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1 AAC Accepts, published online ahead of print on 18 January 2013 Antimicrob. Agents Chemother. doi: /aac Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Title: Virus susceptibility analyses from a phase IV clinical trial of inhaled zanamivir treatment in children infected with influenza 3 Running title: Zanamivir Susceptibility analysis in children Authors: Phillip J Yates 1*, Nalini Mehta 1, Joseph Horton 2, Margaret Tisdale 1. 1 GlaxoSmithKline R&D, Stevenage, UK; 2 GlaxoSmithKline R&D, RTP, USA. * Corresponding author: phil.j.yates@gsk.com Downloaded from on September 26, 2018 by guest 1

2 ABSTRACT A zanamivir post-approval efficacy study was conducted in children (n=279) in Japan during 3 influenza seasons. Pharyngeal swabs (n=714) were obtained for detailed resistance analysis. From 371 cultured viruses, three viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at Day 1(before treatment), one had a N74S amino acid substitution (fold shift of 46), and two ( Day 1 and Day 2) a Q136K amino acid substitution (fold shift of 292 and 301). The Q136K was only detected in cultured virus and not the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. NA population sequencing showed that viruses from twelve subjects had emergent amino acid substitutions but three, with susceptibility data, were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. HA sequencing, showed viruses from 20 subjects had 9 HA amino acid substitutions implicated previously in resistance to NIs in in vitro assays or were close to the Receptor Binding site. Their role in in vivo resistance appears to be less important, but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones were analysed from 90 subjects. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or just chance mutations. 2

3 32 INTRODUCTION Influenza is a respiratory tract infection characterised by seasonal epidemics, widespread morbidity and associated mortality, particularly in at risk groups and during pandemics. Influenza pandemics are caused when a new strain of influenza A virus against which there is little or no existing immunity, emerges in the human population and efficiently transmits from human-to-human. The primary method for prevention of influenza is vaccination but there is a role for treatment of infected individuals with antivirals. There are two classes of antivirals currently available for the treatment of influenza, adamantanes, (adamantine and rimantadine) and neuraminidase (NA) inhibitors (NI). There is widespread resistance to adamantanes and therefore treatment of influenza infection by this class of drugs is not currently recommended by the World Health Organisation (WHO). There are four NA inhibitors currently licensed for treatment and prophylaxis of influenza infection, oseltamivir (Tamiflu), zanamivir (Relenza), peramivir (licensed for treatment in Japan and Korea) and laninamivir (licensed for treatment in Japan and Korea). Oseltamivir is administered orally, zanamivir and laninamivir by oral inhalation and peramivir by injection. One of the factors that can impair the efficacy of NA inhibitors is the development of resistance. Zanamivir was designed to target the highly conserved active site of the influenza neuraminidase and is a close mimic of the natural substrate, 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid (DANA) (1). Zanamivir binds in the NA active site in a similar way to that of DANA which appears to limit the potential for resistance development to zanamivir (2, 3, 4). Resistance to zanamivir in immune-competent patients is rare and has not been observed in more 3

4 than 14,000 subjects participating in treatment and prophylaxis studies (5, 6). However, there has been one resistant virus and three viruses with reduced susceptibility isolated from four immune-compromised patients treated with zanamivir. In one immune-compromised patient with influenza B infection, treated for a prolonged period with inhaled zanamivir, amino acid substitutions in both haemagglutinin (HA) and NA were selected after 15 days treatment (7). During the influenza pandemic of 2009/2010 there were three reports of the selection of viruses with reduced susceptibility to zanamivir in immuno-compromised patients, two of whom were treated with an unlicensed formulation of intra-venous zanamivir (as part of a named patient program) and one with inhaled zanamivir (8, 9, 10). The variants harboured the I223R amino acid substitution in the NA, which conferred a fold change in susceptibility of 45 and 10 with oseltamivir and zanamivir, respectively. In contrast, treatment with oseltamivir has resulted in widespread resistance. In Japan, studies have shown resistance in approximately 1% of adults and 4-18% of children treated with oseltamivir when infected with seasonal (A/H1N1, A/H3N2, and B) influenza virus (11, 12). Different oseltamivir resistance-associated amino acid substitutions have been identified in the different neuraminidase sub-types (N1, N2, and B). The most common oseltamivir resistance amino acid substitution in A/H1N1 viruses is H275Y (H274Y, N2 numbering), and was observed circulating within untreated subjects in many countries in the 2007/2008 influenza season, and by the 2008/2009 northern hemisphere (NH) influenza season was at a level of 95% globally (13, 14, 15). Since the A/H1N1 pandemic started in 2009 there have been sporadic incidences of oseltamivir resistance in the pandemic H1N1 viruses, 596 as of September 2011, all but one harbored the H275Y amino acid substitution (16). There have been clusters of infections with the resistant virus and there have been 4

