Review Influenza virus susceptibility and resistance to oseltamivir

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1 Review Influenza virus susceptibility and resistance to oseltamivir Fred Y Aoki 1, Guy Boivin 2 and Noel Roberts 3 * Antiviral Therapy 12: Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada 2 Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, QC, Canada 3 School of Biosciences, Cardiff University, Cardiff, UK *Corresponding author: Tel: ; robertsna@btinternet.com Oseltamivir phosphate is a prodrug of oseltamivir carboxylate, a highly specific inhibitor of influenza virus neuraminidases. Given that oseltamivir carboxylate binds to highly conserved, essential amino acids in the catalytic site of the enzyme, and that the activity of neuraminidase is critical for virus release from infected cells and subsequent virus spread, the drug was expected to have a low propensity to select for viable resistant mutants. Indeed, viruses with neuraminidase (and haemagglutinin) substitutions conferring reduced susceptibility to oseltamivir have been generated with difficulty in vitro, and these mutants generally have reduced infectivity and transmissibility compared with wild-type virus in animal models. Studies of seasonal influenza isolates collected before the introduction of oseltamivir show an absence of naturally occurring resistance. Few resistant mutants have arisen during clinical trials of oseltamivir in seasonal influenza, with cumulative data from all Rochesponsored studies indicating an incidence of resistance of 0.32% in adults (0.4%, including low-level mutants detected by genotyping alone in mixed virus populations) and 4.1% (5.4%) in children. Higher incidences of resistance were observed in two small Japanese studies, in which children received a different dosing schedule from their Western counterparts. In summary, the overall incidence of influenza virus resistance associated with the seasonal use of oseltamivir is currently low and resistant viruses might be of little clinical significance, except perhaps in immunocompromised individuals. However, continued vigilance, especially of emerging avian H5N1 strains, combined with careful, systematic laboratory-based monitoring, is essential. Introduction Influenza is an acute, febrile disease that causes substantial morbidity [1]. Typically, approximately 5 15% of the population develop influenza annually [2]; the debilitating nature of the disease means that even otherwise healthy adults will generally be confined to bed for 3 7 days and affected by a persistent cough and malaise for approximately 2 weeks [3]. In addition to the morbidity burden, influenza causes around 40,000 deaths each year in the USA alone [4]. Management options for influenza include prevention (using vaccination or chemoprophylaxis with antiviral agents) and treatment with antiviral drugs. However, although vaccination is an important aspect of influenza management, it is not 100% effective and the uptake rates, even in the populations most at risk, are relatively low [5]. Consequently, effective treatment options have an important role in the management of influenza infection. Oseltamivir phosphate is the prodrug of the active metabolite oseltamivir carboxylate, a potent and selective inhibitor of all classes of influenza virus neuraminidase (NA) [6]. Oseltamivir carboxylate binds to essential amino acid residues in the highly conserved active site of the viral NA, thus inhibiting a crucial NA function: release of progeny viral particles from the infected cell [7]. Numerous clinical trials have subsequently established the efficacy of oseltamivir, not only in the treatment of influenza in otherwise healthy adults, children and high-risk groups [8 11], but also for post-exposure prophylaxis in households of which at least one member had influenza-like illness [12,13] and for seasonal prophylaxis [14,15]. As with all antiviral agents, there is potential for the emergence of viruses with decreased susceptibility to oseltamivir [16 19]. Drug resistance is known to be pertinent to influenza viruses through previous 2007 International Medical Press

