Epstein-Barr Virus and Cytomegalovirus

Balance between Herpes Viruses and Immunosuppression after Lung Transplantation

Studies on Epstein-Barr virus and Cytomegalovirus

E.A.M. Verschuuren

Verschuuren, E.A.M. Balance between Herpes Viruses and Immunosuppression after Lung Transplantation Thesis Groningen – with references – with summary in Dutch

Publication of this thesis was financially supported by: ABN/AMRO, Acthelion, Astellas, Novartis, Roche, Wyeth, Guide

Cover: EBV Antigenemia of a lung transplant patient, or a result of a clumsy clinician in the laboratory causing a huge artefact. EBNA-1 = red, CD20 = green, nucleus = blue.

Printed by: Gildeprint, Enschede, the Netherlands ISBN 90-367-2869-X ISBN 90-367-2870-3 digital version © Copyright 2006 E.A.M. Verschuuren All rights reserved. No part of this book may be reproduced or transmitted, in any form or by any means, without written permission from the author.

RIJKSUNIVERSITEIT GRONINGEN Balance between Herpes Viruses and Immunosuppression after Lung Transplantation Studies on Epstein-Barr virus and Cytomegalovirus Proefschrift ter verkrijging van het doctoraat in de Medische Wetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, Dr. F. Zwarts, in het openbaar te verdedigen op woensdag 13 december 2006, om 13:15 uur

Door

Erik Alfons Maria Verschuuren

Geboren op 19 februari 1962 te Breda

Promotores:

Prof. Dr. C.G.M. Kallenberg Prof. Dr. G.H. Koëter Prof. Dr. J.M. Middeldorp

Copromotores:

Dr. W. van der Bij Dr. M.C. Harmsen

Beoordelingscommissie: Prof. Dr. R.J.M. ten Berge Prof. Dr. Mw J.C. Kluin-Nelemans Prof. Dr. F. Miedema

Paranimfen:

Dr. A. de Haan Dr. M.E. Erasmus

Contents

Chapter

Title

Page

1

Aim and outline of this thesis.

9

2

Towards standardization of the human Cytomegalovirus antigenemia assay.

19

3

Effects of changing immunosuppressive regimen on the incidence, duration and viral load of Cytomegalovirus infection in renal transplantation: a single center report.

35

4

Direct quantification of human Cytomegalovirus immediateearly and late mRNA levels in blood of lung transplant recipients by competitive nucleic acid sequence-based amplification.

51

5

Expression dynamics of human Cytomegalovirus immune evasion genes US3, US6, and US11 in the blood of lung transplant recipients.

73

6

Quantitative Epstein-Barr virus (EBV) Serology in Lung Transplant Recipients with primary EBV Infection and/or Post Transplant Lymphoproliferative Disease.

93

7

Dynamics and function of anti Epstein-Barr virus and anti Cytomegalovirus immune responses in the transplant patient: implications for immunosuppression.

111

8

Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of Post Transplant Lymphoproliferative Disease in high risk patients.

133

9

Treatment of Post-Transplant Lymphoproliferative Disease (PTLD) with Rituximab: the Remission, the Relapse and the Complication.

153

10

Epstein-Barr virus reactivation and lung transplant dysfunction.

165

11

Low rate of Bronchiolitis Obliterans Syndrome following Preemptive treatment of Post-Transplant Lymphoproliferative Disease after Lung Transplantation.

183

12

Epstein-Barr virus-DNA load Monitoring Late after Lung Transplantation: A surrogate Marker of the Degree of Immunosuppression and a Safe Guide to Reduce Immunosuppression.

203

13

Summary and future perspective.

223

Nederlandse samenvatting

231

Dankwoord

235

Curriculum Vitae

239

List of publications

241

Chapter 1 Aim and outline of this Thesis Introduction The ideal of clinical transplantation of solid organs is acceptance of the transplant without impairment of host immunity. The struggle to achieve this goal has started with the first transplantation, and, despite the enormous progress in the field of transplantation, we still are far from that ideal. Transplantation of solid organs depends on immunosuppressive effects of toxic medication, in order to inhibit recognition and rejection of the transplanted organ by the immune system. The underlying concept of immunosuppression encompasses that the immune system is suppressed to a level that rejection is prevented and at the same time the immune system remains capable of controlling infections and cancer. In clinical medicine we achieve this balance by starting immunosuppression according to a standard protocol, and adapt the level of immunosuppression guided by clinical sym ptom s of „too m uch‟ or „too little‟ im m unosuppression (that is infections or rejection, respectively). We follow this approach since there is no clinically useful way to measure the capacity of the immune system in such a way that it shows us how to adapt the level of immunosuppression so that rejection is prevented and adequate control of pathogens occurs. Therefor we try empirically to reach a balance in our patients betw een „too m uch‟-immunosuppression, clinically recognized by infectious com plications, and „too little‟ immunosuppression, clinically recognized by rejection of the transplanted organ. O ur ability to “fine -tune” the immunosuppressive medication to the individual needs of the patient determines the outcome of our transplantation protocol. After lung transplantation, not only acute rejection forms an important risk factor for bronchiolitis obliterans syndrome (BOS), the lung transplant presentation of chronic transplant dysfunction, but also infection is widely accepted as a major cause of chronic transplant dysfunction. At the start of the research described in this thesis the percentage of patients developing bronchiolitis obliterans syndrome (BOS), exceeded 40% at 4 years with a median survival of not even 4 years (1). This illustrates our capacity, at that time, to

Aim and outline recognize and adequately respond to the threats to the lung transplant patient. Because rejection after lung transplantation can be patchy and missed by transbronchial biopsies, recognition of infectious complications is of major importance for the treatment of the patient. Often, transplant dysfunction in the absence of (demonstrated) infection is interpreted as rejection and treated as such. This approach only stands if we have sensitive and specific tools to detect infections. This is especially relevant for infectious agents that are already present in the transplant patient and can reactivate whenever immune control fails. This is the case with the herpes viruses of which two are studied in this thesis. Of all herpes viruses these two, namely the Cytomegalovirus (CMV) and the EpsteinBarr virus (EBV), are currently most recognized as a cause of morbidity and mortality after solid organ transplantation. In this study we aimed to develop better tools for monitoring of these viruses and to come to better strategies to treat or prevent the associated morbidity and mortality.

Cytomegalovirus Cytomegalovirus (CMV) infection is the most common viral infection after lung transplantation. It has early and late effects on both the transplanted organ as well as the recipient (2-9). CMV infection can go unnoticed but the initial stage of the infection or reactivation often presents with an acute viral syndrome, with fever and malaise and organ specific symptoms like pneumonitis, hepatitis and entero-colitis (10). Also, CMV has been associated with the development of acute rejection (11,12) The late effects attributed to CMV infection have been the focus of interest during the nineties (13) and raise still much debate (14). The infection can present itself with transplant dysfunction complicating our clinical strategy to adapt immunosuppression according to clinical signs and symptoms. Without a routine monitoring strategy the infection can be mistaken for transplant rejection, and, if treated so, m ay seriously w orsen the patient‟s outcom e (1518). Consequently, routine monitoring of CMV viral load is now widely accepted and performed (19-22). The strategy to deal with CMV infection varies and has evolved over time (23,24). Also the current test arsenal to monitor CMV has grown extensively during the last two decades. It evolved from serology, to monitor the humoral immune response (25), to detection of the virus with shell vial culture after clinical suspicion (26), and, nowadays, to direct assessment of pp65 antigenemia (27) to monitor viral derived proteins, and PCR and NASBA to

10

Chapter 1 measure viral DNA or RNA load (20,28). The different tests used to detect CMV infection were mostly in house developed assays and this complicates comparison between studies, interpretation of results and implementation of multi-centre trials (29,30). A standardization of the most widely used tests was needed and standardization of the CMV antigenemia test forms the second chapter of this thesis. Using this test differences in frequency and severity of CMV reactivation was recognized not only between the different solid organ transplant programs but also over time with the evaluation of immunosuppressive protocols. The impact of different immunosuppressive regimes on frequency and severity of CMV reactivation was studied in chapter 3. A final problem of CMV infection, assessed in this thesis, was the problem of subclinical CMV infection. It has been suggested that chronic subclinical reactivation of CMV causes chronic transplant dysfunction by way of chronic inflammation not only resulting in damage to the transplant but also upregulating alloreactivity (31,32). Using a new method, nucleic acid sequence based amplification (NASBA), which measures viral RNA and thus virus activation/transcription, and not just the presence of the virus, we looked for better tools to monitor CMV activity in the lung transplant patient (chapter 4 and 5).

Epstein-Barr virus With the implementation of new antiviral drugs such as ganciclovir an valganciclovir CMV infection, if clinically managed well, has largely ceased to be a clinical problem (33,34). However, with the use of increasingly more effective immunosuppression, EBV has gained importance as a cause of morbidity and mortality (35-38). In contrast to CMV, EBV has two ways of replication. First, the lytic replication, causing lysis of the host cell, which takes place in the nasopharyngeal cavity and causes shedding of infectious virus with saliva (39). Second, the latent replication when EBV infects B-cells and transforms these cells into large lymphoblasts that can proliferate indefinitely. This leads to the clinical presentation of post transplant lymphoproliferative disease (PTLD) (40). While the lytic replication responds well to antiviral drugs (41), B-cell proliferation does not respond and asks for an alternative approach. Generally, EBV driven B-cell proliferation in the human host is considered a consequence of (iatrogenic) impairment of EBV-specific T-cells (42-46), as EBV specific T-cells have been demonstrated to be most important for control of EBV.

11

Aim and outline When we started the studies reported in this thesis, PTLD was considered a malignancy (47,48). It was recognized only after its clinical presentation as a mass or nodular lesions(37), and was treated with polychemotherapy (49-51). Depending on which organ is transplanted the incidence of PTLD varies between approximately 1% after kidney transplantation up to 10% in lung transplant patients (48,52-54) The diagnosis of PTLD means for a patient a high mortality with a range up to 50-80% (48,52-55). To approach this clinical problem we started various laboratory and clinical studies. First, monitoring tools for EBV were introduced and evaluated as described in chapters 6 to 8. The laboratory studies evolved from EBV specific serology with synthetic peptides to EBV DNA load measurements and measurement of the EBV specific T-cell immunity. Secondly, with the recognition of the presence of CD20 on the cell membrane of B-cells that constitute most PTLD, Rituximab, an anti-CD20 chimeric monoclonal antibody, was successfully introduced in the treatment of PTLD (chapter 9). Clinical observations led to the suspicion that EBV reactivation was associated with transplant dysfunction. This observation is important in the clinical approach of lung transplant patients as transplant dysfunction in absence of an infectious explanation is often regarded as rejection and treated as such. In case of an EBV associated/ induced transplant dysfunction treatment directed on rejection could lead to further over-immunosuppression and possibly PTLD. The relation between EBV and transplant dysfunction was therefore evaluated in chapter 10. Lessons learned from these studies were then implemented in the clinic. In 2001 the immunosuppressive protocol of the lung transplant program was changed and a pre-emptive strategy for PTLD was incorporated in the treatment protocol. This led to what we now call EBV DNA guided immunosuppression, in which the level of immunosuppression is pre-emptively reduced, in case of increasing EBV-DNA load measured in peripheral blood. Primary objective of this pre-emptive approach is reduction in the prevalence of PTLD. The improvement in outcome, particularly the reduction of BOS, which we observed since the introduction of this approach, led to the hypothesis presented in chapter 11. This hypothesis states, that the extent of EBV reactivation, as measured by the peripheral blood EBV DNA load, reflects the balance of the immune system between infection and rejection. As such, peripheral blood EBV DNA load can be used to individualize immunosuppression

12

Chapter 1 after lung (and possibly other solid organ) transplantation. In chapter 11 the results of this pre-emptive approach are described in all adult lung transplant patients transplanted since June 1st, 2001, enabling early intervention in EBV reactivation at the time of initial rise of EBV DNA load, and, thus, in the beginning of EBV reactivation. In chapter 12 the results of this approach, are reported in the adult lung transplant patients transplanted before, and alive at, June 1st, 2001. In this more heterogeneous cohort of patients, safety and efficacy of the pre-emptive approach on patients late after transplantation was studied.

13

Aim and outline

Reference List 1.

Hosenpud JD, Bennett LE, Keck BM, Boucek MM, Novick RJ. The Registry of the International Society for Heart and Lung Transplantation: eighteenth Official Report-2001. J Heart Lung Transplant 2001:20: 805-815.

2.

Fishman JA, Rubin RH. Infection in organ-transplant recipients. N Engl J Med 1998:338: 1741-1751.

3.

Richardson WP, Colvin RB, Cheeseman SH et al. Glomerulopathy associated with Cytomegalovirus viremia in renal allografts. N Engl J Med 1981:305: 57-63.

4.

von Willebrand E, Pettersson E, Ahonen J, Hayry P. CMV infection, class II antigen expression, and human kidney allograft rejection. Transplantation 1986:42: 364-367.

5.

Pouteil-Noble C, Ecochard R, Landrivon G et al. Cytomegalovirus infection--an etiological factor for rejection? A prospective study in 242 renal transplant patients. Transplantation 1993:55: 851-857.

6.

Reinke P, Fietze E, Ode-Hakim S et al. Late-acute renal allograft rejection and symptomless Cytomegalovirus infection. Lancet 1994:344: 1737-1738.

7.

Grattan MT, Moreno-Cabral CE, Starnes VA, Oyer PE, Stinson EB, Shumway NE. Cytomegalovirus infection is associated with cardiac allograft rejection and atherosclerosis. JAMA 1989:261: 3561-3566.

8.

Loebe M, Schuler S, Zais O, Warnecke H, Fleck E, Hetzer R. Role of Cytomegalovirus infection in the development of coronary artery disease in the transplanted heart. J Heart Transplant 1990:9: 707-711.

9.

Kroshus TJ, Kshettry VR, Savik K, John R, Hertz MI, Bolman RM, III. Risk factors for the development of bronchiolitis obliterans syndrome after lung transplantation. J Thorac Cardiovasc Surg 1997:114: 195-202.

10. Fishman JA, Rubin RH. Infection in organ-transplant recipients. N Engl J Med 1998:338: 1741-1751. 11. Sageda 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: 850856. 12. Lowance D, Neumayer HH, Legendre CM et al. Valacyclovir for the prevention of Cytomegalovirus disease after renal transplantation. International Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group. N Engl J Med 1999:340: 14621470. 13. Fishman JA, Rubin RH. Infection in organ-transplant recipients. N Engl J Med 1998:338: 1741-1751. 14. Salvadori M, Rosati A, Di Maria L et al. Immunosuppression in renal transplantation: viral diseases and chronic allograft nephropathy. Transplant Proc 2005:37: 2500-2501. 15. Cope AV, Sweny P, Sabin C, Rees L, Griffiths PD, Emery VC. Quantity of Cytomegalovirus viruria is a major risk factor for Cytomegalovirus disease after renal transplantation. J Med Virol 1997:52: 200-205. 16. Gor D, Sabin C, Prentice HG et al. Longitudinal fluctuations in Cytomegalovirus load in bone marrow transplant patients: relationship between peak virus load, donor/recipient serostatus, acute GVHD and CMV disease. Bone Marrow Transplant 1998:21: 597-605.

