# Presentation: 363827

## Animal Models, A/H5N1 and Oseltamivir

**Arnold S. Monto**
**University of Michigan**
**School of Public Health**
**Ann Arbor, MI**

## Virulence May Determine the Necessary Duration and Dosage of Oseltamivir Treatment for Highly Pathogenic A/VN/1203/04 (H5N1) Influenza Virus in Mice

**H-L. Yen, A. S. Monto, R. G. Webster, E. A. Govorkova*****J. Infect. Dis.***** , accepted.**

## Rationale for the Study

**Influenza viruses may have different sensitivity to NAI *****in vivo*****.**
- A/Bayern/57/93 (H1N1), B/Lee/40 viruses, and A/PR8/34 (H1N1) viruses have different sensitivity to NAI in mice.
**H5N1/04 isolates differ from H5N1/97 isolates****:**
- Antigenicity
- Pathogenicity
**For pandemic preparedness****:**
- Virus used in the study: A/VN/1203/04 (H5N1)
- Human H5N1 isolate, fatal case
- Highly pathogenic in mice and ferrets

- Sidwell et al., 2001; Sturm-Ramirez et al., 2004; Govorkova et al., 2005

##  Sensitivity of A/VN/1203/04 (H5N1) in vitro

## Plasmid-Based Reverse Genetics System for Influenza Virus

- Hoffmann et al., 2000

**PB1, PB2, PA, NP, HA, NA, M1, M2, NS1, NS2/NEP proteins**

## Sensitivity of the Recombinant Viruses in vitro

**NA inhibition assay**** **
- Inhibition of the NA enzymatic activity
- Determine the concentration of the drug that inhibits 50% of NA enzymatic activity **(IC****50****)**

**Virus reduction assay**** **
- Inhibition of virus growth *in vitro*
- Determine the concentration of the drug that inhibits 50% viral growth **(EC****50****)**

## The NA of A/VN/1203/04 Virus is Sensitive to NA Inhibitors in vitro

| 
Reverse Genetics Virus | Mean IC50 ± SD, nM a |  |  | Mean EC50 ± SD, M b  |  |
| --- | --- | --- | --- | --- | --- |
|  | Zanamivir | Oseltamivir
Carboxylate |  | Zanamivir | Oseltamivir
Carboxylate |
| VN/1203 X PR/8 (H1N1) | 0.8  0.1 | 0.4  0.1 |  | 0.9  0.1 | 0.1  0.1 |
| HK/156 X PR/8 (H1N1) | 0.7  0.1 | 4.1 0.2 |  | 0.5  0.1 | 1.0  0.1 |
| PR/8/34 (H1N1) | 0.7  0.1 | 4.5  0.2 |  | 1.1  0.1 | 4.6  1.2 |

**NA Inhibition Assay**

**Virus Reduction Assay**

## Sensitivity of A/VN/1203/04 (H5N1) in vivo

## 5-day Treatment Schedule 

** ****Day 0                                                                     Day3               Day6                Day9                        Day24****     **
**                                                                                                **

**Placebo**
**0.1 mg/kg/d**
**1 mg/kg/d**
**10 mg/kg/d**

**Oseltamivir        **** ****4hrs**** ****       Infect with 5MLD****50                               **
**Treatment                           ****A/VN/1203/04**** ****                                                                                                                                  **

- Virus titers in lungs and brains were determined.

**Twice daily 5 day treatment**

**5-day Treatment Schedule **

| Dosage,
mg/kg/d  | No. survivors/
Total (%) | Mean survival  day  SE b | Hazard ratio
(p-value)  |
| --- | --- | --- | --- |
| 0 (Placebo) | 0 | 9.6  0.3 | 1 |
| 0.1 | 0 | 10.7  0.6 | 0.69 (p=0.41) |
| 1 | 0 | 12.2  1.7 | 0.31 (p=0.06) |
| 10 | 5/10 (50) | 20.1  1.0 | 0.03 (p<0.01) |

- 5-day treatment of oseltamivir at dosages 1 and 10 mg/kg/day were shown to be able to protect mice challenged with **A/HK/156/97** (H5N1) virus (survival rate=100%).** ****                                     **Govorkova et al., 2001

## 8-day Treatment Schedule

**Day 0                                                                       Day3              Day6             Day9                         Day24****     **
**                                                                                                **

**Placebo**
**0.1 mg/kg/d**
**1 mg/kg/d**
**10 mg/kg/d**

**Oseltamivir        **** ****4hrs**** ****       Infect with 5MLD****50                               **
**Treatment                           ****A/VN/1203/04**** ****                                                                                                                                  **

- Virus titers in lungs and brains were determined.

