Original article Valganciclovir prophylaxis against cytomegalovirus impairs lymphocyte proliferation and activation in renal transplant recipients

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1 Antiviral Therapy 2011; 16: (doi: /IMP1879) Original article Valganciclovir prophylaxis against cytomegalovirus impairs lymphocyte proliferation and activation in renal transplant recipients Tomáš Reischig 1 *, Miroslav Prucha 2, Lenka Sedlackova 2, Daniel Lysak 3, Pavel Jindra 3, Mirko Bouda 1, Martin Matejovic 1 1 Department of Internal Medicine I, Charles University in Prague, Faculty of Medicine in Pilsen, Teaching Hospital Pilsen, Pilsen, Czech Republic 2 Department of Clinical Biochemistry and Immunology, Na Homolce Hospital, Prague, Czech Republic 3 Department of Hemato-oncology, Charles University in Prague, Faculty of Medicine in Pilsen, Teaching Hospital Pilsen, Pilsen, Czech Republic *Corresponding author reischig@fnplzen.cz Background: Antiviral prophylaxis against cytomegalovirus has been associated with reduced risk of allograft rejection and improved allograft survival after renal transplantation. This phenomenon might not be fully explained by preventing the indirect effects of cytomegalovirus. The effect of antiviral agents on lymphocyte function in patients treated with modern immunosuppression has not been studied to date. Methods: Adult renal transplant recipients were assigned to 3-month prophylaxis with either valganciclovir (900 mg once daily; n=19) or valacyclovir (2 g four times daily; n=17) as part of an ongoing randomized trial. Subsets of lymphocytes, lymphocyte proliferation and/or cytokine production after in vitro mitogen stimulation were evaluated at the end of prophylaxis and 1 month after withdrawal of antiviral drugs. Results: Lymphocyte proliferation was significantly decreased both after phytohemagglutinine (25% ±15% versus 32% ±18%; P=0.025) and concanavalin A stimulation (17% ±9% versus 25% ±16%; P=0.011) during valganciclovir, but not valacyclovir therapy. Moreover, a lower activated T-cell count (CD3 + HLA-DR + cells) was noted in valganciclovir-treated patients (13% ±10% versus 17% ±12% of total CD3 + T-cells; P=0.005). Conclusions: Valganciclovir suppresses lymphocyte proliferation and activation in patients after renal transplantation. Introduction Indirect effects of cytomegalovirus (CMV) infection have an adverse effect on the long-term outcome of solid organ transplantation [1,2]. In renal transplant recipients, CMV disease and asymptomatic CMV viraemia have been conclusively shown to be independent risk factors for the development of acute allograft rejection [3,4]. Recent studies have shown that CMV viraemia increases the incidence and severity of interstitial fibrosis and tubular atrophy (IFTA) after renal transplantation [5,6]. Consistent with results of the above studies is the evidence of deteriorated graft survival in patients with pre-existing CMV disease or CMV viraemia [7]. Adequate post-transplant CMV-specific T-cell immunity has a protective effect not only prior to the development of CMV disease. In heart transplant recipients, the presence of CMV-specific immunity is associated with a decrease in the incidence of acute rejection and allograft vasculopathy, and with improved graft function in renal transplant recipients [8,9]. Preventive strategies designed to minimize the effect of CMV infection form a part of standard care after solid organ transplantation [10]. Randomized controlled trials have documented a significant decrease in the incidence of CMV disease as well as CMV viraemia following (val)ganciclovir-based prophylaxis and/ or valacyclovir-based prophylaxis in renal transplant recipients [11 15]. A comparable decrease in the incidence of CMV disease can be obtained using preemptive therapy [16 18]. Although the results are inconsistent, only prophylaxis was also associated with a decrease in the incidence of acute rejection even when directly compared with preemptive therapy [14,15,17]. The mechanism of the beneficial action of prophylaxis is generally believed to be based on inhibited CMV 2011 International Medical Press (print) (online) 1227

2 T Reischig et al. replication during therapy with antiviral agents. This explains why the immunomodulatory properties of CMV making up the basis for CMV indirect effects are not involved at all [2,19]. However, this notion is in contrast with the fact that most acute rejection episodes actually precede the development of CMV viraemia [4] and that the decrease of acute rejection in prophylaxis-treated patients is obvious already before the onset of CMV replication in patients with deferred or preemptive therapy [15,17]. Studies in healthy volunteers have shown inhibition of immune functions by ganciclovir [20,21]. These findings formed the basis of our hypothesis that the administration of antiviral agents per se could enhance the effect of immunosuppressive medication and contribute to the decrease of allograft rejection irrespective of suppression of CMV infection. The present study, conducted as part of a