of infection definitions and inhomogeneous prophylaxis. Despite these impediments, findings supporting an important differential impact on CMV disease are accumulating from studies using mTOR inhibitors in comparison with non-mTOR inhibitor-based regimens. This article will summarize a number of these clinical trials, with a focus on whether use of mTOR inhibitors can exert a beneficial impact on CMV infection and disease after kidney transplantation.
Immunosuppressive Therapy as a Risk Factor for CMV Infection and Disease
Immunosuppressive regimens that do not block mTOR can delay CMV-specific immune responses, thereby contributing to the incidence of CMV infection and disease in renal transplant recipients (Table 1). This finding is particularly apparent for lymphocyte-depleting agents, including muromonab-CD3, antithymocyte globulin, and alemtuzumab (22). Further studies have suggested that combinations of specific drug therapies may increase the risk of infection and disease after renal transplantation. For example, cyclosporine-based regimens have been shown to increase the incidence of CMV infection. In the ELITE-Symphony study with 1645 renal transplant recipients, 14.3% and 11.0% of recipients who received standard and low-dose cyclosporine regimens, respectively, reported a CMV infection adverse event, whereas a lower incidence (9.7%) was observed with a low-dose tacrolimus-based regimen (23). Furthermore, in a Spanish Network for Research on Infection in Transplantation (RESITRA) network study involving 1470 renal transplant recipients, cyclosporine was significantly associated with the occurrence of CMV disease (odds ratio [OR] 1.70; 95% confidence interval [CI] 1.10, 2.90) (24). In this study, CMV disease was defined according to predefined RESITRA criteria (for details see Table 1). Using a prospective definition of CMV disease (Table 1), a further study found a higher incidence of CMV disease episodes associated with the use of mycophenolate mofetil (MMF) combination therapies compared with azathioprine-based regimens (25). This finding is supported by the results of several clinical trials that demonstrated that tissue-invasive CMV disease was more commonly observed in transplant recipients who received 3 g/day MMF compared with 2 g/day MMF and azathioprine (26–28).
In contrast to these studies, a significantly higher incidence of CMV infection has been observed following the administration of a thymoglobulin/azathioprine-based regimen in comparison with two basiliximab/MMF combination treatments (Table 1) (29). However, this finding could have been due to the lower cyclosporine dose administered to the recipients who received basiliximab/MMF therapy and/or the potency of depletional induction, emphasizing the complex interactions present in various immunosuppressive regimens.
CMV Findings From Comparative De Novo Studies Evaluating mTOR Inhibitors
CMV findings have been evaluated in several clinical studies comparing the use of sirolimus- or everolimus-based immunosuppressive regimens with non-mTOR inhibitor-based regimens in de novo renal transplantation. The mTOR inhibitor data discussed in this review were obtained from the PubMed biomedical literature database (Tables 2 and 3 for search term and selection criteria details). Only randomized trials were included and no meta-analyses were performed on the publications identified. The studies identified consistently demonstrated low rates of CMV infection with both sirolimus- and everolimus-based regimens in comparison with a number of immunosuppressive regimens (Tables 2 and 3).
Several similarities in study design can be observed across the investigations conducted with both sirolimus and everolimus. In general, the mTOR inhibitors were initially administered within 48 hr of transplantation and CMV prophylaxis was specified in most studies. Various prophylactic treatments were used, including ganciclovir, valacyclovir, acyclovir, valganciclovir, and CMV hyperimmune globulin. Although most of these therapeutics provide effective protection from CMV infection (30), a number of studies have now indicated that oral ganciclovir and valganciclovir are more effective than oral acyclovir or valacyclovir (31–33). CMV infection was typically reported as an adverse event and not prospectively defined, although one study did include an a priori goal of assessing risk of CMV infection (16).
The ELITE-Symphony study compared renal function, efficacy (acute rejection and allograft survival), and relative toxic effects of four immunosuppressive regimens: a standard-dose and a low-dose cyclosporine-based regimen, a low-dose tacrolimus-based regimen, and a low-dose sirolimus-based regimen (23). Despite significantly more rejection and antirejection treatment, the lowest rate of CMV infection was observed with the sirolimus-based regimen (6.1%), followed by the low-dose tacrolimus, low-dose cyclosporine, and standard-dose cyclosporine regimens (9.7%, 11.0%, and 14.3%, respectively; P = 0.003). The tacrolimus results should be interpreted with caution, however, as the standard-dose tacrolimus regimen currently used in many transplantation centers was not included (34).