**Twice daily 8 day treatment**

**8-day Treatment Schedule**

| Dosage,
mg/kg/d  | No. survivors/
Total (%) | Mean survival  day  SE b | Hazard ratio
(p-value)  |
| --- | --- | --- | --- |
| 0 (Placebo) | 0 | 11.0  0.9 | 1 |
| 0.1 | 1/10 (10) | 11.6  0.9 | 0.41 (p=0.06) |
| 1 | 6/10 (60) | 12.1  0.5 | 0.11 (p<0.01) |
| 10 | 8/10 (80) | 13.0  0.0 | 0.04 (p<0.01) |

## Slide 11

## Significantly different from the placebo, p<0.05

**Significantly different from the placebo, p<0.05**
**Sequence analysis (NA and HA1) of the viruses isolated from the mice lungs on days**
**   ****6 and 9 p.i. did not identify resistant variants.**

## Comparison of A/VN/1203/04 and A/HK/156/97 in vivo

## Growth and Infectivity of A/VN/1203/04 and A/HK/156/97 (H5N1) Viruses in Different Hosts

| H5N1Virus | Infectivity a  |  |  |  | Relative Infectivity b |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | Eggs | MDCK cells | Mice |  | 1 MLD50,
EID50  | 1 MLD50,
pfu |
| A/VN/1203/04 | 9.5 ± 0.3 c | 9.1 ± 0.1 c | 9.3 ± 0.3 d |  | 1.6 | 0.6 |
| A/HK/156/97 | 8.0 ± 0.3 | 7.4 ± 0.2 | 6.9 ± 0.4 |  | 12.6 | 3.2 |

-   **1.5~ 2.0 logs differences in infectivity**
**  ****A/VN/1203/04 virus is more virulent than the A/HK/156/97 in mice.**

## Replication Kinetics in vivo

- Virus titers in lungs, brain, and blood were determined

**Replication Kinetics *****in vivo***

## Replication Efficiency of A/VN/1203/04 and A/HK/156/97 (H5N1) Influenza Viruses in Mice

| 

H5N1Virus | Virus Dose/ Mouse  |  | Mean Virus Titer  SD, Log10EID50/mL a |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | EID50 | MLD50 |  | Lungs |  |  |  |  | Brain |  |  |  | Blood |  |  |
|  |  |  |  | 3 d.p.i. | 6 d.p.i. | 9 d.p.i. |  |  | 3 d.p.i. | 6 d.p.i. | 9 d.p.i. |  | 3 d.p.i. | 6 d.p.i. | 9 d.p.i. |
| A/VN/1203/04 | 4.8  | 3 |  | 7.2  0.6 | 7.4  0.6 | 5.75 b |  |  | 2.6  0.9 c | 5.5  1.0 c | 4.5 b |  | 4.3  0.5 | 6.4  0.2 c | NT |
| A/HK/156/97 | 3  | 0.24 |  | 5.4  1.0 | 3.6  3.1 | 3.5  3.0 |  |  | < | < | 0.8  1.3 |  | 0.8  1.3 | 0.8  1.3 | 2.4  2.3 d |
|  | 38  | 3 |  | 6.3  0.5 | 6.3  0.4 | 4.3  0.9 |  |  | 0.8  0.8 | 0.8  0.8 | 1.1  1.9 |  | 2.8  2.4 | 2.4  2.1 | 2.0  1.9 |

**  ****A/VN/1203/04 virus has higher replication efficiency *****in vivo*****.**
**  ****A/VN/1203/04 virus has better ability to disseminate throughout the body.**

## Summary

**Sensitivity *****in vitro***** may not correlate completely with the sensitivity *****in vivo*****, especially for highly pathogenic H5N1 viruses.**
**Higher dosage and longer oseltamivir treatment may be required for the H5N1/04 viruses:**
- 5-day vs 8-day regimens: 2.7 hazard ratio of death (p<0.01)
- Longer treatment significantly reduced the virus titers in the lungs and brain of infected mice.
**A/VN/1203/04 is more virulent than A/HK/156/97 virus *****in vivo*****.**
- Higher viral replication efficiency
- Better ability to disseminate throughout the body
**Viral virulence may determine the necessary treatment dosage and schedule against highly pathogenic influenza viruses****.**

## Resistance development to neuraminidase inhibitors (NAI resistance)
Compromise enzyme functions
Reduction in viral fitness
NA catalytic site
HA receptor binding site
Degree of compromise may vary by mutation (site)

**Compromise enzyme functions**
**Reduction in viral fitness**
**NA catalytic site**
**HA receptor binding site**
**Degree of compromise may vary by mutation (site)**

- http://images.medscape.com/pi/editorial/cmecircle/2002/1840/zangwill/slide11.gif

