1. Establishing a surveillance programme for measurement of Neuraminidase Inhibitor susceptibility Maria Zambon Oct 2006 G224

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

3. Source of Isolates

4. Detection Of Antiviral Resistant Influenza During Treatment Frequency of resistance OseltamivirM2 inhibitor Out-patient adults Out-patient children 0.4% 5.5% ~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

5. 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) 7yr (0/7) Low immunity comparable to pandemic situation ( Kiso et al. Lancet 364: 759, 2004)

6. YearA (H1N1)A (H1N2)A (H3N2)BTotal Resistant IC50 1999-200054 (12)1(0.2)373 (80)37 (8) 1/465 2000-2001402 (48)  100 (12)340 (40) 3/842 2001-2002166 (17)40 (2)409 (39)366 (37)4/980 All years622 (27)40 (2)882 (40)743 (32)7/2287 Global Surveillance 1999-2002 No & (%) Monto et al, 2006

7. 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?

8. Viral Subtype

9. NA mutations, preclinical & clinical G224

10. Influenza Isolates 1996-1999 Log (IC50)

11. H3N2 H1N1 B Fluor Chemi Log IC50 (nM) for Zanamivir and GS4071 by subtype R152K w1 H274Y w1 m1 R292K w1 w2 R152K w1 H274Y w1 E119V R292K w1 w2 L o g I C 5 0 -2 0 2 4 767 139 148 R152K w1 H274Y w1 m1 R292K w1 w2 R152K w1 H274Y w1 E119V R292K w1 w2 L o g I C 5 0 -2 0 2 4 664652127130141143 Z G

12. 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

13. 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 log 10 ) – ↓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

14. Oseltamivir Therapy in H5N1, Thailand and Vietnam, 2004-5 Oseltamivir treatment No. patientsNo. (%) survivors Yes256 (24%) No123 (25%) Writing Committee. NEJM 353:1374, 2005

15. Pharyngeal Viral Loads during Oseltamivir Treatment of H5N1 de Jong et al. NEJM 353:25, 2005

16. 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

17. 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

18. Which drug

19. NA Inhibitor Resistance Profiles NA mutationNA type/ subtype Susceptibility in the NAI assay (fold  ) OseltZanaPeramA-315675 E119VA/N2R (>50)S (1) R292KA/N2R (>1000)S (4-25)R (40-80)S (8) H274YA/N1R (>700)S (1)R (40-100)S (3) R152KBR (>30-750)R (10-100)R (>400)R (150) Mishin et al. AAC 49:4516, 2005; Wetherall et al. AAC 41:742, 2003

20. Assay Methodology

21. Possible methods Cell culture based Enzyme Genotype

22. 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

23. Comparison of Assay Methodology CLF1 Virus TitrationNoYes [Inhib]0.03 – 1000nm0.01 – 5000nm SubstrateNA – starMUN [Substrate]100µm100-200µm Substralet ½MinsHours Assay duration60mins1-2 hours Isolate volume~300µl100-200µl Instrument Cost Luminometer £20 Fluorimeter £10

24. Neuraminidase Inhibitor Susceptibility Screening 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,

25. 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 – Relenza TM contains the active ingredient + lactose Oseltamivir carboxylate – Tamiflu TM 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

26. H3N2 H1N1 B Fluor Chemi Log IC50 (nM) for Zanamivir and GS4071 by subtype R152K w1 H274Y w1 m1 R292K w1 w2 R152K w1 H274Y w1 E119V R292K w1 w2 L o g I C 5 0 -2 0 2 4 767 139 148 R152K w1 H274Y w1 m1 R292K w1 w2 R152K w1 H274Y w1 E119V R292K w1 w2 L o g I C 5 0 -2 0 2 4 664652127130141143 Z G

27. How to interpret data

28. Reference Panel SubtypeVirusMutation CK Titre (pfu/ml) Oseltamivir IC50(nM) Fold Change Zanamivir IC50(nM) Fold Change H3N2 A/Texas/36/91 WT (292R) 1.6x10 7 0.49 4530 0.94 12 292K 3.3x10 6 222011.61 A/Sydney/5/97 WT (119E) 8.5x10 6 0.99 52 1.47 1.5 119V 3.5x10 7 51.932.12 H1N1 A/Wuhan/359/95 WT (274H) 1.5x10 4 0.91 618 0.31 0.6 274Y 1.3x10 4 562.70.49 BB/Memphis/20/96 WT (152R) 2.1x10 6 2.22 569 0.82 189 152K 1.1x10 1263155

29. European Strain IC50 Data: 2004-5 Season

30. Routine Surveillance of UK and European Isolates 2004/5 season Influenza B H3N2 H1N1 Oseltamivir Zanamivir

31. 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 H3N2 + Oseltamivir

32. NI Testing Algorithm NI phenotypic assay in duplicate Repeat NI testing x2 in duplicate Entry of results into central database Genotypic analysis e.g. Sequencing Cloning & sequencing Other methods for mixture analysis IC50/curve in ‘Normal Range’ for season and subtype IC50 >1.6SD above median season and subtype Mean 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

33. 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

34. 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

35. Analysis of Mixtures

36. 292 K

37. B 152

38. 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

39. 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