Two clinical trials compared a sirolimus-MMF-corticosteroid regimen with a tacrolimus-MMF-corticosteroid regimen after renal transplantation (35, 36). The aim of these studies was to investigate recipient and graft survival, acute rejection, renal function, and adverse events. In both studies, the incidence of CMV infection was higher for tacrolimus-treated patients. In the first study, 10 cases of systemic CMV infection were diagnosed in the tacrolimus group (12%) compared with two cases in the sirolimus group (3%; P = 0.02) (35). All of the cases developed in CMV-seronegative recipients who received kidneys from CMV-seropositive donors. In the second study, a single recipient (1.4%) was diagnosed with a CMV infection in the sirolimus group compared with 14 recipients (20%) in the tacrolimus group (P < 0.001) (36).
Two large RESITRA network trials have been analyzed to assess possible risk factors for CMV disease. The results of the study published by San Juan et al. (24) showed that the risk of acquiring CMV disease was significantly lower following the administration of sirolimus (OR 0.27; 95% CI 0.10, 0.78). The positive effect observed with sirolimus treatment was supported by the results of a more recent analysis by Fortun et al. (37). A maintenance regimen that included sirolimus was independently associated with a lower risk of CMV infection (OR 0.16; 95% CI 0.05, 0.54).
As with sirolimus-based regimens, less CMV infection and disease have also been observed with everolimus-based therapy compared with other non-mTOR inhibitor-based regimens. Study A2309, a 24-month Phase IIIb trial, examined the efficacy and safety of two everolimus-based regimens with reduced-exposure cyclosporine and a mycophenolic acid (MPA)-based regimen with standard-exposure cyclosporine (16). The first dose of immunosuppressive therapy was administered within 24 hr posttransplantation. CMV prophylaxis (ganciclovir, CMV hyperimmune globulin, acyclovir, or valacyclovir) was mandatory for all CMV-negative recipients who received a kidney from a CMV-positive donor. The study also aimed to investigate the incidences of CMV syndrome, CMV disease, and CMV infection. At 12 months, lower rates of CMV infection were obtained with both of the everolimus-based regimens compared with the MPA-based regimen: 0.7% and 0.0% for the 1.5 and 3 mg everolimus groups in comparison with 5.9% for the MPA group (Table 3). The incidence of CMV syndrome and CMV disease was also lower for both everolimus arms. Further analysis revealed that the incidence of CMV infection was reduced in recipients with a positive CMV serologic status at baseline, but not in those with a negative CMV status (38).
The efficacy and safety of everolimus-based regimens have been compared with an MMF-based regimen in two 36-month trials: B201 and B251 (11, 17, 39). In both studies, the recipients were randomized to receive treatment with 1.5 mg everolimus, 3 mg everolimus, or MMF. All recipients received concomitant treatment with cyclosporine and steroids as part of their immunosuppressive regimen. In both studies, all high-risk recipients received CMV prophylactic treatment with ganciclovir, CMV hyperimmune globulin, or acyclovir, and the other recipients received CMV prophylaxis according to local practice. In study B201, 5.7%, 8.1%, and 19.9% of recipients had a CMV infection after immunosuppressive treatment with the 1.5 mg everolimus-, 3 mg everolimus-, and MMF-based regimens, respectively (P = 0.0001 for each everolimus regimen in comparison with MMF) (17). In study B251, comparatively lower CMV infection rates of 5.2%, 4.1%, and 6.1% were reported following treatment with the 1.5 mg everolimus-, 3 mg everolimus-, and MMF-based regimens, respectively (11). The disparity between the two studies may have been due to differing local practice of CMV prophylaxis, which contributed to a higher proportion of recipients receiving prophylactic treatment in study B251 than in study B201 (Novartis Pharma AG, data on file). In study B251, CMV prophylaxis was received by 71% of recipients in both the 1.5 mg everolimus and MMF groups, and 78% of recipients in the 3 mg everolimus group. Comparatively, in study B201, 21% of recipients in the 1.5 mg everolimus group, 20% of the recipients in the 3 mg everolimus group, and 23% of the recipients in the MMF group received prophylaxis (40). The variability in CMV infection rates observed in these two studies contributed to CMV infection, CMV disease, and CMV syndrome being prospectively defined in study A2309.