**Oseltamivir Resistance**

## Influenza Viruses Resistant to Oseltamivir and Representing Three Distinct NA Genotypes Isolated by Hoffmann LaRoche from Clinical Studies Employing Oseltamivir as Treatment

| Influenza Virus | Resistance Phenotype | NA Mutation | Evidence of Compromised Activity |
| --- | --- | --- | --- |
| A/Sydney/5/97-like
(H3N2) | Both oseltamivir and zanamivir | *R292K – functional 
(arginine to lysine) | Yes+ , serious effect on NA activity |
| A/Wuhan/359/95-like
(H3N2) | Only oseltamivir | ^E119V – framework 
(glutamic acid to valine) | Yes+, instability of NA but not enzyme activities |
| A/New Caledonia
(H1N1)
[A/Texas/36/91-like] | Only oseltamivir | H274Y – interact with functional (histidine to tyrosine) | Yes+, especially at lower infectious doses |

- *Most common mutation
- ^Least common mutation
- + Carr et al 1999, 2000

## Ferret Model

**Previously established transmission model**
**Best animal model for human influenza**
**Receptor type**
**Course of infection**

## 
To use the ferret transmission model to study selected influenza virus
Infectivity
Transmissibility
Hoffmann-LaRoche supplied viruses
Three pair of influenza isolates generated from clinical studies employing oseltamivir treatment
Each pair consisted of a wild type and a corresponding mutant (with a previously described NAI resistance-conferring mutation)

**To use the ferret transmission model to study selected influenza virus**
**Infectivity**
**Transmissibility**
**Hoffmann-LaRoche supplied viruses**
**Three pair of influenza isolates generated from clinical studies employing oseltamivir treatment**
**Each pair consisted of a wild type and a corresponding mutant (with a previously described NAI resistance-conferring mutation)**

**Study Objectives**

## Ferret Transmission Model

- DONOR

- DONOR

- DONOR

- DONOR

- RECIPIENTS

- RECIPIENTS

- RECIPIENTS

- RECIPIENTS

**16 ferrets for wild type, 16 ferrets for mutant virus**
**Four donors and 12 recipients each for wt and mt**
**Groups of four housed together in cage**

**Ferret Transmission Model**

## Study Design

**Donor ferrets:  intra-nasal inoculation with 0.5 ml mutant or wild type virus, then housed with 3 recipient ferrets**
**Course of infection monitored daily**
**Temperatures**
**Nasal Wash**
**Directigen A **
**Virus titration in MDCK cells**
**Seroconversion **
**Sequence Analysis**

**Nasal washing**

## Comparison of Infectivity and Transmissibility of WT & MUT Pairs:  A (H3N2) R 292K 

| Wild type [WT] and Mutant [MUT] Pairs Isolated from Pre- and Follow-up Specimens from the Same Subject | Infectious Dose | Donor Infection Status | Recipient Infection Status | Sequence Confirmation of WT or MUT NA Genotype |
| --- | --- | --- | --- | --- |
| *A/Sydney/5/97-like (H3N2)
          R292 - WT         | 2.3 TCID50/0.5 ml
 | 4 of 4 | 12 of 12 | WT |
|         *R292K – MUT | Same
 | 2 of 4 | 3* of 6 | *Reversion to WT |

- *Herlocher, et al. 2002.  Antiviral Research, 54: 99-111.

## Comparison of Infectivity and Transmissibility of WT & MUT Pairs:  A (H1N1) H 274Y 

| Wild type [WT] and Mutant [MUT] Pairs Isolated from Pre- and Follow-up Specimens from the Same Subject | Infectious Dose | Donor Infection Status | Recipient Infection Status | Sequence Confirmation of WT or MUT NA Genotype |
| --- | --- | --- | --- | --- |
| A/New Calendonia (H1N1)
          H274 – WT | 1.5 x 10-6
Dilution of stock | 4 of 4 | 12 of 12 | WT |
|           H274Y – MUT  | 1.5 x 10-5
Dilution of stock | 0 of 4
@ day 7 | 0 of 12
@ day 7 |  |
|  | 1.5 x 10-3
Dilution of stock | 4 of 4 | 12 of 12 | MUT |

**Comparison of Infectivity and Transmissibility of WT & MUT Pairs:  A (H1N1) H 274Y**

- *Herlocher, et al. 2002.  Antiviral Research, 54: 99-111.

