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Oseltamivir Therapy for H5N1 Virus Infection

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. Oseltamivir Therapy for H5N1 Virus Infection.

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Oseltamivir Therapy for H5N1 Virus Infection

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  1. 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

  3. Clade 1 Clade 2.1 Clade 2.2 Clade 2.3 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

  4. 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

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

  6. Early Post-exposure Treatment: Survival Infect with 10 or 100 EID50 A/VN/1203/04 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 21 ↔ 4 hrs 5 mg/kg/d (equiv. to half approved human dose of 75 mg bid) 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 * P<0.05

  8. 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 * P<0.05

  9. I418M Detection of Resistant Variants − no mutations; * confirmed by TOPO TA cloning N2 NA numbering (Colman et al, J. Virol.,1993) No changes in Oseltamivir susceptibility in vitro

  10. TOPIC 2Therapeutic Oseltamivir Efficacy: A/Vietnam/1203/04 (H5N1)

  11. Therapeutic Efficacy: Survival 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

  12. 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

  13. Virus Replication in Upper Respiratory Tract and Internal Organs Upper respiratory tract Internal organs Treatment Control * 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 * P<0.05

  14. 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).

  15. 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 1:20-1:40

  16. E277Q E277Q Detection of Resistant Variants H274Y − 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)

  17. TOPIC 3Therapeutic Oseltamivir Efficacy: A/Turkey/15/06 (H5N1)

  18. 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

  19. Virus Replication in Upper Respiratory Tract

  20. 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 * P<0.05

  21. Virus Replication in Internal Organs Treatment Control 2/2 1/2 * * Lung Brain Liver Spleen S. intest. * P<0.05

  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). 

  23. Detection of Resistant Variants R193K − no mutations No changes in Oseltamivir susceptibility in vitro

  24. 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.

  25. Acknowledgements Support: NIAID, NIH (Grants AI-95357, AI-70005 and AI-57570); ALSAC ; F. Hoffmann-La Roche St. Jude Children’s 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.)

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