14

Chapter 1 17. Van der Bij W, Torensma R, Vanson WJ et al. Rapid Immunodiagnosis of Active CytomegaloVirus Infection by Monoclonal-Antibody Staining of Blood Leukocytes. Journal of Medical Virology 1988:25: 179-188. 18. van den Berg AP, van der Bij W, van Son WJ et al. Cytomegalovirus antigenemia as a useful marker of symptomatic Cytomegalovirus infection after renal transplantation--a report of 130 consecutive patients. Transplantation 1989:48: 991-995. 19. Van der Bij W, Torensma R, Vanson WJ et al. Rapid Immunodiagnosis of Active CytomegaloVirus Infection by Monoclonal-Antibody Staining of Blood Leukocytes. Journal of Medical Virology 1988:25: 179-188. 20. Goossens VJ, Blok MJ, Christiaans MH et al. Diagnostic value of nucleic-acid-sequencebased amplification for the detection of Cytomegalovirus infection in renal and liver transplant recipients. Intervirology 1999:42: 373-381. 21. Vanpoucke H, Van Vlem B, Vanholder R, Van Renterghem L. Significance of qualitative polymerase chain reaction combined with quantitation of viral load in the diagnosis and follow-up of Cytomegalovirus infection after solid-organ transplantation. Intervirology 1999:42: 398-404. 22. van den Berg AP, van der Bij W, van Son WJ et al. Cytomegalovirus antigenemia as a useful marker of symptomatic Cytomegalovirus infection after renal transplantation--a report of 130 consecutive patients. Transplantation 1989:48: 991-995. 23. Paya CV, Wilson JA, Espy MJ et al. Preemptive use of oral ganciclovir to prevent Cytomegalovirus infection in liver transplant patients: a randomized, placebo-controlled trial. J Infect Dis 2002:185: 854-860. 24. Lowance D, Neumayer HH, Legendre CM et al. Valacyclovir for the prevention of Cytomegalovirus disease after renal transplantation. International Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group. N Engl J Med 1999:340: 14621470. 25. van der Giessen M, van den Berg AP, Van der Bij W, Postma S, van Son WJ, The TH. Quantitative measurement of Cytomegalovirus-specific IgG and IgM antibodies in relation to Cytomegalovirus antigenaemia and disease activity in kidney recipients with an active Cytomegalovirus infection. Clin Exp Immunol 1990:80: 56-61. 26. Stagno S, Pass RF, Reynolds DW, Moore MA, Nahmias AJ, Alford CA. Comparative study of diagnostic procedures for congenital Cytomegalovirus infection. Pediatrics 1980:65: 251257. 27. Van der Bij W, Torensma R, van Son WJ et al. Rapid Immunodiagnosis of Active CytomegaloVirus Infection by Monoclonal-Antibody Staining of Blood Leukocytes. Journal of Medical Virology 1988:25: 179-188. 28. Emery VC, Sabin CA, Cope AV, Gor D, Hassan-Walker AF, Griffiths PD. Application of viralload kinetics to identify patients who develop Cytomegalovirus disease after transplantation. Lancet 2000:355: 2032-2036. 29. The TH, van den Berg AP, Harmsen MC, van der BW, van Son WJ. The Cytomegalovirus antigenemia assay: a plea for standardization. Scand J Infect Dis Suppl 1995:99: 25-29. 30. Verschuuren EA, Harmsen MC, Limburg PC et al. Towards standardization of the human Cytomegalovirus antigenemia assay. Intervirology 1999:42: 382-389. 31. Lowance D, Neumayer HH, Legendre CM et al. Valacyclovir for the prevention of Cytomegalovirus disease after renal transplantation. International Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group. N Engl J Med 1999:340: 14621470.

15

Aim and outline 32. Sageda 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: 850856. 33. 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: 611-620. 34. Pescovitz MD. Benefits of Cytomegalovirus prophylaxis in solid organ transplantation. Transplantation 2006:82: S4-S8. 35. Schneck SA, Penn I. De-novo brain tumours in renal-transplant recipients. Lancet 1971:1: 983-986. 36. Penn I. The incidence of malignancies in transplant recipients. Transplant Proc 1975:7: 323326. 37. Penn I. Malignant lymphomas in organ transplant recipients. Transplant Proc 1981:13: 736738. 38. Penn I, Brunson ME. Cancers after cyclosporine therapy. Transplant Proc 1988:20: 885-892. 39. Sixbey JW NJR-TNHRPJ. Epstein-Barr virus replication in oropharyngeal epithelial cells. N.Engl.J.Med. 310, 1225. 1984. 40. Paya CV, Fung JJ, Nalesnik MA et al. Epstein-Barr virus-induced posttransplant lymphoproliferative disorders. ASTS/ASTP EBV-PTLD Task Force and The Mayo Clinic Organized International Consensus Development Meeting. Transplantation 1999:68: 15171525. 41. Preiksaitis JK, Diaz-Mitoma F, Mirzayans F, Roberts S, Tyrrell DL. Quantitative oropharyngeal Epstein-Barr virus shedding in renal and cardiac transplant recipients: relationship to immunosuppressive therapy, serologic responses, and the risk of posttransplant lymphoproliferative disorder. J Infect Dis 1992:166: 986-994. 42. Crawford DH, Thomas JA, Janossy G et al. Epstein Barr virus nuclear antigen positive lymphoma after cyclosporine A treatment in patient with renal allograft. Lancet 1980:1: 1355-1356. 43. Thiru S, Calne RY, Nagington J. Lymphoma in renal allograft patients treated with cyclosporine-A as one of the immunosuppressive agents. Transplant Proc 1981:13: 359-364. 44. Nagington J, Gray J., Cyclosporine A immunosuppression, Epstein-Barr antibody, and lymphoma. Lancet 1980:1: 536-537. 45. Hanto DW, Gajl-Peczalska KJ, Frizzera G et al. Epstein-Barr virus (EBV) induced polyclonal and monoclonal B-cell lymphoproliferative diseases occurring after renal transplantation. Clinical, pathologic, and virologic findings and implications for therapy. Ann Surg 1983:198: 356-369. 46. Nalesnik MA, Jaffe R, Starzl TE et al. The pathology of posttransplant lymphoproliferative disorders occurring in the setting of cyclosporine A-prednisone immunosuppression. Am J Pathol 1988:133: 173-192. 47. Starzl TE, Nalesnik MA, Porter KA et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporine-steroid therapy. Lancet 1984:1: 583-587. 48. Penn I. The changing pattern of posttransplant malignancies. Transplant Proc 1991:23: 1101-1103. 49. Swinnen LJ. Durable remission after aggressive chemotherapy for post-cardiac transplant lymphoproliferation. Leuk Lymphoma 1997:28: 89-101. 50. Swinnen LJ, Mullen GM, Carr TJ, Costanzo MR, Fisher RI. Aggressive treatment for postcardiac transplant lymphoproliferation. Blood 1995:86: 3333-3340.

16

Chapter 1 51. Swinnen LJ. Treatment of organ transplant-related lymphoma. Hematol Oncol Clin North Am 1997:11: 963-973. 52. Morrison VA, Dunn DL, Manivel JC, Gajl-Peczalska KJ, Peterson BA. Clinical characteristics of post-transplant lymphoproliferative disorders. Am J Med 1994:97: 14-24. 53. Montone KT, Litzky LA, Wurster A et al. Analysis of Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder after lung transplantation. Surgery 1996:119: 544-551. 54. Raymond E, Tricottet V, Samuel D, Reynes M, Bismuth H, Misset JL. Epstein-Barr virusrelated localized hepatic lymphoproliferative disorders after liver transplantation. Cancer 1995:76: 1344-1351. 55. Chen JM, Barr ML, Chadburn A et al. Management of lymphoproliferative disorders after cardiac transplantation. Ann Thorac Surg 1993:56: 527-538.

17

Aim and outline

18

Chapter 2

Towards standardization of the human Cytomegalovirus Antigenemia assay

Erik AM Verschuuren, Martin C Harmsen, Pieter C Limburg, Wim van der Bij, Arie P van den Berg, Adriana M Kas-Deelen, Boelo Meedendorp, Willem J van Son and T Hauw The.

Intervirology 1999;42:382-389

For the Biomed 2 study group: Prof Dr M Mach, Institut fur Klinische und Molekulare Virologie, Universitat Erlangen, Erlangen Germany, Dr CA Bruggeman, Dept. of Medical Microbiology, University of Limburg Maastricht, the Netherlands, Prof Dr H Einsele, Med Universitätsklinik II, Tübingen, Germany, Prof Dr G Gerna, Servicio di Virologia, IRCCS Policlinico San Matteo, Pavia, Italy, Prof Dr JE Grundy, Dept. of Clinical Immunology Royal Free Hospital School of Medicine, London, United Kingdom , Dr M.P. Landini, Policlinico S. Orsola, Instituto di Microbiologia, Bologna Italy, Prof Dr Th. Mertens, Institut fur Microbiologie, Abt Virologie, Universitat Ulm, Germany, Prof Dr G Palu, Medical School, University of Padova, Italy, Prof Dr T.H. The, Dept. of Clinical Immunology, University Hospital Groningen, the Netherlands and Dr A Volpi, Dept. of Public Health, School of M edicine, U niversity of R om e „T or V ergata‟, Italy.

This study was supported by the European Community Grant no: ERB BMHMCT96-0471(DG 12-SSMA)

Standardization of Antigenemia

Abstract The Human Cytomegalovirus antigenemia (HCMV-Agemia) test has been accepted worldwide as a clinical tool in the diagnosis and management of HCMVassociated syndromes in immuno-compromised patients. The many modifications proposed since the first description by our laboratory make standardisation of the HCMV-Agemia assay necessary to enable multicentre clinical trials. We report the initial work for standardization of the HCMV-Agemia assay. A standard protocol is proposed, the optimal distribution conditions are investigated and the results of the shipment of positive and negative test slides as well as of two sets of coded internal standard slides are discussed. The main conclusions are that standard slides can be distributed at room temperature and that the results of participating laboratories with the coded internal standard slides were strikingly similar in spite of differences in HCMV-Agemia protocols used by participating laboratories.

20

Chapter 2

Introduction Since the Human Cytomegalovirus (HCMV) antigenemia assay (HCMV-Agemia assay) was first described by our laboratory more than 10 years ago (1), numerous studies have confirmed its clinical relevance for the diagnosis of active human HCMV-infection. Its high diagnostic accuracy, rapidity and technical simplicity have made the HCMV-Agemia assay one of the cornerstone methods for the diagnosis and management of active HCMV infection in immune compromised patient (2-6). However, a considerable number of modifications concerning every step in the protocol have been introduced by different laboratories, e.g. the isolation method of polymorphonuclear cells (PMNs), the cell numbers used for preparing slides, the use of cytocentrifuged cell preparations versus methanol spreading, the use of different fixation methods, of different monoclonal antibodies, of chromogenic versus fluorescent detection of positive cells and the reading of the results by only counting the positive cells per slide versus the scoring of positive cells per 50,000 PMNs (7-12). Introduction of these modifications was intended to improve performance of the original assay, but this may lead to increased interlaboratory variation as well. These variations in methodology make comparison of published results difficult and form an obstacle to (future) multicentre trials of antigenemia-directed intervention studies in immune compromised patients with a high risk for fatal HCMV disease. Consequently, a concerted action was initiated to set up a standardization program for the HCMVAgemia test in several collaborating laboratories and clinical centres in the European Community (13). The primary goal was to come to similar test performances in participating laboratories. First step was to make recommendations for a standard protocol and distribute it to the participating centres for modifying their current assay or adopting the standard protocol. Since participating laboratories were reluctant to abandon their local modifications, an alternative goal was set. This became the comparison of the HCMV-Agemia test performances in the participating laboratories on identical control slides. Only then could the decision be taken whether a standardized HCMV-Agemia assay is justified and required for multicentre studies. The second step was the evaluation of the performance of the reference laboratory to see whether the reproducibility of the control slides was adequate. The third step was the investigation of optimal distribution conditions. Effects of temperature and fixation method were analyzed and subsequently

21

Standardization of Antigenemia positive/negative control slides were distributed to be tested in the participating laboratories. The last step discussed in this article is the evaluation of two sets of coded slides to evaluate their usefulness as an internal standard. One set of internal standard slides was made of pp65-positive baculovirus-transfected insect cells. A second set comprised pp65-positive polymorphonuclear cells (PMNs) prepared by cocultivation of normal donor PMNs with productively infected endothelial cells (14;15). In the near future, the interlaboratory variation will be evaluated by distributing control slides made of patient material (external standard) with unknown numbers of positive cells.

Materials and Methods Isolation of leukocytes Two millilitres of EDTA-treated blood was mixed with 1 ml PBS and 1 ml of a 5% dextran (MW 250,000) in 0.9% NaCl and allowed to settle at 1 g sedimentation force at 37C at a 60 angle for 10 min. The leukocyte supernatant was collected and centrifuged at 300 g for 2 min. The erythrocytes were lysed by resuspending the cell pellet in 2 ml cold erythrocyte lysing buffer (NH4CL 155mmol/l, KHCO3 10 mmol/l, Na2.EDTA.3H2O 0.1 mmol/l, pH 7.4) at 4C for 10 min. The cells were washed twice in PBS and then resuspended in PBS and counted. From this a cell suspension of 1.5 x 106 cells/ml was made.

Preparation of cytospin/slides Cytocentrifuge preparations were made using 100 l cell suspensions (input 1.5 x 105 cells) centrifuged at 550 rpm during 5 min (Cytospin 3; Shandon Southern products, Astmoor, United Kingdom). The slides were dried for 15-20 min with a cold blower, wrapped in aluminium foil and stored at -80C until use.

Fixation and staining procedure Cells were fixed with formaldehyde and permeabilized with Nonidet P-40 solution as described before (11). Slides were incubated in duplicate with 50 l 1:5 diluted anti-HCMV- pp65 (C10/C11) (3) for 30 min in a 37C humid chamber, washed twice with PBS, and subsequently incubated with a 50 l HRP conjugated goat Fab anti-mouse IgG (H+L) absorbed with human serum (Protos Immunoresearch, Burlingame, Calif., USA) per spot in a 37C humid chamber for 30 min. After two washings in PBS, the enzyme reaction was performed for15 min with a 3-amino-9-ethylcarbazole (AEC) solution in 0.1 M acetate buffer (pH 4.9)

22

Chapter 2 (AEC 10 mg Sigma chemical Co, St Louis, Mo., USA) dissolved in 4 ml N,Ndimethylformamide and then filled up to 80 ml with acetate buffer, filtered and supplemented with 75 l H2O2 (30% v/v). The slides were then washed with acetate buffer for 10 min and counterstained with hematoxilin Mayer solution (see below for counting all cells), carefully rinsed with tap water and mounted in Kayser glycerine gelatine (Merck 9242).

Quantification of stained slides All PMNs with a yellowish/brown nuclear staining were considered positive. Results were expressed as positive cells per 50,000 by counting all cells using a grid (1) or a semi-automated image analyser (Quantimet 500, Leica) (13).

Storage test Fresh blood samples were taken from 9 solid-organ transplant patients positive in the diagnostic HCMV-Agemia assay, with scores between 10 and 250 per 50,000 PMNs. Six patients had HCMV-Agemia levels between 10 and 50 per 50,000 PMNs, and 3 scored between 50 and 250 per 50,000 PMN. The fixed and unfixed slides of these patients were vacuum sealed and stored in duplicate at room temperature and at -80C, except for one slide of each patient which was stained on the day of sampling. Frozen slides were brought to room temperature with a cold blower before opening the vacuum seal to prevent the formation of condensation on the slides. Staining (and fixation if necessary) was done at days 1, 2, 7, 14 and 21 according to the standard protocol.

