Viral Infection in Transplantation: Indirect Effects Jay A. Fishman, M.D. Infectious Disease and Transplantation Units, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

Viruses may be dangerous . . . .

Three months after renal transplantation: TMP-SMZ allergic patient

Pneumocystis pneumonia: Methenamine silver stain

Failure to respond to appropriate anti-P. carinii therapy

The Growing Family of Viral Pathogens in Transplantation HERPES SIMPLEX VARICELLA ZOSTER EPSTEIN-BARR VIRUS CYTOMEGALOVIRUS HHV6 (& role with CMV) HHV7 (role?) HHV8/KSHV HIV WEST NILE VIRUS RABIES HEPATITIS B and C PAPILLOMAVIRUS POLYOMAVIRUS BK/JC ADENOVIRUS, RSV INFLUENZA, PARAINFLUENZA METAPNEUMOVIRUS PARVOVIRUS B19 SMALLPOX/VACCINIA SARS coronavirus

Effects of Viral Infection in Transplantation DIRECT CAUSATION OF INFECTIOUS DISEASE SYNDROMES Nephrtitis, hepatitis, neutropenia Allograft injury often greater than systemic IMMUNOMODULATORY EFFECTS SYSTEMIC IMMUNE SUPPRESSION -- OI’s CELLULAR EFFECTS - Graft Rejection, GvHD ABROGATION OF TOLERANCE ONCOGENESIS Hepatitis B: hepatocellular carcinoma Epstein Barr Virus: B-cell lymphoma (PTLD) Hepatitis C: splenic lymphoma (villous lymphocytes) Papillomavirus: Squamous cell & anogenital cancer HHV8 (KSHV): Kaposi’s sarcoma, effusion lymphoma

CMV: Direct Effects Endothelial & smooth muscle cells, PBMC Macrophages in the lungs Pancreas? Retina? Virus activated by allogeneic response, antilymphocyte antibodies, TNF (via NF-B), other proinflammatory cytokines AJ Koffron et al, J Virol, 1998, 72:95-103; E. Fietz et al, Transplantation, 1994, 58:675-680; P. Reinke et al, Transplant. Infect Dis, 1999, 1:157-164; M Hummel et al, J Virol, 2001, 75:4814-4822.

CMV Retinitis: Lung Transplant Recipient

Invasive Colitis After Liver Transplant

CMV cecal ulceration in patient with negative antigenemia and PCR assays for CMV

CMV “Indirect Effects”: Possible Mechanisms Upregulation of MHC class II antigens and homolog of MHC class-I (HLA-DR, Fujinami RS, et al. J Virol. 1988;62:100-105. S. Beck, Nature. 1988;331:269-272) Blocks CD8+ (MHC class I) recognition Blocks CMV antigen processing and display (immediate early Ag modification, poor CTL response) Increased ICAM-1, VCAM, cellular myc & fos Inversion of CD4/CD8 ratio (Schooley 1983, Fishman 1984) Increased cytokines: IL-1, TNF, IFN, IL-10, IL-4, IL-8, IL-2/IL-2R, C-X-C chemokines and IL-8 (Kern et al, 1996; CY Tong, 2001) Increased cytotoxic IgM (Baldwin et al, 1983) Stimulation of alloimmune response by viral proteins (Fujinami et al, 1988, Beck et al, 1988) Increased PDGF, TGF Increased granzyme B CD8+ T-cells, -T-cells

CMV: Likely Indirect Effects Indirect effects observed clinically are often uncertain given exogenous immune suppression, rejection, drugs Opportunistic infections Co-factor to EBV in PTLD HHV6 infection Graft rejection (acute and chronic) Vanishing bile duct syndrome (if it exists) Co-factor in Bronchiolitis Obliterans Syndrome Accelerated atherogenesis in cardiac allografts

Opportunistic Infections Promoted by CMV Infection in Transplant Patients Pneumocystis carinii Fungal infections (esp. intra-abdominal transplants): Candidemia and intra-abdominal infection in OLTx; patients with initial poor graft function Aspergillus spp. Role of CMV in promoting fulminant HCV hepatitis rather than direct effect Bacteremia: Listeria monocytogenes Epstein-Barr virus infection (RC Walker et al, CID, 1995, 20:1346-55) HCV: risk for cirrhosis, retransplantation, mortality