randomized trial comparing prophylaxis of CMV disease with valganciclovir and valacyclovir (2VAL Study), was designed to test, in renal transplant recipients treated with modern immunosuppressive protocols, the effects of valganciclovir and valacyclovir on lymphocyte function. Methods Study design Analyses of lymphocyte function made up an integral part of an ongoing prospective randomized open-label single-centre study (2VAL Study). In this study, adult renal transplant recipients have been randomized since November 2007, at a 1:1 ratio, to 3-month prophylaxis with valganciclovir (Valcyte; Hoffman-La Roche, Grenzach-Wyhlen, Germany) at a dose of 900 mg once daily or with valacyclovir (Valtrex; Glaxo Wellcome, Dartford, UK) at a dose of 2 g four times daily with dose reduction depending on renal function [12,17]. Enrolled were patients at risk for CMV (CMV donor/ recipient serostatus of donor-positive/recipient-negative, donor-positive/recipient-positive and donor-negative/recipient-positive). Renal transplantations were performed at the Charles University Teaching Hospital, Pilsen, Czech Republic. The study was approved by the local ethics committee and conducted in compliance with the Declaration of Helsinki. Written informed consent was obtained from all patients. The effect of antiviral agents on lymphocyte function was evaluated in patients enrolled until February 2009 and with a functioning graft at 4 months post-transplant. Lymphocyte subpopulations, proliferation and cytokine production were determined at the end of month 3 post-transplant, at a time when patients were still receiving valganciclovir or valacyclovir, and at 4 months, that is, 1 month after antiviral therapy withdrawal. The magnitude of CMV-specific CD8 + T-cell responses was assessed at the same time. Moreover, quantitative PCR for CMV was performed at weeks 2, 4, 6, 8, 10, 12 and 16 after transplantation. Physicians assessing laboratory results were blinded to the study group of patients. Asymptomatic episodes of CMV viraemia were not treated. Immunosuppression Standard regimen was based on cyclosporine microemulsion (Neoral; Novartis, Basel, Switzerland). Recipients of second or third transplant and/or those with panel reactive antibody of >60% received induction with rabbit antithymocyte globulin (ratg; Thymoglobulin; Genzyme Polyclonals, Marcy L Etoile, France) and tacrolimus (Prograf; Astellas, Killorglin Co., Kerry, Ireland). Graft recipients from highly marginal donors (donation after cardiac death, 70 years old, and/or donors with hypertension or diabetes and significant nephrosclerosis in biopsy) were treated with anti- interleukin (IL)-2R monoclonal antibody (Simulect; Novartis) and low-dose tacrolimus. All groups were given mycophenolate mofetil (Cellcept; Hoffman-La Roche, Basel, Switzerland) and corticosteroids. Lymphocyte proliferation assays Lymphocyte proliferation was assessed using blastic lymphocyte transformation with DNA analysis. A quantity of 200 µl of heparinized whole blood was diluted by 1.8 ml of heated X-VIVO 10 medium (Lonza Group Ltd, Walkersville, MD, USA). A total of 100 µl of blood prepared in this manner was incubated separately with 100 µl of mitogens phytohemagglutinine (PHA) and concanavalin A (Con A; Sigma Aldrich, St Louis, MO, USA) or with the medium for 72 h at 37 C and 5% CO 2. A total of three samples were prepared for each mitogen and medium according to manufacturers instructions. Cell cycle kinetics was measured using a FACS-Canto II device (FACS-DIVA Software; BD Biosciences, San Jose, CA, USA) and analysed with Modfit software. The result was expressed as the percentage of proliferating cells defined as a population of cells in (S) phase and G 2 +M phase of the cell cycle. Quantification of lymphocyte counts and activated T-cells CD3 +, CD4 +, CD8 +, CD19 + and CD16 + /56 + lymphocyte counts were determined using FACS-Canto II (Canto Software, BD Biosciences) and FACS Calibur (Cellquest Software, BD Biosciences) devices. Activated T-cells were defined as CD3 + T-cells expressing HLA-DR. Analyses were made using the BD Multitest IMK Kit (BD Biosciences) containing two BD Multitest Four- Color Reagents (BD Biosciences). Foxp3 + T-cells were quantified using the HU FOXP3 Staining Kit PE (BD Pharmingen, San Diego, CA, USA). Actual measurements were carried out on the FACS Calibur device International Medical Press