Positive negative slides All standard slides were made on the day of blood drawing. Negative cytospots were made with PMN from a normal donor. HCMV-pp65-positive insect cells (Baculovirus expressed) were made as described (13) and mixed with normal donor PMN at a ratio to obtain a score of >100. All slides were dried with a cold blower, wrapped in aluminium foil, vacuum-sealed and stored at -80C until distribution at room temperature (RT).

Preparation of internal standard For the preparation of the reference standard containing pp65-positive cells, two methods were used. Either HCMV-pp65-positive insect cells (Baculovirus expressed) (13) or pp65-positive cells made by coculture of donor PMNs with HCMV-infected endothelial cells (16) were used. To determine the percentage of positive cells in this cellular mixture, a cytospin slide was made and stained according to the standard protocol, and the pp65-positive cells were

23

Standardization of Antigenemia counted. Then the cellular mixture containing the pp65-positive cells was mixed at different ratios with normal donor PMN and cytospins were made (input 1.5 x 105 cells). Ratios between pp65-positive cells and normal PMN were chosen to obtain slides with 0, 1-5, 6-10, 25-50, and >100 pp65-positive cells per 50,000 cells. Slides were dried with a blower, wrapped in aluminium foil vacuum-sealed and stored at -80C until use or distribution. A set of internal standard slides contained 5 coded slides with 2 cytospots each. Six cytospots of each preparation were stained at the reference laboratory to determine the actual number pp65positive cells per 50,000 PMNs. Mailing by courier delivery to the participating laboratories took place at RT, advice was given to stain the slides upon arrival or to store them at -80C until use. Participating laboratories received two sets of coded slides A-E marked with the day of preparation without any further information.

Results Variations in the HCMV-Agemia protocols used by participating laboratories The fixation method of most (5/8) laboratories was formaldehyde/NP-40, 2 centres used acetone and 1 laboratory used methanol/acetone. The monoclonal antibody against the HCMV-pp65 antigen was a pool of C10/C11 (Biotest Ag, Germany, IQ Products , the Netherlands) in 3 centres and 5 centres used an inhouse monoclonal (or a pool of in-house monoclonals) 1 centre used a mixture of an in-house monoclonal and Cinapool (Argene Biosoft) and one centre CINApool only (Table 1).

Storage test Results of experiments on the influence of fixation and temperature on the quality of the slides evaluated at different time points after storage are shown in Fig 1. No detectable loss of signal even after 3 weeks was noticed in any of the slides kept unfixed at -80C (Fig. 1D). Remarkably a decrease of the scores was seen in a minority of the slides fixed with paraformaldehyde and subsequently stored at -80C (Fig. 1C). Similarly fixed slides lost their positivity when stored at room temperature (Fig. 1A). When stored unfixed at room temperature (Fig 1B), no significant loss of signal was observed within the first 2 days of storage and the decline appeared less than with fixed slides stored at room temperature.

24

Chapter 2 Table 1 Variations in the HCMV-antigenemia protocols in the 10 participating laboratories. Fixation method PFA/NP-40 PFA/NP-40 PFA/NP-40 PFA/NP-40 PFA/NP-40 PFA/NP-40 PFA/NP-40 Acetone Acetone Methanol/Acetone

MoAb C10/C11(3) BM 2221 1C3, 2A6 and 4C1(11) C10/C11(3) CINApool2 C10/C11(3) Clone 103 28/771 pp65-331 CINApool + Emmanuel pool4

Staining PO PO IF IF IF IF IF PO PO IF

Quantification per 50,000 PMN per 50,000 PMN per 50,000 PMN per 50,000 PMN per spot per spot per spot per spot per spot per spot

Table 1 : Data are given as stated by participating laboratories. PFA/NP-40= paraformaldehyde/Nonidet P-40. References of monoclonal used are given when available. 1 Reference not available 2 Argene Biosoft cod. 11-002. 3 Clone 10: developed by Dr. J. Booth 4 Emmanuel pool: pool of monoclonal antibodies developed by Dr. Emmanuel

Distribution of positive/negative slides Based on the results described in the previous section, unfixed slides were aluminium wrapped, vacuum sealed and distributed at RT by courier delivery to ensure delivery within 2 days. All participating laboratories reported positive staining of baculovirus transfected HCMV-pp65 pos cells (data not shown). In general cell morphology was maintained well. No false-positive staining was reported by any laboratory.

Internal standard slides In general, the participating laboratories were satisfied with the overall quality of the internal standard slides without problems with the staining procedure (Fig. 2A,B). Data are shown in table 2. Average scores of the slides by our laboratory showed that the internal standard preparations made with the Baculovirus system were 0, 6, 8, 61 and 511.This was close to the objected number of pp65-positive cells per 50,000 PMN (0, 1-5, 6-10, 25-50 and >100).

25

Standardization of Antigenemia Figure 1 Influence of fixation, storage temperature and time on slides of 9 CMV-Agemia-test-positive patients. All slides were wrapped in aluminium and vacuum- sealed. Each line represents the scores of one sample. A) Fixed slides stored at RT. B) Unfixed slides stored at RT. C) Fixed slides stored at -80C. D) Unfixed slides stored at -80C.

The scores of our laboratory with the internal standard slides made with pp65-positive PMN were all within objected ratios (average scores 0, 1, 9, 39 and 680). Seven out of nine participating laboratories returned their data of the coded internal standard slides. No false-positive results were reported. In general, all laboratories were able to detect the pp65-positive spiked cells on the cytospots. With the exception of centre 2, all centres were able to detect an increasing number of pp65-positive cells with an increased number of spiked cells with both types of internal standard slides (Table 2). There was a good correlation between the results of the different laboratories for each slide (Fig 3). The absolute scores of the slides with 0, 1-5, 6-10, 25-50 and >100 pp65-pos insect cells (baculovirus expressed) showed that, of the centres that quantified per 50,000 PMNs, centre 3 (average scores, respectively: 0, 13, 29, 123 and approximately 1450) scored higher than the reference centre (centre 1).

26

Chapter 2 Table 2 Results of the 8 responding laboratories of the coded unfixed internal standard slides distributed at room temperature to the 10 participating laboratories Centre

number of HCMV-pp65-positive cells per 50,000 or per Cytospot* baculovirus expressed pp65-positive insect cells 0

1-5

6-10

25-50

>100

1 (ref)

0

6+2

8+5

61+13

2

0/0

7/3

0/0

17/55

3

0/0

10/16

22/35

11

0

8+3

10+5

1

4

0/0

13/8

13/11

51

0/0

0/7

17/15

61

0/0

19/15

1

7

0/0

81

0/0

pp65-positive PMN made by co-culture 0

1-5

511+141

0

295/584

0/0

-/123

~1500/~1400

0/0

66+13

376+74

0

114/102

890/677

0/0

44/19

646/420

0/0

35/27

107/127

ND/ND

15/13

23/24

124/120 ~1200/~1100

5/2

22/12

56/37

1000/1000

6-10

25-50

>100

1+1

9+3

39+5

680+106

0/0

17/26

5/2

199/142

1/2

8/13

83/75

~670/900

3+2

22+16

59+10

1079+74

0/2

4/8

14/44

596/399

1/0

14/14

57/55

1468/2132

0/0

0/0

4/8

26/27

>200/>200

0/0

0/0

7/9

37/32

~1100/1000

0/0

3/1

10/11

60/48

700/750

Table 2 : Staining was done according to the local protocol of the participating centre and scores of two cytospots (spot A/spot B) are shown. Results are expressed as pp65-positive cells per 50,000 PMNs or pp65-positive cells per cytospot*. Two kinds of pp65-positive cells were used: baculovirus-expressed pp65-positive insect cells and: in vitro generated pp65positive PMNs. Results of the reference centre are expressed as mean +standard deviation (n=6) of both pp65-positive cells per 50,000 PMN and positive cells per cytospot (ND=not done) 1 Counts performed by cytospot

Figure 3 Average results of all participating laboratories with the distributed internal standard slides (Table 2). Results are expressed as pp65-positive cells per 50,000 PMNs if available. For centres that quantified per cytospot results per cytospot were used.

27

Standardization of Antigenemia Figure 2 Photomicrographs of immunoperoxidase stained internal standard slides from the category of >100 pp65-positive cells mixed per 50,000 PMN. A Internal standard slide with pp65 baculovirus-infected insect cells B Internal standard slide with pp65-positive PMNs made by coculture of normal donor PMNs with HCMV infected endothelial cells. Arrows indicate positively stained cells. Note the larger variation in both morphology and staining intensity in the slides spiked with pp65 baculovirus infected insect cells (A) as compared to the slide spiked with pp65-positive PMNs.

28

Chapter 2 The results of centre 3 with the pp65-positive PMN made by coculture of donor PMN with HCMV-infected endothelial cells (average scores of 0, 2, 11, 79 and 785) were at the same level as the results of the reference centre. The results of centre 2 (average scores 0, 5, 0, 36 and 440 for the pp65positive insect cells and 0, 0, 22, 4 and 172 for the pp65-positive PMNs) were neither in line with the expected results no with the results of the other participants for both the internal standard slides made with the pp65-positive insect cells as well as the pp65-positive PMNs. Of the centres that quantified per cytospot centre 5 ( average readings 0, 4, 16, 32 and 533 per cytospot) and centre 8 (average readings 0, 3, 16, 47 and 1000 per cytospot) were comparable with the reference centre (average readings 0, 8, 10, 66, 376 respectively per cytospot) and centre 4 (average readings 0, 11, 12, 108, 784 per cytospot), centre 6 (0, 17, 31, 117 and not done per cytospot) and centre 7 (0, 14, 24, 122 and approximately 1150 per cytospot) scored higher in the Baculovirus transfected pp65-positive cells than the reference centre. With the pp65-positive PMNs, centre 5 (readings of 0, 1, 14, 56 and 1800 per cytospot) and centre 8 (Readings of 0, 2, 11, 54 and 725 per cytospot) had comparable results with those of the reference centre ( readings 0, 3, 22, 59 and 1079 per cytospot), whereas centre 4 (readings 0, 1, 6, 29 and 498 per cytospot), centre 6 (readings of 0, 0, 6, 27 and >200 per cytospot) and centre 7 (readings of 0, 0, 8, 35 and 725 per cytospot) had a lower result than the reference centre. However, since these readings were not quantified per 50,000 PMNs, these results may reflect different cell numbers per spot. The ratios between the preparations with 6-10 and 25-50 pp65-positive cells were very similar (3-5) for the pp65-positive PMN in all laboratories that quantified per cytospot. The ratios were less similar (3-9) for the same centres when the Baculovirus system was used. One centre withdrew from the study, one centre could not interpret the results due to high background staining.

Discussion This study describes the preparation of pp65-positive standard slides and the evaluation of these slides by 8 different laboratories. Although differences in the absolute values were recognized, the results of the readings by the different laboratories were remarkably similar, despite distribution at RT and the differences in HCMV-Agemia protocols used by the participating laboratories. This was especially true for the in-vitro-generated pp65-positive PMNs made by coculture, for which their potential usefulness for standardization was recently described by Gerna et al (16). The main conclusion is that these standard slides

29

Standardization of Antigenemia are well suited for standardization purposes and can be distributed at RT, provided they are wrapped in aluminium, vacuum-sealed, and stored at -80C upon arrival until staining. This enables the different laboratories to evaluate and compare their own assay protocols including differences in fixation techniques. Since the participating laboratories were reluctant to abandon their own modifications of the protocol, it is of major importance to have an internal standard that performs well in all these different laboratories. Optimal storage conditions were evaluated to determine the distribution conditions. These proved to be storage of unfixed slides at -80C. Because of the logistic problems involved with distribution of material at -80C and as no apparent decrease in signal was seen with unfixed slides at RT, we concluded that, for standardization purposes, unfixed slides can be sent at RT. This was confirmed by the distribution of the positive negative slides and later by the results of the internal standard slides. Distribution and evaluation of these internal standard slides showed remarkable results. None of the responding laboratories had false- positive staining. Six out of 8 responding laboratories detected not only the positive cells but also detected the increasing number of spiked cells in the coded slides. This means that in spite of the differences in the protocols the results are remarkably similar. At the start of this concerted action only the baculovirus system was available to make pp65-positive cells. Therefore, this system was chosen to prepare the pp65-positive cells for the positive negative slides of the internal standard. However, during the study it became possible to make pp65-positive PMN in vitro (14, 15). Both methods were used to make internal standard slides. Besides morphological considerations, both types of internal standards have two important differences. The pp65 protein is actively produced in the pp65-baculovirus infected insect cells. During the course of infection, the polyhedrin promoter that drives the expression of pp65 is switched on, with maximal expression reached at late stages of baculovirus infection. As baculovirus infections generally are not synchronous, cells will harbor differing amounts of intracellular pp65. This was observed by the participants as differences in staining intensity of the baculovirus-spiked internal standards (Fig. 2A). Thus the criterium for positivity appeared more difficult to set as compared to pp65-positive PMN and probably has led to more variation in the quantitation results (Table 2). Furthermore, bac-pp65 is a recombinant protein which might influence the staining capacity of some of the anti-pp65 monoclonal antibodies. The use of invitro- generated pp65-positive PMN as internal standard may more closely

30

Chapter 2 resemble the patient situation. Nevertheless, these pp65-positive PMN are also a phenomenon generated in vitro in which the pp65 was acquired by co-cultivation of PMN with HCMV-infected endothelial cells. In contrast to infected cells, these PMN do not produce pp65 (17). As it appears, PMNs generally seem to acquire similar amounts of pp65 upon cocultivation, which is reflected in a more homogeneous staining pattern of the pp65-PMN internal standard (Fig. 2B). This allowed for a lower variation in the quantification of this type of internal standard, making it better suited than pp65 baculovirus-infected cells. When compared to the infection of insect cells with baculovirus, the production of pp65- positive PMNs generated in vitro is more laborious and the transfer protocol needs further optimization. Nevertheless, the use of pp65-positive PMNs generated in vitro is to be the preferred method for production of internal slides. Whether the sensitivity or the threshold of the laboratories differ cannot be decided. This will be investigated with the distribution of external (patient) standard slides in the near future. This will answer the question whether the assay is to be standardized for comparison of the results. In conclusion, this study describes the basic requirements towards the standardization of the HCMV-Agemia assay, and the main results are that unfixed standard slides can be distributed at RT provided they are wrapped in aluminium, vacuum sealed, and stored at -80C upon arrival until staining. The pp65-positive PMNs generated in vitro made by coculture are better suited for standardization purposes than the baculovirus-expressed pp65-positive insect cells. Within individual laboratories, the relative differences between the standard samples were recognized correctly. However, in absolute terms, there were still differences between the participating centres. These differences can be diminished with standard slides as described here so an optimal concordance of test results between different laboratories can be obtained.

31

Standardization of Antigenemia

Reference List 1.

van der Bij W, Torensma, R, van Son, W J, Anema, J, Schirm, J, Tegzess, AM, and The, TH. Rapid Immunodiagnosis of Active Cytomegalovirus Infection by Monoclonal Antibody Staining of Blood Leucocytes. J.Med.Virol. 1988;25(2):179-88.

2.

Boeckh M, Bowden RA, Goodrich JM, Pettinger M, and Meyers JD. Cytomegalovirus Antigen Detection in Peripheral Blood Leukocytes After Allogeneic Marrow Transplantation. Blood 1-91992;80(5):1358-64.

