1. Pathogen inactivation New progress Antonio Piga Hemoglobinopathies Centre Department of Clinical and Biological Sciences University of Torino, Italy antonio.piga@unito.it

2. Mechanism of action of pathogen inactivation

3. Requirements for pathogen inactivation 1.Effective to eliminate pathogens 2.Maintaining the quality of blood products for transfusion 3.Safe 4.Simple and cost-effective to implement in the preparation of blood components

4. Adapted from Busch MP, TRANSFUSION 2006;46:1624-1640 1:100,000 1:1,000,000 1:10,000 1:100 1:1000 Risk per Unit <198419841986198819901992199419961998200020022004200620082010 HIV HCV HBV Revised Donor Deferral Criteria HBsAg Screening HIV Ab Screening NANB Hepatitis Surrogate Testing HCV Ab Screening p24 Ag Testing HCV and HIV NAT HBV NAT WNV NAT T cruzi Ab Screening vCJD Deferral Criteria ICL vCJD PTLVs Monkey Pox SARS H1N1 influenza XMRV SFV Leishmania CHIKV DENV WNV Emerging Infectious Disease Threats T cruzi Declining Risks of major TTVs linked to interventions, BUT accelerating rate of Emerging Infectious Diseases (EIDs) of concern to blood safety

5. Why do infections emerge? Source  New agent (vCJD)  Species jump (HIV, SARS)  Environmental change (Dengue virus)  Failure of control- drug/vaccine resistance and mutations (HBV mutants) Contributing Factors  Behavioral change among humans (HIV)  Inactivation resistant agents (B19, HAV, prions)  Intensive modern farming practices (vCJD)  Susceptible hosts (CMV)  Transport of agents, reservoirs, vectors (WNV)  Population movement (T.cruzi, chikungunya)

6. Movement of Hosts and Vectors courtesy of MP Busch

7. Be a virus, see the world Daily airline traffic patterns

8. Spatial epidemiology and evolution of WNV across N. America derived from phylogenetic analysis of viremic blood donations Pybus OG, PNAS, 2012

9. (Sept. 18, 2012; N of Human Cases = 3,142) West Nile virus (WNV) Clinical Activity reported to CDC,United States, 2012

10. West Nile Virus Cases in EU and Neighbouring, 2011 Source: ECDC

11. Rizzo C, Eurosurveillance, 2012

12. Dengue Viruses Members of the genus Flavivirus Transmitted by the Aedes mosquito; mosquito => human cycle 4 viruses: DENV1, 2, 3, and 4 Over 2.5 billion people live in risk areas for dengue infection

13. Dengue Risk in Puerto Rico Blood Donors 1995 - 2010 Peterson and Biggerstaff Transfusion 2012

14. Dengue Virus, August-October 2012 activity

15. Usutu Virus in Europe 15 ENIVD: European Network for Diagnosis of Imported Viral Diseases Vazquez et al. Euro Surveill. 201116(31) 19935 The icons signify cases of detection of the virus per species Culex pipiens pipiens Flaviviruses (similar to WNV) Enveloped, icosahedral nucleocapsid Ss + RNA strand, 11kb 40-60 nm size Mosquito transmitted Infects birds & horses

16. The discovery curve for human virus species Woolhouse M E et al. Proc. R. Soc. B 2008;275:2111-2115 Rapid pace of viral discovery is providing many “orphan viruses” as candidate pathogens

17. Vazquez et al. Euro Surveill. 2011, 16(31) 19935

18. Vazquez et al. Euro Surveill. 201116(31) 19935

20. EID agent priority matrix Stramer SL, 2009

21. 21 162 cases from 1979-2009 with a 19% fatality rate Herwaldt BH, 2011 Ann Int Med Transfusion-transmitted Babesiosis in the US

22. US Babesiosis – 10 new cases 22

23. Earliest known representation of limb atrophy presumed due to poliomyelitis. Egypt, 1403–1365 BC. from Ny Carlsberg Glyptotek.