3 Lymphocyte function and CMV prophylaxis evaluating 25,000 CD4 + T-cells, and showing the proportion (in percentages) of Foxp3 + CD25 + T-cells among all CD4 + T-cells. Ex vivo cytokine production after mitogen stimulation Lipopolysaccharide (LPS) stimulation was obtained using LPS (Sigma Aldrich) with lyophilisate containing 10 mg of endotoxin, diluted in 10 ml of water for injection and further diluted with RPMI 1640 tissue medium (Sigma Aldrich) to a concentration of 500 pg/ ml. PHA stimulation was obtained using PHA-containing test tubes (Cellestis, Chadstone, Victoria, Australia). In the case of LPS stimulation, 50 µl of heparinized whole blood was incubated for 4 h at 37 C with 50 µl of LPS solution. In the case of PHA stimulation, blood of patients was drawn into test tubes containing PHA and incubated for 24 h at 37 C. Cytokine concentrations in the supernatant were determined using the BD Cytometric Bead Array Human Th1/Th2 Cytokine Kit II containing interferon (IFN)-γ, IL-2, IL-4, IL-6, IL-10, and tumour necrosis factor (TNF)-α (BD Biosciences). Six populations of beads with different fluorescence intensity and with bound antibody specific for the above cytokines were prepared according to manufacturers instructions. After a 3-h incubation, samples were analysed by flow cytometry on a FACS Calibur device and data analysed using CBA Software (BD Biosciences). Quantification of CMV-specific CD8 + T-cells The frequencies of CMV-specific CD8 + T-cells were measured by a whole blood protocol. EDTA blood was stained with antibodies CD8, CD4, CD3 and CD45 (Beckman Coulter, Miami, FL, USA) and the MHC-Ipeptide-tetramers. A set of MHC tetrameric complexes containing HLA A*0201, B*0702, A*0101, B*3501 and A*2402 was used for cytotoxic T-cell detection (itag MHC Tetramer, Beckman Coulter) according to manufacturers instructions. Flow cytometry analysis was performed immediately using a Coulter Epics flow cytometer. Data acquisition and analysis were carried out using System II software. Absolute numbers of HLA tetramer positive T-cells were calculated by multiplying the percentage of CMV-specific CD8 + T-cells by the absolute lymphocyte count. Responses >0.1% were considered specific. Detection of CMV viraemia Quantitative real-time PCR was performed using a commercially available kit (RealArt CMV RG PCR Kit; Qiagen GmbH, Hilden, Germany) according to manufacturers instructions on a Rotor-Gene 2000/3000 system (Qiagen GmbH). DNA was isolated from 200 µl of whole blood using a commercially available kit (NucleoSpin Blood, Macherey- Nagel, Duren, Germany). Study assessments The primary goal of the study was to compare the proportions of activated T-cells, and the rates of lymphocyte proliferation and cytokine production at the time patients were treated by CMV prophylaxis with valganciclovir or valacyclovir with the same parameters after the withdrawal of antiviral agents. A secondary goal was to compare CMV-specific T-cell response and lymphocyte subpopulation counts prior to and after withdrawal of CMV prophylaxis. The effect of individual antiviral agents was analysed separately. Given the small sample size and the potential bias due to differences in the immunosuppression protocol, direct comparisons between the valganciclovir and valacyclovir groups were performed while being well aware of their very limited informative value. Statistical analysis Quantitative parametric data prior to and after withdrawal of CMV prophylaxis were compared using the paired Student s t-test and paired Wilcoxon test in non-parametric distribution. The valganciclovir and valacyclovir groups were compared by Student s t-test and the Mann Whitney U test in non-parametric distribution. Qualitative data were analysed using Fisher s exact test. The incidence of CMV viraemia or disease and acute rejection was calculated using Kaplan Meier curves, with the log-rank test used for comparison. Spearman rank order correlation coefficient was calculated to determine the dependence of lymphocyte function on cyclosporine or tacrolimus levels and prednisone dose. Statistical calculations were made using SPSS for Windows version 3.10 software (SPSS Inc., Chicago, IL, USA) and Statistica 9.0 software (StatSoft, Tulsa, OK, USA). Values of P<0.05 were considered statistically significant. Results Patient characteristics Overall, 42 patients were considered for inclusion. Of these, three were not included: two patients had donor-negative/recipient-negative serostatus and one patient declined to participate. A total of 39 patients were randomized, with transplantation performed in all but one because of a positive cross match test. The intention-to-treat population included 38 subjects, with 19 patients randomized to valganciclovir and valacyclovir prophylaxis each. In the valacyclovir group, graft failure before month 3 occurred in two patients making them ineligible for assessment of the effect of antiviral agents on lymphocyte function (Additional file 1). The average daily dose of valganciclovir at the time of lymphocyte function assessment was 640 ±270 mg, with the respective value for valacyclovir Antiviral Therapy

