ROTARIX® (Rotavirus Vaccine, Live Oral): GSK’s PCV1 Investigation

1. ROTARIX® (Rotavirus Vaccine, Live Oral): GSK’s PCV1 Investigation Barbara Howe, MD Vice President, Director North American Vaccine Development GlaxoSmithKline

2. Why We’re Here • Review data/information from unexpected finding of PCV1 in Rotarix • Finding confirmed in validated lab mid-March • GSK investigation is ongoing • All available data support PCV1 in Rotarix is a manufacturing quality issue not a safety issue

3. Regulatory History • First licensed in Mexico July 2004 • Licensed in U.S. April 2008 • 11 clinical studies • Conducted over 8 yrs in ~ 75,000 subjects • Included large safety study ( > 60,000 subjects) to address risk of IS • Manufactured in compliance with CBER regulations on adventitious agent testing • in accordance with guidance in place at time of licensure • Extensive post-marketing pharmacovigilance ongoing • US and worldwide

4. Providing Infant Protection Against Rotavirus Worldwide • WHO prequalification – Jan 2007 • 69 million doses distributed worldwide; 2.5 million in the US

5. No New Safety Concerns Found by Recent Pediatric Advisory Committee • March 22, 2010 – routine safety review • CBER conclusion: “No new safety concerns identified…” • CBER recommendation: “continued monitoring of Rotarix” • PAC unanimously endorsed CBER conclusion and recommendations • Product reviewed contained PCV1

6. Used ALV as Precedent to Guide Rotarix PCV1 Investigation • Collaborated with experts in porcine viruses and analytical detection methods • 1990’s ALV experience used to inform investigation algorithm • Investigation designed to address: • Source, nature, amount of PCV1 in manufacturing process • Clinical implications • Potential remedial actions

7. Scope of PCV1 Investigation Expanded • IPV-containing vaccines tested for PCV1 • As with Rotarix, PCV1 detected early in manufacturing • NOT found in purified harvest or final container • Due to purification and inactivation of IPV • All other GSK vaccine cell banks tested • NO PCV1 detected

8. Data Shared With Regulatory Authorities Worldwide • EMA, WHO, FDA agree that: • PCV1 not known to cause disease • Rotarix has been studied extensively, presence of PCV1 poses no risk to human safety • Many countries have chosen to continue using Rotarix • Pending further investigation • Because of tremendous need and benefit

9. GlaxoSmithKline Presentation

10. External Expert Prof. Dr. Hans NauwynckDirector Laboratory of Virology,Department of Virology, Parasitology and ImmunologyFaculty of Veterinary MedicineGhent University, Belgium

11. Overview of Porcine Circovirus Type 1 (PCV1) X.J. Meng, M.D., Ph.D. Professor of Molecular Virology Center for Molecular Medicine and Infectious Diseases Virginia-Maryland Regional College of Veterinary Medicine Virginia Polytechnic Institute and State University Blacksburg, Virginia

12. Characteristics of PCV1 Prof. Stewart McNulty Queen's University Belfast, UK Discovered as a contaminant of porcine kidney PK-15 cell line (ATCC CCL-33) (Tischer et al., 1974) Family Circoviridae Small, icosahedral, non-enveloped virus particle of approximately 17 nm in size Single-strand, circular DNA molecule of approx. 1.7 kb genome

13. PCV1 Inactivation Resistant to inactivation at pH3 and by chloroform Heat stable at 70°C for 15 minutes and 60°C for 30 min Variable sensitivity to disinfectants (Royers et al., 2001): no significant titer reduction for some disinfectants such as Nolvasan (chlorhexidine diacetate) and Ethanol 2-4 log titer reduction for other disinfectants such as Sodium hydroxide and Clorox bleach

14. PCV1 is not PCV2 Major genetic difference: differed by about 25% nucleotide sequence identity between PCV1 and PCV2 Major phenotypic difference: PCV1 is not known to cause disease whereas PCV2 causes a disease known as “postweaning multisystemic wasting syndrome (PMWS)” in pigs Neither causes disease in humans PCV2 PCV1 Fenaux et al., 2000