5 four reported incidences of person to person transmission but there was no sustained transmission between individuals, indicating that the variants in the pandemic H1N1 viruses are less fit than the wild-type virus (17, 18, 19, 20). The global incidence of oseltamivir resistance in the pandemic H1N1 strain is approximately 1%, however during the 2010/2011 northern hemisphere influenza season there were several incidences of resistant virus infection in untreated patients suggesting onward transmission of the resistant virus (21). Furthermore, during the 2011 southern hemisphere influenza season in Australia there was a cluster of virus infections with the H275Y amino acid substitution in untreated patients (22, 23). There is therefore cumulative evidence that the oseltamivir resistant virus is becoming readily transmissible and if the H275Y variant replaces all oseltamivir sensitive viruses as occurred with previous seasonal H1N1 virus, subsequent treatment options would be limited. This study was carried out in order to ascertain if resistance to zanamivir was more readily selected in paediatric patients compared to adults. A post-approval study was conducted in Japan during 3 influenza seasons ( ) to monitor for emergence of resistant influenza virus in paediatric patients treated with inhaled zanamivir. Efficacy analysis from this study has been reported elsewhere (24). Analysis was carried out on pharyngeal swabs obtained at Day 1 and during/after treatment. Susceptibility to zanamivir was determined using cultured virus. Population NA and HA sequencing and NA Clonal analyses were carried out directly on swabs and/or cultured virus, to look for new or known resistance associated amino acid substitutions

6 105 MATERIALS and METHODS Compounds. Zanamivir was provided by GlaxoSmithKline, Research and Development (Stevenage, UK) Viruses and cell cultures. A total of 279 paediatric patients in Japan (100 patients in 2006/2007; 79 patients in 2007/2008; 100 patients in 2008/2009) were enrolled in the study and were treated with 10mgs of inhaled zanamivir twice daily for 5 days. Viruses were isolated from pharyngeal swabs taken on Day 1 (prior to treatment) and during- ( Days 2-5) and post-treatment (Days 6-9) timepoints, and were propagated in the absence of zanamivir in Madin Darby Canine Kidney (MDCK) cells by standard techniques and stored at -80 C prior to susceptibility analysis (25). Study procedures. The study was conducted in accordance with all applicable regulatory requirements, including the principles of the Declaration of Helsinki (1996). Before commencement of the study, all relevant study documentation was reviewed and approved by an ethics committee/institutional review board, and all subjects were provided with study information. Written consent was obtained from the legally authorized representative of all paediatric subjects. NA activity inhibition assay. Susceptibility to zanamivir was carried out on MDCK culture supernatants using the NA Star Influenza Neuraminidase Inhibitor Resistance Detection Kit as described by the manufacturers (Applied Bioystems). Viruses with a fold-change in IC 50, compared to a sensitive reference strain, of 2-fold to 10-fold were considered to have reduced susceptibility and those with a fold-change of >10 were resistant Virus gene sequence analysis. Viral RNA was extracted from pharyngeal swabs using guanadinium isothiocyanate as described previously (25,26). The NA and 6

7 HA1 genes were amplified by RT/PCR. RNA was reverse transcribed using SuperScript TM II (Life Technologies Ltd., Paisley, UK) and gene specific primers and amplified using two rounds of PCR with Pfx Platinum DNA polymerase (Life Technologies) and gene specific primers. PCR products were sequenced using gene specific primers. Primer sequences can be provided on request. Amino acid substitutions are shown in relation to the consensus sequence from the respective subtype obtained from the first season of this study. N2 numbering is used throughout except were specified. The accession numbers of NA and HA sequences from all viruses analysed in this study are: KC to KC Clonal analysis. PCR products were cloned using Zero Blunt TOPO PCR cloning kit (Invitrogen) according to manufacturers protocol and sequenced with M13 forward and reverse primers. The mutation rate of the minority species was calculated by the following calculation: Mutation rate of NA mutations = 1/Number of clones analysed x (PCR1 + PCR2), where: PCR1 = Number of nucleotides amplified during 1st round PCR (A/H1N1 = 1408; B = 1396; A/H3N2 = 1424) x Number of 1 st round PCR amplification cycles (35) and PCR2 = Number of nucleotides amplified during 2nd round PCR (A/H1N1 = 1380; B = 1381; A/H3N2 = 1398) x number of 2 nd round PCR amplification cycles. RESULTS Samples Analysed The number of samples analysed, by Susceptibility assays, NA sequencing and HA sequencing have been summarised in Table 1. 7