4 T Reischig et al. Table 1. Baseline characteristics of patients Characteristic Valganciclovir (n=19) Valacyclovir (n=17) P-value Recipient Mean age, years ±sd 46 ±14 47 ± Male gender, n (%) 13 (68) 10 (59) Previous transplantation, n (%) 1 (5) 3 (18) Mean number of HLA mismatches ±sd 3.3 ± ± Pretransplant PRA, % ±sd 6 ±13 8 ± Cytomegalovirus serostatus D + /R -, n (%) 2 (11) 3 (18) D + /R +, n (%) 14 (73) 14 (82) D - /R +, n (%) 3 (16) 0 (0) Donor Mean age, years ±sd 50 ±17 45 ± Donor type (deceased), n (%) 18 (95) 17 (100) Primary immunosuppression CyA plus MMF, n (%) 10 (53) 10 (59) Tac plus MMF, n (%) 9 (47) 7 (41) - Basiliximab, n (%) 8 (42) 3 (18) ratg induction, n (%) 1 (5) 5 (29) CyA, cyclosporine; D, donor; HLA, human leukocyte antigen; MMF, mycophenolate mofetil; PRA, panel reactive antibody; R, recipient; ratg, rabbit antithymocyte globulin; Tac, tacrolimus; +, positive; -, negative. being 6.4 ±1.2 g. The groups did not differ significantly in demographic and immunological parameters, or in CMV serostatus. In patients receiving valacyclovir prophylaxis, there was a trend toward more frequent institution of induction therapy with ratg, whereas valganciclovir group patients were more likely to receive basiliximab (Table 1). Main clinical results By the end of month 4 post-transplant, CMV viraemia with low viral load ( 200 copies/ml) occurred in 3 (16%) valganciclovir group patients with recurrent CMV viraemia (2,850 copies/ml) and in 1 patient after prophylaxis completion. Over the same period, CMV viraemia was detected in 5 (29%; P=0.357) valacyclovir group patients. In four cases, the first episode of CMV viraemia was detected within the prophylaxis period. While all patients had low viral load CMV viraemia ( 200 copies/ml) during valacyclovir therapy, viral load progression (>10,000 copies/ml) was observed in two patients after prophylaxis withdrawal, with the development of CMV syndrome in one of them. At the time of assessing lymphocyte function parameters, CMV viraemia was present only in one valganciclovir patient at month 3, as against two and three patients at months 3 and 4 in the valacyclovir group. The incidence of biopsy-proven acute rejection was higher in the valacyclovir group compared with the valganciclovir group (26% versus 0%; P=0.027). However, when including cases of rejection with a histological pattern of borderline changes, the incidence was similar with 26% and 29% in the valganciclovir and valacyclovir groups, respectively (P=0.771). Graft function was comparable with serum creatinine levels at month 3 (139 ±41 µmol/l versus 132 ±35 µmol/l; P=0.591) and month 4 (143 ±53 µmol/l versus 126 ±38 µmol/l; P=0.276) in the valganciclovir and valacyclovir groups, respectively. The differences in graft function were not statistically significant even when comparing values at months 3 and 4 within the individual groups. Immunosuppression at the time of lymphocyte function evaluation There was no major change in immunosuppressive therapy between months 3 and 4, except for a significant decrease in tacrolimus trough level at month 4 in the valacyclovir group (10.8 ±3.8 versus 7.6 ±2.5 ng/ ml; P=0.006). While prednisone doses were lower at month 4 in either group, the difference was minimal in absolute values. The only statistically significant difference between the valganciclovir and valacyclovir groups was the more frequent use of ratg in the latter as an induction agent and/or antirejection therapy (Table 2). Lymphocyte proliferation The ability of lymphocytes to proliferate during therapy with valganciclovir was significantly poorer compared with the period 1 month after valganciclovir withdrawal (Figure 1). Valganciclovir discontinuation was followed by an increase in the percentage of proliferating lymphocytes after stimulation with PHA (25% ±5% versus International Medical Press

5 Lymphocyte function and CMV prophylaxis Table 2. Immunosuppression at the time of lymphocyte function evaluation Immunosuppression Month 3 Month 4 P-value a Valganciclovir (n=19) Tac-based, n (%) 11 (58) 12 (63) CyA-based, n (%) 8 (42) 7 (37) Mean Tac trough level, ng/ml ±sd 8.9 ± ± Mean CyA trough level, ng/ml ±sd 176 ± ± MMF, n (%) 17 (89) 17 (89) Mean MMF dose, g/day ±sd 1.4 ± ± Mean MPA AUC, g h/l ±sd 54 ±37 ND Mean prednisone dose, mg/day ±sd 10 ±0 9 ± Induction or antirejection ratg, n (%) b,c NA 1 (5) Valacyclovir (n=17) Tac-based, n (%) 12 (71) 13 (76) CyA-based, n (%) 5 (29) 4 (24) Mean Tac trough level, ng/ml ±sd 10.8 ± ± Mean CyA trough level, ng/ml ±sd 185 ± ± MMF, n (%) 16 (94) 16 (94) Mean MMF dose, g/day ±sd 1.5 ± ± Mean MPA AUC, g h/l ±sd 35 ±8 ND Mean prednisone dose, mg/day ±sd 10 ±1 8 ± Induction or antirejection ratg, n (%) b,c NA 8 (47) a P-value for month 3 versus 4. b During first 4 months post-transplantation. c P=0.006 for valganciclovir versus valacyclovir. AUC, area under the concentration curve; CyA, cyclosporine; MMF, mycophenolate mofetil; MPA, mycophenolic acid; NA, not analysed ND, not done; ratg, rabbit antithymocyte globulin; Tac, tacrolimus. Figure 1. Lymphocyte proliferation after mitogen stimulation in patients treated with antiviral prophylaxis and 1 month after prophylaxis withdrawal 40 P=0.025 Percentage of proliferating lymphocytes, mean ±SEM P=0.664 P=0.308 P=0.083 P=0.221 P=0.011 Month 3 Month 4 0 Medium PHA Valacyclovir Con A Medium PHA Valganciclovir Con A Lymphocyte proliferation is shown for patients treated with antiviral prophylaxis (month 3) and 1 month after prophylaxis withdrawal (month 4). Con A, concanavalin A stimulation; PHA, phytohemagglutinine stimulation. 32% ±18%; P=0.025) as well as after stimulation with Con A (17% ±9% versus 25% ±16%; P=0.011). By contrast, no significant changes in lymphocyte proliferation after PHA stimulation (17% ±11% versus 19% ±13%; P=0.308) or Con A stimulation were seen during therapy and 1 month after valacyclovir discontinuation (16% ±10% versus 20% ±12%; P=0.083). When excluding patients with previous ratg therapy, the trend to enhanced lymphocyte proliferation after Con A stimulation after valacyclovir discontinuation Antiviral Therapy

