Kinetics of Host Immune Responses and Cytomegalovirus Resistance in a Liver Transplant Patient

Transcription

1 LIVER TRANSPLANTATION 15: , 2009 ORIGINAL ARTICLE Kinetics of Host Immune Responses and Cytomegalovirus Resistance in a Liver Transplant Patient Kirsten Schaffer, 1 Julie Moran, 3 Margaret Duffy, 3 Aiden P. McCormick, 2 William W. Hall, 3,4 and Jaythoon Hassan 3 1 Department of Microbiology and 2 National Liver Transplant Unit, St. Vincent s University Hospital, Dublin, Ireland; and 3 National Virus Reference Laboratory and 4 Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland Among solid organ transplant (SOT) recipients, donor-seropositive/recipient-seronegative (D /R ) cytomegalovirus (CMV) status is associated with the highest risk of ganciclovir-resistant CMV disease, which has been reported for patients receiving oral ganciclovir but not valganciclovir prophylaxis. We report a case of CMV breakthrough infection in a D /R liver transplant patient while he was receiving oral valganciclovir. Forty samples collected over 6 months were analyzed for the CMV viral load, lymphocyte counts, cytokine levels, and lymphocyte differentiation status. Genotypic resistance testing of the viral UL97 gene was performed when the patient failed to respond. CMV viremia occurred on day 50 post-transplant, and 5 samples taken between days 50 and 85 showed the wild-type UL97 genotype. The appearance of deletion was observed from day 114 post-transplant. Viral loads declined when foscarnet was commenced and remained below 10,000 copies/ml when the lymphocyte count was greater than 1000/ L (P 0.02). T cell responses revealed significant expansion of CD8 terminal effector memory cells. CD4 cells were largely populations of naïve and central memory cells. Circulating interleukin 10 (IL-10) levels correlated with the viral load (P ). Seroconversion occurred on day 230. The CMV viral load in combination with lymphocyte counts and IL-10 may be a predictive marker for the risk of development of resistant CMV disease in D /R SOT patients. Liver Transpl 15: , AASLD. Received March 18, 2009; accepted May 4, Cytomegalovirus (CMV) is an important viral pathogen in solid organ transplant (SOT) recipients. CMV-seronegative recipients receiving a CMV-seropositive organ [ie, donor-seropositive/recipient-seronegative (D /R )] have the highest risk of developing CMV disease. Despite the introduction of antiviral prophylaxis in this patient cohort, approximately 30% of D /R SOT recipients develop delayed-onset primary CMV disease after discontinuation of prophylaxis. 1 Ganciclovir-resistant CMV disease has been reported in up to 7% of SOT patients receiving antiviral prophylaxis. 2 Several genotypic mutations within the viral phosphotransferase gene (UL97) have been identified as conferring phenotypic resistance to ganciclovir. In patients receiving valganciclovir prophylaxis, these mutations have so far been detected only after drug prophylaxis has been terminated. 1,3 Primary CMV infections are controlled by a significant expansion of antiviral CD8 T cells in both primary and chronic infections of healthy and immunosuppressed individuals. 4 CMV-specific CD8 T cells have been found to be substantially enriched in the late differentiation phenotype stage; these are called terminal effector memory cells and are identified by the presence of CD45RA but the lack of CCR7. 4 A greater cyto- Abbreviations: bd, twice a day; CMV, cytomegalovirus; D /R, donor-seropositive/recipient-seronegative; HLA, human leukocyte antigen; IL, interleukin; iv, intravenously; od, every day; po, by mouth; SOT, solid organ transplant; TLC, total lymphocyte count; TNF-, tumor necrosis factor. The study was supported and funded by the National Virus Reference Laboratory and the Department of Medical Microbiology at St. Vincent s University Hospital. Address reprint requests to Kirsten Schaffer, Department of Microbiology, St. Vincent s University Hospital, Elm Park, Dublin 4, Ireland. Telephone: ; FAX: ; kirsten.e.schaffer@ucd.ie DOI /lt Published online in Wiley InterScience ( American Association for the Study of Liver Diseases.