8 152 Virus susceptibility analysis Virus susceptibility to zanamivir was carried out on all cultured viruses. Samples from twenty four subjects out of 279 could not be cultured and therefore were not analysed phenotypically. A total of 371 cultured viruses from 255 subjects were analysed (119 during/after treatment from 111 subjects) (Table 1). IC 50 values were obtained for all samples (Table 2). For samples isolated in the 2006/2007 season, the A/H1N1 isolates had a mean IC 50 of 1.96 ± 5.27 nm, the A/H3N2 isolates of 1.22 ± 0.65 nm and the B isolates of 3.95 ± 1.67 nm. One influenza A/H1N1 virus, isolated at Day 1, (before starting treatment) had a fold-change in susceptibility to zanamivir and oseltamivir of 46 and 2.7 respectively. For samples isolated in the 2007/2008 season the A/H1N1 isolates had a mean IC 50 of 7.88 ± nm and the B isolates 4.8 ± 1.12 nm. Of the 371 viruses, two A/H1N1 viruses isolated on Day 1 and Day 2 (virus was not isolated after Day 2), during the 2007/2008 influenza season were found to be resistant, with IC 50 s of and nm and are discussed in more detail below. The mean IC 50 for A/H1N1 in this season excluding these 2 resistant viruses was 0.96 ± 1.41 nm. For samples isolated in the 2008/2009 season the A/H1N1 isolates had a mean IC 50 of 0.87 ± 0.78nM, the A/H3N ± 0.79 nm and the B isolates 3.92 ± 1.45nM (Table 2 ). NA gene sequencing Of the 279 subjects enrolled in the study, virus from two subjects (5 swabs) were untyped and were not analysed by genotypic analysis. A total of 484 NA sequences (250 A/H1N1; 126 A/H3N2; 108 B) were obtained from 714 swabs from 277 subjects 8

9 (229 during/after treatment from 181 subjects) (Table 1). There were 214 subjects with matched Day 1 and post treatment swabs. Results from this NA sequencing included the three viruses (A/H1N1) described above which showed reduced susceptibility to zanamivir at Day 1 (before commencing treatment). The NA sequence from virus isolated from Subject 1 (2006/2007) revealed an amino acid substitution at N74S (N70S by N1 numbering) with a fold shift in susceptibility. The NA sequence for two viruses (Day 1 and Day 2) from Subject 2 (2007/2008) revealed an amino acid substitution at Q136K (fold shift = 292 and 301) (Table 3). The Q136K amino acid substitution was only detected in cultured virus and not in the swab, indicating that the amino acid substitution arose during in vitro passage (Table 3). Results from this NA sequencing for the remainder of the swabs showed that there were 12 subjects with treatment emergent resistance associated amino acid substitutions one of which was close to the NA active site and three non-emergent resistance associated amino acid substitutions, present in Day 1 samples (Table 4). Three published resistance associated amino acid substitutions (11, 27) were detected within the NA, before, during or after treatment with zanamivir; G248R, N294K and Y155H (Table 4, N2 numbering). The G248R amino acid substitution was present in all viruses isolated from 3 subjects (H1N1), including Day 1, but is reported to only give rise to resistance if present with the I266V, which was not identified in virus from these subjects. Viruses from two subjects harbouring the G248R amino acid substitution did not show elevated IC 50 s. One virus from Subject 1 harboured the G248R along with the N74S amino acid substitution and had an elevated IC 50 as discussed above. One virus analysed in this study contained an 9