24. http://www.aabb.org/Content/About_Blood/Emerging_Infectious_Disease_Agents/appendix2.htm

25. EUFRAT: risk model scheme

26. Summary Testing for TTDs  Expanding the testing for known pathogens has kept the blood safer over time  Each new test adds safety at exponentially increasing costs  Emerging pathogens keep testing invariably on delay and incomplete  Effective and safe pathogen inactivation is the answer

27. - for platelets - for plasma - for red cells Pathogen Inactivation Technologies (PIT)

28. Solheim BG, Transfusion and Apheresis Science, 2008 Pathogen Inactivation Technologies (PIT) for platelets

29. BE SURE. 29 : Routine Customers Canary Islands MartiniqueGuadeloupeFrench Polynesia RéunionChile Routine use at over 100 centers in 18 countries More than 1,000,000 doses transfused Current INTERCEPT Platelet and Plasma Use

30. BE SURE. French National Hemovigilance: Platelet Transfusion 2006 to June 2012 PC TransfusedTransfusion-related sepsis cases (deaths) Conventional PC 1, 601,478 37 (7) INTERCEPT PC115,6480 Agence Francaise de Securite Sanitaire des Produits de Sante (2006-2011). Rapport Hemovigilance 2006. Paris, Afssaps. D Kientz et al. 13th International Haemovigilance Seminar, Amsterdam, The Netherlands February 9 - 11th, 2011 L Corash et al. 14th International Haemovigilance Seminar, Montreal, Canada, April 25-27 2012 P=0.07

31. Solheim BG, Transfusion and Apheresis Science, 2008 Pathogen Reduction Technologies (PIT) for plasma

32. Solheim BG, Transfusion and Apheresis Science, 2008 Pathogen Reduction Technologies (PIT) for plasma

33. Solheim BG, Transfusion and Apheresis Science, 2008 Pathogen Reduction Technologies (PIT) for red cells

34. BE SURE. Pathogen Inactivation for all 3 Blood Components: S-303 Treated Red Blood Cells: William Reed, MD Director, Clinical Research and Medical Affairs Torino and Cagliari September, 2012

35. BE SURE. INTERCEPT RBC: Mechanism of Action S-303 is a nucleic acid-targeted alkylator that quickly diffuses into viruses, bacteria, parasites and blood cells and is designed to react quickly and decompose Glutathione (GSH) is used to quench side reactions of the effector with other biological materials

36. BE SURE. Second Generation INTERCEPT RBC Clinical Process

37. BE SURE. Pathogen Inactivation Efficacy Evaluations **Second generation process, n=4 full RBC units PathogenMean Log Reduction** S. aureus 5.1  0.3 S. marcescens 5.1  0.1 Y. enterocolitica ≥6.8  0.2 E. coli ≥6.7  0.1 HIV >5.9  0.1 Bovine viral diarrhea virus >4.8  0.1 Blue Tongue ≥5.0  0.4 Adenovirus Type 5 >7.4  0.2 PathogenMean Log Reduction* First generation system Plasmodium falciparum >6.8 Babesia microti,>4.9 Trypanosoma cruzi >5.3 West Nile virus >6 Second generation system – tube study XMRV>4 *n=1-3 RBC units or tubes Henschler R, Transfus Med Hemother, 2011

38. BE SURE. Pathogen Inactivation Process Optimization: Reduce interaction of S-303 with RBCs and improve process flexibility First Generation Process 0.2 mM S-303 2 mM GSH (acidic) Compound adsorption device to remove residual S-303 Erythrosol storage solution Second Generation Process 0.2 mM S-303 20 mM GSH (Na salt) Diluent solution for PI Removal of treatment solution and replacement with storage solution Approved RBC storage solutions

39. BE SURE. NEW Phase 1 Clinical, Second Generation: 24- Hour Recovery meets FDA Requirements (n=27) S-303Control 24-hour Recovery (%, Dual Label, Cr-51 and Tc-99m) 88.0 ± 8.590.1 ± 6.9 Subjects with Dual Label Recovery < 75% 11 Median Lifespan (T 50, days)32.8**39.5 AUC (% of cells surviving)22.0*23.9 The 24-hour recovery was similar between groups and met FDA criteria The median lifespan,T50, of Test RBC was within the reference range of 32 to 37 days using 51 Cr label * p<0.05, ** p<0.001 Cancellas et al. Transfusion 2011

