1. Vaccine development for pandemic influenza
John Treanor
University of Rochester

2. Antibody against the HA and NA are major components of influenza immunity
Neuraminidase (NA)
Hemagglutinin (HA)
Cell surface receptor

3. Antibody against the HA and NA are major components of influenza immunity
Functions of HA antibody
Prevention of attachment
Postattachment events
Functions of NA antibody
Prevention of cell spread
Reduced severity of illness
Other mechanisms
M2 antibody
Cellular responses

4. Pandemic influenza vaccine: Background information
Pandemics result from emergence of influenza A viruses with new HA and/or NA
Avian viruses are the source of H1-H16 and N1-N9
H5 and H7 viruses come in two types: highly pathogenic (HPAIV) and low pathogenicity (LPAIV)
The structure of the HA cleavage site determines HP – multiple basic aa = HPAIV
Antibody against the HA is neutralizing and protective – main goal of immunization is to induce HA antibody
There is significant antigenic variation in the HA of current H5 viruses

5. Priority list of avian viruses with pandemic potential
H5: HPAIV, current human cases, high pathogenicity
H7: HPAIV, human outbreaks, generally mild disease
H9: Some documented human cases, generally mild disease
H2: No current human cases, still circulates in birds, proven pandemic potential
H6: No human cases, but contributed genes to current H5 viruses

6. Options for Pandemic Vaccines
Similarity to current vaccines
Inactivated vaccine resembling currently licensed inactivated vaccine (+/- licensed adjuvant)
Live vaccine resembling currently licensed live vaccine
Inactivated vaccines with experimental adjuvants/route of administration
Experimental approaches (DNA vaccines, peptides, vectors)
CLOSER
FARTHER

7. Strategies for production of H5 vaccine seed viruses
Problem: HPAIV are lethal for eggs, must be used under high levels of containment
Use antigenically related LPAIV (e.g., Duck/Singapore/97, H5N3)
Use expressed recombinant protein (e.g., rHA A/HK/156/97)
Use reverse genetics techniques to alter HA cleavage site (e.g., rg A/VN/1203/04 x PR8)

8. Antibody response to egg-grown A/Duck/Singapore/77 (H5N3) without adjuvant
Study day
H5N3 Neutralization GMT
Nicholson et al Lancet 357:1937, 2001

9. Recombinant rHA H5 Vaccine
H5N1 HA
Gene
Recombinant
Baculovirus
Insect cell expressing
rHA
RBCs
Purified rHA H5
SDS-PAGE

10. Two doses of rH5 A/HK/156/97 vaccine resulted in a 50% MN response rate
Percent responding*
* 4-fold or greater increase to a titter of 1:80 with positive WB
Vaccine 19:1732, 2001

11. sanofi pasteur H5N1 vaccine virus
1. Engineer the cleavage site
A/Vietnam/1203/04
2. Change internal genes to PR8
rgA/Vietnam/1203/04xPR8

12. Geometric Mean Titer (GMT)
Serum neutralizing antibody after one and two doses of H5N1 subvirion vaccine
Geometric Mean Titer (GMT) 8 16 32 64
128
Treanor et al NEJM 354:1342; 2006

13. Reverse cumulative distribution of HAI antibody at day 56 (28 days after dose 2)
Percent at above indicated titer
44% (34%, 55%)
Reciprocal HAI titer
Treanor et al NEJM 354:1342; 2006

14. Factors affecting response rates
Receipt of TIV in the previous fall (significantly lower responses)
Age > 40 years (significantly lower responses)
Male (significantly lower rates)
Multivariate analysis pending

15. Modeling of the post-vaccination MN response controlling for prior TIV, gender, dose, visit 1 result, and age
p=.0003
(Adjusted for multiple comparisons)
p=.0057
Adjusted ln neutralization titer at day 56
No TIV
TIV
DMID