6 T Reischig et al. Table 3. Lymphocyte, activated T-cell, and CMV-specific T-cell counts during CMV prophylaxis and 1 month after its withdrawal Lymphocyte subset Month 3 Month 4 P-value a Valganciclovir (n=19) CD3 + T-cells, cells/µl b 1,858 ±1,324 1,773 ±1, Activated T-cells, % c,d 13 ±10 16 ± CD4 + T-cells, cells/µl b 1,150 ±848 1,101 ± Foxp3 + T-cells, % e 6 ±3 6 ± CD8 + T-cells, cells/µl f 688 ± ± CD19 + B-cells, cells/µl 230 ± ± NK cells, cells/µl d 167 ± ± CMV pp65-specific CD8 + T-cells, cells/µl 8 ±13 9 ± Valacyclovir (n=17) CD3 + T-cells, cells/µl b 927 ±775 1,020 ± Activated T-cells, % c,d 19 ±15 28 ± CD4 + T-cells, cells/µl b 541 ± ± Foxp3 + CD25 + T-cells, % 7 ±3 7 ± CD8 + T-cells, cells/µl f 387 ± ± CD19 + B cells, cells/µl 189 ± ± NK cells, cells/µl d 109 ± ± CMV pp65-specific CD8 + T-cells, cells/µl 7 ±18 15 ± Data are expressed as mean ±sd. a P-value for month 3 versus 4. b P<0.05 for valganciclovir versus valacyclovir both at months 3 and 4. c Expressed as percentage of CD3 + HLA-DR + cells out of total CD3 + cell count. d P<0.05 for valganciclovir versus valacyclovir at month 4. e Expressed as percentage of CD4 + CD25 + Foxp3 + cells out of total CD4 + T-cell count. f P<0.05 for valganciclovir versus valacyclovir at month 3. CMV, cytomegalovirus; NK, natural killer. completely disappeared (23% ±9% versus 23% ±11%; P=0.802). The differences between valganciclovir and valacyclovir were statistically non-significant. Previous therapy with ratg inhibited lymphocyte proliferation at month 3 (PHA stimulation 10% ±9% versus 24% ±13%, P=0.006; Con A stimulation 7% ±5% versus 19% ±9%, P=0.002) and at month 4 (PHA stimulation 14% ±12% versus 30% ±17%, P=0.011; Con A stimulation 16% ±12% versus 25% ±15%, P=0.086). There was no correlation between cyclosporine level and the rate of lymphocyte proliferation after PHA or Con A stimulation (PHA r=-0.245, P=0.249; Con A r=-0.008, P=0.970). Similarly, no significant correlation was demonstrated with tacrolimus level (PHA r=-0.274, P=0.062; Con A r=-0.251, P=0.089) and prednisone dose (PHA r=-0.022, P=0.856; Con A r=-0.205, P=0.086). Lymphocyte counts, activated T-cells and CMV-specific T-cell response Valganciclovir and valacyclovir withdrawal was followed by a significant rise in the percentage of activated T-cells (CD3 + HLA-DR + cells out of total CD3 + cells; Table 3). With valganciclovir, the percentage of activated T-cells increased from 13% ±10% to 16% ±12% (P=0.007); the increase with valacyclovir was from 19% ±15% to 28% ±23% (P=0.005). The proportion of activated T-cells at month 4 was higher in the valacyclovir group compared with the valganciclovir group (P=0.036). However, this was due only to the higher proportion of patients with previous ratg therapy in the valacyclovir group. Patients receiving ratg showed a marked rise in activated T-cell count (23% ±18% versus 36% ±25%; P=0.020) at month 4 as against month 3. The percentage of activated T-cells at month 4 was higher in patients with previous ratg therapy (36% ±25% versus 17% ±13%; P=0.024). By contrast, no significant correlation between tacrolimus or cyclosporine trough levels and/or prednisone dose with activated T-cells was demonstrated. No major changes in lymphocyte subsets following the discontinuation of antiviral agents were noted except for a decrease in B-cell count in the valacyclovir group (Table 3). In this particular case, however, the decrease occurred only in those treated previously with ratg (P=0.035); the differences were no longer significant after exclusion of these patients (P=0.146). Absolute CD3 +, CD4 +, CD8 + and natural killer cell counts were significantly decreased in the valacyclovir group because of the higher rates of previous ratg therapy (Table 3). While significant correlations of tacrolimus levels with lymphocyte subsets were also demonstrated, the correlation coefficients were fairly low in all cases (Additional file 1). There were no significant changes in CMV p65-specific CD8 + T-cell counts following valganciclovir or valacyclovir withdrawal. Cytokine production after mitogen stimulation Cytokine production showed considerable variability in either group (Table 4). Moreover, cytokine levels were affected by tacrolimus levels (Additional file 1). At 1 month after valganciclovir withdrawal, there was International Medical Press