## Comparison of Infectivity and Transmissibility of WT & MUT Pairs:  A (H3N2) E 119V 

| Wild type [WT] and Mutant [MUT] Pairs Isolated from Pre- and Follow-up Specimens from the Same Subject | Infectious Dose | Donor Infection Status | Recipient Infection Status | Sequence Confirmation of WT or MUT NA Genotype |
| --- | --- | --- | --- | --- |
| A/Wuhan/359/95-like (H3N2)
          E119 - WT | 1.0 x 10-6
Dilution of stock | 4 of 4 | 11 of 11 | WT |
|           E119V - MUT | 1.0 x 10-6
Dilution of stock | 4 of 4 | 11 of 11 | MUT |

**Comparison of Infectivity and Transmissibility of WT & MUT Pairs:  A (H3N2) E 119V**

- *Herlocher, et al. 2002.  Antiviral Research, 54: 99-111.

## Summary of Transmission Results

**The R292K mutant did not transmit under conditions in which the wild type transmitted.**
**The E119V and H274Y mutants **_**DID**_** transmit, although the H274Y mt required a higher dose.**
**-****	****The sequence of virus from recipient ferret nasal wash revealed the NA mutation was still present.**
**Contribution of HA mutations remains to be determined.**

- Picture:http://www.countrydoctor.co.uk/education/education%20-%20influenza%20article.htm

## Slide 28

## Numbers of Isolates Evaluated During the Three Study Years

**No (%) of Each Type and Subtype for Each Season**

| Year | A (H1N1) | A (H1N2) | A (H3N2) | B | Total |
| --- | --- | --- | --- | --- | --- |
| 1999-2000 | 54 (12) | 1 (0.2) | 373 (80) | 37 (8) | 465  |
| 2000-2001 | 402 (48) | 0  | 100 (12) | 340 (40) | 842  |
| 2001-2002 | 166 (17) | 39 (4) | 409 (42) | 366 (37) | 980  |
| All years | 622 (27) | 40 (2) | 882 (39) | 743 (32) |   2287 |

## Sensitivity of H1N1, H3N2, and H1N2 Extreme Outliers to Both Inhibitors

| Virus Strain/Subtype | Sequence Changes | Oseltamivir
IC50 nM |  | Fold
Difference | Zanamivir
IC50 nM |  | Fold Difference |
| --- | --- | --- | --- | --- | --- | --- | --- |
| H1N1 Outliers |  | Wt# | Variant |  | Wt# | Variant |  |
| Reference strain A/Texas/36/91 | H274Y | 0.4 | 253.9 | 632 | 0.75 | 0.7 | 1 |
| A/Mississippi/3/2001  | H274Y | 0.48 | 157 | 327 | 0.44 | 0.47 | 1 |
| A/New York/24/2001  | G248R
I266V | 0.48 | 64 | 133 | 0.44 | 30 | 68 |
| A/Hokkaido/15/2002  | Y155H | 0.56 | 69 | 123 | 0.27 | 150 | 555 |
| H3N2/H1N2 Outliers |  |  |  |  |  |  |  |
| Reference strain A/Wuhan/359/95 | E119V | 0.3 | 15.6 | 52 | 0.7 | 1.3 | 2 |
| Reference strain A/Sydney/5/97 | R292K | 0.4 | 3,877 | 9692 | 1.8 | 6.7 | 3.7 |
| A/Greece/110/2000  | E41G | 0.43 | 5.07 | 11.7 | 1.55 | 1.67 | 1.1 |
| A/Denmark/25/2002 | Drift only | 0.35 | 4.91 | 14.0 | 1.26 | 1.76 | 1.39 |
| A/Belgium/969/2002  | Q226H | 0.35 | 4.74 | 13.5 | 1.26 | 0.96 | 0.76 |

- # Wild type values are the means for virus type/subtype and drug for the same year.
- Bold indicates value on which outlier status was based.

- Wetherall NT,  et al. J Clin Microbiol. 2003; 41:742-50.

## Sensitivity of B Extreme Outliers to Both Inhibitors

| Virus Strain/Subtype | Sequence Changes | Oseltamivir
IC50 nM |  | Fold
Difference | Zanamivir
IC50 nM |  | Fold Difference |
| --- | --- | --- | --- | --- | --- | --- | --- |
| B Outliers |  | Wt# | Variant |  | Wt# | Variant |  |
| Reference strain B/Memphis/20/96 | R152K | 6.7 | 509.2 | 76 | 3.5 | 33.7 | 9.6 |
| B/New York/39/2001 (+repeat) | I222T | 7.44 | 100 (55) | 13.4 (7.3) | 2.6 | 17 (6) | 6.5 (2.3) |
| B/Perth/211/2001  | D198E | 3.26 | 90 | 26.3 | 1.16 | 7.36 | 6.7 |

- # Wild type values are the means for virus type/subtype and drug for the same year.
- Bold indicates value on which outlier status was based.

- Wetherall NT,  et al. J Clin Microbiol. 2003; 41:742-50.