10 amino acid substitution at position 294 (N294K) of the NA (N2 numbering). The N294S amino acid substitution is a recognised resistance amino acid substitution and has been detected in oseltamivir treated patients infected with influenza A/H3N2 and A/H5N1 (11, 28), and also prior to treatment in a patient infected with influenza B virus (29). The N294S amino acid substitution has been shown to give high level resistance to oseltamivir in influenza A/H1N1, A/H1N1pdm2009 and A/H3N2, decreased susceptibility to oseltamivir in A/H5N1 and B viruses and decreased susceptibility to zanamivir in A/H1N1, A/H3N2 and H5N1 viruses (11, 28, 30, 31). The amino acid substitution detected in this study was N294K in an H1N1 virus isolated on Day 7 after initiating treatment and was not present on Day 1. Virus could not be cultured from this sample for susceptibility monitoring. The N294K has never previously been identified as a zanamivir resistance amino acid substitution, however, data presented here cannot preclude that this amino acid substitution confers resistance to zanamivir. Two influenza A H3N2 viruses from two subjects, Day 1 and Day 3, possessed the Y155H amino acid substitution which has previously been implicated in resistance to NIs in one influenza A H1N1 virus isolate. The Y155H amino acid substitution has been shown to give rise to resistance to oseltamivir and zanamivir, in one H1N1 virus analysed during surveillance studies but not in H3N2 viruses (27). A cultured H1N1 virus in this study with the Y155H amino acid substitution did not have an elevated IC 50 with zanamivir (0.61 nm). One Day 6 virus had a deletion of 93 amino acids between positions 110 to 203 that was not present on Day 1. This region of the NA encompasses part of the active site and deletions in this region would affect the NA activity of the enzyme. NA amino acid substitutions were detected following treatment with zanamivir, that were not present in the same subject s Day 1 sample, at amino acid residues: 10

11 L98L/F (B virus), T69T/I, Y70C, S105N, V114I, S195Y, E229G, N294K, S367N, P380N, P389H (A/H1N1), G373R/G (A/H3N2)(N2- numbering) (Table 4). With the exception of N294K, none of the emergent amino acid substitutions were in the NA active site or have been previously observed in the clinic after treatment with NIs or during surveillance studies. The majority of the emergent variants could not be cultured and therefore no IC 50 values were determined. Two cultured viruses, one harbouring the P389H amino acid substitution and one harbouring both V114I and E229G amino acid substitutions had IC 50 values of 1.7 and 0.22 nm respectively. One influenza B virus cultured on Day 7 with a mixture L98L/F had an IC 50 range of nM, which is within the normal range observed for influenza B viruses. HA gene sequencing 413 HA sequences (186 A/H1N1; 120 A/H3N2; 107 B) were obtained from 714 swabs from 277 subjects (175 during/after treatment from 148 subjects) (Table 1). There were 166 subjects with matched Day 1 and during/post treatment swabs. There were 7 HA amino acid substitutions identified in 18 subjects that have been implicated previously in resistance to NIs (32) in in vitro assays ( H3 numbering: A/H1N1 E75K, A200V, K222R; A/H3N2 - G142R, S262N, A304T; B N145S, N145I). In addition, two amino acid substitutions at 183 and 195 (H3- numbering), in the Receptor Binding Site (RBS) of the HA, were identified in separate viruses from two other subjects. HA amino acid substitutions from 15 of the subjects were present at Day 1 and were therefore not selected as a result of drug pressure. HA amino acid substitutions from 5 subjects did not have a Day 1 sample so it cannot be determined if they were treatment emergent amino acid substitutions. Although the 11

12 role of HA amino acid substitutions in resistance in vitro is well documented their role in vivo is not well understood Clonal Sequence analysis of the Neuraminidase gene Clonal sequence analysis of the neuraminidase was undertaken to determine if minority species of resistant viruses were present in samples taken on or after Day 4 of treatment. A total of 1,682 clones were analysed from 91 swabs (90 subjects) (Table 5). An additional 372 clones were analysed from control viruses isolated on or before Day 3 from 19 subjects (Table 5). Single clones from 12 subjects (from a total of 338 clones analysed) were found to contain amino acid substitutions close to the NA active site on or after Day 4 (Table 6). Two amino acid substitutions identified in clones from two subjects, R152K (A/H1N1) and D198N (A/H3N2) are known to be associated with reduced susceptibility and resistance in influenza B viruses (7, 33). Day 1 viruses from these 2 subjects were subjected to clonal analysis (17 and 22 clones respectively) and did not possess the amino acid substitution detected in their respective during treatment sample. Previous reverse genetics studies using an N2 background have indicated that the R152K amino acid substitution does not give rise to resistance (34) or gives low level resistance but is unstable in N2 (35) but there are no data in an N1 background. It is not clear if the D198N would produce resistance in influenza A strains, although the D198G does reduce susceptibility to zanamivir by 6 fold in A/H1N1pdm2009 virus (31). Two clones from different subjects possessed amino acid substitutions at positions 136 and 143 of the NA which have been associated with resistance to NIs (27, 36, 37). The K143R amino acid substitution gives rise to resistance to 12