7 Lymphocyte function and CMV prophylaxis Table 4. Production of cytokines after stimulation by mitogens during CMV prophylaxis and 1 month after its withdrawal Cytokine Month 3 Month 4 P-value a Valganciclovir (n=19) TNF-α/PHA stimulation, pg/ml 887 ±1, ± TNF-α/LPS stimulation, pg/ml 436 ± ± IFN-γ/PHA stimulation, pg/ml b 1,673 ±1,700 2,024 ±1, IFN-γ/LPS stimulation, pg/ml 2.1 ± ± IL-2/PHA stimulation, pg/ml 263 ± ± IL-2/LPS stimulation, pg/ml 2.8 ± ± IL-4/PHA stimulation, pg/ml 22 ±26 27 ± IL-4/LPS stimulation, pg/ml 2.2 ± ± IL-6/PHA stimulation, pg/ml 4,688 ±924 >5, IL-6/LPS stimulation, pg/ml 753 ±582 2,276 ±1, IL-10/PHA stimulation, pg/ml 67 ± ± IL-10/LPS stimulation, pg/ml 3 ±2 57 ± Valacyclovir (n=17) TNF-α/PHA stimulation, pg/ml 468 ± ±1, TNF-α/LPS stimulation, pg/ml 608 ± ± IFN-γ/PHA stimulation, pg/ml b 556 ±1,017 1,186 ±1, IFN-γ/LPS stimulation, pg/ml 2.8 ± ± IL-2/PHA stimulation, pg/ml 165 ± ± IL-2/LPS stimulation, pg/ml 2.0 ± ± IL-4/PHA stimulation, pg/ml 20 ±29 43 ± IL-4/LPS stimulation, pg/ml 3.1 ± ± IL-6/PHA stimulation, pg/ml 4,666 ±1,212 4,351 ±1, IL-6/LPS stimulation, pg/ml 1,286 ±1,043 1,307 ±1, IL-10/PHA stimulation, pg/ml 54 ± ± IL-10/LPS stimulation, pg/ml 9 ±24 7 ± Data are expressed as mean ±sd. a P-value for month 3 versus 4. b P<0.05 for valganciclovir versus valacyclovir both at months 3 and 4. CMV, cytomegalovirus; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; PHA, phytohemagglutinine; TNF, tumour necrosis factor. an increase in the production of TNF-α (P=0.025) and IL-6 (P=0.001) after LPS stimulation, but not after PHA stimulation. A significant increase in IL-10 was noted after PHA (P=0.040) as well as LPS stimulation (P=0.021). Valacyclovir discontinuation was followed by a statistically significant increase in IFN-γ (P=0.008) and IL-2 (P=0.020) after PHA stimulation. However, the results were affected, in both cases, by correlation with tacrolimus levels, which was lower at month 4 compared with month 3 in the valacyclovir group (Table 2). The higher levels of IFN-γ seen in the valganciclovir group were again due to the higher number of valacyclovir patients with previous ratg therapy, whose IFN-γ production was dramatically decreased (P<0.001). Discussion Our study was the first to evaluate the effect of the antiviral agents valganciclovir and valacyclovir at doses commonly used in CMV prophylaxis on lymphocyte function in an unselected population of renal transplant recipients receiving concomitant modern immunosuppressive therapy. Our data suggest a likely additive immunosuppressive effect of valganciclovir, whereas the effect of valacyclovir on lymphocyte function is negligible. Valganciclovir administration was associated with suppressed lymphocyte proliferation and a decrease in the activated T-cell count. The restricted lymphocyte proliferation was apparent after stimulation by mitogens specific for CD4 + T-cells (PHA) and CD8 + T-cells (Con A). Treatment with valacyclovir, while associated with a decrease in the activated T-cell count, had no major effect on lymphocyte proliferation. Moreover, the changes in activated T-cell count in patients receiving valacyclovir prophylaxis were only evident in the subgroup with previous ratg therapy. The increase in activated T-cell count seems to be due to the dwindling effect of ratg, rather than to valacyclovir discontinuation. Our paper expands on the results of earlier studies showing, consistent with our findings, inhibition of lymphocyte responsiveness to mitogen following ganciclovir administration in healthy individuals [20,22]. Similar to our population of renal transplant recipients, acyclovir had no adverse effect on lymphocyte proliferation, even in healthy individuals [20]. Detailed analyses have shown that the main mechanism whereby ganciclovir affects lymphocyte function is Antiviral Therapy

8 T Reischig et al. impaired DNA synthesis. While directly inhibiting lymphocyte proliferation induced by various stimuli, ganciclovir had no adverse effect on cell growth, survival of unstimulated lymphocytes and did not induce lymphocyte apoptosis [20 22]. Impaired lymphocyte proliferation is due to the antiviral action of ganciclovir, a nucleoside analogue of guanosine, which competitively inhibits the incorporation of deoxyguanosine-triphosphate by viral DNA polymerase [23]. However, its selectivity to viral DNA polymerase is limited, thus explaining the well characterized side effects of ganciclovir related to impaired DNA synthesis, manifesting itself as granulocytopaenia or thrombocytopaenia [24]. Other mechanisms whereby valganciclovir could affect lymphocyte proliferation in our group seem to be unlikely. Our study does not allow evaluation of the effects of valganciclovir or valacyclovir on the pharmacokinetics of concomitant immunosuppressants, which could theoretically affect the resultant immunosuppressive effect. However, clinically relevant interactions have not been reported by earlier studies. By contrast, there is evidence showing antiviral activities of ganciclovir or acyclovir after mycophenolate mofetil administration [25,26]. Similarly, the enhanced proliferation and higher activated lymphocyte count after valganciclovir withdrawal cannot be explained by the immune response to CMV. CMV replication was unusual over the first 4 months, with no difference in CMV-specific T-cell counts at months 3 and 4. Data obtained from our study do not