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

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The 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
2
Oseltamivir 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

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H5N1 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
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Susceptibility 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
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TOPIC 1Early Post-exposure Oseltamivir Therapy
A/Vietnam/1203/04 (H5N1)
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Early 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
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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

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Virus 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
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Detection 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
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TOPIC 2Therapeutic Oseltamivir Efficacy
A/Vietnam/1203/04 (H5N1)
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Therapeutic 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
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Duration 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
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Virus 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

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Immunostaining Virus Detection in the Brain

• Control

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).
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Re-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
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Detection 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)
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TOPIC 3Therapeutic Oseltamivir Efficacy
A/Turkey/15/06 (H5N1)
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Oseltamivir 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

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Virus Replication in Upper Respiratory Tract
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Inflammatory 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
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Virus Replication in Internal Organs
Treatment
Control
2/2
1/2


Lung Brain Liver Spleen S.
intest.
Plt0.05
22
Immunostaining Virus Detection in the Brain

• Control

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). 
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Detection 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
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Summary

• 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.

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Acknowledgements

• 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.)