2 1200 SCHAFFER ET AL. Figure 1. Regimen and dosage of immunosuppressive and antiviral therapy received by the patient post-transplant with the CMV viral load and TLC shown in the graph. The patient was discharged from the hospital on day 22 posttransplant after being treated twice with methylprednisolone pulse therapy (3 1 g od iv) and with 2 doses of daclizumab (first 2 mg/kg and then 1 mg/kg). His immunosuppressive medication at the time of discharge consisted of tacrolimus (5 mg bd), mycophenolate (1.5 g bd), and prednisolone (15 mg od). On readmission to the hospital for intravenous treatment of CMV disease on day 114, his immunosuppression was reduced. His renal function was not compromised while he was on ganciclovir treatment, with serum creatinine levels ranging from 90 to 130 mol/l. Abbreviations: bd, twice a day; CMV, cytomegalovirus; iv, intravenously; od, every day; po, by mouth; TLC, total lymphocyte count. toxic potential has been associated with this CD8 T cell subset, 5 and stimulation with their cognate peptide induces production of antiviral cytokines and chemokines. 6 In SOT patients, increased tumor necrosis factor (TNF- ) and interleukin 10 (IL-10) levels, reflecting an anti-inflammatory response, have been reported with CMV disease. 7 Impaired CMV-specific T cell immunity has been detected in SOT recipients with recurrent, ganciclovir-resistant CMV disease after intravenous (iv) ganciclovir therapy. 8,9 Here we describe a rare case of CMV breakthrough disease in a D /R SOT patient while he was receiving oral valganciclovir prophylaxis and treatment. In order to elucidate the kinetics of CMV disease in this patient, we have also attempted to investigate the primary immunological responses to the virus. PATIENT AND METHODS A 46-year-old CMV-seronegative male [human leukocyte antigen A*01 (HLA-A*01), HLA-A*03, HLA-B*08, and HLA-B*1402] with primary sclerosing cholangitis received a liver transplant from a CMV-seropositive donor. He had received azathioprine for his underlying disease in the years preceding transplantation. His posttransplantation treatment regimen is outlined in Fig. 1. The patient was completely compliant with treatment, as documented in a log book kept by his wife, who is a registered nurse. The donor was a 47-year-old man whose death was accidental and who had no history of antiviral treatment or CMV disease in the past. Written informed consent was obtained from the patient. CMV DNA Extraction, Quantitation, and Resistance Analysis Each specimen was quantified with the Cobas Amplicor CMV Monitor test (Roche Diagnostics, Ltd., West Sussex, United Kingdom). For resistance testing, CMV DNA was extracted from 140 L of plasma with the QiAMP viral RNA extraction kit (Qiagen, Crawley, United Kingdom). pul97 sequencing was undertaken after the amplification of a 700-bp fragment. 10 Each amplified product was sequenced with a PerkinElmer (Massachusetts) 310 genetic analyzer. Unincorporated nucleotides and primers were removed from the amplified products with a rapid purification system (Boehringer Mannheim, Ingelheim, Germany). The sequence was determined by fluorescein-labeled dideoxynucleotides with the sequencing conditions specified in the protocol for the BigDye Terminator Cycle sequencing kit. The CMV-specific oligonucleotides UL97C and UL97D 8 were used as sequencing primers. Sequencing results were confirmed by the Antiviral Resistance Testing Service (West Midlands Public Health Laboratory, Birmingham Heartlands Hospital, Birmingham, United Kingdom). Flow Cytometry Staining Four-color fluorescence was performed on peripheral blood mononuclear cells. Cells ( ) were stained

3 HOST IMMUNE RESPONSES AND CMV RESISTANCE 1201 with CD3 peridinin-chlorophyll protein complex conjugated monoclonal antibody, CD45RA phycoerythrin, CCR7 allophycocyanin, and CD4 or CD8 fluorescein isothiocyanate (Becton Dickinson Biosciences, San Jose, CA). At least 50,000 events were acquired with a FACSCalibur flow cytometer (Becton Dickinson Biosciences). Lymphocytes were gated by forward and sideward scatter, and data were analyzed with the software program CellQuest (Becton Dickinson Biosciences). Measurement of Cytokines/Chemokines The Lincoplex multibead human cytokine kit (Linco Research, St. Charles, MO) was used to measure levels of IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, interferon, and TNF-. The sensitivity of the assay was 3.2 pg/ml for all cytokines and chemokines. Multi-analyte profiling was performed on a Luminex-100 system using an XY platform and Luminex IS 2.2 software. Recombinant IL-10 (R&D Systems, Oxfordshire, United Kingdom) was included in the assay to ensure that there was no cross-detection between CMV IL-10 and human IL-10. Statistical Analysis Correlation coefficients (Spearman) between variables were calculated by linear regression analysis. The statistical analysis was performed with GB-STAT version 9 (Dynamic Microsystems). A P value of less than 0.05 was considered statistically significant. RESULTS