15. In vitro susceptibility of PCV1 to porcine and non-porcine cells Productive infection in porcine kidney PK-15 cell line Non-productive infection in human cell lines (293, Hela, and Chang) (Hattermann et al., 2004) PCV1 replication and gene expression detected Infectious virus particles are not produced (supernatant from infected cells unable to infect naïve cells) Infection of human blood leukocytes (Arteaga-Troncoso et al., 2005) Visualization of some virus-like particles by EM Detection of PCV1 DNA in cells Infectivity was not determined

16. PCV1 infects but does not cause disease in pigs Widespread subclinical infection in pig population : 60-95% seroprevalence in German pigs (Tischer et al., 1995) 26-55% seroprevalence in Canadian pigs (Dulac and Afshar, 1989) PCV1-infected pigs remain clinically healthy PCV1 antigen detected in mesenteric lymph nodes, thymus, lung, liver, spleen and small intestines for 9 days (Allan et al., 1995) PCV1 viremia detected in sera for up to 35 days post-inoculation (Fenaux et al., 2004). No histopathological lesions in lymph nodes, thymus, intestines, tonsil, lung, and livers of PCV1-infected pigs at 21 or 49 days post-inoculation (Fenaux et al., 2004). PCV1 is used as the vector in the first USDA fully-licensed killed vaccine against PCV2(Fenaux et al., 2002, 2004)

17. No credible evidence of human infection by PCV1 PCV1 antibody detected in humans in Germany, likely due to cross-reaction with a related but different agent (Tischer et al., 1995). Loss of binding specificity in ELISA after repeated frozen/thaw and storage of human sera Lower OD values in human sera compared to porcine sera Unable to reproduce Tischer’s results in subsequent studies: PCV1 antibody not detected from 120 humans in Northern Ireland (Allan et al., 2000) PCV1 antibody not detected from 50 swine veterinarians, 6 lab workers, or 33 normal blood donors (Ellis et al., 2000) PCV1 DNA not detected in 1101 human serum, lymph node, blood or urine samples (168 samples from immunocompromised patients) (Hattermann et al., 2004)

18. PCV1 detection in commercial products and human stool PCV1 DNA detected in commercial pepsin but no infectivity in inoculated PK-15 cells or in inoculated pigs (Fenaux et al., 2004) PCV1 DNA detected in 11% (2/18) of the commercial porcine vaccines on the market (Quintana et al., 2005) PCV1 DNA detected in U.S. pork products (Li et al., 2010): 69% (9/13) U.S. pork products contain PCV1 or PCV2 DNA (7 PCV2 sequences, 1 PCV1 sequence, and 1 divergent PCV sequence) PCV1 DNA detected in U.S. human stool samples: 5.3% (13/247) human stool samples positive for PCV1 or PCV2 DNA (Li et al., 2010) Detection of PCV DNA in stool may reflect dietary consumption of PCV-containing pork products (Li et al., 2010)

19. Summary and Conclusion PCV1 infection is widespread in swine PCV1 does not cause any disease in pigs or other animal species including humans There is no concrete evidence of human infection by PCV1 PCV1 detection in commercial products including veterinary vaccines has been reported before PCV1 detected in U.S. pork products

20. GlaxoSmithKline Presentation

21. Routine Rotarix ManufacturingProcess - + + Rotarix vaccine PCV1 DNA Q-PCR (copies per ml) Viral harvest 1010 Rotavirus Expansion on vero cells Purified bulk 109 Final Container 107 + Viral harvest + + Dilution and Filling in Final Container Purified bulk Vero cell line Trypsin Rotarix viral seed PCV1 DNA detection No PCV2 DNA detected