CMV and Graft Dysfunction: Renal Distinguish between studies demonstrating link of CMV to graft dysfunction and improved outcomes with prevention - may be the same or different effects (Herpes Virus Infection Syndrome) CMV Disease causes poor renal graft function at 6 mos and CMV & HHV6 are associated with chronic dysfunction (3 yrs) (CY Tong et al, Transplant. 2002, 74:576-8) Acute but not Chronic allograft rejection is reduced by CMV prevention in liver and kidney (D+/R-) Tx (D Lowance et al, NEJM 1999, 340:1462-70; E. Gane et al, Lancet 1998, 350:1729-33) HHV6 increases CMV infection and OI’s and possibly some acute rejection in renal (A. Humar, Transplant 2002, 73:599-604) & liver recipients (JA DesJardin CID 2001, 33:1358-62; PD Griffiths et al, J Antimicrob Chemother, 2000, 45 sup 29-34) HHV7 associated with increased CMV infection and with acute rejection (IM Kidd et al, Transplant 2000, 69:2400-4)

CMV and Graft Dysfunction: Liver CMV is associated with cirrhosis, graft failure, retransplantation, and death in liver allograft recipients (KW Burak et al, Liver Transplant 2002, 8:362-9) CMV is associated with more aggressive HCV recurrence and fibrosis after OLTx (partially attributed to HHV6) (A. Sanchez-Fueyo et al, Transplant 2002, 73:56-63; N Singh et al, Clin Transplant 2002, 16:92-6; HR Rosen et al, Transplant 1997, 64:721; R. Patel et al, Transplant 1996, 61:1279) Roles of immune suppression, CMV-induced immune suppression & HCV, CMV-induced TGF/fibrosis

Impact of CMV in Heart & Lung Transplantation Obliterative bronchiolitis (BOS) increased in: Serologic R+ and D+ combination Role of asymptomatic CMV replication? CMV infection raises OB to ~60% (Zamora MR. TransID 2001; 3: 49-56 and Am J Tx 2004, 4:1219-1226) Infection of vascular endothelia and smooth muscle cells  Adhesion molecules (VCAM,ICAM,LFA-1,VLA-4)  HLA-DR and MHC Class I mimic  Anti-endothelial Abs? Cytotoxic T-cells?

CMV and Graft Dysfunction: Heart-Lung CMV is associated with Coronary allograft vasculopathy (MT Grattan et al, J Am Med Assoc 1989,261:3562-6) Prophylaxis using CMVIg with ganciclovir reduces cardiac transplant vasculopathy (HA Valantine et al, Circulation 1999, 100:61-6; HA Valentine et al, Transplantation 2001, 72:1647-1652) CMVIG plus DHPG reduced CMV incidence, rejection, and death vs. DHPG alone Coronary Tx vasculopathy reduced Lung and heart-lung recipients had less obliterative brohchiolitis, death from OB, better survival, fewer infections Less PTLD in double Rx CMV disease and D+/R- status are associated with chronic rejection, bacterial and fungal pneumonia, OB and death in Lung Tx (SR Duncan 1992; NA Ettinger 1993; K Bando 1995; RE Girgis 1996; RN Husni 1998) Reduction in BOS and fungus with iv ganciclovir (SR Duncan et al, Am J Crit Care Resp Dis 1994, 150:146-152; DR Snydman NEJM 1987, 317:1049-1054; JA Wagner et al Transplant 1995, 60:1473-7)

Special Risks for CMV in Heart & Lung Transplantation? Lungs: Exposure to environment (stimulation) Lymphatic tissue with graft Macrophage burden with graft Major site of viral “latency” (Balthesen M et al, J Virol 1993; 67:5360-5366) Recurrent infections - stimulation

How best to impact indirect effects? Does prophylaxis delay or prevent CMV infection and disease? - Yes (M Halme Transplant Int 1998, S499-501; JL Kelly et al, Transplant 1995, 59:1144-7) Does prophylaxis delay or prevent CMV-mediated effects? Role of CMV may be uncertain but clinical data support this concept. Do we need to prevent other viral infections? Yes How to best use “screening tests” depends on goal of therapy - prevent CMV disease vs. asymptomatic infection & presumed indirect effects? What is the optimal regimen? Need further data.