3.

van den Berg AP, Klompmaker IJ, Haagsma EB, Scholten-Sampson A, Bijleveld CM, Schirm J, van der Giessen M, Slooff MJ, and The TH. Antigenemia in the Diagnosis and Monitoring of Active Cytomegalovirus Infection After Liver Transplantation. J.Infect.Dis. 1991;164(2):265-70.

4.

The TH, van der Ploeg M, van den Berg AP, Vlieger AM, van der Giessen M, and van Son WJ. Direct Detection of Cytomegalovirus in Peripheral Blood Leukocytes--a Review of the Antigenemia Assay and Polymerase Chain Reaction. Transplantation 1992;54(2):193-8.

5.

Gerna G, Zipeto D, Parea M, Revello MG, Silini E, Percivalle E, Zavattoni M, Grossi P, and Milanesi G. Monitoring of Human Cytomegalovirus Infections and Ganciclovir Treatment in Heart Transplant Recipients by Determination of Viremia, Antigenemia, and DNAemia. J.Infect.Dis. 1991;164(3):488-98.

6.

The TH, van den Berg AP, van Son WJ, Klompmaker IJ, Harmsen MC, van der Giessen M, and Slooff MJ. Monitoring for Cytomegalovirus After Organ Transplantation: a Clinical Perspective. Transplant.Proc. 1993;25(5 Suppl 4):5-9.

7.

Docke WD, Simon HU, Fietze E, Prosch S, Diener C, Reinke P, Stein H, and Volk HD. Cytomegalovirus Infection and Common Variable Immunodeficiency [Letter]. Lancet 1991;338(8782-8783):1597.

8.

Bein G, Bitsch A, Hoye, J, and Kirchner H. The Detection of Human Cytomegalovirus Immediate Early Antigen in Peripheral Blood Leucocytes. J.Immunol.Methods 1991;137(2):175-80.

9.

Revello MG, Percivalle E, Zavattoni M, Parea M, Grossi P, and Gerna G. Detection of Human Cytomegalovirus Immediate Early Antigen in Leukocytes As a Marker of Viremia in Immunocompromised Patients. J.Med.Virol. 1989;29(2):88-93.

10. Jiwa NM, van de Rijke FM, Mulder A, van der Bij W, The TH, Rothbarth PH, Velzing J, van der Ploeg M, and Raap AK. An Improved Immunocytochemical Method for the Detection of Human Cytomegalovirus Antigens in Peripheral Blood Leucocytes. Histochemistry 1989;91(4):345-9. 11. Gerna G, Revello MG, Percivalle E, and Morini F. Comparison of Different Immunostaining Techniques and Monoclonal Antibodies to the Lower Matrix Phosphoprotein (Pp65) for Optimal Quantitation of Human Cytomegalovirus Antigenemia. J.Clin.Microbiol. 1992;30(5):1232-7. 12. Wunderli W, Kagi MK, Gruter E, and Auracher JD. Detection of Cytomegalovirus in Peripheral Leukocytes by Different Methods. J.Clin.Microbiol. 1989;27(8):1916-7. 13. The TH, van den Berg AP, Harmsen MC, van der Bij W, and van Son WJ. The Cytomegalovirus Antigenemia Assay: a Plea for Standardization. Scand.J.Infect.Dis.Suppl 1995;99:25-9. 14. Grundy JE, Lawson KM, MacCormac LP, Fletcher JM, and Yong KL. Cytomegalovirus-Infected Endothelial Cells Recruit Neutrophils by the Secretion of C-X-C Chemokines and Transmit Virus

32

Chapter 2 by Direct Neutrophil- Endothelial Cell Contact and During Neutrophil Transendothelial Migration. J.Infect.Dis. 1998;177(6):1465-74. 15. Revello MG, Percivalle E, Arbustini E, Pardi R, Sozzani S, and Gerna G. In Vitro Generation of Human Cytomegalovirus Pp65 Antigenemia, Viremia, and LeukoDNAemia. J.Clin.Invest 1998;101(12):2686-92. 16. Gerna G, Percivalle E, Torsellini M, and Revello MG. Standardization of the Human Cytomegalovirus Antigenemia Assay by Means of in Vitro-Generated Pp65-Positive Peripheral Blood Polymorphonuclear Leukocytes. J.Clin.Microbiol. 1998;36(12):3585-9. 17. Grefte JM, van der Gun BT, Schmolke S, van der Giessen M, van Son WJ, Plachter B, Jahn G, and The TH. Cytomegalovirus Antigenemia Assay: Identification of the Viral Antigen As the Lower Matrix Protein Pp65 [Letter]. J.Infect.Dis. 1992;166(3):683-4.

33

Standardization of Antigenemia

34

Chapter 3

Effects of changing immunosuppressive regimen on the incidence, duration, and viral load of Cytomegalovirus infection in renal transplantation: a single center report.

Eltjo F. de Maar, Erik A.M. Verschuuren, Jaap J. Homan-vd Heide, Diane M. Kas-Deelen, Danny Jagernath, T. Hauw The, Rutger J. Ploeg and Willem J. van Son.

Transplant infectious diseases, 2002 Jun;4(1):17-24

Changing IS and CMV

Abstract Background. In this retrospective single center study we have evaluated the relation between the immunosuppressive regimen and the incidence and characteristics of Cytomegalovirus (CMV) infection in the setting without CMV prophylaxis from 1989 through 1998. Methods. All (470) first cadaveric renal transplantations in non-sensitized (PRA 600 days after Ltx and IgG against VCA (Y2 axis) became positive almost 800 days after Ltx. No IgG or IgM against EBNA-1 and EA(D) was found.

He had developed IgM and IgG anti-VCA antibodies at 17 weeks after lung transplantation in high titre (Fig. 2) that had gradually decreased. No change in titre was observed shortly before the diagnosis of plasmacytoma. Of 8 patients who did not develop PTLD, one was excluded from serological evaluation due to early death, three developed serological signs of aprimary infection with a partial response consisting of exclusively IgM and IgG to VCA in two patients (patient 7 and 8) and additional IgM to EA(D) in one patient (patient 5). The other four patients remained seronegative during follow up of 10, 20, 27 and 54 weeks.

101

EBV serology and PTLD Seven of 102 (7%) EBV seropositive recipients developed PTLD at 4, 5, 6, 12, 32, 39 and 65 months after Ltx (Table 4). Limited and heterogeneous antibody responses could be demonstrated in 4 of 7 patients, in 2 subjects before the diagnosis of PTLD. No serological changes were seen in the remaining patients. Table 3: H istology, im m unostaining and EB V detection of PT LD ’s. Patient no. 1 1 (relapse) 2

3 4 13 14 15 16 17 18

19

Histology

Immunohistological staining ND

EBV detection

not obtained

-

-

large B-cell blastoid lymphoproliferation plasmacytoma large cell anaplastic B-cell lymphoma large B-cell blastoid lymphoproliferation blastoid cells, some with plasmacytoid differentation large B-cell blastoid lymphoproliferation large B-cell blastoid lymphoproliferation large B-cell blastoid lymphoproliferation

CD20/CD22 IgM, Lambda CD138, IgA (CD20 neg) CD20, CD30, CD45

EBER EBER EBER

ND L26 (CD20)

EBER LMP

L26 (CD20) CD20/22 CD20/22, CD21, mostly kappa, some lambda CD20, CD45, partly CD30

EBNA-2, LMP LMP LMP

Large B-cell blastoid lymphoproliferation large B-cell blastoid lymphoproliferation

monomorfous large cell B-cell lymphoma

failed LMP, EBER

EBER

EBV, Epstein-Barr virus; EBNA, Epstein-Barr nuclear antigen; LMP, Latent membrane protein. Patients 1-4: EBV seronegative before Ltx. Patients 13-19: EBV seropositive before Ltx. Routine immunological staining was done for CD20/CD22 (B-cell), CD45, CD3 (T-cell) and, in case of suspicion of plasmacytoma, for CD138. EBV detection was initially performed by EBNA-2 staining, later monoclonal antibodies against LMP were used and in situ hybridization on EBER

As confirmation of the limited antibody responses in both groups of lung transplant recipients, immunoblotting of consecutive sera was carried out in a number of representative patients. Evaluation of the seronegative recipients with PTLD confirmed the absence of serological response in patient 1 and showed only antibodies against VCA-p18 10 months after Ltx in patient 4 (Fig. 2). Three seropositive recipients with PTLD were evaluated (Fig. 3) and five without PTLD (data not shown). In patient 17, with PTLD, serological reactivation was confirmed by immunoblot. In patient 15 and 16 there was no change in immunoblot pattern at the diagnosis of PTLD. In patient 15, the intensity of the EBNA-1 (P72) signal decreased over time, compatible with an earlier report [Riddler et al.]. The five seropositive recipients

102

Chapter 6 without PTLD showed patterns similar to the patients with PTLD with a relative few EBV reactive bands mainly consisting of the VCA-specific tegument protein P18, reactivation patterns were not detected. Table 4: EBV seropositive Ltx recipients with PTLD Patient no.

Onset of PTLD (months after Ltx)

13 14 15 16 17 18 19

65 39 12 H 6 HH 4 5 32

Significant changes* in antibody titre (weeks before (-) or after (+) diagnosis of PTLD) IgM IgG EA(D) VCA EBNA-1 EA(D) VCA +4 (rise) -2 (rise) +0 (pos) +0 (rise) +0 (pos) +0 (rise) -3 (pos) -

* conversion from negative to positive titre or at least 10EU rise in antibody titre pos = negative becomes positive titer; rise = at least 10 EU rise in antibody titre In patients no. 15 and 16 no serological follow-up (H diagnosis at post-mortem, HH patient died 12 days after diagnosis) - = no serological changes.

Figure 2: Immunoblot analysis of EBV seronegative recipient 1 and 4. Patient 1 shows a faint EBNA-1 band shortly after Ltx due to transfused IgG. No other bands were detected for 14 weeks. In patient 4 after 56 weeks only VCA-P18 was detected.

103

EBV serology and PTLD Figure 3: Immunoblot analysis of EBV seropositive recipients 15, 16 and 17. In patient 15 only anti EBNA-1 antibodies are detected that decrease in time, in patient 16 only preexistent anti VCA-P18 and anti EBNA-1 antibodies were detected and only patient 17 showed antibodies with new specificities after PTLD

104

Chapter 6

Discussion Our data confirm that EBV-seronegative lung transplant recipients have a significant greater risk of post-transplant lymphoproliferative disease (33% vs 7%) than do seropositive recipients. Moreover, two other trends were disease developed earlier in the former recipients (within 3 months in three of four recipients), whereas the earliest post-transplant lymphoproliferative disease in seropositive recipients were diagnosed approximately 6 months after lung transplantation. A further striking feature was the localization of the posttransplant lymphoproliferative disease: whereas the early post-transplant lymphoproliferative disease were almost all localized in the transplanted organ, the late post-transplant lymphoproliferative disease were mostly localized in the gut although disseminated post-transplant lymphoproliferative disease and other organ involvement was also seen. The goals of our study were to evaluate the course and patterns of serological responses to EBV and their impact as a marker for early diagnosis and prognosis of post-transplant lymphoproliferative disease. In previous studies, no diagnostic role for EBV serology, using crude antigen extracts, could be shown. For this reason, we analyzed whether measurements against enriched preparations of synthetic EBV antigens would enable a sensitive detection of relevant antibodies. IgG antibodies against the latent phase antigen EBNA-1 were not detected in any of the patients, not even when immune suppression was lowered after diagnosis of post-transplant lymphoproliferative disease. This may reflect the inability of recipient T cells to kill latently infected EBV-positive B cells effectively, which would normally lead to exposure of the nuclear EBNA-1 and subsequent induction of anti-EBNA1 antibody responses. On the other hand, appearance of anti-VCA IgM and IgG antibodies (directed mainly against the immunodominant BFRF3-encoded p18 tegument protein) would reflect active viral replication in the recipients. However, the limited response to BMRF1encoded EA(D) and other EA and VCA proteins of the lytic phase again indicate insufficient immune responses as none of the patients with primary EBV infection did develop the full spectrum of anti-EBV antibodies characteristic of infectious mononucleosis in the immune competent host [Middeldorp and Herbrink, 1988]. Patient no. 1, who developed post-transplant lymphoproliferative disease 7 weeks after lung transplantation, achieved remission after tapering of immunosuppression and high doses of acyclovir but showed no subsequent antibodies against EBV, a clear example of the impaired serological response after quadruple immune suppression. Only after a second post-transplant

105

EBV serology and PTLD lymphoproliferative disease in the sigmoid did this patient demonstrate seroconversion. Considering cellular immune responses after the reduction of immune suppression as a possible forerunner of the initial remission this case also suggests that humoral immune responses are not an unequivocal reflection of an EBVdirected immune defense. Taken together, the results show that in EBVseronegative recipients antiEBV antibody responses are detectable only after the clinical diagnosis of posttransplant lymphoproliferative disease. In EBVseropositive recipients developing post-transplant lymphoproliferative disease, a rise in antibody titre was seen only in a few of the patients and even in those cases shortly before the diagnosis of post-transplant lymphoproliferative disease to be of diagnostic importance. Consequently, this highly sensitive quantitative serology is not suitable for early diagnosis of post-transplant lymphoproliferative disease. The antibody patterns measured by ELISA were supported by immunoblot analysis. The antibody diversity underlying the anti-EBV antibody response has proved informative for EBV-associated acute, chronic, and malignant diseases in the immune competent host [van der Giessen et al., 1990; Brink et al., 1997]. Immunoblot assays of the sera of our lung transplant recipients showed no other specificity than tested in the peptide-based ELISA, confirming the immunodominant nature of the three peptide reagents used and confirming the very restricted antibody responses in transplant recipients, similarly described by Cen et al. [1993]. Thus, antibodies against the peptide antigens used in the ELISA used in this study may be considered representative of the restricted humoral immune response against EBV in patients with severe immune suppression. In this respect lung transplant recipients are at the extreme of this spectrum. The lack of serological response is somewhat surprising because lung transplant recipients are in general, at least partially, capable of producing antibodies to viral antigens, as detected after CMV infection. Apart from a hampered humoral immune response, this may suggest that EBV-antigens are minimally exposed, probably because they remain associated with the latently transformed cells [Wagner et al., 1995; Babcock et al., 1999]. A possible reason for a suppressed lytic cycle replication and limited presentation of EA and VCA antigens could be the acyclovir prophylaxis that is given as standard treatment to our lung transplant recipients for prevention of a-herpesvirus infection. Acyclovir has been shown to limit productive infection in vitro and reduce oral secretion of EBV in transplant recipients but has no effect on EBV genome replication and proliferation of EBV-transformed B cells [Hanto et al., 1985]. A remaining role for EBV serology could be in guiding clinical decisions, e.g., to decide when to reinstate the normal level of immune suppression after the

106

Chapter 6 patient shows signs of anti-EBV immune reactivity. An extension of the current study group, however, is necessary to substantiate this suggestion. In conclusion, anti-EBV antibody responses after lung transplantation are limited and heterogeneous, quantitative EBV serology is of limited value for the early diagnosis of post-transplant lymphoproliferative disease in both EBVseronegative and -seropositive lung transplant recipients. At best, our results suggest that EBV serology could be used as a marker for protective immunity in EBV-seronegative recipients, but the numbers are small. To monitor EBV-induced lymphoproliferation in the transplant recipient, a direct parameter such as viral load measurement by quantitative polymerase chain reaction (PCR) [Stevens et al., 1999, 2001] is probably more relevant than an indirect parameter such as EBV serology.

