Title: The Antiviral Activity of Oseltamivir against H5N1 Human A/Vietnam/1203/04 and A/Turkey/15/06 Influenza Viruses in Ferrets Elena Govorkova, N.A. Ilyushina, D.A. Boltz, J.L. McClaren, A. Douglas, N. Yilmaz, and R.G. Webster
1The Antiviral Activity of Oseltamivir against
H5N1 Human A/Vietnam/1203/04 and A/Turkey/15/06
Influenza Viruses in FerretsElena Govorkova,
N.A. Ilyushina, D.A. Boltz, J.L. McClaren, A.
Douglas, N. Yilmaz, and R.G. Webster
2Oseltamivir Therapy for H5N1 Virus Infection
- Recommendations for Tamiflu prophylaxis and
treatment are based on the data from seasonal
influenza viruses (H1N1, H3N2, B) -
- H5N1 influenza viruses have a potential for
systemic spread and involvement of multiple
organs - Limited information is available on the efficacy
of oseltamivir against H5N1 infection in the
field - Initial mouse studies suggest that prolonged
oseltamivir treatment is required for most
beneficial protection -
3H5N1 Viruses from Two Clades
Clade 1
- Clade 1 A/Vietnam/1203/04
- Fatal human case
- Lethal infection in ferrets
- Clade 2 Subclade 2 A/Turkey/15/06
- Fatal human case
- Non- Lethal infection in ferrets
A/Vietnam/1203/04
Clade 2.1
Clade 2.2
A/Turkey/15/06
Clade 2.3
4Susceptibility to Oseltamivir in vitro
Enzymatic assay
Plaque reduction assay
EC50 (nM)
IC50 (nM)
A/VN/1203/04 A/Turkey/15/06
A/VN/1203/04 A/Turkey/15/06
A/Vietnam/1203/04 A/Turkey/15/06
14 aa changes in NA 18 aa changes in HA
5TOPIC 1Early Post-exposure Oseltamivir Therapy
A/Vietnam/1203/04 (H5N1)
6Early Post-exposure Treatment Survival
Virus Dose Experimental Group No. dead/ Total no. Day of Death
10 EID50 Treatment 0/3 No
10 EID50 Control 2/3 10, 10
100 EID50 Treatment 0/3 No
100 EID50 Control 3/3 7, 7, 9
Infect with 10 or 100 EID50 A/VN/1203/04
Day 0 Day 1 Day 2 Day 3 Day 4
Day 5
Day 21
5 mg/kg/d (equiv. to half approved human dose of
75 mg bid)
?
4 hrs
Oseltamivir 2x daily for 5 days
7 Virus Replication in Upper Respiratory Tract
10 EID50
100 EID50
Virus titer, log10EID50/ml
Day 3 Day 5 Day 7
Day 3 Day 5 Day 7
Day 3 Day 5 Day 7
Day 3 Day 5 Day 7
Treatment Control
Treatment Control
Plt0.05
8Virus Replication in Internal Organs
100 EID50
10 EID50
Treatment
Treatment
Control
Control
Virus titer, log10EID50/g
1/2
1/2
1/2
1/2
Lung Brain Liver Spleen S.
intest.
Lung Brain Liver Spleen S.
intest.
Note In treatment groups, virus was detected in
1/2 ferrets In control groups, virus
was detected in 2/2 ferrets Plt0.05
9Detection of Resistant Variants
Virus Dose Origin of Sample Amino acid change Amino acid change
Virus Dose Origin of Sample NA HA1
10 EID50 Brain I418M __
100 EID50 Brain Liver Spleen __ __
- no mutations confirmed by TOPO TA cloning
N2 NA numbering (Colman et al, J. Virol.,1993)
No changes in Oseltamivir susceptibility in vitro
10TOPIC 2Therapeutic Oseltamivir Efficacy
A/Vietnam/1203/04 (H5N1)
11Therapeutic Efficacy Survival
Virus Dose Experimental Group No. dead/ Total no. Day of Death
100 EID50 10 mg/kg/d 25 mg/kg/d 3/3 0/3 7, 7, 8 No
100 EID50 Control 3/3 6, 7, 8
Infect with 100 EID50 A/VN/1203/04
Day 0 Day 1 Day 2 Day 3
Day 4 Day 5 Day 6
Day 21
24 hours Delay
10 mg/kg/d (equiv. to approved human dose of 75
mg bid) or 25 mg/kg/d (equiv. to 2.5x human dose
of 75 mg bid)
Oseltamivir 2x daily for 5 days
12Duration of Clinical Signs of Infection
Treatment
Control
3/3 3/3 2/3 1/3 3/3 3/3 3/3 3/3
3/3 3/3
3/3
3/3
Days post-challenge
Days post-challenge
13Virus Replication in Upper Respiratory Tract and
Internal Organs
Upper respiratory tract
Internal organs
Control
Treatment
Virus titer, log10EID50/ml
Virus titer, log10EID50/g
1/2
1/2
Day 3 Day 5 Day 7
Day 3 Day 5 Day7
Treatment Control
Plt0.05
14Immunostaining Virus Detection in the Brain
Treatment
50 microns
Brain was collected 6 days p.i., fixed in 10
neutral-buffered formalin. Cells positive for
viral antigen have a dark-brown granular
appearance viral distribution is shown by red
shading (insets).
