The Science of Influenza
Vaccine Development:
Implications for the Public
Health Practitioner
David Cho, PhD, MPH
Program Officer, Influenza Vaccine Development
Respiratory Disease Branch, DMID, NIAID
Goals of the Presentation
 Describe the basic scientific differences between
seasonal and pandemic influenza
 Explain what researchers are doing to overcome the
challenges that a pandemic strain brings
 Explain what practitioners should consider in
preparation for a pandemic
Characteristics of a Pandemic Influenza
Virus
 Influenza A virus with a novel hemagglutinin or novel
hemagglutinin and neuraminidase in man
 Susceptibility (no neutralizing antibody) to the novel
virus in a large proportion of the population
 Demonstration of the virus to cause and spread
person-to-person in a sustained fashion
Influenza Virus Nomenclature
Source: Subbarao/Murphy
Clinical Burden of Influenza Virus
Morbidity and Mortality
 Previous pandemics
 1918 H1N1 transferred
from birds?: > 40 million
deaths worldwide
 1957 H2N2 avian-
human reassortant:
> 2 million deaths
 1968 H3N2 avian-
human reassortant:
> 1 million deaths
 Seasonal influenza
 Millions of human cases;
hundreds of thousands of
hospitalizations yearly in the
US alone
 Influenza and pneumonia:
7th leading cause of mortality
in the US in 2002
 20,000 to 40,000 deaths
annually US
 250,000 to 500,000 deaths
annually worldwide
Clinical Burden of Influenza Virus
Morbidity and Mortality (continued)
 H5N1 Avian influenza
 Seasonal influenza
 Millions of human cases;
 290+ documented
hundreds of thousands of
human cases
hospitalizations yearly in the
 170+ deaths
US alone
 Influenza and pneumonia: 7th
 Over 3 ½ + years, fewer
leading cause of mortality in
than 100 documented
the US in 2002
cases/ year
 20,000 to 40,000 deaths
annually US
 250,000 to 500,000 deaths
annually worldwide
Cumulative Number of Confirmed Human
Cases of Avian Influenza A/(H5N1) Reported to
WHO 11 April 2007
Country
cases
deaths
Azerbaijan
8
5
Cambodia
7 (1)
7 (1)
China
24 (2)
15 (1)
1
0
Egypt
34 (16)
14 (4)
Indonesia
81 (6)
63 (5)
Iraq
3
2
Lao People’s Dem Rep
2
2
Nigeria
1
1
Thailand
25
17
Turkey
12
4
Viet Nam
93
42
291
172
Djibouti
Total
> 50%
mortality
Cases and countries shown in gold for events since January
2007. WHO reports only laboratory-confirmed cases.
H5N1 outbreaks in 2005 and major flyways of migratory birds
(situation on 30 August 2005)
Mississippi
Americas
flyway
East
Atlantic
flyway
Atlantic
Americas
flyway
Black Sea/
Mediterranean
flyway
Districts with
H5N1 outbreaks
since January
2005
Pacific
Americas
flyway
East Africa
West Africa
flyway
Central
Asia
flyway
East Asia/
Australian
flyway
Sources: AI outbreaks: OIE, FAO, and Government sources. Flyways: Wetlands International
Schematic Version of Influenza Virus
(continued)
 Influenza A subtypes:
 16 Hemagglutnins (HA)
 9 Neuramindases (NA)
 All subtypes: endemic in birds
 H1N1, H2N2, H3N2: endemic in
people
 HA trimers: Binds sialic acid and
fuses viral and cell membranes
 NA tetramers: Removes sialic acid
to prevent adherence to self or cell
during budding
Schematic Version of Influenza Virus
(continued)
 RNA Polymerases (PB1, PB2,
PA) attached to each RNP
 Nucleoprotein (NP) binds RNA
and Matrix protein (M1)
 On viral and infected cell
surface:
 M2 tetramers
 Hydrogen ion channel
 In infected cell:
 NS1
 Binding host proteins
 Role in IFN resistance
Emergence of New Human Influenza
Subtypes
H5N1 Virulence Factors in Mammals
 HA with multibasic amino acid motif (RERRRKKR) at
the HA1-HA2 cleavage site
 Polymerase genes adapted to mammalian host
 1997 H5N1 with PB2 lysine at AA position 627
 2004 H5N1 with polymerases from human source
more virulent in ferrets than same H5N1 with
polymerases from avian source
 NS1 gene adapted for mammalian host
 Inhibition of interferons
 Increased TNF alpha
Source: Salomon et al. JEM 2006;203:689.
What Makes the HA Highly Pathogenic?
Source: Horimoto and Kawaoka. Nature Reviews Microbiology, 2005
Questions?