13 oseltamivir and the Q136K and Q136L amino acid substitutions give rise to resistance to zanamivir. It is not known if the amino acid substitutions identified in this study, Q136H and K143N would give rise to resistance to NIs A further 9 clones from 9 subjects had amino acid substitutions in or close to the NA active site; L134Q, L134P, R224G, E227D, E277G, R292G, R292S (H1N1), R224G (H3N2) and R224G (B) (N2 numbering). Clones from the Day 1 isolate from the same subject did not contain the same amino acid substitution (Table 6). It is unclear whether these single amino acid substitutions could have been amplified as a result of drug pressure or represent chance mutations. The clinical implication of these amino acid substitutions is unclear because the majority of NA active site amino acid substitutions have impaired viral fitness. DISCUSSION NIs are effective drugs for the treatment of influenza infections but the development of resistance is a major factor that could reduce antiviral activity. Previous studies with oseltamivir have shown that the incidence of resistance may be higher in children than in adults. This study was conducted to investigate whether zanamivir resistant viruses were selected in children during treatment. A study was conducted in Japan over three influenza seasons and susceptibility analyses carried out on all isolated viruses. To date there have been no resistant isolates detected in immunocompetent patients treated with zanamivir. In this study three resistant influenza A/H1N1 viruses were detected in samples from 2 Subjects, but were present at Day 1 and therefore did not arise as a result of drug pressure. One pretreatment virus from one subject had reduced susceptibility with an IC 50 of 19.49nM 13

14 (fold shift = 46) and harboured a N74S amino acid substitution (N70S by N1 numbering) in the NA which is outside the active site but gave rise to resistance to zanamivir. Two cultured viruses isolated on Days 1 and 2 from another subject contained the Q136K amino acid substitution which is close to the enzyme active site and confers high level resistance to zanamivir as observed previously (36, 38). This amino acid substitution was only found in cultured virus and not in the original clinical isolate and therefore was selected during in vitro passage rather than by drug pressure. This is consistent with findings from previous studies (36, 38). Although the Q136K amino acid substitution has not been detected in influenza A/H1N1 viruses from original clinical material there has been one report of the amino acid substitution in a clinical specimen of an influenza A/H3N2 virus (39). In this study, virus harbouring the Q136K amino acid substitution was not observed in the swab, but was found to be 100% mutant after just one passage in MDCK cells. This implies that the amino acid substitution may be present in the clinical isolate at a very low frequency and there is a strong selective pressure in favour of this amino acid substitution during growth in cell culture. However, the virus selected in MDCK cells with the Q136K amino acid substitution appeared unfit as it did not grow to a high titre compared to other isolates. Influenza A/H1N1 viruses with the Q136K amino acid substitution may be at a disadvantage as they have never been found in in vitro passage or directly from swabs from patients treated with zanamivir (40). In this study there were an additional 15 viruses from 14 subjects that had identical NA sequences to the clinical isolates from which these two resistant viruses originated, but did not select resistant viruses in culture. Why only the two viruses identified here from this subject underwent host cell selection, and not isolates with identical NA 14

15 sequences from other patients, is not clear but may be associated with other amino acid substitutions present in other genes. If there are compensatory amino acid substitutions that assist the selection of Q136K they do not appear to be in the NA. In previous studies it was shown that viruses with the Q136K amino acid substitution can be transmitted between ferrets and therefore the acquisition of compensatory amino acid substitutions with the Q136K giving rise to a transmissible zanamivir resistant virus cannot be ruled out in the future (36). In addition, the Q136L amino acid substitution was selected in vivo, in zanamivir treated ferrets infected with an influenza A/H5N1 virus (37). The virus harbouring the Q136L amino acid substitution was resistant to oseltamivir and zanamivir with fold changes in susceptibility of 350 and 26 fold respectively. Structurally it is not clear how the Q136K or Q136L amino acid substitution effects resistance to zanamivir as it is located at the base of the active site. The susceptibilities of the zanamivir sensitive isolates were comparable to data obtained previously for sensitive isolates and were in agreement with values obtained for the sensitive subtypes A/H1N1 (mean IC nM), A/H3N2 (mean IC nM) and influenza B (mean IC 50, 2.28nM) (41). NA sequences from samples from twelve subjects showed a difference between the Day 1 sample and their post treatment sample. Eleven out of twelve emergent amino acid substitutions identified here, during zanamivir treatment have not been implicated in resistance to NIs and are not in the vicinity of the NA active site. The mutations were detected by PCR and sequencing, and only three of the samples could be cultured and were shown to be susceptible to zanamivir. The majority of 15