16. Response to H5 compared to inactivated vaccine in children
HAI GMT
Percent
responding
69%
84%
43%
Percent
> 40
50%
69%
44%
Antigen
A/Nanchang/933/95 (H3)
A/Texas/36/91 (H1)
A/Vietnam/1203/04 (H5)
Pre
5.3
4.6
5.1
Post
28.9
70.3
27.7
Responses to H1 and H3 viruses in 36 children 6 mo to 18 mo of age after two doses of TIV (15 mcg per HA) 1996, unpublished
Responses to H5 in 102 adults 18 to 64 years of age receiving two doses of 90 mcg NEJM 354: 1343, 2006

17. A 90 mcg dose in perspective
Doses of licensed and experimental vaccines administered in multi-dose regimens to unprimed individuals
Vaccine
Subvirion H5N1
Tetanus
Pertussis
Hepatitis B
HPV VLP
rPA (Anthrax)
rGP120 (HIV)
Dose (mcg) 90 15
10-40
20-40
50-100
Comment
(based on Lf)
PT component of aP
Experimental
All vaccines, except H5N1, administered with alum or other adjuvants

18. Effects of dose on vaccine availability
Current US manufacturing: ~60M x 45 mcg = 2700M mcg
90 mcg x 2 = 180 mcg = 15M 2-dose vaccines
HHS goal requires 20-fold increase in US manufacturing capacity
Persons vaccinated (M)
Pandemic week

19. Strategies towards improved vaccination against pandemic infuenza
Alternative route of administration (intradermal)
Addition of adjuvants
Booster doses
Live vaccines

20. Aluminum based adjuvants
Category of aluminum salts (e.g., aluminum phosphate, aluminum hydroxide) or mixtures
Mechanism of action not defined, but depends on binding of antigen to the aluminum
Widely used in human vaccines, including pediatrics, licensed in most countries
Not currently used for inactivated influenza vaccines

21. Evaluation of alum adjuvanted vs unadjuvanted (aqueous) H1N1 vaccine in H1 naïve adults (1977)
GMT HI Antibody (% >40) after 9 mcg split H1N1 SC
(58%)
(93%)
(27%)
(70%) 4 16 64
128
GMT
Nicholson J Biolog Standard 7:123, 1979

22. GMT day 42 post vaccination HAI antibody
Effect of aluminum hydroxide on responses to A/VN/1194/04 (H5N1) subvirion vaccine 8 16 32 64
7.5 mcg
15 mcg
30 mcg
Alum
HA dose
GMT day 42 post vaccination HAI antibody
Bresson et al Lancet 367:1367, 2006

23. Immunogenicity of a whole virus vaccine formulated with alum
Dose of whole virion vaccine + alum
Percent with > 4- fold HAI response after 2 doses
Lin et al Lancet 2006

24. Oil-in-water emulsion – MF59
ANTIGEN
No real benefits in young adults, modest improvements in flu response in elderly
Increased local pain and irritation
Licensed in some countries
SQUALENE
TWEEN 80
SPAN 65

25. Significant enhancement of the response to H5N3 virus with MF594 8 16 32 64
H5N3 Neutralization GMT
Vaccine dose group
Nicholson et al Lancet 357:1937, 2001

26. Serum HAI responses to H9N2 subvirion vaccine formulated with or without MF594 8 16 32 64
128
256
GMT HAI antibody day 56
Vaccine Dose Group
Atmar et al CID 43:1135, 2006

27. Additional adjuvants in development
AS03 (GSK)
Adjuvant “A” (sanofi)
CPG (Coley, others)
ISCOM (CSL)
Transdermal LT (Iomai)
Others

28. Strategies towards improved vaccination against pandemic infuenza
Alternative route of administration (intradermal)
Addition of adjuvants
Booster doses
Live vaccines

29. Enhanced antibody responses following a third dose of H5N3 vaccine -/+ MF59
Microneutralization GMT
Stephenson et al, Vaccine 21:1687, 2003