Common Features of H5N1 in Humans
 Contact with sick/dying poultry
 Frequently healthy young person
 Average age <18
 Incubation period 2–4 days from probable exposure
 Presenting symptoms fever, dyspnea, cough
 Diarrhea more common than expected with
influenza
 Leukopenia/lymphopenia/thrombocytopenia
 Metabolic abnormalities
Common Features of H5N1 in Humans
(continued)
 High frequency of progressive pneumonia
 Mostly primary viral
 Occasional contribution of bacteria?
• (Staphylococcus aureus and Haemophilus
influenzae)
 Hepatic necrosis and acute tubular necrosis
 High mortality rate in spite of
antiviral/steroid/antibacterial treatment
Common Features of H5N1 in Humans
(continued)
 Diffuse activation of the innate immune system
(“cytokine storm”) with increased levels of:
 Interleukin 1 beta
 Interleukin 6
 Interleukin 8
 Tumor Necrosis Factor alpha
 Interferon alpha
 Interferon gamma
 Interferon inducible protein 10
 Soluble Interleukin 2 receptor
 Monocyte chemoattractant protein 1
Chest Radiographs of Patient with Severe
H5N1 Influenza Pneumonia: Vietnam, 2004
Source: Tran et al. N Engl J Med 350:1171, 2004
Additional H5N1 Virulence Factors in
Humans
 HA receptor binding
 Two ketosidic linkages of sialic acid to galactose:
alpha 2,3 and alpha 2,6
 Avian HA preference for alpha 2,3 linkage
 Human upper airway predominantly alpha 2,6 linkage
 Human lower airway more abundant in alpha 2,3
linkage
 Possibly contributes to the high incidence of
primary viral pneumonia caused by H5N1
viruses
Source: Shinya et al. Nature 2006;440:435
Evidence for Person-to-Person H5N1
Transmission (Not Sustained)
 Possible instances of infection of health care
workers during 1997 outbreak in Hong Kong
 Family clusters Vietnam, Thailand* and
Indonesia**
 Cluster in Indonesia suggests human to human
to human transmission before the chain
extinguished***
(* Ungchusak et al. N Engl J Med 2005;352:333-340
** Kandun et al. N Engl J Med 2006;355:2186-2194
***Normile Science 2006;312:1855)
Detection of H5N1 Viruses: Lessons from
Recent Experiences
 Throat samples may give higher yield than nasal samples,
but both worth examining
 Rapid tests poor negative predictors and lack specificity
 But microarray methods improving and may provide sensitivity and
specificity
 Polymerase chain reaction (PCR) increases sensitivity but
success depends on the primers used for amplification
 Laboratory confirmation generally accepted
 Viral culture
 Positive PCR for H5N1 RNA
• (see www.cdc.gov/mmwr/preview/mmwrhtml/mm5505a3.htm)
 Positive immunofluoresence using a monoclonal
antibody for H5
 4-fold or greater rise in H5-specific antibody in
paired acute and convalescent sera
Questions?
Antiviral Therapies for Influenza
Antiviral Agents for Treatment of H5N1
Viruses
 Early treatment recommended for suspect cases but
efficacy, optimum dose, and duration uncertain
 Treatment of choice is a neuraminidase inhibitor
 Oseltamivir has been most frequently used
• 5 days treatment of 75 mg twice daily for adults and
dose decreases for children dependent on body
mass is standard
• Higher doses may be considered by some
authorities but no prospective studies
 Oseltamivir resistance during treatment may
not result in resistance to zanamivir
Antiviral Agents for Prophylaxis of H5N1
Viruses
 Oseltamivir 75 mg once daily for 7–10 days may
be considered for significant post exposure
prophylaxis
 But rationale is based on evidence from studies
with other influenza A virus subtypes
 Potential recipients would be poultry
workers/cullers, health care workers,
household contacts
Antiviral Agents for Treatment of H5N1
Viruses
 Zanamivir administered as inhaled powder, which may be
difficult with respiratory symptoms
 Amantadine/rimantadine resistance common in Asian H5N1
viruses
 Possibly from agricultural use of drugs
 Amantadine/rimantadine susceptibility of some recent strains
(African/European/Middle East)
 May be clade specific
 May be a role for M2 inhibitors
 Other drugs (ribavirin and interferon) may also be considered
but no value clearly documented
 Clinical studies in progress




Peramivir (injectable neuraminidase inhibitor)
CS8958 (once daily neuraminidase inhibior)
705 (polymerase inhibitor)
Studies may start soon with FluDase (sialidase to remove viral receptors)
Pandemic Influenza Preparedness:
Complementary Roles Within DHHS
NIH Trials with sanofi pasteur H5N1
A/Vietnam/1203/2004
 Adults (18 – 64y; 7.5, 15, 45, 90ug)*