16 these samples could not be cultured and therefore the effect of these amino acid substitutions on susceptibility to zanamivir could not therefore be determined. The clinical significance of the different amino acid substitutions identified is not clear but most of these may have arisen due to natural variation. One amino acid substitution, N294K, emerged during zanamivir treatment and although N294S is a recognised resistance amino acid substitution, it could not be determined if the N294K affects susceptibility to zanamivir. Reverse genetics analysis will be carried out in the future to ascertain the affect, if any, of N294K on susceptibility. It is noteworthy that all influenza A/H1N1 viruses isolated during the 2008/2009 influenza season harboured the H275Y NA amino acid substitution, which is expected from previous analyses of circulating oseltamivir resistant viruses from this time (13, 14, 15). In contrast, all viruses analysed in this study isolated during 2006/2007 and 2007/2008 influenza seasons were wild-type at position 275 of the NA. Sequencing of the HA gene showed that the majority of viruses analysed did not contain amino acid substitutions in or near the Receptor Binding Site (RBS) of the HA or amino acid substitutions in the HA that have been previously linked to resistance to zanamivir (32). Two amino acid substitutions at positions 179 and 191 in the RBS of the HA were present in Day 1 viruses from different subjects and were therefore not selected as a result of drug pressure. HA amino acid substitutions located in the RBS have been shown to alter affinity of the HA with the cellular receptor and can cause the virus to bypass the NA function completely in cell culture 16

17 (42). Although HA amino acid substitutions are readily selected in vitro the role of HA amino acid substitutions in resistance to NIs in vivo is not fully understood Although resistance to zanamivir is rare, resistance associated amino acid substitutions may be present in clinical isolates as minority species. Population sequencing can only identify minority species down to 25% of the population, which is a major deficiency of this technique and may result in minority species not being identified. In studies with oseltamivir, resistant virus has generally been detected in viruses isolated on or after Day 4 (43, 44). Clonal analysis was therefore carried out in this study on virus samples obtained on Day 4 or beyond. In this study 13 single clones from during-treatment samples from 12 different subjects were found to contain an amino acid substitution in the NA active site or a previously identified resistance associated amino acid substitution. All the amino acid substitutions are close to the NA active site and therefore may have the potential to give rise to zanamivir resistance. None of the amino acid substitutions were detected in the Day 1 sample derived from the respective subject. However, the fact that a single clone was detected in post treatment samples does not mean they were amplified by drug selection but may have arisen by a single chance mutation. The mutation rates of the clones are in the range 0.2 x10-6 to 0.8 x10-6 (Table 7). The error rate of platinum pfx DNA polymerase used in the PCR reactions is 1.6 x 10-6 (45, 46). Therefore, the clonal mutation rate is comparable to the polymerase error rate. Consequently, it is likely that the mutations identified by clonal analysis have 17

18 arisen as a result of polymerase error although it cannot be completely ruled out that they arose during viral replication The clinical implication of the minority mutations is unclear because the majority of NA active site amino acid substitutions have impaired viral fitness and the chance of an impaired virus producing a productive infection is limited (47). Minority species are important in the clinical outcome of patients with chronic infections such as HIV. In a self limiting acute disease such as influenza, the role of minority species is unclear as the virus infection may become eradicated by immune clearance before an unfit virus sub-population can become established. However, the presence of potential resistance amino acid substitutions as minority species may be relevant in certain situations, for example in the treatment of immuno-compromised patients. Further studies are warranted to analyse the phenotypes of some of the NA aminoacid substitutions described in this paper, using reverse genetics in a known genetic background. Acknowledgements The authors would like to thank GSK Japan and the clinical investigators for the design and co-ordination of the clinical study including participating patients for their time and effort in providing samples for this study. Without all of their involvement this study would not have been possible. We would also like to acknowledge the work of Mitsubushi Japan, for the culture of viruses from the swabs and for coordinating the transport of samples from Japan to the UK. In addition, we thank 18