2 FY Aoki et al. experience with the M2 inhibitors amantadine and rimantadine, which generate high rates of resistance [20]. However, as the binding site for oseltamivir on NA is highly conserved and has a critical role in the infection cycle, most mutations in this area appear to be disadvantageous. It is also important to note that, as oseltamivir is active only against influenza NAs, no pressure is exerted on any other respiratory virus to select for resistant variants (unlike the situation with broad-spectrum antibiotics and respiratory bacteria in clinical practice). Based on the authors expertise and a comprehensive review of the available literature, this article assesses the preclinical and clinical data pertaining to influenza virus susceptibility and resistance to oseltamivir. Mechanisms of resistance preclinical studies Because the surface glycoproteins of the influenza virus (NA and haemagglutinin [HA]) have a close functional interrelationship, viral resistance to NA inhibitors can potentially arise through mutations in either the NA or HA [21]. Because the oseltamivir carboxylate molecule contains a bulky side chain, the NA enzyme of influenza A must undergo a conformational change to accommodate it (Figure 1). To do so, the amino acid E276 must rotate and bond with the amino acid R224. Any mutation that inhibits this rotation would be expected to reduce the binding affinity of oseltamivir carboxylate for the NA active site, while still allowing binding of the natural substrate, sialic acid, and NA inhibitors that do not require the conformational change for binding (for example, zanamivir). The R292K and H274Y mutations observed in the clinical setting are known to impede oseltamivir binding in this way [22]. A third mutation, N294S, is also expected to act in this manner [19]. X-ray crystallography also reveals that influenza A viruses carry structurally distinct NAs. This is thought to explain why the implications of the R292K and H274K mutations for oseltamivir susceptibility differ between viral subtypes [22 24]. Other amino acid substitutions at positions outside the active site or remote from the rotation point at position E276 might also influence binding. It is speculated that the fourth mutation identified clinically (at position 119) might allow binding of an additional water molecule, thus interfering with binding affinity of oseltamivir [19]. Influenza A viruses possessing NA enzymes with reduced sensitivity to inhibition by oseltamivir carboxylate have been generated in vitro by continuous passaging of influenza virus in Madin Darby canine kidney (MDCK) cells in the presence of increasing concentrations of the drug (Table 1) [23,25 27]. However, these viruses are generated with difficulty, after many more passages than are required to generate M2 mutants resistant to amantadine or rimantadine [28]. The NA mutations generated were influenzasubtype-specific. An amino acid substitution of arginine for lysine at position 292 (R292K) has been generated in the NA protein of influenza N2 and conferred a 30,000-fold reduction in NA sensitivity to oseltamivir carboxylate [27]. In influenza N1, a histidine-for-tyrosine substitution at position 274 (H274Y) has been generated [23] and, in influenza N9, a glutamic-acidto-valine substitution at position 119 (E119V), Figure 1. Binding of oseltamivir carboxylate to the active site of influenza A neuraminidase and prevention of binding by the R292K mutation A R292 E276 H274 B K292 E276 H274 R224 R224 (A) Oseltamivir carboxylate bound to the active site of influenza A with E276 in its re-oriented position (required to accommodate the hydrophobic side chain of oseltamivir), stabilized by binding to R224 and by interaction with H274. (B) The E276 side chain in its extended position now stabilised by interaction with resistant mutant 292K. This structure (Protein Data Bank ID: 2qwh) was determined by Varghese et al. [78]. There is loss of the hydrophobic pocket and steric hindrance to oseltamivir carboxylate, leading it to bind less tightly under these circumstances. It is also seen that large residues replacing H274 in N1 would create steric hindrance to the E276 reorientation, also with likely loss of any productive stabilizing binding. Figures were reproduced with kind permission of Dr Bradford Graves, F Hoffmann-La Roche. Figures were drawn using the PyMOL Molecular Graphics System (2002, DeLano Scientific, Palo Alto, CA, USA; International Medical Press