 Immune response observed at all dose levels after a single
dose, unadjuvanted vaccine
 2 x 90mcg doses produced most frequent and highest
antibody responses
 April 17, 2007 FDA approval of sanofi vaccine at 90 mcg
dose for persons exposed to H5N1
 Additional studies in elderly (65y+; 45 or 90ug);
children (2–9y; 45ug)
 Immunogenicity results similar to adults
(* Treanor et al. N Engl J Med 2006; 354:1343-1351)
Dose Optimization of Inactivated H5N1
Vaccines: Aluminum Adjuvants
 Controlled trials completed or planned
 CSL Australia: subvirion vaccine +/- AlPO4
 Baxter Austria: whole virus +/- AlOH
 Novartis UK: subunit vaccine +/- AlOH
 Sanofi France and US: subvirion vaccine +/- AlOH in
adult and elderly populations
 Summary
 Vaccines well tolerated with or without aluminum
adjuvant
 Immunogenicity: Aluminum adjuvants do not show a
clear advantage over vaccine alone
Dose Optimization of Inactivated H5N1
Vaccines: Other Adjuvants
 Trials with other adjuvants
 GSK: subvirion vaccine +/- AS (proprietary adjuvant
system)
 Novartis UK: subunit vaccine with MF59 (proprietary
adjuvant system
 Summary
 Vaccines well tolerated with or without adjuvant
but somewhat increased local reactogenicity
 Immunogenicity: Adjuvants result in more
frequent and higher antibody responses
Dose Optimization of Inactivated H5N1
Vaccines: Route
 Trials with other alternate route of administration
 Sanofi subvirion vaccine given intradermal (ID) at reduced
dose or intramuscular (IM) at higher dose
 Summary
 Vaccines well tolerated but increased local reactogenicity
with intradermal administration
 Immunogenicity: High doses IM more immunogenic
than lower doses ID
 Additional studies planned for better direct
comparison of same dose given ID and IM
Source: The WHO Global Influenza Program Surveillance Network
Keeping up with H5N1 Drift: Vaccine
Reference Virus Efforts Underway
 Clade 1 vaccine; trials underway
 Vaccine candidates: A/VN/1203/2004 and A/VN/1194/2004
 Clade 2 - subclade 2 candidates available; vaccine
production ongoing
 CDC: Indonesia/05 (Sanofi US; DHHS)
 NIBSC: A/Turkey/Turkey/1/05
 St. Jude: A/BHG/Qinghai Lake/1A/2005 and
A/WS/Mongolia/244/05
 Clade 2 - subclade 3 candidates in development
 CBER/FDA: A/Duck/Laos/3295/06
 CDC: A/Anhui/1/2005
 St. Jude: A/Japanese White Eye/HK/1038/06
Questions?
What Can We Expect of H5N1 Influenza?
 Since 2003, increasing number of countries in
Africa, Asia, and Europe have documented H5N1
virus in poultry or migratory birds.
 Continued H5N1 evolution, possibly amplified by
uncontrolled transmission in high-density poultry.
 Human cases track exposure to infected poultry
and are accelerating in frequency.
 Clusters and potential human-to-human
spread plus epidemic influenza provide
continuing chance for reassortment.
Hong Kong model for eliminating infected
poultry and preventing human illness
 Agricultural surveillance and action are critical
early steps.
 Enforcement of market sanitation.
 Poultry segregation (quail as asymptomatic
carriers eliminated).
 Vaccination with agricultural vaccine
(asymptomatic infections possible).
 Difficult to implement because of social
and economic concerns.
Annual Influenza Vaccine Production
Bulk
vaccine
production
Millions of
chickens
Global
surveillance
(ongoing)
WHO
strain
selection
Manufacturers
assess growth &
yield of
candidates
Antigenic
relatedness
confirmed
Sheep sera
FDA potency
reagents
Millions of
fertilized
eggs
PHS
strain
selection
Purified
HA
Standard
antigen
~1 rooster for
10 hens
FDA approves
supplement to
license
Coordinated
collaborative
&
complex!
Filled into vials/
syringes
Formulated lots
Generation of high
yield reassortants
“candidates”
? Demand
Distribution/
vaccine use
? Severity of Season
? Recommendations
FDA release
testing
Influenza Vaccine Production Timeline
U.S. Seasonal Influenza Vaccine:
Production and Use
Beyond Eggs and Cell Culture: Research
Efforts to Develop New Technologies
Goal: Develop “agile” vaccine platforms
 DNA
Plasmids – single or multiple gene combinations (HA +
NP + M2); conserved regions; single subtype or multiple
subtypes (H3 + H1 + H5)
 Vector
Adenovirus, alphavirus, salmonella strains
 Recombinant subunit
Expression systems, baculovirus, drosophila
 Peptide vaccines
Synthesized multigenic peptides
 Vector-based vaccines
Influenza Virus and Protein RNAs:
Targets for a “Universal Vaccine”
Source: Subbarao/Murphy
Seasonal
Influenza
Preparedness
Pandemic
Influenza
Preparedness