The new inluenza A virus (H1N1)

and “Mexican Flu”...

towards the next pandemic?

ECCMID Late Braking News

Helsinki, 17th May 2009
Ab Osterhaus
ErasmusMC Rotterdam
The Netherlands
 Human influenza:
three appearances

• Seasonal influenza
(A: H3N2, H1N1; B)

• Avian influenza
(A: H7N7, H5N1…)

• Pandemic influenza
(A: H1N1, H2N2, H3N2…?)
Influenza

- Clinical features -

• Fever >39 °C
• Acute onset
• Myalgia
• Total malaise
• Shivers
• Coughing
• Redness of mucosa in
nose and throat
 The Global Circulation
of Seasonal Influenza A (H3N2) Viruses

Science 2008, Russell et al.

 20th century pandemics

Credit: US National Museum of Health and Medicine

1918: “Spanish Flu” 1957: “Asian Flu” 1968: “Hong Kong Flu”

>50 million deaths 1-4 million deaths 1-4million deaths

A(H1N1) A(H2N2) A(H3N2)

 UK officials declare that pandemic influenza
poses the greatest risk to their population

Cabinet Office UK.

Life expectancy in the USA between 1900-1960
dipped dramatically in 1918 due to the pandemic
The 1918 influenza pandemic had a W shaped
curve, adversely affecting young healthy adults
3000

2500
Specific Death Rate

1911-1917
1918
2000

1500

1000

500

0
<1 1 to 5 to 15- 25- 35- 45- 55- 65- 75- >85
4 14 24 34 44 54 64 74 84
Age Divisions
 Mexican Flu

7000

6500

6000

5500

5000

4500

4000

3500
Cases
Deaths
3000

2500

2000

1500

1000

500

0
03-05-2009 04-05-2009 05-05-2009 06-05-2009 07-05-2009 08-05-2009 09-05-2009 10-05-2009 11-05-2009 12-05-2009 13-05-2009 14-05-2009

17th May: 8480 cases

72 deaths
 In 1957, pandemic influenza
spread worldwide in 6 months

J Am Med Assoc. 1958 Mar 8;166(10):1140-8

 Then & Now…?

1918
>50 million deaths

03-05-2009 – 17-05-2009
72 deaths, and continuing
 Influenza A viruses
• Subtype divisions of influenza A viruses
made on basis of HA (1-16) and NA (1-9)
antigens (all found in migratory birds)

• Genomic nucleic acid consists of 8 RNA

segments,
segments allowing gene reassortment
during mixed infection

• Virulence of influenza virus in birds

depends on presence of presence of series
of basic amino acids adjacent to cleavage
site of the HA polypeptide (H5 and H7
subtypes only)
Influenza A viruses: about wild birds…

…and mammals
Influenza A virus reservoir
 Drift and Shift

Drift

Shift

.
De Jong JC, et al. Influenza virus: a master of metamorphosis. J Infect 2000; 40 (3): 218–28.
 Influenza A (H5N1) virus
-Risk of host adaptation –
“Two different mechanisms”

Sequential mutations Genome reassortment

PB2 PB2 PB2 PB2 PB2 PB2

PB1 PB1 PB1 PB1 PB1 PB1
PA PA PA PA PA PA
HA HA HA HA HA HA
NA NA NA NA NA NA
NP NP NP NP NP NP
M M M M M M
NS NS NS NS NS NS

De Jong et al., Nature 1997

Claas & Osterhaus, Nature Medicine 1998
 Swine influenza viruses

• Influenza A viruses of H1N1, H1N2, H3N1 and H3N2 subtypes are

enzootic in pigs worldwide (most common is the H1N1 subtype)

• Most are reassortants with swine and/or human and/or avian genes

• Highly contagious acute respiratory disease in pigs

• Morbidity tends to be high and mortality low (1-4%)

• In pig, the virus spreads by aerosols and direct & indirect contact

• In Europe they differ in their antigenic and genetic make up from those
in North America

Vincent AL, et al. Adv Virus Res 2008; 72: 127-154. - Van Reeth K. Vet Res 2007; 38 (2): 243-260.0
 Swine Influenza A(H1N1) : in USA 1976
Swine flu outbreak (H1N1) in Fort Dix,
New Jersey, USA,
>200 cases with serious illness and one
death
>40 million people were vaccinated
Vaccination stopped after >500 cases of
Guillain-Barre syndrome were reported
(30 deaths)

December 2005 - February 2009:

12 human infections with swine
influenza were reported from 10 states
in the United States

Isolated severe cases of re-assortant H1N1

swine flu cases in the Netherlands

Sencer DJ, et al. Emerg Infect Dis 2006; 12 (1): 29-33.