3 Susceptibility and resistance to oseltamivir partially in combination with R292K and a likely variant R305Q, giving a >6,000-fold decrease in NA susceptibility to oseltamivir carboxylate, was generated [26]. Similar selection experiments with influenza B virus and oseltamivir carboxylate did not result in any NA mutations [25]. This is consistent with the fact that when oseltamivir carboxylate binds to NA of influenza B, the major rotation of E276 does not occur and hence the mechanisms of resistance described above are not operative (unpublished data). Several research groups have used reverse genetics to study the effects of site-specific mutations in the influenza NA gene (Table 1) [23,29 31]. One group introduced six specific NA mutations, equivalent to those selected in vitro by zanamivir and oseltamivir, at position 116 on the NA gene of an influenza B virus (equivalent to position 119 on influenza A N2), and at positions 291 and 149 (equivalent to positions 292 and 152 in N2 numbering) [30]. The NA enzymes of all recombinant viruses were resistant to oseltamivir carboxylate, with NA enzyme 50% inhibitory concentrations (IC 50 s) fold higher than those for wild-type NAs. However, the NA activity from the R291K and E116D mutant viruses was low compared with that of wild type (6% and 29%, respectively), and NA protein stability was reduced. In addition, and with the exception of the virus containing the E116G mutation, all viruses containing the altered NA enzymes had reduced replicative capacity in cell culture [30]. Another group found that a recombinant H1N1 viral NA containing the H274Y mutation was 200-fold less sensitive to inhibition by oseltamivir carboxylate than wild-type NA, whereas a recombinant H3N2 virus containing the same mutation did not display reduced susceptibility [23]. Additionally, in an N1 NA, H274Y and H274F mutations resulted in a Table 1. Summary of observed or engineered mutations and their effect on susceptibility to oseltamivir carboxylate in vitro and viral fitness in vitro or in vivo Effect on susceptibility to oseltamivir carboxlyate Impact on viral fitness versus Approach Mutation versus wild type wild type In vitro passaging in MDCK R292K K i 30,000-fold higher Reduced NA activity cells, influenza A/H3N2 [27] 10,000-fold reduction in infectivity Reverse genetics, influenza H274Y or H274F (H1N1) K i 300-fold higher Reduced NA activity A/H1N1 or H3N2 [23] IC fold higher H274Y (H3N2) None Not assessed H274N or H274G or H274S Unchanged or slightly Not assessed or H274Q (H1N1) increased In vitro passaging in MDCK E119V plus R292K or R305Q K i 1,100-fold higher Not assessed cells, influenza A/N9 [26] EC 50 6,000-fold higher In vitro passaging in MDCK None Not assessed Not assessed cells, influenza B [25] Reverse genetics, E116G IC fold higher Reduced NA activity influenza B [30] Reduced NA stability E116D or R149K or R291K IC >300-fold higher Reduced NA activity Reduced NA stability Reduced replicative capacity E116A or E116V IC 50 >300-fold higher Reduced NA activity Reduced NA stability Reverse genetics, influenza H274Y IC fold higher Reduced NA activity A/H1N1 [29] E119Q IC 50 9-fold higher Reduced NA activity Reverse genetics, influenza R118K Not assessed NA activity too low for independent growth A/H3N2 [31] R292K IC 50 >60,000-fold higher Reduced NA activity E227D Not assessed NA activity too low for independent growth R371K IC fold higher Reduced NA activity R152K No change in sensitivity Reduced NA activity R224K IC 50 >4,000 fold higher Markedly reduced NA activity E276D IC fold higher Reduced NA activity D151E IC fold higher Reduced NA activity EC 50, 50% effective concentration; IC 50, 50% inhibitory concentration; MDCK, Madin-Darby canine kidney; NA, neuraminidase. Antiviral Therapy 12:4 Pt B 605

4 FY Aoki et al. fold reduction in sensitivity, while H274N, H274G, H274S and H274Q mutations resulted in unchanged or slightly increased enzyme sensitivity to oseltamivir carboxylate [23]. In another study, influenza A (H1N1) viruses containing NA gene mutations previously associated with reduced susceptibility to NA inhibitors in N1, N2 and N9 viruses were generated [29]. Viruses containing the R292K and E119G, E119V, E119A, or E119D NA mutations (previously observed only in N2 or N9 viruses) could not be generated. An H274Y mutant in influenza A N1 was approximately 400-fold less sensitive to enzyme inhibition by oseltamivir carboxylate, whereas an E119Q mutant showed only a ninefold reduction in sensitivity. Again, the replicative ability of both mutant viruses in cell culture was impaired relative to wild-type [29]. Another group used site-directed mutagenesis to generate seven mutations in conserved