Objectives: Few data regarding viral replication in patients receiving belatacept are available. The aim of this single-center study was to compare the incidence of viral infections (cytomegalovirus, Epstein Barr virus, BK virus, and JC virus), in 62 de novo kidney transplant patients enrolled in the BENEFIT studies, receiving either belatacept (n=42) or cyclosporine (n=20).

Materials and Methods: By means of polymerase chain reaction, belatacept-treated patients were tested for cytomegalovirus, Epstein-Barr virus, BK virus, and JC virus infections monthly for 36 months, monthly for the first 6 months, and then quarterly for 36 months in cyclosporine-treated patients. Additional samples were obtained when a viral infection was suspected.

Results: The number of positive cytomegalovirus, BK virus, or JC virus viremias over the number of polymerase chain reactions performed through all 3 years was similar in both groups. Conversely, over the 3-year study, the number of positive Epstein-Barr virus viremias over the number of Epstein-Barr virus polymerase chain reactions performed was significantly higher in the belatacept group (76% vs 50%; P = .047). The number of Epstein-Barr virus primary infection was similar in both groups, while the number of Epstein-Barr virus reactivation was higher in the belatacept group.

Conclusions: Epstein-Barr virus replication occurs more often in patients receiving belatacept, than it does in those receiving cyclosporine.

Key words : Belatacept, Cytomegalovirus, Epstein Barr virus, BK virus, JC virus, Kidney transplant

Introduction

Kidney transplant, the standard of care for patients with end-stage renal disease, improves survival and quality of life.¹ Although advances in posttransplant immunosuppressive management have reduced the rates of acute rejection and improved 1-year outcomes, commensurate improvements in long-term kidney-allograft survival have not been observed.^2,3 The commonly used calcineurin inhibitors (CNI), effective for immunosuppression, may contribute to late allograft loss and death. Calcineurin inhibitors are potentially nephrotoxic and have adverse effects on blood pressure, lipid levels, and glucose homeostasis.^4,5 Thus, CNIs promote cardiovascular disease, the most common cause of death among transplant recipients, as well as interstitial fibrosis and tubular atrophy, and are the most common cause of late graft loss among surviving patients.^6,7

Current long-term outcomes for kidney transplant recipients indicate a need for selective immuno-suppressants that improve long-term patient and allograft survival by avoiding the chronic toxicities of nonselective immunosuppressive therapies. Multiple small molecules and biological agents are currently being studied.8 Blockade of T-cell costimulation is a promising approach^9,10 and has been intensively investigated since the development of belatacept.

Belatacept, a selective costimulation blocker, binds to the surface costimulatory ligands (CD80 and CD86) of antigen-presenting cells. In the context of antigen recognition (signal 1), the interaction of CD80 and CD86 with the surface costimulatory receptor, CD28, of T cells (signal 2), is required for the latter’s full activation. Blockade of signal 2 inhibits T-cell activation, thereby promoting anergy and apoptosis.11 Belatacept is a human fusion protein that combines the extracellular portion of cytotoxic T-lymphocyte–associated antigen 4 (CTLA4) with the constant-region fragment (Fc) of human IgG1 (CTLA4Ig).^12-14

Several phase 2 and phase 3 clinical trials,^15-18 which are investigating the use of belatacept in de novo kidney transplants, found that belatacept was effective at preventing acute rejection and preserving kidney function by avoiding CNI-induced nephrotoxicity. It also minimizes metabolic adverse events, which could result in improved long-term cardiovascular outcome. However, the incidence of Epstein-Barr virus (EBV)-induced posttransplant lymphoma disease (PTLD) is numerically greater in patients receiving belatacept compared with those treated with cyclosporine^17-18; this has mainly occurred in recipients who are EBV-seronegative whereas their donor is EBV seropositive.

Viral infections are quite common in transplant patients receiving immunosuppressive drugs. For instance, cytomegalovirus (CMV) infections are more frequently observed in patients receiving CNIs compared with those receiving mammalian target of rapamycin inhibitors (mTOR-I)¹⁹ and in patients receiving mycophenolic acid compared with azathioprine.²⁰ Incidence of BK virus (BKV) replication and nephropathy is significantly higher in patients receiving tacrolimus than it is in those receiving cyclosporine.²¹ However, few data regarding viral replication and infection in patients receiving belatacept are available.