 A/California/04/2009 is a reassortant of
4 influenza A virus subtype H1N1 strains

http://www.hhs.gov - http://www.who.int. -
http://www.cdc.gov
 ~ 1998
PB2,PA:
Triple reassortant

~ 1968 ~ 1998
PB1:
N-America

~ 1918 Classical swine

HA, NP, NS:

~ 1979
NA, MA: Eurasian swine Eurasia

A/California/4/2009
PB2
PB1
PA
HA
NP
The “Mexican flu” virus NA
MA
NS
INFLUENZA A VIRUS
Recent zoonotic transmissions from birds

Subtype Country Year # Cases # Deaths

H7N7 UK 1996 1 0
H5N1 Hongkong 1997 18 6
H9N2 SE-Asia 1999 >2 0
H5N1 Hongkong 2003 2? 1
H7N7 Netherlands 2003 89 1
H7N2 USA 2003 1 0
H7N3 Canada 2004 2 0
H5N1 SE-Asia/M-East/ 2003-9 >400 >250*
Europe/W-Africa *CFR >60%

H5N1: high pathogenicity

but low transmissibility
Bird markets
- Asia 1997 -
Confirmed H5N1 avian influenza virus
endemic areas (poultry and wild birds) since 2003
H7N7 in The Netherlands
- Fatal case chest X-ray upon admission -

What determines the pathogenicity

of avian/pandemic influenza viruses
for mammals/humans?

Clues from:
Spanish flu virus
H5 viruses
H7 viruses
 Influenza A virus:
major determinants of pathogenicity for mammals

Spanish flu (H1) virus:

PB2 E627K (Taubenberger et al., 2005)
HA (Kobassa et al., 2004)
NA (Tumpey et al., 2005) replication in absence trypsine
NS1 (Geiss et al., 2002) IFN pathway (in mice with Mx1 gene)
H7 viruses (LPAI/HPAI)
PB2 E627K (Munster et al., 2007; Shinya et al., 2007)
HA (Munster et al., 2007) attachment pattern
H5 viruses (HPAI)
PB2 E627K (Hatta et al., 1997)
PB2 D701N (Li et al., 2005) duck virus in mice
HA (van Riel et al., 2006; Shinya et al 2006) attachment pattern
NS1 (Seo et al., 2002)
PB1-F2 is believed to be an important determinant
of the virulence of the influenza virus
•The second RNA segment of the influenza
virus genome encodes two proteins, PB1
and PB1-F2. The latter protein is believed to
be an important determinant of virulence of
influenza virus. Can we learn anything about
the virulence of the new influenza virus
H1N1 strains from a study of this protein?

•During influenza virus infection, PB1-F2 is

targeted to the mitochondria, where it
induces a form of cell death known as
apoptosis. Experiments in a mouse model of
influenza virus infection have shown that
PB1-F2 regulates lethality of the virus.

PB1-F2 is currently missing from A/H1N1/California

Conenello, G., Zamarin, D., Perrone, L., Tumpey, T., & Palese, P. (2007). A Single Mutation in the PB1-F2 of
H5N1 (HK/97) and 1918 Influenza A Viruses Contributes to Increased Virulence PLoS Pathogens, 3 (10) DOI:
10.1371/journal.ppat.0030141
MCAULEY, J., HORNUNG, F., BOYD, K., SMITH, A., MCKEON, R., BENNINK, J., YEWDELL, J., &
MCCULLERS, J. (2007). Expression of the 1918 Influenza A Virus PB1-F2 Enhances the Pathogenesis of Viral
and Secondary Bacterial Pneumonia Cell Host & Microbe, 2 (4), 240-249 DOI: 10.1016/j.chom.2007.09.001
Influenza A virus evolution
- H5N1 The next step ? –
- H1N1 Mexican…Ro > 1.4
Avian influenza A H5N1 virus
- HA: Receptor specificity -

Shinya et al.,
Nature 440, 2006

Van Riel et al.,

Science 312, 2006

Van Riel et al.,

Am J Pathol 171, 2007
 Current status could progress from pandemic
alert (phase 3) to full pandemic in a few months
Estimated Current Phase of the Pandemic
Post- Pandemic
Inter-Pandemic Period Pandemic Alert Period Pandemic Period Period

H5N1 H1N1
Post-
Phase 1 Phase 2 Phase 3 Phase 4 Phase 5 Phase 6 Pandemic
Period

• No new • No new • Human • Small clusters • Large clusters • Increased and • Return to inter-
influenza influenza infections with of human-to- of human-to- substantial pandemic period
subtypes in subtypes in new influenza human trans- human trans- transmission in
humans humans subtypes mission mission general pop.

• Low risk of • Animal • Rare human- • Infected • Infected • Global

human infection influenza to-human areas areas still pandemic
from influenza subtype poses transmission highly localized risk
present in substantial risk of disease localized
animals to humans

How long is phase likely to last?