107

EBV serology and PTLD

References 1.

Walker,R.C., Marshall,W.F., Strickler,J.G., Wiesner,R.H., Velosa,J.A., Habermann,T.M., McGregor,C.G., and Paya,C.V., Pretransplantation assessment of the risk of lymphoproliferative disorder. Clin.Infect.Dis. 20, 1346-1353, 1995.

2.

Walker,R.C., Paya,C.V., Marshall,W.F., Strickler,J.G., Wiesner,R.H., Velosa,J.A., Habermann,T.M., Daly,R.C., and McGregor,C.G., Pretransplantation seronegative Epstein-Barr virus status is the primary risk factor for posttransplantation lymphoproliferative disorder in adult heart, lung, and other solid organ transplantations. J.Heart Lung Transplant. 14, 214-221, 1995.

3.

Aris,R.M., Maia,D.M., Neuringer,I.P., Gott,K., Kiley,S., Gertis,K., and Handy,J., Posttransplantation lymphoproliferative disorder in the Epstein-Barr virus-naive lung transplant recipient. Am.J.Respir.Crit Care Med. 154, 1712-1717, 1996.

4.

Murray,R.J., Kurilla,M.G., Brooks,J.M., Thomas,W.A., Rowe,M., Kieff,E., and Rickinson,A.B., Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J.Exp.Med. 176, 157168, 1992.

5.

Preiksaitis,J.K., Diaz-Mitoma,F., Mirzayans,F., Roberts,S., and Tyrrell,D.L., Quantitative oropharyngeal Epstein-Barr virus shedding in renal and cardiac transplant recipients: relationship to immunosuppressive therapy, serologic responses, and the risk of posttransplant lymphoproliferative disorder. J.Infect.Dis. 166, 986-994, 1992.

6.

Riddler,S.A., Breinig,M.C., and McKnight,J.L., Increased levels of circulating Epstein-Barr virus (EBV)-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of posttransplant lymphoproliferative disease in solid-organ transplant recipients. Blood 84, 972-984, 1994.

7.

McKnight,J.L., Cen,H., Riddler,S.A., Breinig,M.C., Williams,P.A., Ho,M., and Joseph,P.S., EBV gene expression, EBNA antibody responses and EBV+ peripheral blood lymphocytes in posttransplant lymphoproliferative disease. Leuk.Lymphoma 15, 9-16, 1994.

8.

Rogers,B.B., Conlin,C., Timmons,C.F., Dawson,D.B., Krisher,K., and Andrews,W.S., Epstein-Barr virus PCR correlated with viral histology and serology in pediatric liver transplant patients. Pediatr.Pathol.Lab Med. 17, 391-400, 1997.

9.

Abedi,M.R., Linde,A., Christensson,B., Mackett,M., Hammarstrom,L., and Smith,C.I., Preventive effect of IgG from EBV-seropositive donors on the development of human lympho-proliferative disease in SCID mice. Int.J.Cancer 71, 624-629, 1997.

10. Nadal,D., Guzman,J., Frohlich,S., and Braun,D.G., Human immunoglobulin preparations suppress the occurrence of Epstein- Barr virus-associated lymphoproliferation. Exp.Hematol. 25, 223-231, 1997. 11. van Grunsven,W.M., Spaan,W.J., and Middeldorp,J.M., Localization and diagnostic application of immunodominant domains of the BFRF3-encoded Epstein-Barr virus capsid protein. J.Infect.Dis. 170, 13-19, 1994. 12. Middeldorp J.M, van Benthem E van Grunsven W et al. Synthetic combi-peptides containing immunodominant epitopes of EBNA-1, EA(D) or VCA marker proteins and their utilization in Epstein-Barr virus serodiagnostics. The imminologist (4th int symp clinical immunology.Abstr:7). suppl 1:56, 21-21. 1997. 13. Cooper,J.D., Billingham,M., Egan,T., Hertz,M.I., Higenbottam,T., Lynch,J., Mauer,J., Paradis,I., Patterson,G.A., and Smith,C., A working formulation for the standardization of nomenclature and for clinical staging of chronic dysfunction in lung allografts. International Society for Heart and Lung Transplantation. J.Heart Lung Transplant. 12, 713-716, 1993.

108

Chapter 6 14. Brink,A.A., Dukers,D.F., van den Brule,A.J., Oudejans,J.J., Middeldorp,J.M., Meijer,C.J., and Jiwa,M., Presence of Epstein-Barr virus latency type III at the single cell level in posttransplantation lymphoproliferative disorders and AIDS related lymphomas. J.Clin.Pathol. 50, 911-918, 1997. 15. van der,Giessen M., van den Berg,A.P., van der,Bij W., Postma,S., van Son,W.J., and The,T.H., Quantitative measurement of Cytomegalovirus-specific IgG and IgM antibodies in relation to Cytomegalovirus antigenaemia and disease activity in kidney recipients with an active Cytomegalovirus infection. Clin.Exp.Immunol. 80, 56-61, 1990. 16. Middeldorp,J.M. and Herbrink,P., Epstein-Barr virus specific marker molecules for early diagnosis of infectious mononucleosis. J.Virol.Methods 21, 133-146, 1988. 17. van Grunsven,W.M., Nabbe,A., and Middeldorp,J.M., Identification and molecular characterization of two diagnostically relevant marker proteins of the Epstein-Barr virus capsid antigen complex. J.Med.Virol. 40, 161-169, 1993. 18. Cen,H., Williams,P.A., McWilliams,H.P., Breinig,M.C., Ho,M., and McKnight,J.L., Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders. Blood 81, 13931403, 1993. 19. Wagner,H.J., Hornef,M., Middeldorp,J., and Kirchner,H., Characteristics of viral protein expression by Epstein-Barr virus- infected B cells in peripheral blood of patients with infectious mononucleosis. Clin.Diagn.Lab Immunol. 2, 696-699, 1995. 20. Babcock,G.J., Decker,L.L., Freeman,R.B., and Thorley-Lawson,D.A., Epstein-Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients [In Process Citation]. J.Exp.Med. 190, 567-576, 1999. 21. Hanto,D.W., Frizzera,G., Gajl-Peczalska,K.J., and Simmons,R.L., Epstein-Barr immunodeficiency, and B cell lymphoproliferation. Transplantation 39, 461-472, 1985.

virus,

22. Stevens,S.J., Vervoort,M.B., van den Brule,A.J., Meenhorst,P.L., Meijer,C.J., and Middeldorp,J.M., Monitoring of Epstein-Barr virus DNA load in peripheral blood by quantitative competitive PCR. J.Clin.Microbiol. 37, 2852-2857, 1999.

109

EBV serology and PTLD

110

Chapter 7

Dynamics and function of anti-Epstein-Barr virus and anti-Cytomegalovirus immune responses in the transplant patient: implications for immunosuppression

Running head: Immune respons against EBV and CMV after transplantation

Erik AM Verschuuren1, Servi JC Stevens2, Wim van der Bij1, Martin C Harmsen3, Ieneke van der Gun3, Aalzen de Haan3, Wim Timens3, Gerard Koeter1, Jaap M Middeldorp2, Cees.G.M. Kallenberg4 and T Hauw The4.

Department of 1 Pulmonary Diseases, 3Pathology and Laboratory Medicine, 4 Internal medicine University Medical Centre Groningen, p.o. Box 30.001, 9700 RB Groningen, the Netherlands. 2 Department of Pathology, University Hospital Vrije Universiteit, de Boelelaan 1117, 1081 HV, Amsterdam, the Netherlands

EBV and CMV immune responses

Abstract We studied the development of primary immune responses to Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in a CMV and EBV naive lung transplant patient in relation to virus parameters and clinical symptoms. Primary CMV infection readily resolved after treatment with ganciclovir and after seroconversion CMV antigenemia remained negative during follow-up. Despite treatment with ganciclovir primary EBV infection presented with posttransplantation lymphoproliferative disease (PTLD). PTLD was reversed by lowering immunosuppression. However, even after seroconversion PTLD relapsed twice with high levels of EBV DNA. EBV specific CD3+,CD8+, CD69+, InterferonGamma producing-T-cells became detectable at 10 weeks after lung transplantation (Ltx) and rem ained positive at significant levels. “Ex vivo” EBV specific lysis of autologous PTLD-derived lymphoblastoid cell-line could be shown by 51C r release assay. “In vivo” function w as suggested by rapid rem ission of PT LD after reduction of the immunosuppression. The EBV DNA load in peripheral blood as determined by quantitativecompetitive-PCR fluctuated with immunosuppression and not with the use of antiviral drugs. We here show the development of immune responses to CMV and EBV despite a high level of immunosuppression early after Ltx. After seroconversion primary CMV infection resolved but EBV infection resulted in relapsing PTLD in spite of substantial num bers of EBV specific C T L‟s. W e suggest that EBV activated C T L‟s w ere not restricted in number but in function due to immunosuppression, reflecting differences in immune control against CMV and EBV infected cells.

112

Chapter 7

Introduction Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) are ubiquitous human herpesviruses that persist lifelong after primary infection. While major advances in the monitoring and treatment of CMV have been made, EBV remains a source of morbidity and mortality after solid organ transplantation(1). Since monitoring of CMV became possible several strategies to control CMV after solid organ transplantation have been described. These range from treatment of clinical disease to pre-emptive and prophylactic antiviral treatment. With the availablity of antiviral drugs effective against CMV such as ganciclovir and foscarnet these treatment strategies have been proven to be highly succesfull (2). Antiviral drugs, however, are only effective against the productive replication cycle of herpesviruses. Because CMV only has a productive way of replication, CMV is susceptible to these drugs. Additionally, the productive replication cycle enholds that upon host cell lysis new virus particles are exposed to the humoral immune system and CMV neutralising antibodies have been described (3). EBV infected cells, however, have both a productive and a non-productive replication cycle. The latter functions by EBV-mediated proliferation of infected B-cells, whereas EBV lytic replication is mainly restricted to mucosal epithelia (4). In the healthy virus carrier EBV-neutralizing antibodies are readily detectable but EBV-specific-cytotoxic T-cells (EBV-CTLs) are thought to be predominantly responsible for the lifelong control of non-productive EBV replication (5,6). If the balance between EBV-driven proliferation and EBV specific immunity is disturbed, e.g. due to the use of immunosuppression, uncontrolled B-cell proliferation may result, ultimately leading to post transplant lymphoproliferative disease (PTLD) and outgrowth of malignant lymphoma (7). Because T-cells are responsible for transplant rejection they are the major targets for immunosuppression. This immunosuppression is both directed at the function of the T-cells, e.g. by using calcineurin inhibitors (cyclosporine A, tacrolimus), as well as at the numbers of T-cells, e.g. by using anti T-cell antibodies (ATG, OKT-3) and proliferation inhibitors (azathioprine and Mycophenolate Mofetil). Consequently, the intensity and spectrum of the T-cell directed immunosuppression correlates with development of PTLD (8). While antiviral drugs have been shown to be effective against the productive replication cycle of EBV (9), the effect on the proliferative replication cycle is controversial (10,11). Treatment strategies for EBV should thus be

113

EBV and CMV immune responses focussed on restoring the balance between EBV driven B-cell proliferation and Tcell mediated EBV-specific immunity. In allogeneic bone marrow transplantation (BMT), with rapid tapering of iatrogenic immunosuppression, the impairment of EBV control lies in the limited numbers of EBV-CTLs present at early stages post BMT. In this situation adoptive transfer of EBV-CTLs, either by bulk donor lymphocyte infusion or infusion of in vitro expanded EBV-CTLs, has been demonstrated to be effective (12-16). After solid organ transplantation, especially after induction therapy with anti-T-cell antibodies, EBV-CTLs will initially be absent in the circulation. Because most EBV-seropositive patients do not develop PTLD, they must be able to restore functional EBV-CTLs in time. In solid organ transplant patients who develop PTLD however, it is not directly clear whether the impairment in EBV control is a consequence of impaired function or decreased numbers of EBV-CTLs, because the immunosuppression used in these patients afflicts both. Also, the time necessary for EBV-CTLs to develop after primary infection or to restore their function in EBV-seropositive patients during immunosuppression is not known and the selective effects of different immunosuppressive regimens on cellular immune responses are not known. This, however, has implications for the choice between the therapeutic strategies for PTLD. On the one hand, when the numbers of EBV-CTLs are too low or absent as suggested by Haque et al (16,17), this would be a strong argument for adoptive transfer of EBV-CTLs. On the other hand, in case of functional impairment of adequate numbers of EBV-CTLs, the therapeutic goal should be to improve EBV-CTL function by reduction or modification of immunosuppression. Adoptive transfer of EBV-CTLs would then be a less logical treatment strategy. To explore these options we studied the development of the humoral and cellular immune responses against CMV and EBV in a CMV and EBV seronegative lung transplant recipient who was transplanted with a transplant from a CMV and EBV positive donor. This patient had many known risk factors for PTLD, that is: primary EBV- and primary CMV infection and use of r-ATG induction therapy (18). We hypothesized that, in case adequate num bers of EB V C T L‟s could be dem onstrated in this patient, the development of relapsing PTLD must have been due to the impaired function of EBV C T L‟s.(19,20).

114

Chapter 7

Patient, Materials and Methods Patient and Treatment A 24 years old CMV-seronegative and EBV-seronegative patient with cystic fibrosis received a bilateral lung transplant from a CMV and EBV seropositive donor. He received standard immunosuppression (Fig. 1A) including rabbit-Anti-Thymocyte Globulin (r-ATG,3 mg/kg, 3 times postoperatively)( Merieux, France), azathioprine (1.5-3 mg/kg/day), cyclosporine-A (Neoral, Novartis)(dose adjusted to trough levels of 400 g/l within 3 weeks tapered to trough levels of 150 g/l), prednisolone (3 times 125 mg the first day, 0.2 mg/kg/day from day 2 to the third month and 0.1 mg/kg/day thereafter). Co-trimoxazole was given as Pneumocystis Jerovici (previously Carinii) prophylaxis (960 mg eod) and Acyclovir (200 mg qd) was given as prophylaxis for alphaherpesviruses. Acute rejection episodes were diagnosed on clinical signs and symptoms and chest x-ray findings with histology if clinically indicated, and treated with pulse therapy methylprednisolone (500 or 1000 mg iv for 3 days). Ongoing transplant dysfunction was treated by replacement of azathioprine with Mycophenolate Mofetil (cellcept, Roche) from week 37 onwards. Figure 1A: Immunosuppression and antiviral therapy. CsA = cyclosporine-A, MP= pulse therapy methylprednisolone, GCV= ganciclovir, ACV= acyclovir

115

EBV and CMV immune responses Cytomegalovirus-related disease was treated with ganciclovir I.V. (Cymevene, Roche) until pp65-antigenemia levels dropped below limit of detection (21). PTLD episodes were treated by decreasing cyclosporine A trough levels to 50% (75-100 g/l) combined with high-dose aciclovir administration (800 mg, 5 doses/day) or Valaciclovir (1000 mg tid).

CMV antigenemia and CMV serology CMV antigenemia was determined as described previously (22,23). IgM and IgG antibodies against HCMV were determined weekly by a semi-quantitative ELISA using alkaline glycine-extracted HCMV antigens obtained from HCMV AD169 infected fetal fibroblasts and in parallel on an extract of mock-infected fibroblasts (24).

