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Establishing a surveillance
programme for measurement
of Neuraminidase Inhibitor
susceptibility
Maria Zambon
Oct 2006
G224
Practicalities
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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
0.4%
~30%
Out-patient children
5.5%
~30%
Inpatient children
18%
80%
Immunocompromised
Yes
>33%
Surveillance
0.1-1%
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)
Global Surveillance 1999-2002
No & (%)
Year
A (H1N1)
A (H1N2)
A (H3N2)
B
Total
Resistant
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
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
G224
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
Influenza Isolates
Log (IC50)
1996-1999
Log IC50 (nM) for Zanamivir and GS4071 by subtype
4
R292K
Fluor
H274Y
R152K
R152K
E119V
2
Log IC50
R292K
w1
w1
0
w1
m1
w2
w1
w2
H274Y
w1
w1
-2
767 767
139 139
148 148
4
R292K
R152K
H274Y
2
R152K
Log IC50
Chemi
E119V
w1
R292K
0
w2
m1
w1
w1
w2
w1
H274Y
w1
w1
-2
664 652
127 130
Z
G
H3N2
H1N1
141 143
B
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
Susceptibility in the NAI assay (fold )
NA type/
subtype
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
Virus Titration
[Inhib]
Substrate
[Substrate]
Substralet ½
Assay duration
Isolate volume
Instrument
Cost
CL
F1
No
0.03 – 1000nm
NA – star
100µm
Mins
60mins
~300µl
Luminometer
£20
Yes
0.01 – 5000nm
MUN
100-200µm
Hours
1-2 hours
100-200µl
Fluorimeter
£10
35000.00
40000.00
30000.00
35000.00
25000.00
30000.00
25000.00
RFU
20000.00
15000.00
20000.00
Oseltamivir Conc (nM)
292R
292K
119E
VC
0.0153
0.0610
0.2441
0.9766
3.9063
15.6250
62.50
250
4000
VC
0.0153
0.0610
0.2441
0.9766
3.9063
0.00
15.6250
0.00
62.50
5000.00
250
5000.00
1000
10000.00
1000
15000.00
10000.00
4000
RFU
Neuraminidase Inhibitor
Susceptibility Screening
Zanamivir Conc (nM)
119V
292R
292K
119E
119V
292R: 0.59nM
292K: >4000nM
292R: 0.6nM
292K: 15.9nM
119E: 0.74nM
119V: 66.63nM
119E: 0.95nM
119V: 1.47nM
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
Inhibitors
Zanamivir – RelenzaTM contains the active ingredient + lactose
Oseltamivir carboxylate – TamifluTM contains the prodrug oseltamivir
phosphate, cannot be used in assays as needs activation to oseltamivir
carboxylate. Need MTA from Roche.
Equipment
Any fluorimeter can be used for MUNANA, substrate stable for hours
Any 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
Log IC50 (nM) for Zanamivir and GS4071 by subtype
4
R292K
Fluor
H274Y
R152K
R152K
E119V
2
Log IC50
R292K
w1
w1
0
w1
m1
w2
w1
w2
H274Y
w1
w1
-2
767 767
139 139
148 148
4
R292K
R152K
H274Y
2
R152K
Log IC50
Chemi
E119V
w1
R292K
0
w2
m1
w1
w1
w2
w1
H274Y
w1
w1
-2
664 652
127 130
Z
G
H3N2
H1N1
141 143
B
How to interpret data
Reference Panel
Subtype
Virus
A/Texas/36/91
H3N2
A/Sydney/5/97
H1N1
B
A/Wuhan/359/95
B/Memphis/20/96
Mutation
CK Titre
(pfu/ml)
Oseltamivir
IC50(nM)
WT
(292R)
1.6x107
0.49
Fold
Change
Zanamivir
IC50(nM)
0.94
4530
12
292K
6
3.3x10
2220
11.61
WT
(119E)
8.5x106
0.99
1.47
119V
3.5x107
51.93
2.12
WT
(274H)
1.5x104
0.91
0.31
52
1.5
618
4
0.6
274Y
1.3x10
562.7
0.49
WT
(152R)
2.1x106
2.22
0.82
152K
1.1x10
569
1263
Fold
Change
189
155
European Strain IC50 Data: 2004-5
Season
Switzerland
Sweden
Spain
Slovenia
Slovakia
Portugal
Poland
Norway
Netherlands
Latvia
Italy
Ireland
Iceland
Germany
France
Finland
Denmark
Czech Republic
Belgium
Austria
Zanamivir
Country
Country
Oseltamivir
Switzerland
Sweden
Spain
Slovenia
Slovakia
Portugal
Poland
Norway
Netherlands
Latvia
Italy
Ireland
Iceland
Germany
France
Finland
Denmark
Czech Republic
Belgium
Austria
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32
Number of Isolates
Number of Isolates
Normal Range
Top 5%
IC50
1
3
5
7
9
1.
1.
1.
1.
1.
or
e
9
0.
3
5
7
9
1.
1.
1.
1.
28
24
30
24
20
25
IC50
IC50
M
8
4
3
or
e
3.
3.
6
2
2.
2.
0
8
5
4
3
1.
20
1.
25
or
e
1
6
0.
25
1
2
30
M
9
1.
0
0.
0
7
5
5
4
0.
10
0.
15
3
20
0.
Number of Isolates
10
0.
15
1
35
Number of Isolates
20
0.
1.
9
M
or
e
1.
7
1.
5
1.
3
1.
1
0.
9
0.
7
0.
5
0.
3
IC50
M
7
8
0.
12
5
16
3
6
Number of Isolates
9
0.
IC50 (nM)
0.
1
72
or
e
12
1
28
Number of Isolates
M
66
60
54
Number of Isolates
15
0.
or
e
30
28
26
24
48
42
36
30
24
18
12
6
0
18
0.
IC50 (nM)
22
20
18
16
14
12
Oseltamivir
M
Zanamivir
10
8
6
4
2
0
Number of Isolates
Routine Surveillance of UK and
European
Isolates
2004/5
season
H3N2
H1N1
Influenza B
15
10
5
0
0
20
16
12
8
4
0
Identification and Analysis of Mixed
Strains
•
•
•
•
Depends heavily on
criteria determining
10000.00
normal range
1000.00
1.6SD above median=
100.00
top 5% (red line)
3SD above median=
10.00
unusually high IC50
1.00
(black line)
0.10
OR: calculate 95th
0
percentile after removing
major outliers
H3N2 + Oseltamivir
20
40
60
80
100
120
140
160
NI Testing Algorithm
IC50 >1.6SD above
median season
and subtype
Repeat NI testing x2
in duplicate
NI phenotypic
assay in duplicate
Mean IC50 in
‘Normal Range’ for
season and subtype
Mean IC50
>1.6SD above
median season
and subtype
Genotypic analysis
e.g. Sequencing
Cloning & sequencing
Other methods for mixture analysis
IC50/curve in ‘Normal
Range’ for season
and subtype
Entry of results
into central database
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 subtype
Known resistance mutations 274, 198
? New resistant variants Y155H
10-15 fold > mean IC50 subtype
Altered susceptibility viruses
E41G, Q226H, I222T
No 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