The use of an mTOR inhibitor as part of the immunosuppressive regimen could also be considered in recipients with CMV infection resistant to antiviral therapy. In a study of nine renal transplant recipients who had ganciclovir-resistant CMV infection (41), a rapid decrease in antigenemia levels was observed after conversion to sirolimus and ganciclovir administration, and none of the recipients experienced acute rejection or CMV recurrence.
CMV Findings From Comparative Conversion Studies Evaluating a Switch to mTOR Inhibitors
Other investigations have shown that the beneficial findings observed with mTOR inhibitors in the de novo setting may not be apparent in immunosuppression conversion studies following renal transplantation. In the recently completed ZEUS study, rates of CMV infection were similar after immunosuppressive therapy with cyclosporine and delayed everolimus (19% and 18%, respectively) (42). Recipients were randomized in ZEUS to continue with cyclosporine or receive an everolimus-based regimen following an initial 4.5 months of immunosuppressive therapy. In the CONCEPT study, similar incidences of CMV infection were reported with a cyclosporine-based regimen or a delayed sirolimus-based regimen (6% and 4%, respectively) (43). In this study, all the recipients initially received immunosuppressive treatment with a cyclosporine-based regimen before being randomized after 3 months to continue with cyclosporine or switch to a sirolimus-based regimen. In the CALLISTO study, when maintenance everolimus was compared with de novo everolimus, CMV infection rates of 6.8% and 1.5%, respectively, were observed for recipients who received everolimus 5 weeks after transplantation after 4 weeks of therapy with MMF and recipients who received everolimus the day after renal transplantation (44). A retrospective study also showed that the use of sirolimus-based maintenance immunosuppression was not associated with a reduced incidence of CMV disease (OR 0.76; 95% CI 0.30, 1.90) (45).
In contrast, statistically lower CMV infection rates were obtained with a sirolimus-based regimen compared with a cyclosporine-based regimen in the SMART immunosuppression conversion study (7.3% and 28.2%, respectively; P = 0.0016) (46). This finding may have been due to the fact that the switch to sirolimus-based therapy occurred shortly after transplantation (10–24 days posttransplant).
Anti-CMV Mechanisms of Action of mTOR Inhibition
As only a limited number of preclinical studies have investigated the anti-CMV mechanisms of mTOR inhibitors, several potential molecular mechanisms may account for the anti-CMV potency of mTOR inhibitors at the cellular level (Fig. 1).
Activation of mTOR in host cells is essential for CMV to successfully propagate translation of viral proteins, even under conditions that normally block mTOR activity, such as cellular stress, which is regularly associated with the process of viral entry (18, 47). A recent study with the mTOR kinase inhibitor Torin1, which blocks both mTORC1 and mTORC2 (48), demonstrated the complex involvement of mTOR during CMV infection (49). Torin1 is capable of not only decreasing the accumulation of viral DNA but also dramatically reducing the levels of the pUL99 viral late protein (18). Furthermore, it was demonstrated that inhibition of mTORC1 prevented the accumulation of immediate early, early, and late viral proteins (49). However, blocking mTORC1 activity at very early time points after viral infection resulted in the most profound effects on viral translation and overall infection efficiency compared with later time points. These results indicate a dynamic relationship of mTORC1 activation and CMV infection that is also potentially susceptible to the effects of pharmacologic mTOR inhibition.
Further studies on the multifunctional role of mTOR within the immune system suggested that mTOR inhibitors may also exert their antiviral effect by influencing immune-mediated responses. Interestingly, recent studies have demonstrated that mTOR inhibitors regulate CD8 memory T-cell formation, because inhibition of the mTOR pathway enhanced not only the quantity but also the quality of virus-specific CD8+ T-cells (20, 50). Using sophisticated models, a recent investigation demonstrated that the environment in which an antigen is presented alters the influence of sirolimus on antigen-specific T-cell expansion (20, 51). Following sirolimus monotherapy, the antigen-specific CD8+ T-cell response was inhibited in response to graft transplantation and augmented in response to viral or bacterial pathogens.
