CMV and Herpesviruses Michael Boeckh, M.D. Fred Hutchinson Cancer Research Center University of Washington Seattle, WA, USA

Human Cytomegalovirus Beta herpesvirus Seroprevalence 50-95% Establishes latency Sites of latency –Mononuclear cells –Polymorphonuclear cells –Tissue (e.g. lung )

CMV Seroprevalence in the US Staras et al. Clin Inf Dis, 2006

Transmission of CMV Saliva –Toddlers Blood products Transplanted organs Breast milk Trans-placental Sexual contact –Semen –cervical secretion

CMV Clinical Disease in Healthy Subjects Mononucleosis Hepatitis Meningoencephalitis Myocarditis Interstitial pneumonia

Impact of CMV in Immunocompromised Hosts Stem Cell Transplant Disease (Pneumonia, GI Disease, Graft Failure etc.) Mortality Hospitalization Donor Pool Other infections ? GvHD Solid Organ Transplant Disease (Syndrome, Hepatitis, Pneumonia etc.) Other Infections Hospitalization Donor Pool Rejection HIV Disease (Retinitis, GI, CNS etc.) Hospitalization Blindness Mortality Quality of Life Treatment- or Prophylaxis-Related Adverse Events

Congenital CMV Acquisition during pregnancy % of life birth Appr. 105 symptomatic High morbidity of disease –Leading cause of CNS maldevelopment in children Hearing loss Mental retardation Jaundice Microcephaly Seizure Long-term sequelae

CMV Pneumonia in HCT Recipients 1970s and early 1980s: CMV disease is the leading infectious cause of death after HCT 85-90% fatality rate

Time to CMV Reactivation and Disease after HCT Any CMV AG/DNA = Reactivation CMV Disease

pp65 AG/DNA CMV viral load Days after Transplantation CMV Viral Load Endpoints o o o o o o o peak Area Under the Curve (AUC) Initial Load (Any Reactivation) Preemptive Antiviral Treatment

CMV Disease Pneumonia Gastrointestinal disease Retinitis Encephalitis Hepatitis Marrow suppression Immunosuppressive effects Rejection GvHD

Late CMV Disease in HCT Clinical Manifestations < Day 100 >Day 100 Retinitis Marrow failure Encephalitis Sinusitis Cystitis Boeckh & Marr 2002

CMV: Current Issues Transplantation Drug toxicity HCT > SOT Survival disadvantage of seropositive patients undergoing URD/TCD TxMainly HCT Late DiseaseHCT = SOT Drug resistance SOT > HCT

Granulocytes Days after Transplantation ProphylaxisPre-emptive Antigenemia DNA/RNA Current Prevention Strategies CMV

CMV Prevention Strategies ProphylaxisIndirect Effects Preemptive TherapyDirect Effects

CMV Prevention in HCT Recipients History Prophylaxis Preemptive Therapy

Antiviral Drug Time Preemptive Therapy PCR, pp65 AG Pp67 mRNA RTC Ganciclovir+ Foscarnet+ Cidofovirnot tested Valganciclovirpilot study

Start of Ganciclovir Based on –Threshold correlating with disease –In vivo replication dynamics Highly immunosuppressed patients have a shorter replication time

Start of Ganciclovir Antigenemia: low/moderate risk CMV D-/R- Autologous > 1 mg/kg1/slide Autologous < 1 mg/kg5/slide

Viral Doubling Time in vivo relative to Degree of Immunosuppression Weeks Log 10 CMV Weeks FastSlow

Viral Doubling Time in vivo relative to Degree of Immunosuppression Weeks Log 10 CMV Weeks FastSlow R+ or D+/R- allograft < 1 mg/kg steroids and no T cell depletion All other allos CD34-s autos Cord blood: very fast

CMV Viral Load Assay Variability Quantitative DNA assays have lower variability than antigenemia assay Coefficient of variation of most DNA assays < 0.3 Viral load increases of > 0.5 log 10 likely to indicate true increase

Preemptive Therapy Threshold Levels for Starting Therapy RiskThreshold Very High< day 100:Any level > day 100:1000 copies/mL High< day 100: 100 copies/mL* > day 100: 1000 copies/mL Low< day copies/mL* > day copies/mL * Repeat after 2-3 days if less and treat if next value > 5x baseline

Other Key Points Stop of preemptive therapy: –One negative test Valganciclovir –After day 100: ok for induction –Before day 100: generally IV induction