residues in the NA active site of an H3N2 virus (R118K, R371K, E227D, R152K, R224K, E276D and D151E) [31]. These residues were chosen because they directly interact with NA inhibitors but have not been reported to be associated with resistance. The mutations all significantly reduced NA activity compared with wild-type virus, and the recombinant viruses differed in replication efficiency. The growth of the R118K and E227D viruses in MDCK cells was compromised to such a degree that a concentrated homogenous virus population could not be prepared, and no further analyses could be performed. The NA activity of the other five viruses was ~ fold lower than wild type. The R152K mutation did not confer resistance to oseltamivir carboxylate, and the R371K, E276D, D151E and R224K viruses were 45-, 15-, and >4,000-fold less sensitive in enzyme inhibition assays, respectively. However, the R224K virus was extremely genetically unstable [31]. The reduction in NA activity and stability, along with the reduction in whole virus replicative ability associated with these modifications, demonstrates that reductions in inhibitor binding affinity are associated with reduced enzyme function and therefore reduced viral fitness. The integrated actions of viral HA and NA in ensuring the release of progeny virus mean that resistance to NA inhibitors might also arise through mutations in the HA. Mutations that decrease the affinity of HA for sialic acid will make the release of progeny virus less dependent on NA activity and thus less sensitive to NA inhibitors as a class. Viruses with HA mutations conferring reduced susceptibility to NA inhibitors have been generated in vitro [21,27]. In one study, after the eighth passage of an influenza A H3N2 virus, a virus with two HA mutations (A28T and R124M) emerged and was 8.6-fold less susceptible to oseltamivir carboxylate [27]. In another study, two viruses with HA mutations emerged. These carried G143E and N199S, respectively, and both viruses also carried a R292K NA mutation [21]. By antiviral assay in MDCK cells, these mutants were more resistant to oseltamivir carboxylate than a mutant carrying the R292K NA mutation alone [21]. However, these HA mutations are unlikely to be predictive of what might be seen in the clinic. This is because most in vitro studies are performed in MDCK cells, in which the sialic acid linkage is predominantly α2,3. Changes in HA that reduce the binding affinity to sialic acid on MDCK cells might reduce sensitivity to oseltamivir in vitro, but might not change the affinity to human respiratory cells that predominantly have α2,6 sialic acid linkages, as discussed below. Methods for the detection of resistance in clinical isolates It is widely acknowledged that standard cell lines such as MDCK do not provide a reliable test system for the antiviral potency of NA inhibitors as a class against clinical isolates [32 34]. This is because the sensitivity of influenza virus to NA inhibitors in cell culture does not reflect NA enzyme inhibition sensitivity, and this is particularly relevant for clinical isolates of low passage number. It is thought that the problem results from a mismatch in receptor types between human airway epithelial cells and the cell culture systems [35,36]. Human airway epithelial cells contain mainly sialylα2,6-galactosyl receptors (the receptors for human influenza viruses in the human respiratory tract), and small numbers of sialyl-α2,3-galactosyl receptors [35,36]. The concentration of sialyl-α2,6-galactosyl receptors in cell lines used in the laboratory is relatively low, leading to progeny viruses (if they are clinical isolates and therefore not adapted to culture conditions) being poorly bound via HA to the cell surface and thus able to detach and infect other cells without the requirement for NA cleavage activity [35,36]. This means that viral replication in cell culture is relatively insensitive to NA activity and hence NA inhibition, and therefore leads to false-positive resistance results. An attempt to address this has been made through the development of MDCK cell lines stably transfected with the gene of human 2,6-sialyltransferase [35,36]. Initial studies with these cell lines suggest that they give a more clinically relevant indication of the antiviral potency of NA inhibitors than the parent MDCK cells, particularly for clinical isolates that are adapted to infect the sialyl-α2,6-galactosyl receptors predominating in the human respiratory tract [35 37]. However, use of these cell lines to screen for clinical isolates with resistance to NA inhibitors is yet to be reported. Given their operational ease, enzyme inhibition assays remain the preferred option, despite their International Medical Press