The aim of this single-center study was to compare the incidence of viral infections, notably CMV, EBV, BKV, and JC virus (JCV), in de novo kidney transplant patients receiving either belatacept-based or cyclosporine-based immunosuppression.

Materials and Methods

The population in this study represents the 62 patients enrolled at our transplant center in Toulouse in the BENEFIT (Belatacept Evaluation of Nephro-protection and Efficacy as First-line Immuno-suppression Trial) and BENEFIT-EXT (Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial—EXTended criteria donors) studies between December 2005 and May 2007, who were followed for at least 3 years. BENEFIT and BENEFIT-EXT are 3-year, randomized, partially blinded, phase 3 studies in which patients have been randomized 1:1:1 to receive a more intensive regimen of belatacept, a less intensive regimen of belatacept, or cyclosporine as the primary maintenance immunosuppressive therapy. The study was blinded to patients and study personnel with respect to assignment of belatacept-dose regimen, and was open-labeled with respect to allocation to belatacept or cyclosporine, primarily because of the need for therapeutic-dose monitoring in cyclosporine-treated patients. Each patient was treated with an induction of basiliximab (20 mg IV on days of transplant and 4, oral mycophenolate mofetil (MMF) (2 g/d) in 2 divided doses, and steroids (tapered to ≥ 2.5 mg/d by posttransplant month 6). In the BENEFIT study, patients who had received an extended-criteria donor-kidney transplants were excluded, unlike in the BENEFIT-EXT study, which enrolled this category of patient.^17,18 All patients were given sulfametho-xazole/trimethoprim for pneumocystis jirovecii prophylaxis for the first 6 months posttransplant.

By means of molecular tools, all patients were tested prospectively for CMV, EBV, BKV, and JCV infections. Whole blood samples were collected monthly for 36 months just before belatacept infusion in the belatacept-treated patients, and monthly for the first 6 months, then quarterly for 36 months during follow-up visits in cyclosporine-treated patients. Additional samples were obtained when a viral infection was suspected. Cytomegalovirus prophylaxis by valganciclovir was given for the first 3 months posttransplant in CMV-seropositive recipients, as well as in CMV-seronegative recipients whose donor was CMV seropositive. Viral infection was defined as a positive DNAemia. Viral disease was defined as the presence of a positive DNAemia with clinical symptoms.

Virologic methods
Quantitative real-time PCRs for CMV, EBV, BKV, and JCV were performed according to previously published methods.^22-24

Statistical analyses
Analyses were performed according to intention-to-treat, with data included from all patients who underwent a transplant. Reported values represent either means (± SD) or median (ranges). Quantitative variables were compared using the t test. Proportions were compared using the Fisher exact test. A value of P ≤ .05 was considered statistically significant.

Results

Characteristics and disposition of patients
A total of 62 patients were included: 42 of these were randomized into the belatacept regimen (group 1, and either more intensive or less intensive), and 20 into the cyclosporine (group 2) regimen (Figure 1). Recipient-baseline and donor-baseline characteristics were well balanced. The mean age of recipients was 48.4 ± 13.4 years in group 1 and 47.5 ± 15 years in group 2 (P = NS). There were 25 males in group 1 and 14 in group 2 (P = NS). Mean duration of dialysis was 44.6 ± 30.4 months in group 1 and 43.1 ± 28.8 months in group 2 (P = NS). All patients had negative panel-reactive antibodies, and all were recipients of a first kidney transplant. The numbers of HLA A/B mismatches (2.4 ± 0.94 in group 1 and 2.3 ± 0.98 in group 2), HLA DR/DQ mismatches (1.44 ± 1.99 in group 1 and 1.6 ± 1.35 in group 2), and numbers of total HLA mismatches (4.38 ± 1.6 in group 1 and 3.9 ± 1.8 in group 2) were similar in both groups.

There was no difference in mean donor age between either group (47.02 ± 15.1 in group 1 and 47.6 ± 15.9 in group 2). Twenty-two donors in group 1 were males compared with 11 in group 2 (P = NS), and 37 donors were deceased donors in group 1, compared with 20 in group 2 (P = NS).