EBV serology Serum samples were obtained twice weekly and at every visit to the outpatient clinic and stored at -20˚C until use. O ne serum sam ple before transplantation and all sera until week 25 were examined in one assay. EBVspecific IgG and IgM antibodies were determined as described before (25). In short the test procedure was as follows. Polystyrene micro titer plates were incubated during 48 hours at 4° C with the peptide solution (1 g/ml in 0.1mM carbonate buffered solution, pH 9.6) and subsequently blocked with bovine serum albumin (BSA 3%) and sucrose (5%) in phosphate buffered saline, dried at room temperature and stored at 4° C until use. Plates were washed and sera diluted in incubation buffer containing 0.01 M Tris, 0.3 M NaCl, 0.05% Tween-20 and 2% BSA (pH7.5). Sera were diluted 1:100, 1:200, 1:400 and 1:800 for IgG and IgM. Sera were incubated on an ELISA shaker for 45 minutes at room temperature. Plates were washed and conjugates were added. (IgG conjugate, Goat-anti-human IgG-Peroxidase labeled (De Beer, the Netherlands), IgM conjugate, Goat-anti-human IgM-peroxidase labeled (Pasteur, France)) Plates with conjugates were incubated for 30 minutes at room temperature and washed. Substrate, 100 micro liter of 0,3g/l OPD in citrate buffered solution (pH=5.05) with 0,0002% H2O2, was added and after 20 minutes 100 l of 1M H2SO4 was added to stop the reaction. Plates were read at 490nm. In every assay four EBV-negative control sera were tested to determine cutoff values. T he am ount of antibody present in the patients‟ serum w as expressed in (arbitrary) Elisa Units (EU)(24).

116

Chapter 7

DNA isolation Whole blood samples for EBV DNA detection were taken weekly after transplantation. One ml of fresh unfractionated whole blood was lysed in 9 ml of NASBA lysis buffer (5 M guanidine isothiocyanate, 1.2 % Triton X-100, 20 mM EDTA, 0.1 M Tris-HCl. pH 6.4; Organon Teknika, Boxtel, the Netherlands) and stored at -80C until use. DNA was isolated from 1 ml of lysate by silica-based extraction as described previously (26). One negative control (water) was included for each run of ten whole blood samples.

Quantitative Competitive EBV-DNA PCR The experimental approach for determination of EBV DNA load in clinical specimens has been described previously (27). Briefly, the DNA equivalent of 5 l whole blood or serum was amplified in a qualitative EBNA-1 PCR. EBV-DNA load in PCR positive samples was subsequently determined by quantitative competitive EBNA-1 PCR (Q-PCR) as described (27). DNA quality of whole blood samples was checked by -globin PCR according to Roda Husman et al (28). To ensure the validity of the results, several precautions were taken to avoid contamination of the PCR (29). As control for accuracy and reproducibility of quantification, a fixed amount of WT plasmid DNA was quantified in each experiment in duplicate. In addition, all samples were screened blindly and appropriate negative and positive controls for DNA isolation, preparation of PCR master mix and EIA detection were included (one negative control for each ten tested samples and one positive control per experiment).

Isolation of PBMCs, expansion of tumor cells ex vivo and production of LCL Blood samples for isolation of peripheral blood mononuclear cells (PBMC) were taken before transplantation and at regular time intervals after transplantation. PBMC were isolated using Lymphoprep density gradients (Nycomed, Oslo, Norway) and cryopreserved in liquid nitrogen until further use. Upon use, PBMC were thawed and resuspended in RPMI supplemented with 10% heat-inactivated FCS (RPMI/FCS). Previous testing showed no loss of functionality with this procedure (data not shown). The patient-derived EBV-transformed lymphoblastoid cell-line (LCL) was expanded from (tumor cell-positive) biopsies by culturing a small sample of PTLD biopsy material, obtained during the diagnostic open lung biopsy, in RPMI/FCS containing 0.5 µg cyclosporine A per ml. Growth of cells was observed within 3 weeks of culturing and a stable cell culture was obtained. HLA-typing

117

EBV and CMV immune responses demonstrated that the outgrowing tumor cells were of recipient origin. Control LCLs were obtained by infection and transformation of B-cells from healthy controls with the B95-8 EBV strain in vitro using standard procedures.

Measurement of EBV activated T-cells by flowcytometry Recipient PBMC 2x106were added to polystyrene tubes (Greiner) containing 4 ml RPMI/FCS only (unstimulated samples) or 2x106 irradiated biopsy-derived autologous LCL in RPMI/FCS. If sufficient recipient PBMC were available, the PBMC were also stimulated with 25 ng/ml Phorbol Myristate Acetate (PMA, Sigma) and 1 g/ml ionomycin (Sigma) to serve as a positive control. Unstimulated and LCL-stimulated samples were incubated at 37C and 5% CO2 overnight. Brefeldin A (Sigma; 10 g/ml) was added to these cultures only after the initial 6 hr of culture. PMA/ionomycin-stimulated samples were incubated for 4 hr in the immediate presence of Brefeldin A (10 g/ml). After stimulation, samples were washed with PBS containing 1% BSA, and resuspended in a small volume PBS containing 1% BSA and 5% human AB serum. Then, surface markerspecific monoclonals were added (aCD3-CyQ; IQ Products, Groningen, The Netherlands, and aCD8-A PC ; Pharm ingen) and cells w ere incubated for 15‟ at room temperature. Next, FACS lysing solution (Becton Dickinson) was added and cells were incubated for 10‟ at room tem perature. T hey w ere then spun dow n, resuspended in FACS Permeabilizing Solution (Becton Dickinson) and incubated for 10‟ at room tem perature. A fter w ashing, C D 69-specific PE-labeled antibody (Coulter), Gamma-IFN-specific FITC-labeled antibody (Beckton Dickinson) or FITC-labeled isotype-matched control antibody (IQ Products) was added and cells w ere incubated for 30‟. A fter w ashing, stained cells w ere analyzed on a flowcytometer (Coulter EPICS Elite) and data were analyzed by WinList 3D (Verity Software House inc, Maine USA). Usually, over 20.000 counts (CD8 T cells) were collected for the analysis. As controls, EBV-seronegative and EBV seropositive healthy volunteers were tested.

Calculation of the absolute numbers of EBV-specific CD3+, CD8+, IFN-γ+ T -cells Total numbers of lymphocytes were determined in peripheral blood by routine methods. The percentage of CD3+, CD8+ T-cells within the lymphocyte population was analyzed by flowcytometry. Also the percentage CD3+,CD8+,CD69+, IFN-γ+ EBV specific T-cells within the CD3+,CD8+ T cell population was analyzed by flowcytometry as described above. Absolute numbers of EBV-CTLs were then calculated by multiplying total number of lymphocytes

118

Chapter 7 with the percentage of CD3+, CD8+ T-cells, and the percentage of CD3+, CD8+, CD69+, INF-γ+ T -cells, respectively (Table 1). Table 1: Calculation of absolute numbers of EBV-activated-CD8+ T-Cells. White blood cell counts (WBC) and lymphocyte counts were determined in peripheral blood. Percentages of CD3+, CD8+T-cells were analyzed by flowcytometry. Of these CD3+, CD8+ T-cells percentages of EBV-activated T-cells were analyzed by determining CD69-positive and Interferon-γ (IFN -γ ) positive cells after stimulation with cultured autologous EBV-LCLs. Absolute numbers of EBVactivated CD3+, CD8+ T-cells were than calculated by multiplying total number of lymphocytes with CD3+, CD8+ percentage and CD69+, IFN-γ+ percentage. N D = not done Period WBC Lymfocytes % CD3+ & % CD69+ & Absolute numbers Chromium after Ltx 109/L 109/L CD8+ IFN-γ + of CD3+, CD8+, Release CD69+, IFN-γ + T assay cells/L blood Pre Ltx

11.8

ND

19

0.025

ND

7% 7

5 weeks

2.2

0.198

12

0.02

0.05 x 10 /L

ND

10 weeks PTLD

5.2

0.364

13

1.12

5.3 x 107/L

30%

17 weeks 24 weeks 35 weeks 39 weeks 60 weeks

6.1 5.7 8.7 7.1 14.9

1.15 1.23 0.94 1.19 1.25

33 45 50 51 40

0.91 0.54 0.30 0.71 0.25

7

55%

7

50%

7

ND

7

43%

7

ND

34.5 x 10 /L 29.9 x 10 /L 14.1 x 10 /L 43.1 x 10 /L 12.5 x 10 /L

Cytotoxicity assay The capacity of the T cells to lyse the EBV-positive tumor cells was measured after in vitro prestimulation. Briefly, recipient T cells were isolated from cryopreserved and thawed PBMC using Lymphokwik-T (One Lambda, Montpellier, France). Then 2x106 T cells, together with 1x106 irradiated (20 Gy) PBMCs as feeder cells, were added to 1x105 autologous irradiated biopsy-derived LCL cells in a total volume of 2 ml RPMI/FCS. On days 3, 6 and 9, 0.5 ml of medium was replaced with fresh RPMI/FCS medium containing 10 U IL-2/ml. At day 12, the expanded T cells were harvested and added to 51Cr-labeled autologous and allogeneic control LC L cells (see before) at effector: target ratio‟s of 50:1, 25:1 and 12.5:1. After 4 hr of incubation, supernatants were harvested and the released 51Cr release was measured in a Packard gamma-counter. 51Cr release was measured in six individual wells per condition. Spontaneous release and maximum release were determined by incubating the labeled cells in medium alone or 5% Triton X-100 in PBS, respectively. Specific release was calculated 119

EBV and CMV immune responses using the following formula: % specific lysis = (experimental release spontaneous release) / (maximal release - spontaneous release). No lysis of the allogeneic control LCL cells was observed in these assays.

Results Clinical course of the patient and diagnosis of PTLD episodes Shortly after transplantation 2 rejection episodes were treated with pulse Methyl Prednisolone (Fig. 1A, Fig. 1B). The first treatment was given because of lowgrade fever and persistent pleural effusion and the second also because of lowgrade fever without signs of infection. CMV infection was diagnosed at week 3 and successfully treated with ganciclovir (see below). At 10 weeks after lung transplantation the patient presented with multiple nodules of 1-2 cm in size on the routinely obtained chest X-ray. Lung function was still improving (Fig. 1B) and there were no clinical symptoms of infection or rejection. An open lung biopsy revealed a PTLD by demonstrating a proliferation of EBER positive, CD20 positive and IgM kappa-positive large blastoid cells. After reduction of immunosuppression (trough level of cyclosporine to 75ng/L) all nodules disappeared within 2 weeks. Lung function decreased however gradually thereafter, and 23 weeks after lung transplantation pulse methylprednisolone was given because of suspected rejection. Cyclosporine A trough level was increased to standard maintenance level (150 ng/L). Lung function initially stabilized but then decreased further and at 27 weeks after transplantation a bronchoscopy was performed. Transbronchial biopsies showed active bronchiolitis obliterans and, to a minor extent, small aggregates of small and large lymphocytes. The large lymphocytes were EBER-positive, CD20 positive B-cells, histologically considered as residual PTLD. Presumptive diagnosis was ongoing rejection and some residual PTLD. The active bronchiolitis was treated with pulse methylprednisolone. Lung function, however, did not improve and FEV1 values decreased gradually. Azathioprine was converted to Mycophenolate Mofetil (cellcept, Roche), but FEV1 only stabilized after another pulse methylprednisolone at 42 weeks and increased maintenance dose of prednisolone. Patient then withdrew from control until week 58. At this time all blood samples were retrospectively analyzed for EBVDNA load. An increase in EBV-DNA load in the peripheral blood (Fig. 2A, see below) raised suspicion of ongoing PTLD.

120

Chapter 7 Figure 1B: Lung function depicted as FEV1 in absolute volume and as % of baseline. Baseline lung function is calculated according to ISHLT criteria: the average of the 2 best FEV1 results with at least 3 weeks interval. BOS= Bronchiolitis Obliterans Syndrome IS= Immunosuppression, MP= pulse therapy methylprednisolone

Figure 2A: Results of EBV-qPCR. Results are expressed as genomes/ml.

At 58 weeks after lung transplantation a third episode of PTLD was diagnosed. In the transbronchial biopsies aggregates of lymphoid cells were seen again. In these aggregates CD20+, EBER positive cells were demonstrated. Liver function tests deteriorated and the cyclosporine level rose to toxic levels. All

121

EBV and CMV immune responses hepatotoxic medication was stopped and patient died soon thereafter due to respiratory failure. Permission for postmortem was not obtained.

Cytomegalovirus pp65-antigenemia and serology (Fig. 3) In the immediate post-transplant period the patient developed a primary CMV infection. CMV-antigenemia became positive 3 weeks after Ltx and ganciclovir iv was started (5 mg/kg bd). There were no clinical signs or symptoms of C M V disease except for a m inor rise in body tem perature (37,4˚C ). C M V antigenemia reached a peak of 76 pp65 positive cells /50,000 and became negative 7 weeks after Ltx. Figure 3: CMV antigenemia and Serology. After anti-CMV IgM and IgG became positive CMV antigenemia remained negative.

Ganciclovir was continued until CMV-antigenemia was negative for 2 weeks and then stopped (week 9). Ganciclovir was also given from week 13 to 16 because of a positive CMV antigenemia result during rejection treatment. CMV antigenemia remained negative thereafter. IgM anti-CMV antibodies were detectable from week 3 on. IgG anti-CMV antibodies became positive 13 weeks after Ltx.

EBV-DNA load dynamics in peripheral blood (Fig. 2B) The patient also developed a primary EBV infection as confirmed by EBVserology (Fig. 2B). EBV-DNA became detectable in week 6 at 7,000 EBV DNA copies/ml blood during treatment with ganciclovir iv. It remained detectable in week 8 and 9 at this level (7,200 and 8,600 EBV DNA copies/ml blood), until

122

Chapter 7 ganciclovir was stopped when CMV antigenemia was negative for 2 weeks. In week 10 the patient presented with PTLD, accompanied by a high viral load of 66,600 EBV-DNA copies/ml blood and was admitted in our hospital. Upon admission high dose aciclovir was started (1000 mg qd) because of suspected PTLD. One week later this diagnosis was confirmed by open lung biopsy and subsequently immunosuppression was decreased. Clinical course and EBV DNA load is described above. Figure 2B: Results of EBV serology. Only anti-VCA antibodies were detected.