19 Sundip Modha, GSK, Stevenage, for carrying out purification of clonal plasmids using the Qiagen extraction robot References 1. von Itzstein M, Wu W-Y, Kok GB, Pegg MS, Dyason JC, Jin B, van Phan T, Smythe ML, White HF, Oliver SW, Colman PM, Varghese JN, Ryan DM, Woods JM, Bethell RC, Hotham VJ, Cameron JM and Penn CR Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature 363: Collins PJ, Haire LF, LinYP, Liu J, Russell RJ, Walker PA, Skehel JJ, Martin SR, Hay AJ and Gamblin SJ The mechanism of H5N1 Influenza resistance to Tamiflu and implications for drug stockpiles. Nature 453(7199): Smith BJ, McKimm-Breshkin JL, McDonald M, Fernley RT, Varghese JN, and Colman PM Structural Studies of the Resistance of Influenza Virus Neuramindase to Inhibitors. J. Med. Chem., 45, Varghese JN, Smith PW, Sollis SL, Blick TJ, Sahasrabudhe A, McKimm- Breschkin JL and Colman PM Drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase. Structure, 6(6): Laforce C, Man CY, Henderson FW, McElhaney JE, Hampel FC, Bettis R, Kudule L, Harris J, Yates P, Tisdale M, Webster A Efficacy and Safety of Inhaled Zanamivir in the Prevention of Influenza in Community-Dwelling High-Risk Adult and Adolescent Subjects: A 28-Day, 19

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27 Table 1. Number of swabs analysed and results obtained for 1) cultured virus susceptibility using NA enzyme assay, and 2) genotyping directly from swabs for a) the NA gene, and b) the HA gene Sample description NA Susceptibility NA genotypes HA genotypes Swabs (Analysed/Taken) 371/714 b 484/714 b 413/714 b Subjects (With Match samples a /Total) 108/ / /277 Treatment swabs (Analysed/taken) 119/ / /437 Day 1 252/ / /277 Day 2 c (Analysed/taken) 39/68 61/68 56/68 Day3 c (Analysed/taken) 49/123 80/123 63/123 Day 4 c (Analysed/taken) 19/70 37/70 25/70 Day 5 c (Analysed/taken) 7/38 20/38 15/38 Day 6/7 d (Analysed/taken) 5/95 26/95 14/95 Day 8/9 d (Analysed/taken) 0/38 5/38 2/38 Influenza A/H1N1 166/ / /391 Influenza A/H3N2 105/ / /174 Influenza B 100/ / /149 a Matched samples are a baseline and a during or post treatment sample from the same subject. b Includes 5 untyped swabs from 2 subjects. c During treatment. d Post treatment 27

28 Table 2 Zanamivir susceptibilities determined using the NA Star assay, for the different influenza subtypes isolated by cell culture. SUBTYPE Range IC 50 (nm) Influenza season 2006/ / /2009 Mean IC 50 ± SD Mean IC 50 ± SD (nm) Range IC 50 (nm) Range IC 50 (no. Of isolates) (nm) (no. Of isolates) (nm) 28 Mean IC 50 ± SD (nm) (no. Of isolates) No. Totals 7.88 ± (82) ±5.27 (13) a A/H1N ± ± 0.78 (71) 166 A/H3N ±0.65 (72) NA NA (0) ± 0.79 (33) 105 B ±1.67 (71) ± 1.12 (2) ±1.45 (27) 100 a IC 50 excluding resistant viruses, see Table 3.

29 Table 3 Genotypic (NA) and phenotypic analysis (NA Star assay) of influenza A/H1N1 resistant viruses Subject No. Season Visit Source IC 50 (nm) ZMV FC IC 50 (nm) 29 OSV /2007 Day 1 Swab na na na na /2007 Day 1 Cultured 19.5 a 46 a FC Amino Acid Substitution (N2 numbering) N74S,P82S,K130R,I187M,G248R,M266I,T286I, N347D,G357D,I370L,I397V,I454T b N74S,P82S,K130R,I187M,G248R,M266I,T286I, N347D,G357D,I370L,I397V,I454T b /2008 Day 1 Swab na na na na A86V,V94I,N208K,Q216K,G331R,S452G /2008 Day 2 Swab na na na na A86V,V94I,N208K,Q216K,G331R,S452G /2008 Day 1 cultured A86V,V94I,Q136K,N208K,Q216K,G331R,S452G /2008 Day 2 cultured A86V,V94I,Q136K,N208K,Q216K,G331R,S452G a 12.9 nm, FC=31 on re-test, b Four viruses with the same sequence but without the N74S did not show reduced susceptibility to zanamivir, na not applicable, FC- fold change, Resistance associated amino acid substitution in bold.

