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Transcript of Establishing a surveillance programme for measurement of Neuraminidase Inhibitor susceptibility...
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