15Re-infection with Lethal H5N1 Virus Dose
Re-infection with 100 EID50 A/VN/1203/04
Infect with 100 EID50 A/VN/1203/04
All animals survived lethal challenge
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5
Day 6 Day 21
24 hours Delay
25 mg/kg/d
Oseltamivir 2x daily for 5 days
HI titers 120-140
16Detection of Resistant Variants
H274Y
Dose Origin of Sample Amino acid change Amino acid change
Dose Origin of Sample NA HA1
10 mg/kg/d Nasal wash Lungs V116A (1/20) H274Y (1/10) V178I (1/20) __
Nasal wash __ ND
25 mg/kg/d Brain H274R (1/10) E277Q (1/10) ND
- no mutations ND not done Detected by TOPO TA
cloning and analysis of individual plaques
No changes in Oseltamivir susceptibility in vitro
N2 NA numbering (Colman et al, J. Virol.,1993)
17TOPIC 3Therapeutic Oseltamivir Efficacy
A/Turkey/15/06 (H5N1)
18Oseltamivir Treatment A/Turkey/15/06 Virus
- Oseltamivir treatment
- Initiation 24 hours p.i.
- Duration 5 days twice daily
- Dose 10 mg/kg/d
Non-Lethal challenge 106 EID50 A/Turkey/15/06
- Note. 10 mg/kg/d in a ferret model equiv. to
approved human dose of 75 mg bid
19Virus Replication in Upper Respiratory Tract
20Inflammatory Responses in Upper Respiratory Tract
Protein concentration
of Inflammatory cells
Cell count (number x 106/ml)
3 5
7 Days post-challenge
3 5 7 Days
post-challenge
Plt0.05
21Virus Replication in Internal Organs
Treatment
Control
2/2
1/2
Lung Brain Liver Spleen S.
intest.
Plt0.05
22Immunostaining Virus Detection in the Brain
Treatment
50 microns
Brain was collected 6 days p.i., fixed in 10
neutral-buffered formalin. Cells positive for
viral antigen have a dark-brown granular
appearance viral distribution is shown by red
shading (insets).
23Detection of Resistant Variants
R193K
Dose Origin of Sample Amino acid change Amino acid change
Dose Origin of Sample NA HA1
10 mg/kg/d Nasal wash - R193K
- no mutations
No changes in Oseltamivir susceptibility in vitro
24Summary
- Oseltamivir treatment (25 mg/kg/d) protects
ferrets against lethal challenge and further
re-infection with A/VN/1203/04 (H5N1) virus - Oseltamivir (10 mg/kg/d) reduces lethargy of
animals, inhibits inflammation and blocks
A/Turkey/15/06 (H5N1) virus spread to internal
organs - Virulence may affect the antiviral treatment
schedule and higher oseltamivir dosages may be
required against more pathogenic virus - Early oseltamivir treatment is crucial for
protection against highly pathogenic H5N1
influenza viruses - Oseltamivir-resistant variants were not detected
by direct sequencing. Analysis of individual
viral clones detected a minor population of
clones carrying NA and/or HA mutations, although
changes in drug susceptibility were not
determined.
25Acknowledgements
- Support NIAID, NIH (Grants AI-95357, AI-70005
and AI-57570)
ALSAC F. Hoffmann-La Roche - St. Jude Childrens Research Hospital
- Robert G. Webster
- Natalia Ilyushina
- David Boltz
- Lana McClaren
- Oseltamivir Expert Working Group
- Frederick G. Hayden Noel Roberts
- Arnold S. Monto James Smith
- Albert D.M.E. Osterhaus Ron
A.M. Fouchier - T.D. Nguyen (Vietnamese Ministry of Agric. and
Rural Health Develop.) - Neziha Yilmaz (Virology and NIC of Turkey Refic
Saydam Hygiene Inst.)