Other components of the innate immune system, including γδ T-cells, may also be affected by the administration of mTOR inhibitors. A dramatic expansion in the number of γδ T-cells occurs in the peripheral blood of renal allograft recipients following the development of a CMV infection (52). Additionally, γδ T-cells are capable of killing CMV-infected target cells, producing interferon-γ, and limiting CMV propagation in vitro (52, 53). Recent investigations have demonstrated that the introduction of sirolimus increased the proliferation of treated and antigen-exposed γδ T-cells in vitro (54).
The inhibition of mTOR may affect innate immune cells such as monocytes, macrophages, and dendritic cells (20, 55). The production of pro-inflammatory cytokines such as interleukin (IL)-12 and IL-23 is substantially increased, whereas the classical anti-inflammatory cytokine IL-10 is suppressed after sirolimus treatment (55, 56). Recent studies investigating the growth of Epstein-Barr virus-positive lymphomas have suggested that sirolimus may exert its effect on IL-10 by inhibiting the phosphorylation of the mTOR substrate p70-S6 kinase (57, 58). Several investigations have discovered that early events associated with the invasion of CMV trigger host production of pro-inflammatory cytokines such as IL-12 (59–61). This effect is countered by CMV through the production of a viral homolog of IL-10 and the suppression of host IL-12 production, which limits the production of TH1-specific interferon-γ producing T-cells (55, 62, 63). Interestingly, a further study also identified a relationship between CMV reactivation after kidney transplantation and a single nucleotide polymorphism in the host IL-12p40 gene (64). These findings suggest that the blocking of mTOR through the administration of everolimus and sirolimus might inhibit the potent viral host evasion strategies used by CMV.
The occurrence of CMV infection and disease is associated with significant clinical illness, allograft loss, and mortality after renal transplantation (65). Despite encouraging results using candidate CMV vaccines (66), antiviral therapy remains the mainstay of patient management for CMV. However, several studies indicate that specific immunosuppressive agents may contribute to the incidence of CMV infection and disease observed in renal transplant recipients.
The evidence summarized above indicates that the mTOR inhibitors may actually decrease the incidence of CMV infection and disease experienced by transplant recipients relative to other protocols. CMV infection in de novo renal transplant recipients was significantly lower following the use of sirolimus-based regimens in comparison with both cyclosporine- and tacrolimus-based regimens (15, 23, 35, 36, 67). Likewise, significantly less CMV infection has been observed after immunosuppressive treatment with everolimus in comparison with MMF (17, 40).
In light of the relationship between mTOR inhibitors and the reduced risk of CMV infection in de novo renal transplant recipients, large Phase III clinical trials are required to evaluate the relative risk of infection and disease with mTOR inhibitors in comparison with other immunosuppressive therapies (cyclosporine, tacrolimus, and MPA). CMV infection as a clinical end point should be rigorously defined and the prospective incidence of infection should be investigated, along with the prevalence of CMV syndrome and disease.
In keeping with the findings in the renal transplantation setting, mTOR inhibitor-based regimens are also associated with a reduced incidence of CMV infection and disease in cardiac and hepatic transplant recipients. A significantly lower incidence of any CMV event was observed for everolimus plus reduced-exposure cyclosporine in comparison with MMF plus standard-exposure cyclosporine in de novo cardiac transplant recipients (8.8% and 32.5%, respectively; P < 0.001) (68). Significantly lower rates of CMV infection, CMV syndrome, and organ involvement were also observed. A low incidence of CMV disease (2%) was reported after the use of a prednisone-free, sirolimus-based immunosuppressive regimen in 150 liver transplant recipients (69). The incidence of CMV disease was lower than in the other recipient groups who had received conventional CMV prophylactic treatment.
In conclusion, clinical evidence from comparative studies demonstrate that early use of mTOR inhibitor-based regimens can reduce the incidence of CMV infection and so impact on the immediate and long-term clinical sequelae associated with CMV. Further investigation of this observation should include randomized trials with homogeneous antiviral prophylaxis, standardized definitions, and adequate power to confirm or refute the observations.
The authors thank Mike Parsons of Complete Health Vizion for provision of medical writing assistance; this assistance was funded by Novartis Pharma AG. The authors thank Dr. Heike Schwende, Medical Communication, Novartis Pharma AG, for review of the manuscript.
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