Decades Years Months Weeks 1-2 years

http://www.who.int/entity/csr/resources/publications/influenza/WHO_CDS_CSR_GIP_2005_ 5/en/index.html
 Declaration of a phase 5 on April 30th, 2009

• The WHO raised its pandemic alert level to 4, verifying

human-to-human transmission of the new influenza A
(H1N1) virus. April 28th, 2009
• WHO influenza expert Dr. Keiji Fukuda pointed out that
it is too late to contain the new influenza A (H1N1) virus

« Containment is not a feasible operation. Countries should now

focus on mitigating the effect of the virus »

Phase 4 Phase 5
28 April 2009 30 April 2009
 Three options for the
new influenza A (H1N1) virus:

- disappear spontaneously (unlikely?)

- cause a mild pandemic (Asian flu like?)

- cause a severe pandemic

(after mutation/re-assortment)
 INFLUENZA: are we prepared?
- medical interventions –
NB: societal interventions
• Surveillance
Humans: seasonal surveillance is the basis
Animals: “early warning” and “rapid response”

• Antiviral therapy
Preventive and therapeutic…resistance development ?

• Vaccination
Cornerstone of prevention…pre-pandemic vaccination?
General Diagnosis New Influenza A (H1N1):
clinical evaluation and sampling for lab investigation
 Symptoms of the new
influenza A (H1N1)

http://www.hhs.gov - http://www.who.int. - http://www.cdc.gov

General Diagnosis New Influenza A
(H1N1)

• A confirmed case of new influenza A

(H1N1) virus infection is defined as an
individual with laboratory confirmed
new influenza A (H1N1) virus infection
by one or more of the following tests:
– Real-time RT-PCR
– Viral culture
– Four-fold rise in new influenza A
(H1N1) specific VN antibodies

http://www.hhs.gov - http://www.who.int. - http://www.cdc.gov

 New Influenza A (H1N1):
infectious period

• The infectious period for a confirmed case of new

influenza A (H1N1) virus infection is defined as
1 day prior to 7 days after onset.
onset

• The new influenza A (H1N1) virus infection in humans

is most contagious during the first five days of the
illness although some people, most commonly
children, can remain contagious for up to 10 days,
days
immunocompomised up to 1-3 months.
months

http://www.hhs.gov - http://www.who.int. - http://www.cdc.gov

 Network of outbreak assistance laboratories
in addition to NIC
Minimal requirements
National Influenza Centre
Outbreak assistance
•24 hours, 7 days a week
laboratories availability
•Minimum of 100 samples a day,
during 2-3 months
•Able to unpack samples possibly
containing BSL-3 organisms
•Routine molecular diagnostics
•Internal control used for routine
diagnostics
•8 hours turnaround time
•Willing to participate in quality
control programs and to share
results
 New Influenza A (H1N1):
Case Summary Form for case-based
data collection

For the first time ever,

the evolution of a new
influenza virus can be
monitored in real time !!!

http://www.who.int.
 INFLUENZA: are we prepared?
- medical interventions –
NB: societal interventions
• Surveillance
Humans: seasonal surveillance is the basis
Animals: “early warning” and “rapid response”

• Antiviral therapy
Preventive and therapeutic…resistance development ?

• Vaccination
Cornerstone of prevention…pre-pandemic vaccination?
 Structure of Influenza A Virus

Orthomyxovirus

Neuraminidase
(9 subtypes)

Viral RNA

M2 M1 protein
protein
Haemagglutinin
(16 subtypes)
Influenza virus replication cycle:
options for antiviral intervention
strategies
 Antiviral Agents for Influenza

Class/agent Brand name Route

M2 inhibitors
Amantadine Symmetrel PO
Rimantadine Flumadine PO

NA inhibitors
Zanamivir Relenza Inhaled
Oseltamivir Tamiflu PO
(Peramivir) … …
 Production Capacity for Tamiflu®
450
400
400
Millions
of 350
treatment
courses 300

250
190
200

150
100
55
50 18 27
5.5
0
'99 - '02 2003 2004 2005 2006 2007
 Two Fatal Oseltamivir Resistant Influenza A (H1N1)
Infections in Immunocompromised patients during the
2007/2008 Influenza Season in The Netherlands

Figure 3. Chest Radiographs and Computed Tomography of Patient 2 taken at

Different Time Points during Hospitalization
 INFLUENZA: are we prepared?
- medical interventions –
NB: societal interventions
• Surveillance
Humans: seasonal surveillance is the basis
Animals: “early warning” and “rapid response”

• Antiviral therapy
Preventive and therapeutic…resistance development ?