EBV-activated CD3+, CD8+ T-cells, Cytotoxicity assay and EBV antibody response (Table 1, Fig. 2B, Fig. 2C) In order to detect T-cell-mediated EBV-imunity, PBMC from an EBV seronegative healthy volunteer and 2 EBV seroposotive healthy volunteers were stim ulated w ith autologeous and allogeneic EB V LC L‟s and subsequently tested for CD69 expession and IFN-γ production in C D 3+, C D 8+ T -cells as measured by flowcytometry. Using this test no CD3+, CD8+, CD69+, IFN-γ+ T -cells could be demonstrated in the EBV seronegative healthy control whereas 1.10% and 1.17% of the CD3+, CD8+ T-cells, obtained from the two EBV seropositive healthy controls, expressed CD69 and produced IFN-γ after stim ulation w ith autologeous EBV-LC L‟s. T his corresponds w ith results of T -cell mediated EBV immunity described by Kuzushima et al (30). In the blood of the patient, 0.025% and 0.02% CD3+, CD8+, CD69+, IFN-γ+ T cells were detected, at the time of transplantation and 5 weeks after

123

EBV and CMV immune responses transplantation respectively, upon ex vivo stimulation with autologous EBV-LCLs. These negative results were consistent with the EBV-seronegative status of the patient before transplantation. Ten weeks after Ltx, 1.12 % CD3+, CD8+, CD69+, IFN-γ+ T -cells were detected, which co-incided with the time the patient presented with PTLD. After 17 weeks 0.94 % and thereafter between 0.71 and 0.25 % CD3+, CD8+, CD69+, IFN-γ+ T -cells were detected. Absolute numbers of CD3+, CD8+, CD69+, IFN-γ+ T -cells were calculated per liter of peripheral blood. Before Ltx and after 5 weeks EBV-activated-CD3+, CD8+ T-cells were considered negative. At presentation of PTLD at 10 weeks 5.3 x 107/L CD3+, CD8+, CD69+, IFN-γ+ T -cells further increasing to 34.5 x 107/L at 17 weeks after Ltx were detected, paralleled by the resolution of the first PTLD episode. During follow-up CD3+, CD8+, CD69+, IFN-γ+ T -cells remained detectable but decreased gradually corresponding with the increase in immunosupression, rise of EBV DNA load and relapses of PTLD. Figure 2C: Ex vivo EBV-specific cytotoxicity and absolute numbers of EBV activated-CD3+, CD8+,CD69+,IFN-γ+ T -cells as calculated from peripheral blood lymphocyte count. In vitro EBV-specific lysis of autologeous EB V LC L’s w as 25% at 10 weeks (presentation of PTLD) and rose to 55% at 17 weeks (after reduction of immunosuppression and disappearance of PTLD). It was 50 and 43% at week 24 and 39 respectively

The pattern observed in the development of CD3+, CD8+, CD69+, IFN-γ+ T cells corresponded with the pattern in EBV-specific lysis as measured by chromium release assay. Before transplantation the EBV specific lysis was, as expected, very low (7 % lysis). Specific lysis increased to 30 % in week 10, when the patient presented with PTLD and increased further to 55 % in week 17 and remained high with 43 % in week 40. This showes that functional EBV-specific T124

Chapter 7 cells remained present throughout the patient follow-up, despite the relapse of PTLD and rise in EBV DNA load. EBV-VCA specific IgM and IgG antibodies were negative prior to Ltx and became detectable in week 12 and 21 respectively, confirming a primary EBVinfection and reflecting the patients immune responses to EBV antigens in vivo. No antibodies were detected against EA(d) or EBNA-1 during the follow-up period showing only a limited serological response to EBV antigens.

Discussion In this case study we noticed a clear dichotomy between the development of immunity against CMV and EBV. Our patient was seronegative for both CMV and EBV prior to transplantation and received an organ from a CMV and EBV seropositive donor. He subsequently developed a primary infection for both CMV and EBV during the initial phase of high immunosuppression, given after lung transplantation. While CMV was easily controlled with a pre-emptive strategy using ganciclovir, EBV was never controlled in this patient and ultimately might have caused his death. This difference might be explained by the ways of replication for both viruses. CMV replicates in a lytic productive fashion exposing both structural and infected cellassociated antigens to cellular and humoral immune system, leading to the rapid induction of cytolytic and virus neutralizing effector mechanisms. EBV however associates with latent replication by inducing proliferation of infected B-cells with only limited lytic virus replication. This latent lymphoproliferation enables EBV to multiply its genome with minimal exposure to a virus-neutralizing humoral immune response. This may in part explain why in our patient EBV escaped from immune control. Analysis of the source of EBV-DNA in this patient revealed an exclusive cellular origin, as no cell-free EBV-DNA was detected in plasma or serum, not even at the highest peaks of EBV DNA-emia (14). Although VCA-IgM and VCA-IgG responses were detected, these developed rather late and their contribution to effective immune control remains undefined. The absence of anti-EBNA1 antibody responses may be a reflection of poor cellular immunity as suggested by previous publications (31,32). This also illustrates the importance of functional cellular immune responses for the control of EBV. When analysing the cellular immune response against EBV we noticed that EBV-responsive CD3+, CD8+ T-cells developed within 10 weeks and that these EBV-activated-CTLs were functional upon further stimulation ex vivo, as reflected by their capacity to lyse autologous PTLD-derived LCL. Clinically their functionality in vivo was suggested by the rapid regression of all PTLD lesions

125

EBV and CMV immune responses when immunosuppression was decreased. So we conclude that EBV-specific cytotoxic T-cells (EBV-CTLs) developed already 10 weeks after transplantation in spite of high level of induction therapy with r-ATG and maintenance immunosuppression. However, despite the primed cellular immune status, subsequent PTLD episodes developed, as reflected by increasing EBV-DNA levels and presence of muliple EBV-positive cells in biopsy specimens. This suggests a functional deficiency in the circulating T-cells to counteract the growth potential of EBV-infected B-cells, which may reach a doubling time of 56 hours in vivo (14) Although we found between 5.3 and 43.1 x 107 EBV-activated-CTLs/L (30), these numbers are considerably higher than reached by the adoptive transfer of ex vivo cultured EBV-CTLs (12,16,33). This raises the question wether the detected EBVspecific T cells were truly functional in vivo. To explain this paradox, it may be assumed that the EBV-specific CTLs we detected by flow cytometric detection of intracellular IFN- production display a „functional diversity‟. T his „functional diversity‟ has previously been illustrated for EB V -specific T cell lines which showed an inverse relationship between IFN- production and cytotoxic potential (34,35). This could mean that the EBV-specific cytotoxic T cells, as detected in the 51-chromium release assay, arise from a different subpopulation of CD8 T cells than EBV specific T cells with IFN- producing capacity, which we demonstrated by flow cytometry. On the other hand, effector CD8 T cells usually display both perforin/granzymes and IFN- (36). It is of interest to note that Van Baarle et al found that increasing numbers of IFN- producing EBV specific Tcells rather than increasing numbers of EBV tetramer-positive T cells predicts good antiviral responses and reduction of EBV viral load in immunocompromised patients, such as HIV carriers (37). Finally, it can be argued that a combined low proliferative and functional capacity of the EBV-reactive T-cells, caused by the immunosuppressive therapy, contributed to the failure to control EBV. Summarizing, we think that the EBV-CTLs in our patient were not impaired in numbers, but in their in vivo function, most likely due to the effects of maintenance immunosuppression. Apparently this dysfunction of T-cells is still adequate to control CMV infection, once acute replication is suppressed by antiviral drug treatment. An alternative explanation for the impaired EBV control might be that recurrent PTLD were due to a variant EBV strain with mutations in important CD8-Tcell epitopes, thus escaping immune control (38). Additionally, chronic (low-grade) CMV infection with accumulation of large clones of replicative senescent T-cells may have hampered the development and maintenance of an effective T-cell mediated EBV specific immune response (39,40)

126

Chapter 7 In the studies on adoptive transfer of EBV-CTLs for PTLD Khanna et al (33) describe a lung transplant recipient with primary EBV infection and PTLD. All PTLD lesions disappeared after infusion of in-vitro (in absence of immunosuppressive) expanded EBV-CTLs. In this study PTLD also relapsed (33), indicating that the restoration of the number of EBV CTLs alone was not enough and led only to a temporary control of EBV as observed in our patient. Thereafter maintenance immunosuppression again impaired their in vivo function. This indicates that, in the long term, not only restoration of numbers but also restoration of function should be the therapeutic objective. Reduction of immunosuppression should thus be part of any treatment strategy for EBV after solid organ transplantation. The results of the EBV-DNA Q-PCR clearly reflect the fragile balance between EBV and the impaired immune system after solid organ transplantation. These observations also point to a crucial difference in the long term control of CMV and EBV infection respectively, the latter being more dependent on T-cell mediated functions. Initially, the patient developed a primary EBV infection associated with low levels of circulating EBV-DNA that developed into a PTLD 10 weeks after Ltx. Reduction of CsA led to a complete remission and EBV-DNA disappeared from the peripheral blood. The acute rejection that was triggered by this decrease in immunosuppression, was treated with pulsed methylprednisolone (MP) and an increase of CsA. Immediately EBV became detectable again (Fig 2A), suggesting a profound effect of immunosuppressive drugs on the EBV-CTL control of EBV. Because all samples were retrospectively analyzed this was not known at that time and the subsequent transplant dysfunction was interpreted as ongoing rejection and therefore treated again with pulse MP. This clearly demonstrates the need for careful and parallel monitoring EBV-DNA levels. This patient also illustrates the contrast in the effects of antiviral medication between EBV and CMV infection. The primary CMV infection was rapidly controlled by ganciclovir therapy which effectively blocks lytic viral replication and viral spread. After CMV-specific antibodies became detectable in the peripheral blood CMV-antigenemia remained negative and no CMV related morbidity was seen. This is in contrast to the EBV primary infection. Initial rise in EBV-DNA levels occurred during ganciclovir treatment, and EBV-specific antibodies became detectable only after the first episode of PTLD. Thereafter two relapses of PTLD were seen and EBV-DNA remained detectable in the circulation despite continued high dose (val-)aciclovir. The explanation of this contrast between the two herpesviruses is believed to be twofold. First, CMV mainly produces a lytic viral infection after which the virus has to spread

127

EBV and CMV immune responses systemically to infect distant organs and cells. EBV on the other hand remains latent while driving infected cells (B-cells) into proliferation. This way, the EBV multiplication is relatively shielded from the humoral immune response. Secondly, the pre-emptive use of ganciclovir reduces CMV replication levels which may allow even a suboptimal T-cell system to maintain control, whereas EBV replication is relatively resistant to antiviral drugs because EBV uses the cellular DNA polymerase to multiply the latent genome (41) and lytic cycle associated phosphokinases are not induced. It is suggested that EBV infection require a more optimal T-cell system for effective short- and long-term control (42). In conclusion we suggest that not the lack in EBV-CTL numbers but a lack in function induced by immunosuppression is responsible for PTLD after solid organ transplantation. Under the same therapeutic regimen, CMV infection can be controlled effectively. Regarding the fragile balance between EBV viral load and levels of immunosuppression we suggest that pre-emptive treatment guided by EBV-DNA levels should be the strategy of choice. This approach should include reduction of immunosuppression in relation to EBV-CTL function. Furthermore, studies are required to clarify the mechanistic differences in long-term control of CMV and EBV infection, including TCR-diversity and effector functions of humoral immunity.

Acknowledgements We thank the laboratory of transplant Immunology of the University Hospital Groningen for the collection of the whole blood samples.

128

Chapter 7

References 1.

Opelz G, Dohle B. Lymphomas after solid organ transplantation: A collaborative transplant study report. American Journal of Transplantation 2004:4: 222-230.

2.

Snydman DR. Counterpoint: Prevention of Cytomegalovirus (CMV) infection and CMV disease in recipients of solid organ transplants: The case for prophylaxis. Clinical Infectious Diseases 2005:40: 709-712.

3.

Shimamura M, Mach M, Britt WJ. Human Cytomegalovirus infection elicits a glycoprotein M (gM)/gN-specific virus-neutralizing antibody response. J Virol 2006:80: 4591-4600.

4.

Shannon-Lowe CD, Neuhierl B, Baldwin G, Rickinson AB, Delecluse HJ. Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells. Proc Natl Acad Sci U S A 2006:103: 7065-7070.

5.

Moss DJ, Wallace LE, Rickinson AB, Epstein MA. Cytotoxic T cell recognition of Epstein-Barr virusinfected B cells. I. Specificity and HLA restriction of effector cells reactivated in vitro. Eur J Immunol 1981:11: 686-693.

6.

Wallace LE, Rickinson AB, Rowe M, Epstein MA. Epstein-Barr virus-specific cytotoxic T-cell clones restricted through a single HLA antigen. Nature 1982:297: 413-415.

7.

Savoie A, Perpete C, Carpentier L, Joncas J, Alfieri C. Direct correlation between the load of Epstein-Barr virus-infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood 1994:83: 2715-2722.

8.

Opelz G, Henderson R. Incidence of non-Hodgkin lymphoma in kidney and heart transplant recipients [see comments]. Lancet 1993:342: 1514-1516.

9.

Preiksaitis JK, Diaz-Mitoma F, Mirzayans F, Roberts S, Tyrrell DL. Quantitative oropharyngeal Epstein-Barr virus shedding in renal and cardiac transplant recipients: relationship to immunosuppressive therapy, serologic responses, and the risk of posttransplant lymphoproliferative disorder. J Infect Dis 1992:166: 986-994.

10. Starzl TE, Nalesnik MA, Porter KA et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporine-steroid therapy. Lancet 1984:1: 583-587. 11. Green M, Kaufmann M, Wilson J, Reyes J. Comparison of intravenous ganciclovir followed by oral acyclovir with intravenous ganciclovir alone for prevention of Cytomegalovirus and Epstein-Barr virus disease after liver transplantation in children. Clin Infect Dis 1997:25: 1344-1349. 12. Rooney CM, Smith CA, Ng CY et al. Use of gene-modified virus-specific T lymphocytes to control Epstein- Barr-virus-related lymphoproliferation. Lancet 1995:345: 9-13. 13. Nalesnik MA, Rao AS, Furukawa H et al. Autologous lymphokine-activated killer cell therapy of Epstein-Barr virus-positive and -negative lymphoproliferative disorders arising in organ transplant recipients. Transplantation 1997:63: 1200-1205. 14. Rooney CM, Smith CA, Ng CY et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998:92: 15491555. 15. Papadopoulos EB, Ladanyi M, Emanuel D et al. Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation [see comments]. N Engl J Med 1994:330: 1185-1191. 16. Haque T, Amlot PL, Helling N et al. Reconstitution of EBV-specific T cell immunity in solid organ transplant recipients. J Immunol 1998:160: 6204-6209.

129

EBV and CMV immune responses 17. Haque T, Thomas JA, Parratt R, Hunt BJ, Yacoub MH, Crawford DH. A prospective study in heart and lung transplant recipients correlating persistent Epstein-Barr virus infection with clinical events. Transplantation 1997:64: 1028-1034. 18. Walker RC, Marshall WF, Strickler JG et al. Pretransplantation assessment of the risk of lymphoproliferative disorder. Clin Infect Dis 1995:20: 1346-1353. 19. Cockfield SM, Preiksaitis JK, Jewell LD, Parfrey NA. Post-transplant lymphoproliferative disorder in renal allograft recipients. Clinical experience and risk factor analysis in a single center. Transplantation 1993:56: 88-96. 20. Ho M, Miller G, Atchison RW et al. Epstein-Barr virus infections and DNA hybridization studies in posttransplantation lymphoma and lymphoproliferative lesions: the role of primary infection. J Infect Dis 1985:152: 876-886. 21. van den Berg AP, Tegzess AM, Scholten-Sampson A et al. Monitoring antigenemia is useful in guiding treatment of severe Cytomegalovirus disease after organ transplantation. Transpl Int 1992:5: 101-106. 22. van der Bij W, Torensma R, van Son WJ et al. Rapid immunodiagnosis of active Cytomegalovirus infection by monoclonal antibody staining of blood leucocytes. J Med Virol 1988:25: 179-188. 23. van den Berg AP, van der Bij W, van Son WJ et al. Cytomegalovirus antigenemia as a useful marker of symptomatic Cytomegalovirus infection after renal transplantation--a report of 130 consecutive patients. Transplantation 1989:48: 991-995. 24. van der Giessen M, van den Berg AP, van der BW, Postma S, van Son WJ, The TH. Quantitative measurement of Cytomegalovirus-specific IgG and IgM antibodies in relation to Cytomegalovirus antigenaemia and disease activity in kidney recipients with an active Cytomegalovirus infection. Clin Exp Immunol 1990:80: 56-61. 25. Verschuuren E, van der BW, de Boer W, Timens W, Middeldorp J, The TH. Quantitative EpsteinBarr virus (EBV) serology in lung transplant recipients with primary EBV infection and/or posttransplant lymphoproliferative disease. J Med Virol 2003:69: 258-266. 26. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der NJ. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990:28: 495-503. 27. Stevens SJ, Vervoort MB, van den Brule AJ, Meenhorst PL, Meijer CJ, Middeldorp JM. Monitoring of Epstein-Barr virus DNA load in peripheral blood by quantitative competitive PCR. J Clin Microbiol 1999:37: 2852-2857. 28. Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ. The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 1995:76 ( Pt 4): 1057-1062. 29. Kwok S, Higuchi R. Avoiding false positives with PCR [published erratum appears in Nature 1989 Jun 8;339(6224):490]. Nature 1989:339: 237-238. 30. Kuzushima K, Hoshino Y, Fujii K et al. Rapid determination of Epstein-Barr virus-specific CD8(+) T-cell frequencies by flow cytometry. Blood 1999:94: 3094-3100. 31. Cen H, Williams PA, McWilliams HP, Breinig MC, Ho M, McKnight JL. Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders. Blood 1993:81: 13931403. 32. Stevens SJ, Blank BS, Smits PH, Meenhorst PL, Middeldorp JM. High Epstein-Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology. AIDS 2002:16: 993-1001. 33. Khanna R, Bell S, Sherritt M et al. Activation and adoptive transfer of Epstein-Barr virus-specific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc Natl Acad Sci U S A 1999:96: 10391-10396.