The rate of immunosuppressive discontinuation of study medication during the 36 months posttransplant follow-up was significantly different between the 2 groups: 14% in group 1 and 50% in group 2 (P = .005). Of the 6 group 1 patients who discontinued the study, 3 were switched to tacrolimus for steroid-resistant acute rejection, 1 had posttransplant lymphoproliferative disorder (PTLD), 1 had colon cancer, and 1 had ganciclovir-resistant CMV disease. Of the 10 group 2 patients who discontinued the study, 7 were switched to tacrolimus for steroid-resistant acute rejection, and 3 were switched to mTOR-I for CNI-related toxicity (Figure 1).

Patient-survival and graft-survival rates
There was no statistical difference between the incidence of graft-survival and patient-survival rates and the time from transplant until patient death or graft loss. Graft losses, 1 from group 1 and the other from group 2, were due to noncompliance to immunosuppression medications intake. One group 1 patient died of central nervous system lymphoma and one group 2 patient died of septic shock.

Incidence of cytomegalovirus infection
Cytomegalovirus donor (D) and recipient (R) status, notably D+/R+, D+/R–, D-/R+, and D–/R-, number of patients on CMV prophylaxis, and duration of prophylaxis were comparable between both groups. The incidence of CMV infection was 47% in group 1 and 45% in group 2 (P = NS). Primary infection rate in D+/R– CMV-status patients was 54% in group 1 and 45% in group 2 (P = NS). Seventy-six percent of CMV seropositive recipients in group 1 and 65% from group 2 had reactivation of CMV (P = NS).

Resistance to ganciclovir was comparable between both groups. There was no significant difference between the 2 groups concerning the time since transplant until the first positive CMV DNAemia was detected, or the time from prophylaxis withdrawal until the first CMV DNAemia was detected. The number of positive CMV DNAemias over the numbers of CMV PCRs performed during the first, second, and third years, and through all 3 years, as well as the number of treated CMV DNAemias over the numbers of CMV PCRs performed during the first, second, and third years, and through all 3 years, were similar in both groups (Table 1). These results were not changed when these 5 parameters were compared in D+/R– patients from the 2 groups.

Incidence of Epstein-Barr virus infection
Epstein-Barr virus donor and recipient serostatus, that is, D+/R+, D+/R–, D–/R+, and D–/R–, were similar in both groups. Primary infection occurred in 2 of the 3 EBV D+/R– belatacept-treated patients and 2 of the 4 EBV D+/R– cyclosporine-treated patients (P = NS). Rate of EBV reactivation was numerically different in both groups: 78% in group 1 vs 50% in group 2 (P = .056). Rate of EBV infection (primary infection or reactivation) was statistically higher in the belatacept group, that is, 76% vs 50% (P = .047). There was no significant difference between the 2 groups concerning the time from transplant until detection of the first positive EBV DNAemia. The number of positive EBV DNAemias over the numbers of EBV PCRs performed during the first, second, and third years, and through all 3 years, as well as the number of treated EBV DNAemias over the numbers of EBV PCRs performed during the first, second, and third years, and through all 3 years, were similar in both groups (Table 2). Over the 3-year period there were 2 cases of PTLD in group 1 in EBV seropositive patients and none in group 2. Both cases of PTLD were located within the central nervous system (CNS); both were EBV-related. One patient was a 71-year-old man who was seropositive for CMV and EBV at transplant, who had CMV reactivation at month 10 posttransplant, and EBV reactivation at month 3. He did not have an acute rejection of his graft. Diagnosis of B-cell CNS lymphoma was made by month 13 and he died soon after. The second patient was 44 years old, seropositive for EBV at transplant with reactivation at month 8, was seronegative for CMV with a seropositive CMV donor, and had a flair-up of CMV disease at month 6 posttransplant. He was treated, at 2 months posttransplant, with anti-thymocyte globulins for steroid-resistant rejection. He had JC viremia at posttransplant month 15 and was diagnosed with B-cell CNS lymphoma by M16. He was treated by chemotherapy, radiotherapy, and withdrawn of all immunosuppressants except the steroids. At the time of this writing, he is alive 5 years later with a functioning graft.

Incidence of BK virus infection
A rate of 4.7% of belatacept-treated patients developed BKV nephropathy compared with 5% who were treated with cyclosporine (P = NS). Median time from transplant until detection of the first positive BKV DNAemia was 8 months in the belatacept group compared with 4 months in the cyclosporine group (P = NS). The number of positive BKV viremias over the numbers of BKV PCRs obtained during the first, second, and third years, and through all 3 years, were similar in both groups (Table 3).