30 Table 4 Emergent and resistance associated amino acid substitutions detected in the NA of influenza virus isolates Subject No. Season Type/ Sub type IC 50 (nm) Incidence 30 Amino Acid Substitution (N2 numbering) 3, /07 H1N a Non-emergent G248R d /07 B b Emergent, Day 7 L98L/F /08 H1N1 NC Emergent, Day 7 T69T/I /08 H1N1 NC Emergent, Day 3 S367N /08 H1N1 NC Emergent, Day 3 S195Y /09 H1N1 NC Emergent, Day 7 N294K e /09 H1N1 NC Emergent, Day 6 DEL /09 H1N1 NC Emergent, Day 4 P380N /09 H1N1 NC Emergent, Day 7 Y70C /09 H1N Emergent, Day 3 V114I, E229G /09 H1N1 NC Emergent, Day 2 S105N /09 H1N1 1.7 Emergent, Day 6 P389H /09 H3N Non-emergent Y155H d /09 H3N2 NC Emergent, Day 4 G373R/G a Range of 5 viruses from 2 subjects, b Range of 2 viruses, c Range of 2 viruses, d (27), e (20), NC not cultured.

31 Table 5 The total number of samples analysed by clonal analysis of the NA Number of swabs (Number of clones) Pretreat ment During-Treatment Post-Treatment Season Strain Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8 Day9 31 Total Analysed Total Treated Analysed Total Controls Analysed Total Treated Analysed Day 4 and after 2006/07 H1N1 2 (27) (27) 1 (13) (24) 5 (91) 3 (64) 2 (27) 3 (64) 2006/07 H3N2 2 (47) 2 (7) 0 4 (13) 6 (72) 4 (17) (156) 16 (109) 4 (54) 14 (102) 2006/07 B 1 (30) 0 1 (34) 9 (90) 4 (40) 3 (33) 1 (14) (241) 18 (211) 2 (64) 17 (177) 2007/08 H1N1 7 (174) 1 (0) 0 9 (270) 8 (209) 3 (126) 5 (146) 5 (102) 1 (31) 39 (1058) 32 (884) 7 (174) 31 (884) 2007/08 B (0) (0) 1 (0) 0 1 (0) 2008/09 H1N1 2 (26) (150) 0 4 (114) 3 (60) 1 (0) 0 17 (350) 15 (324) 2 (26) 15 (324) 2008/09 H3N (21) 1 (1) 2 (13) (35) 6 (35) 0 6 (35) 2008/09 B 1 (27) (96) (123) 4 (96) 1 (27) 5 (123) Totals 15 (331) 3 (7) 1 (34) 37 (667) 20 (335) 16 (303) 10 (220) 6 (102) 2 (55) 110 (2054) 95 (1723) 19 (372) 91 (1682)

32 Table 6 Summary of potential NA resistance amino acid substitutions identified by clonal analysis Season Subject No. Visit Sub- Type Number of clones with mutation Mutation rate Amino acid substitution (N2 numbering) /07 18 Day1 H1N1 0/ /07 18 Day4 H1N1 1/ x 10-6 R152K 2006/07 19 Day1 H3N2 0/ /07 19 Day5 H3N2 1/ x 10-6 R224G 2006/07 20 Day1 H3N2 0/ /07 20 Day5 H3N2 1/ x 10-6 D198N 2007/08 21 Day1 H1N1 0/ /08 21 Day6 H1N1 1/ x 10-6 E227D 2007/08 21 Day6 H1N1 1/ x 10-6 R224G 2007/08 22 Day1 H1N1 0/ /08 22 Day8 H1N1 1/ x 10-6 L134Q 2007/08 23 Day1 H1N1 0/ /08 23 Day7 H1N1 1/ x 10-6 E277G 2007/08 24 Day2 H1N1 ND 2007/08 24 Day9 H1N1 1/ x 10-6 R292G 2007/08 25 Day1 H1N1 0/ /08 25 Day4 H1N1 1/ x 10-6 R292S 2007/08 26 Day1 H1N1 0/ /08 26 Day4 H1N1 1/ x 10-6 L134P 2008/09 9 Day1 H1N1 0/ /09 9 Day4 H1N1 1/ x 10-6 L139P,K143N 2008/09 27 Day1 H1N1 0/ /09 27 Day4 H1N1 1/ x 10-6 Q136H 2008/09 28 Day1 B 0/ /09 28 Day4 B 1/ X 10-6 R224G ND not done. 32