5 Susceptibility and resistance to oseltamivir inability to detect variants resistant through HA mutations. At present, resistance in clinical isolates continues to be best assessed by NA enzyme inhibition phenotypic assay supported by genotypic analysis, and by an HA genotypic assay with receptor binding studies. In Roche-sponsored clinical trials, the primary assay used to detect resistance was the NA enzyme phenotypic assay. Viral NA levels in nose and throat swab samples are generally too low to assay for NA activity, meaning the virus must first be expanded in cell culture. Additionally, the standard assay [38] was modified in Roche-sponsored clinical trials to increase sensitivity because, on expansion, virus containing resistant NA might not replicate as well as wild-type virus and the resistant NA might also have compromised enzymatic activity, making it more difficult to detect. Both of these characteristics have been observed in preclinical studies [23,24,27,29 31,39]. An NA genotypic analysis was then used to validate the phenotypic analysis. As there is the possibility that the expanded viral population does not accurately reflect the primary swab sample, the genotypic analysis was performed before and after expansion in cell culture (to reveal any expansion artefacts) as well as before and after treatment in the oseltamivir-treated group (to determine drug-induced changes) and the placebotreated group (to ascertain the incidence of random variants). Additionally, sequence analysis was used to assess the likelihood of any HA mutations (particularly in the receptor binding site); this sequencing was performed on primary swab samples obtained before and after treatment from oseltamivir- and placebotreated patients. Distinguishing natural variants from resistance mutations in clinical studies As discussed above, the strategy for assessing viral resistance during oseltamivir clinical trials included sequencing of the NA and HA genes both before and after treatment for both oseltamivir-treated patients and placebo recipients. Where possible, sequencing was performed directly on nasopharyngeal swab samples, as well as on the viruses after expansion in cell culture, to ascertain whether the expanded virus used for phenotypic assays faithfully reflected the virus population in the patient. Sequence-predicted amino acid changes in both NA and HA were noted between pre- and post-treatment isolates from oseltamivirtreated patients and placebo recipients. Thus, the virus appeared to be spontaneously changing on passage in the patient within a 4 6-day period. The incidence of changes was the same in viruses from both oseltamivir-treated patients and placebo recipients and therefore appeared to be independent of drug effects. The incidence was about one amino acid change in every fifth sequence pair for both NA and HA. Thus, for example, from two studies of the treatment of adults and one paediatric treatment study, 98 pre- and post-treatment HA sequence pairs were obtained from swab samples for patients carrying H3N2 viruses. There was an amino acid variation between the pairs in the case of 7/34 (21%) samples from placebo recipients and 12/64 (19%) samples from oseltamivir-treated patients. No individual variant occurred more than once. Only one amino acid change was in or near the sialic acid binding site (Y137S), and might have been interpreted as conferring resistance to oseltamivir, but this was observed only in a virus from a placebo recipient. Variations in NA were also seen at a similar frequency in samples from both treated patients and placebo recipients. Moreover, the overall incidence of variants arising in these studies is consistent not only with the reported error rate for influenza polymerase of mutations per nucleotide per infection cycle [40] (assuming infectious cycles between pre- and post-treatment samples and about 50% of nucleotide changes resulting in an amino acid change [41]) but also with the genetic variation rate reported for HA1 changes over time for a persistent H1N1 infection in an immunodeficient child [41]. These observations strongly suggest that natural variants can