Incidence of JC virus infection
Over the 3-year period, 5 patients were found to have JCV viremia, and all were within the belatacept group, although there was no statistical difference between either group in the number of positive JCV viremias over the number of JCV PCRs performed in the first, second, and third years, or through all 5 years. All patients had other viral infections: 4 cases of CMV, 4 of EBV, and 1 mixed EBV, CMV, and BKV. None of the patients with JCV DNAemias developed progressive multifocal leukoencephalopathy.

Maintenance immunosuppression combined with belatacept or cyclosporine
There was no imbalance in dose levels of either mycophenolate mofetil or of steroids in either group. In group 1, nine patients stopped MMF: 5 because of CMV infection, 2 because of BKV nephropathy, 1 for PTLD, and 1 after graft loss. In group 2, three patients stopped MMF: 2 because of BKV nephropathy and 1 for graft loss (data not shown).

Kidney function
Glomerular-filtration rate was estimated by creatinine clearance using the modification of diet in renal disease equation. Creatinine clearance was numerically higher through the total 3-year follow-up of group 1 patients; however, the difference was statistically significant between the 2 groups only at 1 year posttransplant, that is, 56 ± 13 mL/min (belatacept group) vs 45 ± 18 mL/min (cyclosporine group; P = .009). There was no statistical imbalance in either the incidence of acute rejection (steroid-sensitive, steroid-resistant, or humoral) or the time from transplant to the occurrence of the first rejection between the 2 groups (Table 4).

Discussion

The number of solid-organ transplants conducted has increased worldwide since the first successful human-kidney transplant was performed in 1954.²⁵ As immunosuppressive agents and graft-survival rates have improved, infection and malignancy have become the main barriers to disease-free survival after organ transplant.²⁶ As a result of the growing population of immunosuppressed patients with prolonged survival, an increased incidence and spectrum of opportunistic infections is now observed among other viral infections.²⁷ The aim of this study was to look, by means of a systematic and very frequent PCR monitoring, at the effect of belatacept used as a maintenance therapy in kidney transplant patients, on CMV, EBV, BKV, or JCV infections, compared with cyclosporine therapy.

Cytomegalovirus is the most serious virologic agent to affect transplant recipients. The incidence of CMV infection is around 60% and the incidence of CMV disease is 25%.²⁸ Most infections begin 1 to 6 months after transplant. Despite antiviral prophylaxis among high-risk recipients, particularly CMV-seronegative recipients of organs from CMV-seropositive donors (CMV D+/R–), a large number (estimated range, 15%-38%) still develop delayed onset CMV disease soon after completion of antiviral prophylaxis.²⁹ The serologic status of pretransplant recipients determines both the incidence and severity of disease.^30,31 The clinical effects of CMV infection are the infectious syndromes produced by the virus itself, predisposition to super-infections by opportunistic pathogens, and the virus’s possible implications in allograft dysfunction, new onset diabetes mellitus, and increased patient mortality.^32,33

Cytomegalovirus infection has been associated with increased immunosuppression exposure to induction with anti-thymocyte globulins and muromonab, and to the occurrence of rejection (and its treatment). Alemtuzumab induction, together with tacrolimus therapy, increases the incidence of CMV infections when compared with tacrolimus plus MMF, but none of these infections were tissue invasive.³⁴ In some studies, treatment with MMF increased the incidence of CMV infection as compared with placebo or AZA or everolimus.^35,36 Glotz and associates³⁷ observed that, in anti-thymocyte globulin induction with tacrolimus versus sirolimus maintenance therapy, the rate of CMV infection was statistically higher in the tacrolimus group. Webster and associates³⁸ observed that when mTOR inhibitors replace antimetabolites, the risk of CMV infection was reduced by 51%. In the present study, in which a systematic PCR monitoring was performed, the incidence of CMV infection over 3 years was comparable between both groups, and was concordant with the results of the belatacept phase 2 and phase 3 trials,¹⁵ in which there was no mandatory monitoring for CMV infection. In these later studies, only CMV infections declared by the investigators were considered.