• Vaccination
Cornerstone of prevention…pre-pandemic vaccination?
Current seasonal
flu vaccines

1 dose (trivalent):

1 chicken egg!
Seasonal influenza vaccine
formulations

Source: IFPMA website

Pandemic influenza vaccines
- Key issues -

• Response time
(currently >6 months)

• Production capacity
(currently 700 million doses seasonal world-wide,
1-2 billion doses pandemic?)

• Efficacy/Safety
(animal and human trials with prototype vaccines)

NB: Regulatory preparedness

Pandemic influenza vaccines
- Improvements in production systems -

Influenza A virus surveillance in wild birds

Strain selection
Reverse Genetics

Seed-strain preparation

Production systems

Cell culture: MDCK, Vero, PERC-6, others:

Continuous availability of production substrate
 Structure of Influenza A Virus

Orthomyxovirus

Neuraminidase
(9 subtypes)

Viral RNA

M2 M1 protein
protein
Haemagglutinin
(16 subtypes)
Pandemic influenza vaccines
- Room for improvement -

small changes; easy to incorporate

Strain selection

Seed-strain preparation

Production systems

New targets; identify correlates of protection

Improve efficacy: adjuvants & delivery systems

larger changes; longer path before implementation?

Assessment of pre-pandemic H5N1 clinical trials
Type of vaccine
Split vaccine no adjuvant
Split/subunit vaccine with alum
Whole virus (egg) with alum
Subunit with MF59 adjuvant
Whole virus Vero cell culture, no
adjuvant
Split vaccine with AS adjuvant
Compliance with EU
licensing criteria
Need two doses of 90 µg
Need two doses of 30-45 µg
Need two doses of 10-15 µg
Need two doses of 7.5 µg
Need two doses of 7.5 µg
Need two doses of 3.8 µg

Data presented at WHO meeting, February 2007

(Sanofi Pasteur, 4 Companies in Jp, CSL, Microgen, Sinovac, GSK, Novartis,Baxter)
 Adjuvanted H5N1 influenza vaccine is highly
immunogenic at a very low antigen dose

100

3.8 µg H5N1
Seroconversion rate (%)

75
7.5 µg H5N1
15 µg H5N1

50 30 µg H5N1
40
CHMP criterion

25

0
No adjuvant Adjuvanted No adjuvant Adjuvanted
Post first dose Post second dose

Leroux-Roels et al. Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised
controlled trial. Lancet 2007; 370 (9587): 580–89.
 Ferrets vaccinated with AS-adjuvanted
H5N1 split candidate vaccine
• 2 immunisations of ferrets at D0 and D21 (H5N1 A/Vietnam/1194/04 split virus /
AS)
• Heterologous challenge (wild-type virus A/Indonesia/5/05, 105 TCID50) at D49
• Post challenge results at D5

Dead Alive % Survival

Pooled controls
(15 µg Antigen
12 0 0
only or AS only)
1.7 µg H5N1 –
AS
1 5 83
3.8 µg H5N1 –
AS
0 6 100
7.5 µg H5N1 –
AS
0 5 100
15 µg H5N1 – AS
0 6 100
Baras et al., PLoS One 2008
 Evaluation MVA-Flu-HA in
cynomolgus macaques

0 28 56 60

Vaccines Read-out
ƒ MVA-HA (A/Vietnam/1194/04)1
•Serology*
ƒ MVA (wildtype)1
•Clinical
ƒ PBS
signs
Viruses •Virus titers
ƒ A/Vietnam/1194/04 •Pathology
ƒ A/Indonesia/5/05
Kreijtz et al., JID 2008
Reduction of virus replication in the lungs

A/VN/1194/04
Day 2
A/IND/5/05
 CONCLUSIONS 1/2

Current pandemic “H1N1 threat” is serious

Three options for the virus:

- disappear spontaneously
(unlikely?)

- cause a mild pandemic

(“Asian flu” like?)

- cause a severe pandemic

(after mutation/reassortment with
H3N2, H1N1, or H5N1 viruses?)
 CONCLUSIONS 2/2

Global pandemic preparedness should focus on:

• Surveillance and diagnostics

• Social distancing

• Use of antiviral stockpiles (resistance monitoring)

• Production of pandemic vaccines (flexible approach)

NB: equitable distribution?

Acknowledgements
Erasmus MC ViroNovative BV
Vincent Munster James Simon
Emmie de Wit Viroclinics BV
Debby van Riel Koert Stittelaar
Theo Bestebroer
Ger van der Water Sanofi Pasteur
Geert van Amerongen GSK
Robert Dias d’Ullois Roche
Marion Koopmans Novartis
Martin Schutten Solvay
Jan de Jong Nobilon
Charles Boucher
Guus Rimmelzwaan
Bart Haagmans
Thijs Kuiken
Ron Fouchier