130

Chapter 7 34. Picker LJ, Singh MK, Zdraveski Z et al. Direct demonstration of cytokine synthesis heterogeneity among human memory/effector T cells by flow cytometry. Blood 1995:86: 1408-1419. 35. Nazaruk RA, Rochford R, Hobbs MV, Cannon MJ. Functional diversity of the CD8(+) T-cell response to Epstein- Barr virus (EBV): implications for the pathogenesis of EBV- associated lymphoproliferative disorders. Blood 1998:91: 3875-3883. 36. Hamann D, Roos MT, van Lier RA. Faces and phases of human CD8 T-cell development. Immunol Today 1999:20: 177-180. 37. van Baarle D, Hovenkamp E, Callan MF et al. Dysfunctional Epstein-Barr virus (EBV)-specific CD8(+) T lymphocytes and increased EBV load in HIV-1 infected individuals progressing to AIDSrelated non-Hodgkin lymphoma. Blood 2001:98: 146-155. 38. Gottschalk S, Ng CY, Perez M et al. An Epstein-Barr virus deletion mutant associated with fatal lymphoproliferative disease unresponsive to therapy with virus-specific CTLs. Blood 2001:97: 835-843. 39. Fletcher JM, Vukmanovic-Stejic M, Dunne PJ et al. Cytomegalovirus-specific CD4+ T cells in healthy carriers are continuously driven to replicative exhaustion. J Immunol 2005:175: 82188225. 40. Vescovini R, Telera A, Fagnoni FF et al. Different contribution of EBV and CMV infections in very long-term carriers to age-related alterations of CD8+ T cells. Exp Gerontol 2004:39: 1233-1243. 41. Andersson J, Skoldenberg B, Ernberg I, Britton S, Henle W, Andersson U. Acyclovir treatment in primary Epstein-Barr virus infection. A double- blind placebo-controlled study. Scand J Infect Dis Suppl 1985:47: 107-115. 42. Yao QY, Ogan P, Rowe M, Wood M, Rickinson AB. Epstein-Barr virus-infected B cells persist in the circulation of acyclovir-treated virus carriers. Int J Cancer 1989:43: 67-71.

131

EBV and CMV immune responses

132

Chapter 8

Frequent monitoring of Epstein-Barr virus DNA load in unfractionated whole blood is essential for early detection of Post Transplant Lymphoproliferative Disease in high risk patients*

Servi JC Stevens#, Erik AM Verschuuren#, Inge Pronk, Wim van der Bij, Martin C Harmsen, T Hauw The, Chris JLM Meijer, Adriaan JC van den Brule & Jaap M Middeldorp

Blood 2001;97(5):1165-1171

# These authors contributed equally to this publication

*

This work was supported by European Community Grant no. 1C-18-CT960132.

Early detection of PTLD

Abstract Posttransplant Lymphoproliferative Disease (PTLD) is a frequent and severe Epstein-Barr virus (EBV)-associated complication in transplant recipients that is caused by iatrogenic suppression of T-cell function. The diagnostic value of weekly EBV DNA load monitoring was investigated in prospectively collected unfractionated whole blood and serum samples of lung transplantation (LTx) recipients with and without PTLD. In PTLD patients, 78% of tested whole blood samples were above the cut-off value of quantitative competitive polymerase chain reaction (Q-PCR) (greater than 2000 EBV DNA copies per ml blood), with the majority of patients having high viral loads before and at PTLD diagnosis. Especially in a primary EBV-infected patient and in patients with conversion of immunosuppressive treatment, rapid increases in peripheral blood EBV DNA load diagnosed and predicted PTLD. In non-PTLD transplantation recipients, only 3,4% of the whole blood samples was above the cut-off value (P90%) of EBV in the general population and the presence of detectable EBV-DNA in blood up to months before the clinical manifestations of PTLD (6,8) suggest that PTLD is the tip of the iceberg of EBV reactivation and that EBV reactivation is more common than previously recognized. A striking feature of PTLD is, on the one hand, its correlation with increase in immunosuppression and, on the other, its treatment that consists primarily of reduction of immunosuppression. The usual explanation is that the increase in immunosuppression, given because of rejection, results in EBV reactivation and PTLD (9). In our previous observations, however, we frequently observed that EBV infection was already present before rejection therapy was started, and suggestions of a relation between transplant dysfunction and Epstein Barr virus have been made (10-12). Therefore, we suggest an alternative explanation, namely that EBV reactivation, if unrecognized, might mimic rejection, and thus, leads to rejection treatment, over-immunosuppression and, ultimately, PTLD. The implementation of routine EBV-DNA detection in blood samples has allowed us to explore this hypothesis. The present report describes the retrospective analysis of blood levels of EBV-DNA in relation with transplant function and histolopathological findings in 29 lung transplant recipients.

Materials and Methods Patients The initial study population consisted of 34 consecutive recipients of a primary bilateral or unilateral lung transplant. Five patients were excluded 167

EBV and Transplant dysfunction because of early death (primary graft failure in 3 patients, lethal hemorrhage and complicated relapse of tuberculosis in one patient each). The remaining 29 patients all survived for more than one year and a complete follow up was available from all of them. Induction treatment included 3 gifts of rabbit-antithymocyte-globulin (Merieux, France), 3 mg/kg, during the first 10 days after transplantation. Maintenance immunosuppressive regimen consisted of cyclosporine-A (Neoral, Novartis, Switzerland), azathioprine (2 mg/kg/d), and prednisolone (0.2 mg/kg/d with subsequent tapering to 0.1 mg/kg/d). Cyclosporine-A treatment aimed at a trough level of 400 ng/ml directly after transplantation, as determined by highperformance liquid chromatography, which was tapered in 3 weeks to reach a trough level of 150 ng/ml. Acute rejection was treated with a 3 days course of 500 to 1000 mg methylprednisolone intravenously daily. All patients received acyclovir, 4 daily doses of 200 mg orally, for herpes prophylaxis, and co-trimoxazole, 960 mg orally, on alternate days for Pneumocystis Jerovici (formerly Carinii) prophylaxis. No Cytomegalovirus (CMV) prophylaxis was given at that time.

Diagnostic protocol and routine follow-up Graft function was determined by formal spirometry according to ATS/ERS guidelines, with emphasis on forced-expiratory-volume-in-one-second (FEV1). Measurements were done at least twice weekly during hospitalization and at every outpatient visit, which were initially weekly and gradually reduced to once every three months one year after lung transplantation (Ltx). Acute allograft rejection was diagnosed clinically in case of deteriorating pulmonary function after exclusion of infection. Histologic diagnosis of rejection was defined according to ISHLT criteria (13). EBV-DNA load was measured retrospectively in, for that purpose, stored blood samples. Results were not available at the time of treatment, and, as a consequence, did not influence clinical management. CMV infection or reactivation was monitored by testing for CMV antigenemia (14) and CMV serology (15). Active CMV infection was defined as the presence of pp65positive cells in the CMV antigenemia test. Bronchoscopies were routinely performed within 6 weeks after lung transplantation, at 6 months intervals, and when clinically indicated. Bronchoscopy included bronchoalveolar lavage (BAL) and transbronchial biopsies, as described previously(16). All BAL samples were cultured for (myco)bacteria, fungi and yeast, and subjected to auramine and silver staining for detection of Mycobacterium Tuberculosis and Pneumocystis Jerovici, respectively.

168

Chapter 10

Blood sampling for EBV-DNA From 1997 until 2001, whole blood samples of all lung transplant recipients were prospectively collected by venapuncture during the first postoperative year, weekly during admission and at every outpatient visit. For EBV PCR analysis 1 ml of EDTA-treated whole blood was mixed thoroughly with 9 ml of NASBAlysisbuffer (Organon Technika,Oss, The Netherlands) and stored at -80°C until use (11). Serially obtained samples of our study population were retrospectively tested for EBV DNA.

Quantitative EBV-DNA PCR The quantitative EBV-DNA PCR was carried out as described previously (6). In short, one ml of freshly obtained unfractionated whole blood was lysed in 9 ml of lysisbuffer and stored at -80C until use. DNA was isolated from 1 ml of lysate by silica-based extraction. The DNA equivalent of 5 l whole blood or serum was amplified in a qualitative EBNA-1 PCR. When the qualitative EBNA-1 PCR tested positive EBV-DNA load was subsequently determined by quantitative competitive EBNA-1 PCR (Q-PCR). The cut-off value used in Q-PCR was 2000 copies/ml blood of EBV-DNA load, which is the detection limit of this assay. DNA quality of whole blood samples was checked by -globin PCR(6).

EBV-DNA load in relation to transplant (dys)function EBV reactivation was considered present when the number of EBV-DNA copies exceeded the detection limit. As a marker of the severity of EBV reactivation the peak DNA load of an EBV reactivation episode was used. Peak values of EBV reactivation were related to changes in FEV1 (defined as the difference between the average of the last two FEV1 values measured within 3 months before EBV-DNA tested positive and the FEV1 value measured at the time EBV-DNA became positive). Care was taken to exclusively include FEV1 values taken prior to, i.e. not influenced by, a diagnostic or therapeutic intervention (e.g. bronchoscopy or rejection treatment). For this part of the analysis blood samples and routine lung function data available after LTx were used. To further substantiate the relation between EBV and transplant (dys)function we evaluated the response to rejection treatment, that is pulse methylprednisolone, in the presence or absence of positive testing for EBV DNA.

EBV-DNA load and histopathology All routine biopsies were re-evaluated, especially for EBER positive cells in biopsies taken during the presence of positive blood tests for EBV-DNA. EBV in

169

EBV and Transplant dysfunction biopsies was analyzed by EBER1/2 in situ hybridization (8). When necessary, additional EBER-ISH was performed. Biopsy specimens of lesions suspected of PTLD were stained by immunohistochemistry as described before (8).

Statistical analysis SPSS software (release 12.0.2) was used (Spearmans rho test) to determine the correlation between EBV-DNAemia and changes in FEV1.

Results EBV-DNA load Twenty-nine out of the 34 consecutive LTx recipients survived the initial phase after transplantation and were included in the study. Patient data are given in table 1. Of these 29 patients, 18 patients remained EBV-DNA negative throughout the observation period. The median number of samples tested per patient in this group was 9 (range 8-17). Eleven patients became EBV-DNA positive. T hese patients w ere divided in a group w ith „low ‟ EB V D N A load and a group w ith „high‟ EBV D N A load . This was arbitrarily based on the peak level of EBV DNA load (Table 1). T he EB V D N A „low ‟ group consisted of 6 patients w ith 13 EBV DNA positive samples out of 96 samples tested, and an EBV-DNA load up to 14,600 genom es/m l w hole blood. T he EBV „high‟ gro up consisted of 5 patients with 67 EBV DNA positive samples out of 100 samples tested, and peak levels of EBV DNA ranging from 22,900 to 137,600 EBV-DNA genomes/ml (Table 1).

EBV-DNA and transplant function The 11 patients who became EBV DNA positive presented 20 episodes of EBV reactivation. Five episodes started shortly after transplantation before routine lung function testing had started. For one episode the first EBV DNA positive sample had been taken elsewhere and lung function testing was not available from the start of the EBV DNA positive episode. From the remaining 14 episodes, lung function data were available from before the start of the episode, and, thus, the relation between EBV reactivation and changes in pulmonary function could be evaluated. Appearance of EBV DNA coincided in time with decrease in FEV1 in 10 out of the 14 episodes (in 8 patients) (median 19%, range 2-35%) (representative examples are given in Fig. 1a-c).

170

Chapter 10 Table 1: Patient data and results of EBV DNA testing. (AT-def= Alpha-1antitrypsin deficiency). Other indications were Bronchiectasies (3), Pulmonary fibrosis (1), Primary pulmonary hypertension (1) and Lymfangioleiomyomatosis (1) No=number, pt=patient. EBV neg

No of patients

EBV pos Low

high

18

6

5

13 (5)

3 (2)

1

Indications for lung transplantation Emphysema (AT-def) Cystic Fibrosis

4

Other

1

3

2

39 (13-55)

47 (34-55)

48 (21-61)

Median age (range, yrs) No of patients with EBV mismatch No of EBV DNA pos. samples (samples tested) Median no of EBV DNA pos samples /pt (range) Median no of samples tested per patient (range) peak EBV DNA load (genomes/ml whole blood)(range) Median EBV DNA load

2

0

0

1

0(187)

13(96)

67(100)

0 9 (8-17)

1 (1-6) 14(10-29)

13 (4-23) 20(13-29)

NA

2,400-14,600

22,900-137,600

NA

6,500

98,400

No of PTLD

1

0

3

No of patients with CMV mismatch

1

1

3

31(1.7)

14(2.3)

15(3)

No of episodes with rejection treatments (mean/pt)

Response to Rejection treatment and EBV DNA load Overall, from one month after Ltx on, 60 courses of rejection treatments were given to the 29 patients. There was a gradual increase in the number of rejection treatment courses given in the “high” EB V D N A group (1.7, 2.2 and 3.0 rejection treatm ent episodes per patient in the EBV D N A negative, the EBV D N A “low ” and th e EBV D N A “high” group, respectively, although this was not statistically significant). Twenty-nine episodes of rejection treatment were recorded in the EBV DNA positive groups. Response to rejection treatment was defined as increase of FEV1 within a period of one month after starting treatment. Failure was defined as a decrease in FEV1 below the level of the FEV1 value at the time rejection therapy was started or as ongoing rejection based on the clinically judged necessity to give another rejection treatment within one month. 171

EBV and Transplant dysfunction Results are depicted in figure 2a. The patients with EBV reactivation received 29 rejection treatment couses. Twelve were given at a time that EBV DNA was present and resulted in only 3 responses. In contrast, 17 courses of rejection treatment were given to 7 of the former 12 patients at a time that EBV DNA was absent. A response to treatment was noted after 12 out of these 17 courses (p