The immunosuppressive treatment regimen received by the patient following liver transplantation is detailed in Fig. 1. His immediate postoperative course was complicated by episodes of acute cellular rejection for which he received 2 courses of methylprednisolone followed by daclizumab therapy. Because of his CMV D /R status, CMV prophylaxis with oral valganciclovir (900 mg daily) was started on day 10 post-surgery. On day 50 post-transplantation, the CMV polymerase chain reaction became positive with a viral load of 6520 copies/ml. As liver function tests remained normal and the patient was feeling well, outpatient treatment with valganciclovir (900 mg twice a day by mouth) was initiated. His viral load fell to 1715 copies/ml by day 80 post-transplantation, but by day 114, the patient complained of general weakness, fatigue, and diarrhea. At this stage, his CMV viral load had increased to 25,265 copies/ml. He was admitted to the hospital for intravenous ganciclovir treatment, and his immunosuppression was reduced. Immunohistochemical staining of a colonic biopsy showed cells positive for CMV. Despite intravenous ganciclovir treatment, the CMV viral load continued to rise to 49,510 copies/ml on day 123, and CMV resistance testing revealed the characteristic UL97 gene deletion in amino acid positions 594 and 595. The patient was switched to intravenous foscarnet treatment and discharged home after 3 weeks, having improved clinically. When this article was written, he remained well with good graft function. A retrospective analysis of earlier specimens revealed wild-type CMV sequences up to day 85 post-transplantation. Specimens from day 114, day 120, and day 123 showed a mixture of wild-type virus and virus harboring the deletion The unusual kinetics of CMV resistance development prompted us to investigate his immune response in more detail. Viral loads remained below 10,000 copies/ml when the white cell count exceeded 6000/ L and the lymphocyte count was greater than 1000/ L (Fig. 1). During the course of the illness, white cell counts ranged from 600 to 17,200/ L, and lymphocyte counts ranged from 600 to 3400/ L. The total lymphocyte count (TLC) showed a significant negative correlation with the viral load (r 0.41, P 0.02; data not shown). Lymphocyte subset analysis on days 136 and 300 showed a reduction in the CD3 CD4 count from 1050/ L to 836/ L, whereas a doubling of the CD3 CD8 lymphocyte count from 345/ L to 792/ L was observed, resulting in a change of the CD4 /CD8 ratio from 3 to 1. An analysis of the differentiation status showed that 45% of CD3 CD8 cells were terminal effector memory cells (CD45RA CCR7 ), whereas 35% of CD3 CD4 cells were naïve (CD45RA CCR7 ) and 28% were central memory cells (CD45RA CCR7 ). CMV seroconversion was delayed to day 230 post-transplant. A significant correlation of circulating IL-10 levels with the viral load (r 0.72, P ) was observed (data not shown). TNF- levels correlated with IL-10 (r 0.35, P 0.03). Levels of IL-1, IL-2, interferon, IL-4, and IL-6 were slightly raised and ranged between 20 and 80 pg/ml; however, treatment with foscarnet reduced the levels of these cytokines below 20 pg/ml. The following additional immunological investigations were all within the normal range: immunoglobulin G subclasses, complement C3 and C4 levels, classical pathway CH 100, alternate pathway AP 100, and protein electrophoresis. DISCUSSION We report here a case of ganciclovir-resistant CMV breakthrough disease occurring in an SOT patient while he was receiving oral valganciclovir prophylaxis and treatment. A ganciclovir-resistant CMV prevalence of 2.9% (3/103) has been reported in SOT recipients (excluding lung transplant recipients) on oral ganciclovir prophylaxis. 3,11 The UL97 mutations that had been detected in patients during valganciclovir prophylaxis did not confer phenotypic resistance, 12 and a recent study undertaken among 225 CMV D /R SOT recipients on valganciclovir prophylaxis observed no CMV breakthrough disease. 1 Because of its low bioavailability, oral ganciclovir achieves suboptimal circulating levels, which can facilitate resistance development. The oral bioavailability of valganciclovir is about 10-fold higher, providing drug

4 1202 SCHAFFER ET AL. exposure comparable to that of intravenous ganciclovir and thus making resistance development less likely. 13 Sequencing on day 85 post-transplantation revealed only wild-type virus in this patient. In subsequent specimens (days 114, 120, and 123), a mixture of mutant virus sequences and wild-type sequences was detected. Because of the limited sensitivity of the adopted sequencing approach, we cannot determine if mutant virus was present at undetectable levels in previous specimens (before day 114) and whether viral resistance arose during the prophylactic or therapeutic regimen of valganciclovir. In agreement with the observed time course of resistance development in our patient, Emery and Griffiths 14 estimated that a mutant virus would require 92 to 103 days to dominate in a viral population in primary CMV infection in an SOT patient. It is generally believed that the development of ganciclovir resistance in SOT recipients is favored by (1) D /R CMV status, (2) a high viral load generating a greater likelihood of selection of the resistant virus, (3) prolonged exposure to ganciclovir, and (4) intensive immunosuppression. Interestingly, ganciclovir resistance has not always been associated with clinical CMV disease or failure to respond to ganciclovir treatment, and this further highlights the importance of host factors in the outcome of CMV infection. 