allow for a conclusive evaluation of the effects of valganciclovir or valacyclovir on cytokine production. Unlike lymphocyte proliferation and activation, there was a correlation between cytokine levels and the level of exposure to tacrolimus. Although an increase in the rate of production of virtually all cytokines reaching statistical significance in the case of IL-10, IL-6 and TNFα, particularly after the discontinuation of valganciclovir, became obvious, most of the changes might have admittedly been due to a decrease in tacrolimus levels. Moreover, ganciclovir did not inhibit IL-2 and IFN-γ production in healthy individuals in previous trials, and there is no clear known mechanism through which cytokine production could be affected by ganciclovir [22]. The issue of the clinical relevance of the immunosuppressive action of valganciclovir in renal transplant recipients should be addressed by future studies. A beneficial consequence could be mitigation of the indirect effects of CMV as a result of suppression of CMV-induced local inflammation within the allograft. Improved long-term graft survival with ganciclovir prophylaxis has been demonstrated in a randomized study [18]. This may be roughly linked to mitigation of the long-term impact of CMV disease treated by more effective immunosuppression, as was the case of mycophenolate mofetil compared with azathioprine [27]. It should be noted that our data cannot call to question the beneficial effect of CMV prophylaxis due to suppression of CMV viraemia or disease. CMV viraemia or intragraft CMV infection are associated with an increased incidence of IFTA and deteriorated graft function and survival [5,6,28,29]. By contrast, the immunosuppresive effect of valganciclovir might delay the development of CMV-specific immunity, which is additionally suppressed by the absence of antigenic stimuli following the suppression of CMV viraemia during prophylaxis. Consistent with this is the high incidence of late-onset CMV viraemia and CMV disease upon prophylaxis completion, truly rare occurrences when using the preemptive approach [16,17,30]. Late-onset CMV disease is associated with a higher risk of allograft loss or mortality [31]. Additionally, adequate CMV-specific immunity reconstitution is protective against the development of acute and chronic rejection [8,9]. Interestingly, a long-term comparison of valganciclovir prophylaxis with preemptive therapy did not document any advantage of prophylaxis in terms of restraining the indirect effects of CMV [32]. The present study has some major limitations, which have to be highlighted to allow for proper interpretation of results. Our results were substantially affected by the high proportion of ratg-treated patients in the valacyclovir group. After excluding ratg-treated individuals, the number of patients receiving valacyclovir treatment is too small precluding conclusive analyses. The absence of a major effect of valacyclovir on lymphocyte function in our study, while consistent with data obtained from healthy individuals, should not automatically foresee the same outcome when administering valacyclovir concomitantly with immunosuppressive medication [20]. Another drawback of our study was the determination of lymphocyte proliferation only after non-specific stimulation. The clinical effect of the immunosuppressive action of valganciclovir would have been more properly evaluated after stimulation by donor lymphocytes or CMV peptides. In conclusion, valganciclovir administration as part of CMV disease prophylaxis in renal transplant recipients treated with calcineurin inhibitor-based immunosuppression in combination with mycophenolate mofetil inhibits lymphocyte proliferation and activation. A similar immunosuppressive effect has not been documented with valacyclovir. However, the bias caused by ratg use in valacyclovir-treated patients precludes convincing conclusions. Further studies are warranted to assess the effect of valganciclovir or valacyclovir on cytokine production. Acknowledgements The study was supported by research project number MSM , Replacement of and Support to Some Vital Organs, awarded by the Ministry of E ducation, Youth and Physical Training of the Czech International Medical Press

9 Lymphocyte function and CMV prophylaxis Republic. The authors thank Eliska Cagankova and Milada Brozova for their assistance in data collection. Disclosure statement The authors declare no competing interests. Additional file Additional file 1: A flow diagram of participants in this study and a table illustrating significant correlations of tacrolimus and/or cyclosporine level and prednisone dose with lymphocyte counts and lymphocyte function parameters can be accessed via com/uploads/documents/avt-11-oa-2002_reischig_ Addfile_1.pdf References 1. Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med 2007; 357: Reischig T. Cytomegalovirus-associated renal allograft rejection: new challenges for antiviral preventive strategies. Expert Rev Anti Infect Ther 2010; 8: Sagedal S, Nordal KP, Hartmann A, et al. The impact of cytomegalovirus infection and disease on rejection episodes in renal allograft recipients. Am J Transplant 2002; 2: Reischig T, Jindra P, Svecova M, Kormunda S, Opatrny K, Jr., Treska V. The impact of cytomegalovirus disease and asymptomatic infection on acute renal allograft rejection. J Clin Virol 2006; 36: Reischig T, Jindra P, Hes O, Bouda M, Kormunda S, Treska V. Effect of cytomegalovirus viremia on subclinical rejection or interstitial fibrosis and tubular atrophy in protocol biopsy at 3 months in renal allograft recipients managed by preemptive therapy or antiviral prophylaxis. Transplantation 2009; 87: Smith JM, Corey L, Bittner R, et al. Subclinical viremia increases risk for chronic allograft injury in pediatric renal transplantation. J Am Soc Nephrol 2010; 21: Sagedal S, Hartmann A, Nordal KP, et al. Impact of early cytomegalovirus infection and disease on long-term recipient and kidney graft survival. Kidney Int 2004; 66: Tu W, Potena L, Stepick-Biek P, et al. T-cell immunity to subclinical cytomegalovirus infection reduces cardiac allograft disease. Circulation 2006; 114: Nickel P, Bold G, Presber F, et al. High levels of CMV- IE-1-specific memory T cells are associated with less alloimmunity and improved renal allograft function. Transpl Immunol 2009; 20: Kotton CN, Kumar D, Caliendo AM, et al. International consensus guidelines on the management of cytomegalovirus in solid organ transplantation. Transplantation 2010; 89: Brennan DC, Garlock KA, Singer GG, et al. Prophylactic oral ganciclovir compared with deferred therapy for control of cytomegalovirus in renal transplant recipients. Transplantation 1997; 64: Paya C, Humar A, Dominguez E, et al. Efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant 2004; 4: Humar A, Lebranchu Y, Vincenti F, et al. The efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients. Am J Transplant 2010; 10: Lowance D, Neumayer HH, Legendre CM, et al. Valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. N Engl J Med 1999; 340: Reischig T, Jindra P, Mares J, et al. Valacyclovir for cytomegalovirus prophylaxis reduces the risk of acute renal allograft rejection. Transplantation 2005; 79: Khoury JA, Storch GA, Bohl DL, et al. Prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients. Am J Transplant 2006; 6: Reischig T, Jindra P, Hes O, Svecova M, Klaboch J, Treska V. Valacyclovir prophylaxis versus preemptive valganciclovir therapy to prevent cytomegalovirus disease after renal transplantation. Am J Transplant 2008; 8: Kliem V, Fricke L, Wollbrink T, Burg M, Radermacher J, Rohde F. Improvement in long-term renal graft survival due to CMV prophylaxis with oral ganciclovir: results of a randomized clinical trial. Am J Transplant 2008; 8: Freeman RB, Jr. The indirect effects of cytomegalovirus infection. Am J Transplant 2009; 9: Heagy W, Crumpacker C, Lopez PA, Finberg RW. Inhibition of immune functions by antiviral drugs. J Clin Invest 1991; 87: Battiwalla M, Wu Y, Bajwa RP, et al. Ganciclovir inhibits lymphocyte proliferation by impairing DNA synthesis. Biol Blood Marrow Transplant 2007; 13: Bowden RA, Digel J, Reed EC, Meyers JD. Immunosuppressive effects of ganciclovir on in vitro lymphocyte responses. J Infect Dis 1987; 156: Crumpacker CS. Ganciclovir. N Engl J Med 1996; 335: Cvetković RS, Wellington K. Valganciclovir: a review of its use in the management of CMV infection and disease in immunocompromised patients. Drugs 2005; 65: Gimenez F, Foeillet E, Bourdon O, et al. Evaluation of pharmacokinetic interactions after oral administration of mycophenolate mofetil and valaciclovir or aciclovir to healthy subjects. Clin Pharmacokinet 2004; 43: Neyts J, Andrei G, De Clercq E. The novel immunosuppressive agent mycophenolate mofetil markedly potentiates the antiherpesvirus activities of acyclovir, ganciclovir, and penciclovir in vitro and in vivo. Antimicrob Agents Chemother 1998; 42: Giral M, Nguyen JM, Daguin P, et al. Mycophenolate mofetil does not modify the incidence of cytomegalovirus (CMV) disease after kidney transplantation but prevents CMV-induced chronic graft dysfunction. J Am Soc Nephrol 2001; 12: Reischig T, Nemcova J, Vanecek T, et al. Intragraft cytomegalovirus infection: a randomized trial of valacyclovir prophylaxis versus pre-emptive therapy in renal transplant recipients. Antivir Ther 2010; 15: Helanterä I, Koskinen P, Finne P, et al. Persistent cytomegalovirus infection in kidney allografts is associated with inferior graft function and survival. Transpl Int 2006; 19: Singh N. Prevention of cytomegalovirus in organ transplant recipients: cross roads between antiviral and antitumor immunity. Transplantation 2010; 90: Arthurs SK, Eid AJ, Pedersen RA, et al. Delayed-onset primary cytomegalovirus disease and the risk of allograft failure and mortality after kidney transplantation. Clin Infect Dis 2008; 46: Spinner ML, Saab G, Casabar E, Bowman LJ, Storch GA, Brennan DC. Impact of prophylactic versus preemptive valganciclovir on long-term renal allograft outcomes. Transplantation 2010; 90: Accepted 4 March 2011; published online 11 August 2011 Antiviral Therapy