oseltamivir therapy for h5n1 virus infection

The Antiviral Activity of Oseltamivir against The Antiviral Activity of Oseltamivir against

H5N1 Human A/Vietnam/1203/04 and H5N1 Human A/Vietnam/1203/04 and

A/Turkey/15/06 Influenza Viruses in FerretsA/Turkey/15/06 Influenza Viruses in Ferrets

Elena Govorkova,Elena Govorkova, N.A. Ilyushina, D.A. Boltz, J.L. McClaren, N.A. Ilyushina, D.A. Boltz, J.L. McClaren,

A. Douglas, N. Yilmaz, and R.G. WebsterA. Douglas, N. Yilmaz, and R.G. Webster

Recommendations for Tamiflu prophylaxis Recommendations for Tamiflu prophylaxis and treatment are based on the data from and treatment are based on the data from seasonal influenza viruses (H1N1, H3N2, B)seasonal influenza viruses (H1N1, H3N2, B)

H5N1 influenza viruses have a potential for H5N1 influenza viruses have a potential for systemic spread and involvement of multiple systemic spread and involvement of multiple organsorgans

Limited information is available on the Limited information is available on the efficacy of oseltamivir against H5N1 infection efficacy of oseltamivir against H5N1 infection in the fieldin the field

Initial mouse studies suggest that prolonged Initial mouse studies suggest that prolonged oseltamivir treatment is required for most oseltamivir treatment is required for most beneficial protectionbeneficial protection

Oseltamivir Therapy for H5N1 Virus InfectionOseltamivir 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 caseNon- Lethal infection in ferrets

H5N1 Viruses from Two Clades

Clade 1

Clade 2.1

Clade 2.2

Clade 2.3

Clade 1

Clade 2.1

Clade 2.2

Clade 2.3

A/Vietnam/1203/04A/Vietnam/1203/04

A/Turkey/15/06A/Turkey/15/06

0

5

10

15

20

25

30

35

0

2

4

6

8

10

A/VN/1203/04 A/Turkey/15/06A/VN/1203/04 A/Turkey/15/06 A/VN/1203/04 A/Turkey/15/06A/VN/1203/04 A/Turkey/15/06

Susceptibility to Oseltamivir Susceptibility to Oseltamivir in vitroin vitro

Enzymatic assayEnzymatic assay Plaque reduction assayPlaque reduction assay

ICIC505

0 (n

M)

(n

M)

EC

EC

5050

(nM

) (

nM

)

A/Vietnam/1203/04 A/Vietnam/1203/04 A/Turkey/15/06A/Turkey/15/06

14 aa changes in NA14 aa changes in NA18 aa changes in HA18 aa changes in HA

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

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 21 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 21

Oseltamivir 2x daily for 5 daysOseltamivir 2x daily for 5 days

Infect withInfect with10 or 100 EID10 or 100 EID5050

A/VN/1203/04 A/VN/1203/04

5 mg/kg/d (equiv. to half approved human dose of 75 mg bid)5 mg/kg/d (equiv. to half approved human dose of 75 mg bid)

↔↔4 hrs4 hrs

Early Post-exposure Treatment: SurvivalEarly Post-exposure Treatment: Survival

VirusVirus DoseDose

Experimental Experimental GroupGroup

No. dead/No. dead/Total no.Total no.

Day of Day of DeathDeath

10 EID10 EID

5050

Treatment Treatment 0/30/3 NoNo

ControlControl 2/32/3 10, 1010, 10

100 EID100 EID5050

TreatmentTreatment 0/30/3 NoNo

ControlControl 3/33/3 7, 7, 97, 7, 9

0

1

2

3

4

5

6

7

Treatment ControlTreatment Control

Vir

us

tite

r, l

og

10E

ID50

/ml

Day 3 Day 5 Day 7Day 3 Day 5 Day 7 Day 3 Day 5 Day 7Day 3 Day 5 Day 7 Day 3 Day 5 Day 7 Day 3 Day 5 Day 7

Virus Replication in Upper Respiratory TractVirus Replication in Upper Respiratory Tract


10 EID10 EID5050 100 EID100 EID5050

* P<0.05* P<0.05

* * ** * * * * ** * *

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Vir

us

tite

r, lo

gV

iru

s ti

ter,

log

1010E

IDE

ID505

0/g/g

10 EID10 EID5050100 EID50

Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.