Summary: Effects of Antiviral Agents on Allograft Injury Valacyclovir in kidney recipients  50%  in rejection Oral ganciclovir in heart, liver, kidney recipients   trend in rejection Prophylactic IV ganciclovir in heart recipients  long-term benefit in  of vasculopathy Unfortunately, we do not know much about the effect of antivirals at this point. Valacyclovir, the valine ester of acyclovir, is more effective than placebo in terms of prophylaxis, and it has been shown to reduce rejection rates among kidney recipients.1 Ganciclovir is an extremely potent anti-CMV drug. When administered for 1 month to heart transplant recipients, IV ganciclovir provided a long-term benefit as evidenced by a decrease in vasculopathy in seropositive recipients.2 To date, a trend toward a decrease in rejection has been seen with oral ganciclovir.3,4 Lowance D, et al, for the International Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group. N Engl J Med. 1999;340:1462-1470. Valantine HA, et al. Circulation. 1999;100:61-66. Ahsan N, et al. Clin Transplant. 1997;11:633-639.

Indirect Effects of CMV: Summary Renal: Decreased early graft function and some chronic dysfunction (increased by HHV6 and HHV7) Acute but possibly not chronic allograft rejection is reduced by CMV prophylaxis Liver: CMV associated with cirrhosis, graft failure, need for retransplantation & death More aggressive HCV recurrence and fibrosis after OLTx (partially attributed to HHV6) CMV disease is preventable Heart: cardiac allograft vasculopathy Reduced by ganciclovir +/- CMV Ig Lungs: CMV and D+/R- associated with Bronchiolitis Obliterans Syndrome, infection, death Reduced by iv ganciclovir +/- CMV Ig Pancreas: Not studied (no CMV yet in islets)

Indirect Effects: Other Viruses Cytomegalovirus: best studied, global immune suppression, increased graft rejection Hepatitis B and C: increased incidence of opportunistic infection Epstein-Barr virus: link to non-Hodgkin's lymphoma Parvovirus B19: elaboration of cytokines, autoimmune effects, Cellular apoptosis RSV, Coronavirus, influenza: ciliary injury, local suppression

“Herpes Virus Infection Syndrome” in Transplant Patients HHV6 and HHV7 are risk factors for CMV disease, invasive fungal infection (DH Dockrell et al, Transplant. 1999, JID, 1997; S. Chapenko et al, Clin Transplant 2000, 14:486-92; CY Tong et al, Transplant 2000, 70:213-6; IM Kidd et al, Transplant 2000, 69:2400-4) CMV D+/R- and D-/R+ groups with invasive disease more likely to have co-infection with HHV6, HHV7) HHV6 and HHV7 associated with positive CMV antigenemia in liver Tx (I. Lautenschlager, J. Clin Virol 2002) HHV6 and CMV associated with more severe recurrence/fibrosis with HCV (A. Humar et al, Am J Transplant 2002) Ganciclovir reduces load of all 3 viruses (JC Mendez et al, JID, 2001; 183:179-184) while CMVIg does not (JA Desjardins et al, JID, 1998, 178:1783-6).

Examples: Parvovirus B19 Ubiquitous virus (50-90%) of children and adults. Spread by contact, transfusion. Peak incidence in late Winter and early Spring. DNA virus, 5600 bp. Features: Tropism for erythroid cells via P-glycoprotein (glyboside) Causes elaboration of cytokines Cellular apoptosis (caspase 3 inhibition via down-regulation of Bcl-2) Cellular cytotoxicity in vitro (in vivo?)

