Establishing a surveillance programme for measurement of Neuraminidase Inhibitor susceptibility

Maria Zambon

Oct 2006

G224

Practicalities

• Source of isolates• Viral subtype• Assay type• Which drug• Which control viruses• How to interpret data• Phenotype vs Genotype• Analysis of mixtures

Source of Isolates

Detection Of Antiviral Resistant Influenza During Treatment

Frequency of resistance

Oseltamivir M2 inhibitor

Out-patient adults

Out-patient children

0.4%

5.5%

~30%

~30%

Inpatient children 18% 80%

Immunocompromised

Surveillance

Yes

0.1-1%

>33%

Roberts N. Phil Trans R Soc Lond 356:1895, 2001

Kiso et al. Lancet 364: 759, 2004

Resistance to Oseltamivir in Children

• Feb – Mar 2002, Jan – Feb 2003 (Japan)• 50 children(median age 3-7), treated twice

daily(4mg/kg/day)• 33 positive for H3N2 after treatment• NA resistance mutation in 9(18%) from 4 days p.t.;

R292K(6), G119V(2), N294S(1). Mutant only(3); mutant+wt(6)

• < 1yr (1/9); 1yr (4/12); 2-6yr (4/22); >7yr (0/7)• Low immunity comparable to pandemic situation ( Kiso et al. Lancet 364: 759, 2004)

Year A (H1N1) A (H1N2) A (H3N2) B TotalResistant

IC50

1999-2000 54 (12) 1(0.2) 373 (80) 37 (8) 1/465

2000-2001 402 (48) 100 (12) 340 (40) 3/842

2001-2002 166 (17) 40 (2) 409 (39) 366 (37) 4/980

All years 622 (27) 40 (2) 882 (40) 743 (32) 7/2287

Global Surveillance 1999-2002

No & (%)

Monto et al, 2006

Conclusions 1 1999-2002

• Detection of resistant variants at very low level

• No evidence of increased frequency over time/shift susceptibilities

• Novel mutations• Relationship between phenotype and

genotype to be further defined• Role of HA in altered NI susceptibility?

Viral Subtype

NA mutations, preclinical & clinical

Inhibitor Subtype Mutation Selection In vitro In vivo

Enzyme Function

Z A/N2,B A/N9 B B

E119G R292K R152K D149N

+ + - -

- - + -

stability <20% activity 3-5% activity <1% activity

O A/N2 A/N2/N9 A/N1 B

R292K E119V H274Y D198N

+ - + -

+ + + +

<20% activity reduced activity reduced activity reduced activity

G224

Influenza Isolates

1996-1999

Log (

IC5

0)

H3N2 H1N1 B

Fluor

Chemi

Log IC50 (nM) for Zanamivir and GS4071 by subtype

R152K

w1

H274Yw1

m1

R292K

w1w2

R152K

w1

H274Y

w1

E119V

R292K

w1w2

Log

IC

50

-2

0

2

4

767 767 139 139 148 148

R152K

w1

H274Yw1

m1

R292K

w1w2

R152K

w1

H274Y

w1

E119V

R292K

w1w2

Log

IC

50

-2

0

2

4

664 652 127 130 141 143

Z G

Oseltamivir Resistance N2, Japan, 2003-4

• Single season survey of NAI resistance– ~ 6M treatment courses (or ~5% of population) – Outpatient isolates from 74 public health labs– Phenotypic susceptibility by NAI assay

• 3/1,180 (0.3%) of influenza A(H3N2) isolates resistant– 2 E119V, 1 A292K

• Very low frequency of resistance in community isolates despite substantial oseltamivir use– Likely due to low-level transmission of resistant

variants and not primary NA inhibitor resistance

Neuraminidase Inhibitor Susceptibility Network.

WHO Weekly Epi Record, April 29, 2005

Oseltamivir Resistance In N1 Neuraminidase

• Single nucleotide substitution (His274Tyr) → ↓oseltamivir susceptibility (≥ 400–fold) • Frequency drug therapy of N1:

– H1N1: children 16% (7/43), adults 4% (2/50)– H5N1: 2/8 (25%)

• Reduced replication in cell culture (> 2.0 log10)– ↓infectivity in mouse (1,000-fold) and ferret (>10-fold)– Variable ↓ pathogenicity in ferret

• Transmissible in ferret model

Ives et al. Antiviral Res 5:307, 2002

Herlocher et al. JID 190:1627, 2004

Oseltamivir Therapy in H5N1, Thailand and Vietnam, 2004-5 Oseltamivir treatment

No. patients No. (%) survivors

Yes 25 6 (24%)

