1. a New Era Begins Live attenuated intranasal Influenza Vaccine

2. Some Considerations for Evaluation of a Live Vaccine Safety Efficacy Risk:Benefit Basis of Attenuation Transmission/ genetic stability Time to Development Cost

3. Structure of Influenza Virus (Type A and B) Viral Genome 8 Negative-Sense RNA Segments Viral Non-Envelope Proteins: PB1, PB2, PA Polymerase Complex NP Matrix (M1) Non-Structural Proteins NS1, NEP M2 Neuraminidase (NA) Viral Envelope Proteins: Hemagglutinin (HA)

4. Influenza Epidemiology / Burden of Disease

5. Influenza Most common medically attended acute respiratory illness Fever, chills, myalgia, cough, sore throat, nasal congestion, headache, malaise Annual U.S. disease burden  20-50M people infected  20,000-50,000 deaths  70M lost work days and 38M lost school days  Societal costs nearly $12B MMWR 2001; American Lung Association, 3/01

6. Influenza-related Morbidity and Mortality <5 5-9 10-14 15-19 20-24 25-34 35-44 45-54 55-64> 65 Pneumonia-Influenza Mortality 120 80 40 0 Age (Years) 80 40 0 30 20 10 0 Per 100,000 Per 10,000 Per 100 Hospitalizations Medically Attended Illness Rates Glezen WP. Epidemiol Rev. 1996; 18(1),64-76. 20 60

7. Vaccination is primary method for prevention of disease  Trivalent inactivated vaccine given IM  Recommended 6-24 months, >50 years  Virus “drift” dictates annual vaccination Two manufacturers  Aventis Pasteur  Chiron (Powderject/Evans/Medeva) 80- 90M doses sold annually in U.S.  Growing at 7% Inactivated Influenza Vaccine

8. Current Trivalent Inactivated Influenza Vaccine (TIV)  93 million doses of TIV sold in U.S. for 2002/2003, but  25M-50M annual influenza infections in the US  70M lost work days, 38M missed school days  Limitations of TIV vaccine program  150 million healthy people not vaccinated  <10% of healthy children, < 30% of healthy adults <30% of high risk children  Annual injection required  A live Flu vaccine meets a major public health need  Ease/ acceptance of administration

9.  Live, cold-adapted, attenuated influenza virus vaccine: current indication –healthy individuals 5- 49 years of age  Delivered by large particle intranasal mist  Trivalent blend of 10 7.0 infectious particles of each strain: A/H1N1, A/H3N2, B  Induction of systemic IgG/IgA, local secretory IgA and cytotoxic T cell responses  > 20 clinical trials completed; >25,000 subjects First live attenuated Nasal Flu Vaccine- FluMist™

10. 19671985198919951976 Johnson 1963 - 69 Nixon 1969 - 74 Ford 1974 - 77 Carter 1977 - 81 Reagan 1981 - 89 Bush 1989 - 93 Clinton 1993 - 01 By 2002, 25 separate influenza strains in over 28,000 people tested as CAIV reassortants John Maassab describes cold adapted influenza viruses First human studies with 6:2 CAIV reassortants Four year efficacy study of CAIV/TIV First FluMist tm trial 2003 Bush, Jr. 2001- Live Influenza Vaccine Milestones: The First 36 years FDA VRBPAC Review - #1 FDA VRBPA Review - #2 Licensure 6/17/03 Aviron formed

11. Derivation of Cold-Adapted A & B Master Donor Viruses Maassab, Nature; 1967 36 o C 33 o C 30 o C 25 o C PCK Cells Multiple Passages At Reduced Temperatures Plaque Purify 25 o C PCK Cells Plaque Purify Egg Amplification MDV- ca,ts,att WT

12. Derivation of New Master Virus Strain 6:2 Master Virus Strain (ca, ts, att) New Wild Type StrainMaster Donor Virus Co-infect Cells Six genes from MDV confer ca, ts, att phenotype Hemagglutinin and Neuraminidase Genes from Wild Type

13. In a Time & Motion study* of 497 pediatric subjects receiving intranasal flu vaccine, 12/497, or 2% cried * Unpublished data Intranasal Administration of FluMist tm

14. Clinical Studies Flumist tm

15. Efficacy Trial Healthy Children Study AV006 Randomized, double-blind, placebo-controlled two year trial 1,602 healthy children ages 15-71 months at entry Active surveillance with illness cultures Primary endpoint: culture-confirmed influenza Circulating strains  A/Wuhan/359/95 (H3N2) and B/Beijing/184/93-like in Year 1  A/Sydney/05/97 (H3N2), a mismatched strain in Year 2

16. Safety of FluMist in Indicated Populations (Ages 5-49 years) Generally safe and well tolerated Most common side effects are mild and temporary  Runny nose and other various cold-like symptoms No related serious adverse events

17. Pediatric Efficacy Trial (AV006): Results Year 1Year 2*Years 1 & 2 Virus (n = 1602) (n = 1358) A 95% 87%* 92% B 91% 100% 91% Either 93% 87% 92% *H 3 N 2 mismatched Belshe et al. N Engl J Med. 1998; 338:1405 Belshe et al. J Pediatr. 2000; 136:168

18. % Seroconversion (>=4-fold rise) HAI Antibody to H3N2 Strains Two dose regimen in seronegative children Belshe et al., 2000 P<0.001

19. % Seroconversion (>=4-fold rise) P<0.001 Antibody to H3N2 After One Dose of CAIV-T or TIV in Seronegative Infants --------HAI assay-----------Neutralization assay-- - Wyoming strain is a Fujian-like antigenic variant to the Panama vaccine strain

20. FluMist Effectiveness Against Febrile Upper Respiratory Illness in Healthy Adults EndpointReductionp-value a Occurrence of:21.9 %0.010 Episodes of:23.6 %<0.0001 Days b of:24.8 %<0.0001 Illness Associated Days of: Missed Work Health-care Provider Visits Prescription Antibiotics OTC Medication Use 28.4 % 40.9 % 45.2 % 28.0 % <0.0001 <0.0001 a Unadjusted for multiple comparisons. b Days per 1,000 participants per 7-week outbreak period.