30. Evaluation of priming with an antigenic variant: schematic of study design
1998
2005
UR-98012
A/HK/156/97
rH5
DMID
Healthy recipients of any rH5
rgA/VN/1203/04
H5-PRIMED
Placebo
25 ug x 2
45 ug x 2
90 ug x 2
90 ug x 1 +
10 ug x 1
Placebo excluded
90 ug x 1
37 subjects
This diagram shows a schematic of the study design. H5-primed subjects were derived from our original study, denoted as UR 98012, which was conducted in In that study, subjects were randomized to receive placebo or rH5 at either two doses of 25 mcg, two doses of 45 mcg, two doses of 90 mcg, or one dose of 90 mcg followed by one dose of 10 mcg, at intervals of 21, 28, or 42 days.
Subjects who received any active vaccine were considered H5-primed and were recruited to participate in the current study (DMID ). All subjects received a single dose of 90 mcg of A/VN/1203/04 vaccine in open label fashion.
Their results are compared to those of H5-naïve individuals who had received two doses of 90 mcg of A/Vietnam/1203/04 vaccine in study DMID
147 subjects
DMID
Vaccine 19:1732, 2001
rgA/VN/1203/04
H5-NAIVE
90 ug x 2
103 subjects
NEJM 354:1343, 2006
CLADE 3
CLADE 1

31. Serum neutralizing (NT) antibody responses following one or two doses of H5 vaccine in naïve subjects or following a single dose in H5 vaccine-primed subjects 4 8 16 32 64
128
256
H5 NAIVE
H5 PRIMED
DMID
DMID
GMT NT Antibody
90 mcg
5.1
8.3
22.9
6.4
93.8
68.9
This slides show the neutralizing antibody responses in H5-naïve subjects shown in the left and the H5-primed subjects shown in the right, with 95% confidence intervals.
Neutralizing titers after a single 90 mcg dose in H5 primed subjects were also higher than after one or even two doses of 90 mcg in vaccine naïve subjects

32. Advantages and disadvantages of priming
Current strategies generally depend on two doses (naïve population)
Logistically difficult in face of pandemic
Requires twice as much vaccine
Pre-priming would require less vaccine when pandemic occurs, easier response, possibly some protection
Requires decision to administer potentially unnecessary vaccine
Priming vaccine would not match pandemic strain, but multiple priming experiences might broaden response

33. Strategies towards improved vaccination against pandemic infuenza
Alternative route of administration (intradermal)
Addition of adjuvants
Booster doses
Live vaccines

34. Rapid attenuation of new antigenic variants by genetic reassortment
PB1
PB1
PB2
PB2 PA
PB1
PB2 PA NP M NS PA HA HA NA NA NP HA NA NP M M NS NS
ATTENUATED DONOR VIRUS
WILD-TYPE H5N1 VARIANT
ATTENUATED H5N1 VACCINE VIRUS

35. Live vaccine is especially efficacious in unprimed, immunologically naïve subjects
Attack rate (%)
Antigenic relatedness of vaccine and circulating virus
89.2 (67.7 to 97.4)
79.2 (70.6 to 85.7)
16.1 (-7.7 to 34.7)
Relative protective efficacy of CAIV compared to TIV
A/H3N2
A/H1N1 B
Belshe et al NEJM 356:685-96, 2007

36. Live vaccines
Conventional CAIV are highly immunogenic in susceptible populations
Higher levels of protection
Potential use of low doses
Induction of mucosal immunity might reduce shedding, halt transmission
Broader cross protection
Overattenuation is possible
Concerns about transmission
But…..

37. Experimental Approaches
DNA Vaccines
Live viral vectors (e.g., adenovirus, alphavirus replicons)
Expressed protein/virus-like particles
Cross protective peptides/epitopes
Nasal inactivated vaccines
Alternative live vaccines

38. H5 vaccines: summary
Likely licensure of sanofi 90 mcg vaccine is only a first step
Multiple simultaneous approaches to improved vaccines are being explored
Advances in H5 vaccines may be translated into better control of seasonal influenza