40. BE SURE. NEW Phase 1 Study, Second Generation: In Vitro RBC Characteristics-- Day 35 (n=27) AttributeS-303Control Total Hemoglobin (g/unit) 1 52.7 ± 3.6Not measured Hemolysis (%) 2 0.24 ± 0.110.22 ± 0.13 Spun Hematocrit (%) 2 59.7 ± 2.4*62.4 ± 3.6 ATP concentration (mmol/g Hb) 2 3.34 ± 0.80*3.59 ± 0.81 pH (at 37 o C) 2 6.404 ± 0.039*6.483 ± 0.122 Extracellular Potassium (mM) 2 39.5 ± 3.6*52.7 ± 6.5 1 Measured prior to storage 2 Measured after 35-days of storage * p-value <0.05 The total hemoglobin, hemolysis and hematocrit meet the requirements for leukoreduced RBC in additive solution The ATP concentration is well over the critical threshold of 2.0 mmol/g Hb ( Hess JR, Greenwalt TG. Transf Med Rev., 16 (4), 283-295 (2002))

41. North A, Transfusion, 2011

42. BE SURE. Clinical Studies Europe: Acute and chronic anemia separately Chronic anemia – thalassemia major Acute anemia – cardiac surgery US: thalassemia and sickle cell anemia Chronic transfusion Final design will require Hgb increment data from Europe and US

43. Italian Thalassemia Study Phase 3 study of efficacy and safety of S-303 treated RBC components Investigators: A Piga - Torino R Galanello – Cagliari 43

44. Design Randomized, controlled, double ‑ blind, crossover study to evaluate both efficacy and safety of S ‑ 303 treated RBC components in 70 subjects 4 transfusion cycles for Test and 4 for Control 2 additional wash-n cycles for each period Statistical hypothesis of non-inferiority Non-inferiority margin of 15% 44

45. Italian Thalassemia Study Transfusion-dependent thalassemia major patients (n = 70) Randomized, controlled, double ‑ blind, crossover study Statistical hypothesis of non-inferiority 1° efficacy endpoint = Hemoglobin usage 1° safety endpoint = Immunogenicity with repeat exposure 45 INTERCEPT 4 txns 2 txns* Control 4 txns 2 txns* 4 txns 2 txns* 4 txns 2 txns* INTERCEPT Control n=35 Screen, randomize n=35 Each patient is on study ~9-12 months * Patients receive 2 wash-in transfusions followed by 4 transfusions of INTERCEPT or control.

46. Secondary Efficacy Endpoints Hb percent decline per day Hb increment 1-h post-Tx (Adjusted for mass of Hb transfused and body weight) 46

47. Data and Safety Monitoring Board A Cohen - thalassemia (Philadelphia) L Pierelli – transfusion medicine (Rome) T Peyrard - immunohematology (Paris) 47

48. Proposed U.S. Phase III – chronic RBC transfusion INTERCEPT n=41 Group A – Efficacy & Safety Assessment (n=82, cross-over) 1° efficacy endpoint = Hemoglobin usage Screen, randomize n=41 6 txns 2 txns* Control 6 txns 2 txns* 6 txns 2 txns* 6 txns 2 txns* INTERCEPT Control INTERCEPT n=218 Group B – Safety Assessment Only (n=291, 3:1 ratio) Screen, randomize n=73 3 txns Control 3 txns * Group A patients receive 2 wash-in transfusions followed by 6 transfusions evaluated for efficacy. Safety Assessment: Groups A & B (n=373) 1° safety endpoint = % patients with S- 303 Abs with clinically significant hemolysis

49. BE SURE. New RBC Biologic Component for Chronic Tranfusion? Pathogen inactivated WBC inactivated (without gamma damage) Phenotyped/genotyped Very low plasma content Defined hemoglobin (gms) Defined Fe (mg) Diminished HLA and/or RBC alloimmunization potential?

50. Acknowledgements Michael P Busch Blood Systems Research Institute University of California, San Francisco, USA William Reed Nina Mufti Anne North Cerus Corporation Concord, California USA Richard Benjamin American Red Cross Washington, DC, USA Renzo Galanello University of Cagliari Cagliari, Italy Filomena Longo Alessandro Sandri Simona Roggero Marianna Genisio Hemoglobinopathies Center, University of Torino Torino, Italy