Case 58 yo women, 9 months after NM PBSCT for ALL in 1 st remission Early posttransplant complications –2 episodes of CMV reactivation (+ one pre-Tx) –Acute GvHD –RSV URI Now severe GI GVHD, diagnosed 4 weeks ago –1 mg/kg steroids, FK506 and beclomethasone dipropionate Admitted with respiratory failure requiring intubation

Case – continued CMV reactivation 7 weeks prior to admission: –plasma PCR 1100 treated with valganciclovir, –switched to 450 mg/day after one week, –switched back to acyclovir prophylaxis a 2 days prior to admission Lab: Crea 0.2, bili 23.4, AST 390 PCP prophylaxis: –atovaquone 1500 mg/day, –acyclovir 800 mg BID, –fluconazole 400 mg Other medications – prednisone 2 mg/kg – beclomethasone dipropionate, FK506 for GI GVHD

Case – Questions What would be your differential diagnosis? 1.PCP 2.CMV pneumonia 3.Respiratory virus pneumonia 4.Bronchiolitis obliterans 5.All of the above

Case – Questions The patient was started on broadspectrum antibiotics (imipenem, vancomycin, gentamicin, levofloxacin). Which additional agents would you start empirically? 1.High-dose TMP-SMX 2.Ganciclovir induction therapy 3.Voriconazole 4.Oseltamivir 5.1 and 2 6.All of the above

Case – Additional Information Patient received TMP-SMX and ganciclovir empirically Additional diagnostic IT aspirate: GPC, GPR, GNR (mixed flora), yeast BAL: –Gram stain: GPC, GNR –Viral DFA; negative for ADV, RSV, FLU, PIV, HMPV –PCP DFA: negative –CMV shell vial: pending –Respiratory 12-virus multiplex PCR: pending –Aspergillus GM: positive (index 2) –Aspergillus PCR: pending –Legionella: negative CMV PCR (plasma): 2 million copies/mL

Case – Interim Working Diagnosis Presumed CMV pneumonia –Drug resistance possible if not probable Pulmonary aspergillosis Bacterial pneumonia

Utility of Galactomannan Detection in BAL Hematopoietic Cell Transplantation Becker et al. Br J Haematol 2003 Musher et al. J Clin Microbiol 2004 Maertens et al. Clin Infect Dis 2009 Hematologic malignancies Hsu et al. BMC Inf Dis 2010 Bergeron et al. Chest 2010 Lung transplantation Husein et al. Transplantation 2007 Pascaloto et al. Transplantation 2010

Case – Questions How would you adjust treatment? Switch to foscarnet Add foscarnet Add voriconazole Add both foscarnet and voriconazole No change

Foscarnet and voriconazole added

Ganciclovir Resistance against CMV Prevalence 2010 SOT >>> HCT

Ganciclovir Resistance against CMV Prevalence 2010 SOT >>> HCT However…..

Low immune status Drug induced Severe IS (e.g. TCD, HD steroids) D+/R- CD4 count Subclinical CMV load Low drug levels Prolonged Adminstration Resistance Ganciclovir Resistance Not one single factor is responsible

CMV Drug Resistance High Risk Situation Viral Load Months after Transplantation Ganciclovir Ganciclovir-experienced patient (prophylaxis, preemptive therapy, pre-transplant use), especially with low doses Increase of viral load > 2 weeks High risk transplant setting Lung, K-P transplant (D+/R-) HCT (severe TCD or immunosuppression, e.g. haplo Tx)

CMV Drug Resistance Low Risk Situation Viral Load Months after Transplantation Ganciclovir Ganciclovir-naïve patient Increase of viral load during the first 2-3 weeks of therapy Low risk setting (R+, kidney Tx, liver Tx, heart Tx, HCT)

CMV Drug Resistance Mutation Map: UL97 and UL54 Chou S, Rev Med Virol 2008 UL97 UL54

CMV Drug Resistance Diagnosis Increases of viral load as surrogate marker for resistance –drug-naïve subjects, early during treatment, low risk setting (R+): drug resistance unlikely increases most likely due to the underlying immunosuppression –after significant exposure (especially low-dose), high risk setting More likely True viral load increase: > 0.5 log 10 (> 3x baseline) Testing: direct genotypic testing if resistance is suspected –UL97 gene: CMV, maribavir –UL 54 gene: foscarnet, cidofovir, ganciclovir (high level)

CMV Drug Resistance Management Strategies Switch to alternative drug –Ganciclovir Foscarnet Cidofovir (some cross-resistance) –FoscarnetGanciclovir Cidofovir Reduce immunosuppression (if possible)