1/2 1/2 1/21/2

Note: In treatment groups, virus was detected in 1/2 ferretsNote: In treatment groups, virus was detected in 1/2 ferrets In control groups, virus was detected in 2/2 ferretsIn control groups, virus was detected in 2/2 ferrets* P<0.05

TreatmentTreatment

ControlControl

TreatmentTreatment

ControlControl

Virus Replication in Internal OrgansVirus Replication in Internal Organs

** ** ** ** **

Detection of Resistant VariantsDetection of Resistant Variants

VirusVirus

DoseDose

Origin ofOrigin of

SampleSample

Amino acid changeAmino acid change

NANA HA1HA1

10 10 EIDEID5050 BrainBrain I418M*I418M* ____

100 100 EIDEID5050 BrainBrain

LiverLiver

SpleenSpleen

____ ____

− − no mutations; * confirmed by TOPO TA cloningno mutations; * confirmed by TOPO TA cloning

I418MI418M

N2 NA numbering (Colman et al, J. Virol.,1993)N2 NA numbering (Colman et al, J. Virol.,1993)

No changes in Oseltamivir susceptibility No changes in Oseltamivir susceptibility in vitroin vitro

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

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 21 Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 21


Infect withInfect with100 EID100 EID5050

A/VN/1203/04 A/VN/1203/04

10 mg/kg/d (equiv. to approved human dose of 75 mg bid) 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)or 25 mg/kg/d (equiv. to 2.5x human dose of 75 mg bid) 24 hours24 hoursDelayDelay

Therapeutic Efficacy: SurvivalTherapeutic Efficacy: Survival

VirusVirus DoseDose

Experimental Experimental GroupGroup

No. dead/No. dead/Total no.Total no.

Day of Day of DeathDeath

100 EID100 EID5050

10 mg/kg/d10 mg/kg/d


3/33/3

0/30/3

7, 7, 87, 7, 8

NoNo

ControlControl 3/33/3 6, 7, 86, 7, 8

Duration of Clinical Signs of InfectionDuration of Clinical Signs of Infection

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Fever

Weight loss

Lethargy

Inappetence

Labored breathing

Diarrhea

Neurol. Symptoms

Death

Days post-challengeDays post-challenge

TreatmentTreatment ControlControl

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Fever

Weight loss

Lethargy

Inappetence

Labored breathing

Diarrhea

Neurol. Symptoms

Death

Days post-challengeDays post-challenge

3/33/3

3/33/3

2/32/3

1/31/3

3/33/3

3/33/3

3/33/3

3/33/3

3/33/3

3/33/3

3/33/3

3/33/3

0

1

2

3

4

5

6

Vir

us

tite

r, lo

g10

EID

50/m

l

Day 3 Day 5 Day 7

Day 3 Day 5 Day7

Virus Replication in Upper Respiratory TractVirus Replication in Upper Respiratory Tract and Internal Organsand Internal Organs


0

1

2

3

4

5

6

Lungs FrontBrain

RearBrain

Spleen Liver

Vir

us

tite

r, lo

g10

EID

50/g

Upper respiratory tractUpper respiratory tract Internal organsInternal organs

1/2 1/2

ControlControlTreatmentTreatment

*

* P<0.05

** ** **

Immunostaining: Virus Detection in the BrainImmunostaining: Virus Detection in the Brain

ControlControl TreatmentTreatment

Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Cells positive for viral antigen have a dark-brown granular appearance; Cells positive for viral antigen have a dark-brown granular appearance; viral distribution is shown by red shading (insets).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 21Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 21


Infect withInfect with100 EID100 EID5050

A/VN/1203/04 A/VN/1203/04

25 mg/kg/d 25 mg/kg/d 24 hours24 hoursDelayDelay

Re-infection with Lethal H5N1 Virus DoseRe-infection with Lethal H5N1 Virus Dose

Re-infection withRe-infection with100 EID100 EID50 50

A/VN/1203/04A/VN/1203/04

HI titers 1:20-1:40HI titers 1:20-1:40

All animalsAll animalssurvived lethalsurvived lethal

challengechallenge


DoseDose Origin ofOrigin of

SampleSample


NANA HA1HA1


Nasal washNasal wash

Lungs Lungs

V116AV116A(1/20)(1/20)