21. Medically Attended Events in Children and Adolescents - Kaiser Study (AV019) Randomized 2:1, double-blind, placebo-controlled Safety of FluMist in 9,689 children Outcomes examined from diagnosis in database over 42 days >1,500 comparisons made without statistical adjustment  Emergency Department, Clinic, Hospital, and Combined  1-17 years, 1-8 years, 9-17 years, 18-35 mo., 12-17 mo.

22. Occurrence of Asthma (AV019) AgeFluMist n/N Placebo n/N Rate per 1000 person-months FluMist / Placebo Binomial Relative Risk (90% CI) 1 – 1758 / 647330 / 32164.58 / 4.77 0.96 (0.66, 1.40) P=.422 1 – 845 / 376921 / 18685.05 / 4.76 1.06 (0.69, 1.66) P=.418 18–35 mos. 16 / 7282 / 3699.30 / 2.29 4.06 (1.29, 17.86) P=.019 12–17 mos. 1 / 1713 / 902.51 / 14.43 0.17 (0.01, 1.17) P=0.67

23. Asthma by Day Following Vaccination for Participants 18–35 Months of Age Relative Risk: 4.06 - but no temporal relationship within 42 days 0 1 2 3 4 5 6 0481216202428323640 Day post Vaccination FluMist 0 1 2 3 Placebo FluMist Placebo

24. Viruses detection rates in asthma/wheezing episodes in children % Episodes Adapted from S Johnston, et al, BMJ,1995.

25. Genetic Basis of Attenuation Flumist tm

26. Derivation of Cold-Adapted A & B Master Donor Viruses 36 o C 33 o C 30 o C 25 o C PCK Cells Multiple Passages At Reduced Temperatures Plaque Purify 25 o C PCK Cells Plaque Purify Egg Amplification MDV- ca,ts,att WT Sequence

27. 5 Amino Acid Changes Confer ts and att Phenotypes PB2 PB1 PA NP M NS 821 (N) 1195 (K) 1766 (E) 146 (D) PB2 PB1 PA NP M NS 821 (S) 1195 (E) 1766 (G) 146 (G) non-tsts 2005 (T) 2005 (A)

28. Shedding / Transmission Flumist tm

29. Shedding and Infectivity of CAIV Following challenge, % shedding, mean peak titer and mean duration of shedding are higher in young children than adults HID 50 lower in young children compared to adults Population% Shedding Mean Peak Titer (Log 10 TCID 50/ mL) Mean Duration (days) Children67 - 912.1 – 3.44.5 – 9.0 Adults14 - 600.7 – 1.80.6 – 1.9 Virus Shedding PopulationHID 50 (Log 10 TCID 50 ) Children2.5 – 4.6 Adults4.9 – 6.4 Virus Infectivity Reviewed In Murphy, Inf. Dis. In Clin Prac, 1993 Reviewed In Murphy & Coelingh, Viral Immunol, 2002

30. Finnish Daycare Trial Wyeth, unpublished data Randomized (1:1), double-blind, placebo- controlled 197 children age 8 months - 36 months 51 playgroups  Average of 4.1 study children per playgroup  Children attended daycare > 3 days/week and > 4 hours/day  Each placebo child exposed to an average of 1.9 vaccinees

31. Shedding of influenza A and B in Children – Finnish Trial Type B Shed from Vaccinee Type B Shed from Placebo Calculated transmission probability 0.006

32. Coding Changes Observed in Viruses Shed from Children Do Not Overlap with ts or att loci PB2 PB1 PA NP M NS 821 (N) 1195 (K) 1766 (E) 146 (D) 2005 (T) 340 (V) 198 (E) 1304 (C/S) 789 (F-M2) PB2 PB1 PA NP M NS 499 (H/Q) 399 (V/A) 1286 (P/H) 571 (M/V) 718 (M) MDV-AMDV-B ts/att locus Coding change observed in >1 clinical isolate 1320 (V/M)

33. Conclusions/ Issues 1)Flumist tm is safe and effective in healthy children and adults 2)Flumist tm phenotypes (ts, att) are multigenic and stable 3)Flumist tm has a low transmission rate 4)The road from invention to licensure is long and unpredictable, as is the road to acceptance 5)Major issues to be resolved : a. Relationship of Flumist tm to asthma b. Safety and efficacy in 50-64 year olds c. reverse genetics to select new vaccines d. concurrent vaccination: safety/ efficacy e. cost f. Heterotypic immunity

34. Optimal Indication for a Live Attenuated Influenza Vaccine Healthy children and healthy adults ages 6 months to 65 years

35. “The End of the Vaccination Man Era”

36. Acknowledgements A) Medimmune VaccinesC) Investigator Community K Coelingh G. Kemble B. Belshe R. Spaete K. Nichol E. Hoffmann S. Black H. Jin J. Maassab P. Mendelman Others Clinical Group B) NIH B. Murphy R. Chanock The VTEUs Others