CMV Drug Resistance Management Strategies In refractory situations (viral load increases, clinical deterioration): –Reduce immunosuppression if feasible –Consider dose increase if possible (West P et al. Transplant Infect Dis 2008) –Consider combination therapy Continue ganciclovir in addition to foscarnet (Emery et al. PNAS 2008)

CMV Drug Resistance Management Strategies In refractory situations – continued : –Consider alternative agents (alone or in combination) CASE REPORTS ONLY – NO CONSISTENT EVIDENCE Leflunomide (Avery RK et al. BMT 2004; Battiwalla M et al. TID 2007) Artesunate (Shapira et al., CID, 2008; Effert et al. CID, 2008) – not available everywhere –Consider alternative immunosuppressive agents Sirolimus (Chou S, Rev Med Virol, 2008)

CMV Drug Resistance Management Strategies In refractory situations – continued : –Consider experimental drugs if available: High dose maribavir –Alone –In combination with foscarnet or cidofovir (not ganciclovir) CMX-001 (lipid cidofovir)

Summary: CMV Drug Resistance Increases of viral load as surrogate marker for resistance –drug-naïve subjects, early during treatment, low risk setting: Drug resistance unlikely, increases most likely due to the underlying immunosuppression –after significant exposure (especially low-dose), high risk setting More likely, true increase: > 0.3 log 10 Testing: direct genotyping if resistance is suspected Alternative treatment while awaiting results

Case – Follow-up Final BAL results: –Aspergillus GM and PCR positive –CMV SV positive, UL97 mutation detected –Pseudomonas aeruginosa (> 10 4 cfu/ml) –No evidence of Respiratory virus disease PCP Legionellosis Mycobacterial disease Patient died of refractory respiratory failure

Nakamae et al. ASH 2007 abstract Ganciclovir-related Neutropenia Not Reduced after Non-myeloablative Conditioning Adj. HR 1.1, P=0.52

Reduction of GCV or VGCV- related Neutropenia Strategies Limit use of marrow-toxic drugs –Hold/replace concomitant medications (e.g. TMP- SMX, MMF, Imatinib) Preemptive use of G-CSF –Studied in HIV-infected patients (Dubreuil-Lemaire et al. Eur J Haematol 2000, Kuritzkes et al. AIDS 1998) Foscarnet (Reusser et al. Blood 2002) –Equivalent to IV GCV for CMV disease-free survival –Less neutropenia Cidofovir: no randomized trials

Diagnostic Test Ganciclovir/FSC (e.g. PCR > 1000 copies/mL) days 100 days Prevention of Late CMV Disease Currrent Strategies Duration of monitoring: until 6-12 m after HCT – Detectable CMV-specific T cell function – No or minimal systemic immunosuppression No or minimal systemic steroids No anti-T cell agents No DLI – Several negative surveillance assays

Control of CMV Future Strategies Novel anti-CMV drugs –Maribavir T cell therapy Vaccination strategies

Summary Current anti-CMV strategies have reduced the incidence of CMV disease but –A mortality disadvantage persists in high-risk seropositive recipients –Breakthrough disease continues to occur –Toxicity remains a problem. New strategies include –Novel drugs, e.g. maribavir –Combined virologic and immunologic monitoring –T cell therapy –Vaccination

Herpesviruses 2010 Transplantation VZVHCT: Long-term acyclovir works No issues with drug resistance Long-term adherence is a problem When to stop prophylaxis? SOT: Zoster also occurs in approximately 8-12% no general recommendation for ACV use

Varicella Zoster Virus Courtesy of Galvin and D’Alessandro

VZV after HCT Disseminated and Visceral Disease Dissemination in 36%, mainly during the 1 st year Visceral VZV: appr. 1/250 seropositive recipients –Symptoms –RUQ or back pain –Sometimes vomiting, diarrhea, bloody emesis –NO SKIN LESIONS at onset (may occur after h, maybe rare, always disseminated) –Hallmark: rapidly rising LFT’s (transaminitis > 1000) –Diagnosis: VZV DNA by PCR in plasma, biopsy –Management: empiric use of high-dose IV acyclovir –Outcome: often fatal even with early treatment

Year of Transplantation B Introduction of Acyclovir prophylaxis Autologous Transplant Recipients Allogeneic Transplant Recipients Introduction of Acyclovir prophylaxis V Erard et al. Blood 2007 VZV Disease after HCT Acyclovir 800 mg twice daily