No 12 3 (25%)

Writing Committee. NEJM 353:1374, 2005

Pharyngeal Viral Loads during Oseltamivir Treatment of H5N1

de Jong et al. NEJM 353:25, 2005

Oseltamivir Treatment Failure in H5N1• Late initiation - pulmonary injury• Prolonged viral replication - primary infection, replication

competence, immune evasion• Altered pathogenesis

– Viral virulence factors– Extra-pulmonary dissemination– Pro-inflammatory host immune responses

• Inadequate dose regimen– Inadequate absorption (diarrhea, GI dysfunction)

• Antiviral resistance emergence

Summary

• Little evidence of existence of naturally resistant virus isolates

• The precise orientation and the immediate surrounding residues of conserved NA site differs between subtypes (? different enzymes)

• Drug binding will not be identical across all subtypes

• Resistance ‘strategy’ not identical between subtypes

Which drug

NA Inhibitor Resistance Profiles

NA mutation NA type/ subtype

Susceptibility in the NAI assay (fold )

Oselt Zana Peram A-315675

E119V A/N2 R (>50) S (1) S (1) S (1)

R292K A/N2 R (>1000) S (4-25) R (40-80) S (8)

H274Y A/N1 R (>700) S (1) R (40-100) S (3)

R152K B R (>30-750) R (10-100) R (>400) R (150)

Mishin et al. AAC 49:4516, 2005; Wetherall et al. AAC 41:742, 2003

Assay Methodology

Possible methods

• Cell culture based

• Enzyme

• Genotype

NI Susceptibility Screening Methodology

• Based on methodology developed by Potier et al (1979)

• Fluorometric: measures level of 4-methyumbelliferone cleaved by

influenza NA from the fluorogenic substrate 2’-(4-methyumbelliferyl)-

α-D-N-acetylneuraminic acid (MUNANA) (Sigma-Aldrich)

• Viruses are pre-titrated to ensure equivalent NA activities are

compared against inhibitors

• IC50 values (concentration of inhibitor required to reduce NA activity

by 50%) are calculated using curve fitting software

• Chemi-luminescent substrate available (Applied Biosystems) and

methodology is under development

Comparison of Assay Methodology

CL F1

Virus Titration No Yes

[Inhib] 0.03 – 1000nm 0.01 – 5000nm

Substrate NA – star MUN

[Substrate] 100µm 100-200µm

Substralet ½ Mins Hours

Assay duration 60mins 1-2 hours

Isolate volume ~300µl 100-200µl

InstrumentCost

Luminometer £20

Fluorimeter £10

Neuraminidase Inhibitor Susceptibility Screening

0.00

5000.00

10000.00

15000.00

20000.00

25000.00

30000.00

35000.00

40

00

10

00

25

0

62

.50

15

.62

50

3.9

063

0.9

766

0.2

441

0.0

610

0.0

153

VC

Oseltamivir Conc (nM)

RF

U

292R 292K 119E 119V

0.00

5000.00

10000.00

15000.00

20000.00

25000.00

30000.00

35000.00

40000.00

4000

1000 25

0

62.5

0

15.6

250

3.90

63

0.97

66

0.24

41

0.06

10

0.01

53 VC

Zanamivir Conc (nM)

RF

U

292R 292K 119E 119V

292R: 0.6nM 292K: 15.9nM

119E: 0.95nM 119V: 1.47nM

292R: 0.59nM 292K: >4000nM

119E: 0.74nM 119V: 66.63nM

Weatherall et al,

Availability of reagents for laboratories setting up NI assays

Substrates• Fluorescent assay MUNANA commercially available• Chemiluminescent (CL) assay – NA-star now available in kit form

InhibitorsZanamivir – RelenzaTM contains the active ingredient + lactoseOseltamivir carboxylate – TamifluTM contains the prodrug oseltamivir

phosphate, cannot be used in assays as needs activation to oseltamivir carboxylate. Need MTA from Roche.

EquipmentAny fluorimeter can be used for MUNANA, substrate stable for hoursAny luminometer can be used, but reaction half life 5 mins. For multiple

samples need automatic addition of enhancer.