15 Our patient fulfilled the D /R status as one of the major risk factors. His viral load in the weeks leading up to the detection of resistance remained comparatively low, ranging from 1715 to 28,200 copies/ml. He had received 104 days of valganciclovir (40 days at 900 mg every day by mouth and 64 days at 900 mg twice a day by mouth). We did not measure ganciclovir serum levels and can therefore only assume that in the absence of malabsorption or noncompliance, serum levels achieved by valganciclovir would have been satisfactory. Over the same time period, tacrolimus serum levels on standard dosing were achieved, and the patient gained weight; this indicated satisfactory gastrointestinal absorption. Nevertheless, the CMV viral load failed to decrease significantly after a therapeutic dose of valganciclovir was started on day 50 (Fig. 1), although sequencing analysis of the specimen from day 50 revealed only wild-type CMV sequences. At this stage, he had already received significant amounts of antiviral and immunosuppressive drugs, which potentially impaired his lymphocyte response and allowed ongoing viral replication in the presence of the drug facilitating resistance development. One could speculate that earlier initiation of intravenous ganciclovir, once the viral load failed to clear with treatment doses of valganciclovir, could have prevented resistance development. It was noted that once CMV DNAemia was present, lymphocyte counts remained suppressed. Evidence exists that CMV controls lymphocyte damage by priming a substantial proportion of T cells for apoptosis in order to reduce the immune response against infected cells. 16 Antivirals such as ganciclovir and foscarnet are known in vitro to enhance bcl-2 expression and prevent apoptosis. 17 CMV IL-10, which has 27% homology with human IL-10, has been shown to directly inhibit dendritic cell maturation and function while enhancing IL-10 production. 18 The finding of a strong positive correlation between the viral load and circulating IL-10 levels as well as a significant negative correlation between IL-10 and TLC supports the important role of IL-10 in suppressing the host immune response and driving the response away from a dominant T helper 1 like antiviral response. In the current study, the hallmark of specific T cell immunity was observed by significant expansion of the CD3 CD8 T cell subpopulation, which largely consisted of terminal effector memory cells. Previous studies have shown that the emergence of CD4 effector memory T helper cells precedes the appearance of anti- CMV antibodies, 19 and because the CD3 CD4 T cells were mostly of the naïve and central memory phenotype, T cell help for B cell responses was delayed as the patient seroconverted more than 7 months after transplantation. In agreement, delayed CD4 T cell help has previously been reported in patients following kidney transplantation. 19,20 This report highlights the complex interactions between the host immune response (TLC and IL-10), the immunosuppressive and antiviral treatment, and the virus itself in a case of CMV breakthrough infection. Because of the complexity of the disease, a combination of factors is likely to have contributed to the development of resistance observed in our patient, and this emphasizes the necessity of individualizing the management of D /R SOT recipients with respect to the prevention and management of CMV disease. In addition to antiviral prophylaxis, pre-emptive CMV therapy has been successfully used to prevent CMV disease in D /R SOT recipients. The experience with our patient demonstrates also that CMV resistance development and breakthrough infection are possible during oral valganciclovir prophylaxis or treatment, and this needs to be considered in the management of D /R SOT recipients. ACKNOWLEDGMENT The authors thank Dr. Osman (Antiviral Resistance Testing Service, West Midlands Public Health Laboratory, Birmingham Heartlands Hospital, Birmingham, United Kingdom) for the UL97 gene sequencing analysis. REFERENCES 1. Eid AJ, Arthurs SK, Deziel PJ, Wilhelm MP, Razonable RR. Emergence of drug-resistant cytomegalovirus in the era of valganciclovir prophylaxis: therapeutic implications and outcomes. Clin Transplant 2008;22: Limaye A, Corey L, Koelle DM, Davis CL, Boeckh M. Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants. 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