H274YH274Y(1/10)(1/10)

V178IV178I(1/20)(1/20)

____

Nasal washNasal wash ____ NDND


BrainBrain H274RH274R(1/10)(1/10)

E277QE277Q(1/10)(1/10)

NDND

− − no mutations; ND – not doneno mutations; ND – not doneDetected by TOPO TA cloning and analysis of individual plaquesDetected by TOPO TA cloning and analysis of individual plaques

H274YH274Y

E277QE277QE277QE277Q

N2 NA numbering (Colman et al, J. Virol.,1993)N2 NA numbering (Colman et al, J. Virol.,1993)


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

Oseltamivir treatment:Oseltamivir treatment:

Initiation – 24 hours p.i.Initiation – 24 hours p.i.

Duration – 5 days twice dailyDuration – 5 days twice daily

Dose – 10 mg/kg/dDose – 10 mg/kg/d

Oseltamivir Treatment: A/Turkey/15/06 VirusOseltamivir Treatment: A/Turkey/15/06 Virus

Note. 10 mg/kg/d in a ferret model equiv. to approved human dose of 75 mg bid Note. 10 mg/kg/d in a ferret model equiv. to approved human dose of 75 mg bid

Non-Lethal challengeNon-Lethal challenge10106 6 EIDEID5050 A/Turkey/15/06 A/Turkey/15/06

Virus Replication in Virus Replication in Upper Respiratory TractUpper Respiratory Tract

0

1

2

3

4

5

10 mg/kg/day

Control

# of Inflammatory cells# of Inflammatory cells

Inflammatory Responses in Upper Inflammatory Responses in Upper Respiratory TractRespiratory Tract

3 5 73 5 7Days post-challengeDays post-challenge

0

2

4

6

8

1010 mg/kd/day

Control

Cel

l co

un

t (n

um

ber

x 1

0C

ell c

ou

nt

(nu

mb

er x

1066 /

ml)

/ml)

3 5 73 5 7Days post-challengeDays post-challenge

Protein concentrationProtein concentration

****

**

**

**

* P<0.05

Virus Replication in Internal OrgansVirus Replication in Internal Organs

Lung Brain Liver Spleen S. intest.Lung Brain Liver Spleen S. intest.0

0.5

1

1.5

2

2.5

3

TreatmentTreatment

ControlControl

2/22/2 1/21/2

** **

* P<0.05

Immunostaining: Virus Detection in the BrainImmunostaining: Virus Detection in the Brain

ControlControl TreatmentTreatment

Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Brain was collected 6 days p.i., fixed in 10% neutral-buffered formalin.Cells positive for viral antigen have a dark-brown granular appearance; Cells positive for viral antigen have a dark-brown granular appearance; viral distribution is shown by red shading (insets). viral distribution is shown by red shading (insets).

50 microns


DoseDose Origin ofOrigin of

SampleSample


NANA HA1HA1

10 mg/kg/d10 mg/kg/d Nasal wash Nasal wash

── R193KR193K− − no mutationsno mutations


R193KR193K

SummarySummary

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 Oseltamivir-resistant variants were not detected by direct sequencing. Analysis of individual viral clones detected a sequencing. Analysis of individual viral clones detected a minor population of clones carrying NA and/or HA mutations, minor population of clones carrying NA and/or HA mutations, although changes in drug susceptibility were not determined.although changes in drug susceptibility were not determined.

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 IlyushinaNatalia Ilyushina

David BoltzDavid BoltzLana McClarenLana McClaren

Oseltamivir Expert Working GroupOseltamivir Expert Working Group Frederick G. Hayden Noel RobertsFrederick G. Hayden Noel RobertsArnold S. Monto James SmithArnold S. Monto James Smith

Albert D.M.E. Osterhaus Ron A.M. FouchierAlbert 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.)

AcknowledgementsAcknowledgements

oseltamivir therapy for h5n1 virus infection

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