NISN selected the chemiluminescent assay due to its higher sensitivity,

Fluorescent assay is the most practical for regional laboratories Kit format CL assay offers new possibilities for regional laboratories

H3N2 H1N1 B

Fluor

Chemi

Log IC50 (nM) for Zanamivir and GS4071 by subtype

R152K

w1

H274Yw1

m1

R292K

w1w2

R152K

w1

H274Y

w1

E119V

R292K

w1w2

Log

IC

50

-2

0

2

4

767 767 139 139 148 148

R152K

w1

H274Yw1

m1

R292K

w1w2

R152K

w1

H274Y

w1

E119V

R292K

w1w2

Log

IC

50

-2

0

2

4

664 652 127 130 141 143

Z G

How to interpret data

Reference PanelSubtype Virus Mutation

CK Titre (pfu/ml)

OseltamivirIC50(nM)

Fold Change

ZanamivirIC50(nM)

Fold Change

H3N2

A/Texas/36/91

WT (292R) 1.6x107 0.49

45300.94 

12292K 3.3x106  2220 11.61 

A/Sydney/5/97

WT (119E) 8.5x106  0.99

521.47 

1.5119V 3.5x107  51.93 2.12 

H1N1  A/Wuhan/359/95

WT (274H) 1.5x104  0.91

6180.31 

0.6274Y 1.3x104  562.7 0.49 

B B/Memphis/20/96 

WT (152R) 2.1x106  2.22

5690.82 

189152K 1.1x10   1263 155 

European Strain IC50 Data: 2004-5 Season

Zanamivir

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32

AustriaBelgium

Czech RepublicDenmark

FinlandFrance

GermanyIcelandIreland

ItalyLatvia

NetherlandsNorwayPoland

PortugalSlovakiaSlovenia

SpainSweden

Switzerland

Cou

ntry

Number of Isolates

Routine Surveillance of UK and European Isolates 2004/5 season

0

3

6

9

12

15

18

IC50 (nM)

Num

ber

of Is

olat

es

0

4

8

12

16

20

24

28

IC50 (nM)

Num

ber o

f Iso

late

s

0

5

10

15

20

25

30

35

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

Mor

e

IC50

Nu

mb

er

of

Iso

late

s

0

5

10

15

20

25

30

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

Mor

eIC50

Nu

mb

er

of

Iso

late

s

0

4

8

12

16

20

24

28

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

Mor

e

IC50N

um

be

r o

f Is

ola

tes

0

5

10

15

20

25

0.2

0.6 1

1.4

1.8

2.2

2.6 3

3.4

3.8

Mor

e

IC50

Nu

mb

er

of

Iso

late

s

Influenza B H3N2 H1N1

Oseltamivir

Zanamivir

Identification and Analysis of Mixed Strains

• Depends heavily on criteria determining normal range

• 1.6SD above median= top 5% (red line)

• 3SD above median= unusually high IC50 (black line)

• OR: calculate 95th percentile after removing major outliers

0.10

1.00

10.00

100.00

1000.00

10000.00

0 20 40 60 80 100 120 140 160

H3N2 + Oseltamivir

NI Testing Algorithm

NI phenotypic assay in duplicate

Repeat NI testing x2 in duplicate

Entry of results into central database

Genotypic analysise.g. Sequencing

Cloning & sequencingOther methods for mixture analysis

IC50/curve in ‘Normal Range’ for season

and subtype

IC50 >1.6SD above median season

and subtypeMean IC50 in

‘Normal Range’ for season and subtype

Mean IC50 >1.6SD above

median season and subtype

Sequencing of a percentage

of ‘normal range’ isolates for baseline

Naturally occurring altered susceptibility isolates

• There may be altered susceptibility due to normal drift mutations

• These mutations are different to those observed after drug treatment, and are not necessarily in conserved residues

• Although there is high conservation of residues in and around the active site, there are clearly also secondary structural effects outside the active site which can affect drug binding

• Structural data is needed to understand how these background residues affect the active site

Genotype:phenotype relationship

>20 fold mean IC50 subtypeKnown resistance mutations 274, 198? New resistant variants Y155H

10-15 fold > mean IC50 subtype Altered susceptibility virusesE41G, Q226H, I222TNo known mutations

Analysis of Mixtures

292 K

B 152

B/Perth/211/2001 Not from drug treated patient

Medium resistance to oseltamivir and BCX-1812, low resistance to zanamivir in CL assay

Resistance to all three inhibitors in Fluorescent assay

Initial sequencing by 2 independent laboratories did not detect any mutation

Subsequent sequencing of cloned NA and plaque purified virus mixed population, with D198 in sensitive and D198E mutation in resistant population

Practicalities for European/national surveillance

• Source of isolates• Viral subtype• Assay type• Which drug• Which control viruses

• How to interpret data• Phenotype vs Genotype• Analysis of mixtures