Parvovirus B19 - Myocarditis Lymphocytic myocarditis in an 11 mo child (Papadogiannakis et al, CID 2002: 35, 1027) Patchy necrosis of myocardium Mononuclear cell infiltration of myocardium, lungs Normal bone marrow Anemia, lymphocytic myocarditis 2 mos post cardiac transplantation

Parvovirus: Mechanisms P-receptor binding Erythroid precursors (fetal anemia, hydrops?) Giant pronormoblasts with intranuclear inclusions, vacuoles Megakaryoblasts (platelets) Endothelial cells (vasculitis) Myocardial cells (myocarditis uncommon but may be rapid and severe in normal hosts) (Porter et al, Lancet 1988; 1, 535-6; Naides & Weiner, Prenat Dx 1989; 9: 105-14) Autoimmune via -T-cells? (Eck et al, Am J Surg Path 1997; 21: 1109-12) Lower level of virus in heart vs other unaffected tissues (Murray et al, Hum Path 2001, 32:342-5) CD8+ cell recruitment (Tolfuenstam et al, J Virol. 2001; 75:540)

Respiratory Viruses

RSV: Pediatric Liver Transplant Recipients 3.5% of 493 children >5 years; median, 20 months old 76% nosocomial; median, 24 days posttransplantation Tachypnea 65%, cough 53%, fever 53%, wheeze 29%, pneumonia 35% Coinfections: bacteremia/fungemia 35%, fungal/bacterial pneumonia 24%, CMV 24% No ribavirin therapy: mortality 12% Peigue-Lafeuille H, Gazuy N, Mignot P, Deteix P, Beytout D, Baguet JC. Severe respiratory syncytial virus pneumonia in an adult renal transplant recipient: successful treatment with ribavirin. Scand J Infect Dis 22:87-9, 1990. Pohl C et al. J Infect Dis. 1992;165:166-169.

Parainfluenza in Lung Transplant Recipients Median onset, 2.1 years (range, 0.6-5) posttransplantation Presenting symptoms Cough 71% Shortness of breath 64% Fever 17% Pneumonia 17% 18 (82%) had concurrent rejection; 32% progressed to bronchiolitis obliterans 9 of 10 treated with ribavirin survived Vilchez RA, et al. Clin Infect Dis. 2001;33:2004-2008. Wendt CH et al. J Heart Lung Transplant. 1995. 14:479-485. Hayden FG et al. Antivir Ther. 1996;1:51-56.

Indirect Effects and Respiratory viral infections RVI  increased risk of 2.1 fold for development of Aspergillus infections.(13) RVI  risk factor for graft rejection, particularly chronic graft rejection in lung transplants.(14-19) RVI of the lower respiratory tract, but not the upper tract, predispose to bronchiolitis obliterans syndrome (BOS) (RR, 2.3; 95% CI, 1.1-4.9).(14) Rat lung transplant model of Sendai virus infection, a virus related to parainfluenza virus (PIV) links lower tract disease and BOS.(20) Seasonal trend to BOS that peaks shortly after the peak of winter respiratory viral infections.(5) Marr KA et al. Blood 2002;100(13):4358-66; Billings JL et al. J Heart Lung Transplant 2002;21(5):559-66; Chakinala MM, Walter MJ. Semin Thorac Cardiovasc Surg 2004;16(4):342-9.; Garantziotis S et al. 2001;119(4):1277-80; Khalifah AP at al. Am J Respir Crit Care 2004;170(2):181-7; Vilchez RA et al. Am J Transplant 2003;3(3):245-9; Winter JB et al. Transplant. 1994;57(3):418-22).

Where Do Indirect Effects Stop and Where Does Oncogenesis Start?

Viral Oncogenesis and Proliferative Events in Transplantation CMV: Early arteriosclerosis, role in PTLD. Epstein Barr Virus: PTLD (B-cell), Hodgkin’s, T-cell (Asia), Burkitt’s (c-myc, P. falciparum), Cofactor in Kaposi’s? Papillomavirus: Squamous, Anogenital Cancers HHV8/KSHV: Kaposi’s sarcoma, Castleman’s ds. Hepatitis B (C?): Hepatocellular carcinoma Hepatitis C: Splenic, non-Hodgkin’s lymphoma (immunostimulation of villous B-lymphocytes) HTLV-1: Adult T-cell leukemia & lymphoma BK virus: Ureteric smooth muscle proliferation JC virus: PML, neuroglial tumors?

