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

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 Oseltamivir Therapy for H5N1 Virus Infection

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 H5N1 Viruses from Two Clades Clade 1 Clade 2.1 Clade 2.2 Clade 2.3 A/Vietnam/1203/04 A/Turkey/15/06

A/VN/1203/04 A/Turkey/15/06 Susceptibility to Oseltamivir in vitro Enzymatic assay Plaque reduction assay IC 50 (nM) EC 50 (nM) A/Vietnam/1203/04 A/Vietnam/1203/04 A/Turkey/15/06 A/Turkey/15/06 14 aa changes in NA 18 aa changes in HA

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

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 21 Oseltamivir 2x daily for 5 days Infect with 10 or 100 EID 50 A/VN/1203/04 5 mg/kg/d (equiv. to half approved human dose of 75 mg bid) ↔ 4 hrs Early Post-exposure Treatment: Survival Virus Dose Dose Experimental Group No. dead/ Total no. Day of Death 10 EID 50 Treatment0/3No Control2/3 10, EID 50 Treatment0/3No Control3/3 7, 7, 9

Treatment Control Virus titer, log 10 EID 50 /ml Day 3 Day 5 Day 7 Virus Replication in Upper Respiratory Tract Virus Replication in Upper Respiratory Tract Treatment Control 10 EID EID 50 * P<0.05 * * *

Virus titer, log 10 EID 50 /g 10 EID EID 50 Lung Brain Liver Spleen S. intest. 1/2 Note: In treatment groups, virus was detected in 1/2 ferrets In control groups, virus was detected in 2/2 ferrets In control groups, virus was detected in 2/2 ferrets * P<0.05 Treatment Control Treatment Control Virus Replication in Internal Organs *****

Detection of Resistant Variants VirusDose Origin of Sample Amino acid change NAHA1 10 EID 50 BrainI418M*__ 100 EID 50 BrainLiverSpleen____ − no mutations; * confirmed by TOPO TA cloning I418M N2 NA numbering (Colman et al, J. Virol.,1993) No changes in Oseltamivir susceptibility in vitro

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

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 21 Oseltamivir 2x daily for 5 days Infect with 100 EID 50 A/VN/1203/04 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) 24 hours Delay Therapeutic Efficacy: Survival Virus Dose Dose Experimental Group No. dead/ Total no. Day of Death 100 EID mg/kg/d 25 mg/kg/d 3/30/3 7, 7, 8 No Control3/3 6, 7, 8

Duration of Clinical Signs of Infection Days post-challenge Treatment Control 3/33/32/31/33/33/33/33/3 3/33/3 3/3 3/3

Virus titer, log 10 EID 50 /ml Day 3 Day 5 Day 7 Virus Replication in Upper Respiratory Tract and Internal Organs and Internal Organs Treatment Control Virus titer, log10EID50/g Upper respiratory tract Internal organs 1/2 ControlTreatment * * P<0.05 ***

Immunostaining: Virus Detection in the Brain ControlTreatment 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). 50 microns

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 21 Oseltamivir 2x daily for 5 days Infect with 100 EID 50 A/VN/1203/04 25 mg/kg/d 24 hours Delay Re-infection with Lethal H5N1 Virus Dose Re-infection with 100 EID 50 A/VN/1203/04 HI titers 1:20-1:40 All animals survived lethal challenge

Detection of Resistant Variants Dose Origin of Sample Amino acid change NAHA1 10 mg/kg/d Nasal wash LungsV116A(1/20)H274Y(1/10)V178I(1/20)__ __ND 25 mg/kg/d BrainH274R(1/10)E277Q(1/10)ND − no mutations; ND – not done Detected by TOPO TA cloning and analysis of individual plaques H274Y E277QE277Q N2 NA numbering (Colman et al, J. Virol.,1993) No changes in Oseltamivir susceptibility in vitro

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

Oseltamivir treatment: Initiation – 24 hours p.i. Duration – 5 days twice daily Dose – 10 mg/kg/d Oseltamivir Treatment: A/Turkey/15/06 Virus Note. 10 mg/kg/d in a ferret model equiv. to approved human dose of 75 mg bid Non-Lethal challenge 10 6 EID 50 A/Turkey/15/06

Virus Replication in Upper Respiratory Tract

# of Inflammatory cells Inflammatory Responses in Upper Respiratory Tract Days post-challenge Cell count (number x 10 6 /ml) Days post-challenge Protein concentration * * * * * * P<0.05

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

Immunostaining: Virus Detection in the Brain ControlTreatment 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). Cells positive for viral antigen have a dark-brown granular appearance; viral distribution is shown by red shading (insets). 50 microns

Detection of Resistant Variants Dose Origin of Sample Amino acid change NAHA1 10 mg/kg/d Nasal wash ─R193K − no mutations No changes in Oseltamivir susceptibility in vitro R193K

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.

Support: NIAID, NIH (Grants AI-95357, AI 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 Frederick G. Hayden Noel Roberts Arnold S. Monto James Smith Albert D.M.E. Osterhaus Ron A.M. Fouchier 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.) Acknowledgements