22. Vero cell Line P124 Ancestor of Rotarix viral seed Rotarix viral seed PCV1 Detected in StartingMaterials of Rotarix ATCC - Potential source: Porcine –Derived Trypsin used for cell propagation GSK 1980 + + Master Cell Bank P133 Working Cell Bank P136 PCV1 DNA detection 1983 1993 - + 1999

23. Investigation StrategyBased on ALV Precedent Is PCV1 signal associated with presence of: Viral particles? Viral particles capable of infecting permissive cells? Viral particles capable of productive infection in human cells? Viral particles capable of causing infection in human infants? Manufacturing Clinical

24. Investigation StrategyBased on ALV Precedent Is PCV1 signal associated with presence of: Viral particles? Viral particles capable of infecting permissive cells? Viral particles capable of productive infection in human cells? Viral particles capable of causing infection in human infants? Manufacturing Clinical

25. Investigation StrategyBased on ALV Precedent Is PCV1 signal associated with presence of: Viral particles? Viral particles capable of infecting permissive cells? Viral particles capable of productive infection in human cells? Viral particles capable of causing infection in human infants? Manufacturing Clinical

26. PK15 PCV1 Virus Viral Infection Viral Gene Expression (mRNA detected by RT-PCR) Negative control: Medium Negative (Ct value : >40)* Positive control: PCV1 Positive (Ct value : 26.8) * (LOD 1-10 CCID50) Infectivity Assay Used Natural Host CellsPermissive PK15 (Porcine Kidney) cell lines PK15 Rotarix Purified Bulk Positive (Ct value : 28.6)* (1500 doses) *A Cycle threshold (Ct) value of 40 will indicate that the tested sample is negative. Presence of infective viral particles demonstrated

27. Infective Viral Particle Titration Assay Vero cell line Rotarix Viral Harvest 1010 PCV1 DNA copies per ml 10 x dilution Immunodetection anti-PCV1 protein antibody 7 days Vero cell monolayer PCV1 titer in viral harvest : 102 CCID50 per ml →estimated3 CCID50 of PCV1 per Rotarix dose Low level of infective viral particles in final container can not be excluded

28. Infectivity in Permissive CellsDoesn’tMeanInfectivity in Humans Is PCV1 signal associated with presence of: Viral particles? Viral particles capable of infecting permissive cells? Viral particles capable of productive infection in human cells? Viral particles capable of causing infection in human infants? Manufacturing Clinical

29. Can PCV1 Induce Productive Infectionin Human Cells? PCV1 virus ? PK15 Human Cell Infectious viral particles PCV1 virus Viral gene expression ? Hattermann et al, Xenotransplantation 11:284 2004 18 human cell lines tested: Chang liver, FL, 293, Hep2, RH, CaCo, Hela, Ma23, Rd., Wil2, THP1, Jurkat, Molt4, C8166, CEM, U937, H9, Human PBMCs « Although PCV1 gene expression and DNA replication took place in human cells, the infection is non-productive »

30. Can PCV1 Induce Productive Infectionin Human Cells? Presently no evidence indicating that PCV1 associated with Rotarix can undergo productive infection in human cell lines

31. Additional investigations

32. PCR Results on Other Cell Banks Negative for PCV1 and PCV2 DNA

33. IPV Production DifferentFrom Rotarix Process IPV Expansion on vero cells Purified bulk (chromatography) Viral harvest Inactivation Dilution and Filling in Final Container Vero cell line IPV viral seed

34. Estimated clearance of Purification step – 104 DNA copies Inactivation step – ongoing Polio Virus Containing Vaccine Investigation

35. Rotarix Manufacturing Investigation Conclusions • GSK is in the process of evaluating long term manufacturing changes to remain in compliance with regulations • PCV1 material associated with presence of competent PCV1 viral particles • PCV1 viral particles present in very low amount in Rotarix Final container • Estimated titer: 3 CCID50 of PCV1 per dose • No evidence PCV1 associated with Rotarix can undergo productive infection in human cell lines