HHV8/KSHV Risk Factors: Risk of KS increased with positive HHV8 serology (RR: 4.9-28.4) Immune Status: Risk of KS increased by CyA vs. Aza Risk of KS increased by MMF vs. Aza HHV8 activated by steroids in vitro (BCBL-1) Antilymphocyte antibodies - Relative Risk=11.3 Non-neoplastic lymphoproliferative (plasmacytoid) disorders (Matsushima, Am J Surg Path 1999, 23:1393-1400) and PEL (Dotti, Leukemia 1999, 13:664-70) post-TX Many proliferative disorders

PTLD and EBV Infection in Transplantation 28-49-fold increase in transplant recipients above age-matched controls. 55% “Benign” polyclonal B-cell proliferation (“infectious mono”) 30% Polyclonal or oligoclonal +/- early malignant transformation 15% Extranodal, monoclonal B-cell disease (Based on Ig rearrangements or EBV genome termini) Of non-Hodgkin’s lymphomas: 87.0% arose in B-lymphocytes, 12.6% of T-cell origin, 0.4% null cell origin.

EBV-associated malignancies Burkitt’s lymphoma (endemic, sporadic) CNS lymphoma (compromised host) B-cell lymphoma* (PTLD) T-cell lymphoma (nasal, angiocentric) Primary effusion lymphoma (AIDS, HHV8) Hodgkin’s* (mixed cellularity) Nasopharyngeal carcinoma (Lymphoepthelioid) Smooth muscle tumor (compromised host) *Broad array of EBV latency proteins are expressed (contrast other EBV-associated tumors).

Colitis 36 year old male status post heart transplant with abdominal pain, diarrhea, no h/o bowel disease.

EBV Colitis? Uncommon Syndrome? In Situ Hybridization: Edematous colonic mucosa, focal ulceration and granulation tissue with bizarre stromal cells; No viral cytopathic changes are identified, and immunostains for CMV, HSV-1 and HSV-2 are negative. In-situ hybridization for EBV (EBER probe) reveals occasional positive cells in the epithelium. The pattern of staining in most cells is suggestive of cytoplasmic staining by endocrine cells. THE LYMPHOID CELLS ARE NEGATIVE.

Other Indirect Effects

Why is tolerance difficult to produce? It is worth recalling that the adaptive immune system (specificity and memory) was (likely) developed to protect against infectious challenges, not allografts. Successful (animal) tolerance induction has generally been achieved in mice relatively free of long-lived memory T-cells. These are usually pathogen-free mice capable of generating anergy, deletion (apoptosis), suppression (Tregs) or immune deviation (non-harmful phenotype) of naïve T-cells.

Heterologous Immunity What is the effect of prior infectious exposures to immune responses to allografts? Virally induced alloreactive memory provides a barrier to transplantation tolerance (AB Adams et al, JCI 2003:111:1887-95) and may  autoimmunity Alloreactive T-cells are activated by viral infections (Yang H and Welsh RM JI 1986: 1186-1193; Braciale TJ et al J Exp Med 1981, 153:1371-76; Chen HD 2001, Nat Imm 2:1067-76) Allo-cross reactivity of CMV and EBV Pre-existing alloreactive memory T-cells increase rejection rate (Heeger PS et al. JI 1999, 163:2267-75)

Heterologous Immunity Viral infections  alloreative memory T-cells These CD8+ central memory T-cells confer resistance to tolerance induction The level of resistance to tolerance induction is related to the number of prior infectious exposures Resistance can be adoptively transferred Studied vaccinia virus, vesicular stomatitis virus, lymphocytic choriomeningitis virus

Longer term implications? The role of “persistent” infection in alloimmune responses is under study Down-regulation of immune responses to virus over time (Treg CD4+ cells) also decrease anti-tumor responses (e.g., HCV, CMV, murine Friend virus) Other viruses escape immune responses by altering cytokine responses (pox & herpes) or hiding (papillomavirus) These infections may serve as models for the host response to allografts (e.g., Zinkernagel)

Hemorrhagic Fever Viruses What’s Next? Variola Vaccinia Smallpox Ebola Marburg Agents of Bioterrorism Hemorrhagic Fever Viruses Unknown The most lethal agents known to man Ebola: mortality of 88% (in some outbreaks) Marburg: 25% Lassa: 5-10% Depending who you read…. To date, reservoirs of these two agents are unknown (By comparison, mortality of pneumonic plague: 40-70%) SARS

Thank you for inviting me. I would be happy to answer questions. If I can help: jfishman@partners.org