Acyclovir Prophylaxis Extended during IS No ACV (N=932) 1 yr ACV (N=1117) 1 yr + ACV (N=586) P<0.001 P=0.01 V Erard et al. Blood 2007

Acyclovir Prophylaxis Extended during IS: No Rebound VZV No ACV (N=932) 1 yr ACV (N=1117) 1 yr + ACV (N=586) P<0.001 P=0.01 V Erard et al. Blood 2007

VZV Prophylaxis How long is enough ? Thomson et al. BMT 2005 –200 mg BID until d/c of IS and CD4 count > 200/uL –Breakthrough during prophylaxis: 1/247 (0.4%) –26/64 (40%) developed herpes zoster a median of 135 after d/c (range ) –93% dermatomal After HCT After d/c of ACV

Herpesviruses 2010 Transplantation HHV-6HCT: High complication rate in cord blood recipients Encephalitis Other disease endpoints not well defined Drug toxicities with currently available agents SOT: rarely a problem

HHV-6 Most recipients seropositive Variant A < B (PBL) but high frequency of A in plasma Early reactivation (first month) Viremia occurs in 30-70% Clinical manifestations –CNS disease (encephalitis): best evidence –Marrow suppression Delayed platelet engraftment –Rash, fever, sinusitis –Interstitial pneumonia ? Association with GvHD, rejection ? Impact on mortality ?

Risk Factors Age Underlying Disease Cord blood transplantation HLA match of transplantation Sex match of transplantation Anti-CD3 monoclonal antibodies Glucocorticoids

HHV-6 & Clinical Endpoints Zerr et al. CID 2006

HHV-6 and Encephalitis Following HCT >100 cases described in literature Clinical criteria –Delirium +/- seizures –HHV-6 DNA detected in CSF +/- viremia –No other identified etiology

HHV-6 Encephalitis Allogeneic Present median D +24 (12-39) Delirium characterized by: –Short–term memory loss –Confusion –Disorientation –Depressed consciousness Seizures in 50%

HHV-6 Encephalitis CSF findings: –pleocytosis uncommon –elevated protein >50% MRI abnormal in 9/12 –7 mesial temporal lobes/hippocampus (*short term memory loss*) All treated with FSC +/- ganciclovir

HHV-6 Encephalitis ( ): Outcomes 4 (29%) died within 1 month 10 (71%) survived >1 month –8 Persistent cognitive deficits Self report: memory/concentration issues Severe cognitive impairment

HHV-6 Treatment Drugs with antiviral activity –Foscarnet –Ganciclovir –Cidofovir

HHV-6 Take Home Points HHV-6 reactivation occurs in appr. 40% of patients HHV-6 is associated with –Encephalitis –Possibly also with delayed platelet engraftment and GvHD Long-term sequelae of CNS disease are common

Herpesviruses 2010 Transplantation EBVPTLD in high-risk patients » HCT: T cell depletion »SOT: seromismatch, anti T cell ABs HCT: »Surveillance and preemptive therapy with rituximab (non-randomized) »T cell therapy (non-randomized) HSVNo real issue: no increase of drug resistance with long-term acyclovir (if the dose is high enough) HHV-7No confirmed disease association HHV-8Rarely Kaposi sarcoma

HSV Infection after HCT Take Home Points HSV reactivation is common ACV prophylaxis now commonly applied –Minimum 30d –At FHCRC: minimum 100 days, > 1 year if VZV positive –Highly effective, eliminates wildtype and ACV-resistant HSV –Therapy of resistant disease: IV foscarnet (renal toxicity) V Erard et al. JID 2007 All HSV disease ACV-resistant HSV disease

Cidofovir Lipid Conjugates Lipid esters under development as oral therapy for smallpox virus –Also have activity vs. human herpesviruses, adenovirus, BKV In vitro activity enhanced 2 o 100-fold increases in intracellular levels –Rapid association with membrane phospholipids (vs. pinocytosis for CDV) Ciesla SL et al. Antiviral Res 2003;59:163 CMX001 – Chimerix, Inc.

CMX001 Potency against dsDNA Viruses VirusCell Line CidofovirCMX001 Enhanced Activity EC 50 (µM) Variola majorVero Vaccinia VirusHFF HCMV(AD169)MRC BK VirusWI HSV-1MRC HHV-6HSB AdenovirusHFF HPV 18HeLa HPV 11 A EBV Dardi> >4250 Provided by Chimerix Inc.