Epstein-Barr virus is a herpes virus with a double-stranded DNA core that targets B lymphocytes. The critical effect of EBV in organ-transplant recipients is its role in the pathogenesis of posttransplant lymphoproliferative disorders (PTLD). In adult solid-organ recipients, PTLD has been reported in up to 2.3% of kidney transplants.³⁹ Over a 10-year period, the risk of PTLD in kidney transplant recipients was 12-fold higher than it was in the general population.⁴⁰ It was highest during the first year, after which the rate of PTLD decreased.^40,41 It is associated with a reported mortality of 40% to 60%.⁴² The risk factors for PTLD include pretransplant EBV serologic status and immunosuppression. Allograft recipients who are EBV-seronegative before the transplant, and receive an organ from a seropositive donor, are at the greatest risk. Induction or rejection therapy, with monoclonal or polyclonal antibodies, increases the risk of PTLD,^40,43 whereas MMF maintenance immuno-suppression has been associated with a significantly decreased risk of PTLD, perhaps because of its role in preventing EBV DNAemia.^43,44

In a pooled belatacept analysis of 1425 intent-to-treat patients (more intensive belatacept, n=477; less intensive belatacept, n=472; cyclosporine, n=476), with a median follow-up of 2.4 years, a total of 15 cases of PTLD, including 8 cases that involved the CNS, were reported.⁴⁵ Of the 15 PTLD cases, 8 occurred in the more intensive belatacept treatment, 5 in the less intensive belatacept therapy, and 2 in patients receiving cyclosporine. Of the 8 CNS PLTD cases, 6 were reported in the more intensive belatacept group and 2 were from the less intensive belatacept group.⁴⁵ Risk factors for PTLD included primary EBV infection after transplant into a seronegative recipient of an allograft from a seropositive donor, allograft rejection, and exposure to antilymphocyte preparations, and CMV coinfection.⁴⁵

In our study, the number of EBV infection, that is, primary infection or reactivation, was significantly higher in the belatacept group. However, EBV seronegative patients from the belatacept group did not have a greater number of EBV primary infections compared with EBV seronegative patients receiving CNIs. Conversely, there was a higher rate of EBV reactivations in seropositive patients from the belatacept group compared with those receiving CNIs. Two patients in our population had PTLD: both were within the belatacept group and both had it localized within the CNS. Both cases of PTLD were located within the CNS; both were EBV-related. One patient died before any specific therapy could be given. The other patient is still alive 5 years later with a functioning graft; he was treated by chemotherapy, radiotherapy, and withdrawal of all immuno-suppressants except the steroids. Because belatacept does not selectively target alloreactive T lymphocytes, and must be combined with classic immuno-suppressive drugs. It therefore increases the risk for infectious and neoplastic complications, particularly posttransplant lymphoproliferative disorders.⁴⁶

The use of potent immunosuppressive therapies has coincided with the emergence of polyomavirus infection and nephropathy, which are associated with the BK virus. The prevalence of BKV viruria and BKV viremia in renal-transplant recipients is around 40% and 12%.²³ BK virus nephropathy, which affects about 8% of recipients, manifests as an asymptomatic gradual rise in serum creatinine levels and results in allograft loss or permanent dysfunction in 40% to 60% of cases.⁴⁷ The diagnosis is based on detection of viruria, viremia, and histologic findings from a kidney biopsy.⁴⁸ Immunosuppression with tacrolimus and MMF has been considered the most important risk factor for infection.⁴⁹ However, it has been reported with any triple-drug regimen, and perhaps the overall load of immunosuppressive drugs is the most important risk factor.⁵⁰ Our results showed an incidence of 5% BK nephropathy in both groups, with no significant difference in the incidence of BKV viremia between either group.

There is little information on JCV infection in renal transplant patients. The seroprevalence of JCV reaches about 50% by the age of 60 to 69 years.²⁴ Five of our patients had JCV viremia and were in the belatacept group, but the incidence between the 2 groups was not statistically significant. All patients had other viral infections: 2 cases of CMV, 2 of EBV, and 1 mixed EBV, CMV, and BKV. No patient developed progressive multifocal leukoencephalopathy, regardless of the viral load. Detection of JCV may indicate an over-immunosuppressed patient.

Limitations of the study include its relatively small size and its lack of statistical power. There was also the potential for observer bias, as with any open-label study.

In conclusion, belatacept as a maintenance therapy in kidney transplant patients does not increase the incidence of CMV, BKV, or JCV infections compared with cyclosporine therapy, but it is associated with increase reactivation of EBV.

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