36. RotarixManufacturing Investigation Conclusions Is PCV signal associated with presence of: Viral particles? YES Manufacturing YES Viral particles capable of infecting permissive cells? Viral particles capable of productive infection in human cells? NO Viral particles capable of causing infection in human infants? Clinical

37. GlaxoSmithKline Presentation

38. Investigation Strategy Is PCV1 signal associated with presence of: Viral particles? Viral particles capable of infecting permissive cells? Viral particles capable of productive infection in humancells? Viral particles capable of causing infection in human infants? Manufacturing Clinical

39. Clinical Testing Approach • Blinded retrospective laboratory evaluations on archived samples from completed Rotarix clinical trials • Selected studies required to meet the following criteria: • Placebo controlled • Collection of pre/post vaccination sera and stool samples at pre-determined time points

40. Selected Studies • Samples tested from 4 completed studies • Conducted in Africa, Asia, Europe and Latin America • Infants aged 6 to 12 weeks at time of first dose • 3 studies in healthly infants, 1 study in HIV positive infants • Samples from 80 subjects included in the evaluation • 40 Rotarix and 40 placebo recipients

41. Clinical Testing Objectives • To evaluate the presence of PCV1 DNA and pattern of detection in stool samples collected at pre-determined time points after a single dose of Rotarix or placebo • To evaluate if infants receiving 2 or 3 doses of Rotarix or placebo develop an immune response to PCV1 as assessed by the presence of antibodies against PCV1

42. Clinical Sample Selection Criteria • Subject enrollment number • The first 10 Rotarix and first 10 placebo recipients enrolled in each study with adequate samples • Sample requirements • Stool samples • Availability of pre-vaccination and ≥ two post dose 1 samples • At least 100 µL of residual volume • Sera samples • Availability of pre vaccination and post dose 2 (or 3) • At least 125µL of residual volume

43. Overview of Selected Clinical Studies (N= 80 Infants)

44. Laboratory Assays: Stool • Quantitative polymerase chain reaction (Q-PCR) to detect PCV DNA • Two replicates tested for each sample Neg/Neg → “Negative” Pos/Pos → “Positive” Pos/Neg Indeter/Neg Indeter/Indeter 3rd replicate tested • Final result: “Positive”, “Negative” or “Inconclusive” • If Q-PCR positive or inconclusive, sequencing performed to confirm PCV1 virus identity

45. Laboratory Assays: Serum • IPMA (ImmunoPeroxydase Monolayer Assay) to detect anti-PCV1 antibody response • Uses PCV1 infected PK-15 cells • Developed by Prof Hans Nauwynck (Ghent University) • Previously used to detect anti-PCV1 in pig serum • Adapted for testing of human serum samples • Pig serum used as a positive control Negative serum Positive serum (Control) Courtesy of Prof Hans Nauwynck (Ghent University)

46. Analysis Plan • Descriptive analyses without hypothesis testing • Sample size selected based on the feasibility of generating data for committee review • 3 weeks available for assay set up and sample testing • 300 stool samples for Q-PCR (+ sequencing) • 160 serum samples for IPMA testing

47. Considerations for Data Interpretation of PCV1 DNA in Stool • Some examples of possible detection patterns: Pre Day 3- 7 Day 15 Possible Interpretation - - - No evidence of viral replication + - + Dietary exposure - - + - + ++ Consistent with viral replication Transient passage of PCV - 1 DNA • Must consider serology results when interpreting stool testing results

48. Summary of Clinical Results (1) **1 sample non-interpretable due to high background *Confirmed as PCV1 sequence in Rotarix DNA detection pattern suggests transient GI tract passage of PCV1

49. Summary of Clinical Results (2) *Confirmed as PCV1 sequence in Rotarix DNA detection pattern suggests transient GI tract passage of PCV1

50. PCV DNA Findings in Individual Rotarix Recipients (Stool Samples) In each infant pattern is consistent with transient DNA passage + = PCV1 sequence confirmed NA= not available *Indeter/Neg → Indeter (3rd replicate testing) → Inconclusive