arise both in NA and HA in an untreated patient over the course of infection. It is therefore of paramount importance to distinguish these phenotypically from true resistance mutations when studying genotypic changes in virus from drug-treated patients. Random variants, adaptations and/or changes in population proportions appearing as artefacts of viral expansion in cell culture (required for phenotypic assay) were also seen in the oseltamivir studies and have been reported previously by others [42,43]. These changes must also be distinguished and correctly interpreted in resistance studies. Oseltamivir resistance in clinical isolates Studies of influenza isolates collected before the NA inhibitors oseltamivir and zanamivir were introduced into clinical practice showed no evidence of naturally occurring resistance among over 1,000 isolates collected worldwide [44]. Similarly, no resistance was observed in over 3,000 pre-treatment isolates collected in clinical trials of oseltamivir (unpublished data). There was also no resistance to oseltamivir carboxylate or zanamivir detected in 267 influenza A and B viruses isolated over one season in France, prior to the introduction of NA inhibitors in clinical practice there [45]. Similarly, no resistance was detected in 42 influenza A Antiviral Therapy 12:4 Pt B 607

6 FY Aoki et al. and 23 influenza B viruses isolated from untreated individuals in Canada during the first year in which NA inhibitors were available [46]. Furthermore, in an Australian study, when IC 50 values for the NA enzymes of 245 influenza A and B viruses isolated before and after the introduction of NA inhibitors were compared, full sensitivity to inhibition by oseltamivir carboxylate was retained [47]. During the first 3 years of NA inhibitor use ( ), only 0.33% (8/2,287) of isolates collected worldwide had a >10-fold decrease in susceptibility to oseltamivir [48]. It appears that none of the isolates with decreased sensitivity were from patients treated with NA inhibitors, so these viruses were the result of transmission of virus from treated patients, natural variants with lower sensitivity than the norm or, in some instances, assay variation. Of the eight isolates with decreased sensitivity, only one H1N1 virus contained an NA mutation previously found in clinical trials to confer resistance to oseltamivir (H274Y); this virus was 327-fold less sensitive than wild type. Two other H1N1 isolates were 133-fold (G248R plus I266V) and 123-fold (Y155H) less sensitive, whereas three H3N2 or H1N2 (E41G, Q226H and genetic drift only) and two influenza B (I222T and D198E) viruses were ~11 26-fold less sensitive [48]. In another surveillance study, 1,050 isolates collected worldwide during the and influenza seasons were analysed for susceptibility to oseltamivir [49]. Influenza A H1N1, H1N2 and H3N2 viruses showed high levels of susceptibility (mean IC 50 s: nm). Very few of the isolates had IC 50 values that were classified as statistical outliers (numbers not given) and, of the isolates sequenced, none contained any of the NA mutations at positions 119, 152, 274 or 292 that have been associated with resistance to either oseltamivir or zanamivir in clinical treatment isolates [49]. Use of oseltamivir in the season was greatest in Japan. Isolates from untreated Japanese patients were analysed to determine the incidence of resistance in the circulating virus population [39]. Four (0.4%) of the 1,180 influenza A (H3N2) isolates tested were oseltamivir resistant. Three of these variants had mutations previously shown to confer resistance (two E119V and one R292K). In a case report, a mixed influenza B viral population with reduced susceptibility to oseltamivir was isolated from an infant with no history of treatment or contact with NA inhibitors [50]. The population contained a mixture of wild-type virus and virus with a D197E substitution (D198E using influenza A N2 numbering); the latter was 11.8-fold less sensitive to oseltamivir carboxylate (the same isolate as was included in the report by Monto et al. [48]). Infection with influenza B viruses is associated with a lesser therapeutic response in patients. In otherwise healthy persons with influenza A infection, the therapeutic efficacy of oseltamivir has been established in large, placebo-controlled field trials [9 11,51]. Oseltamivir therapeutic efficacy in patients with influenza B infection has been suggested by one placebo-controlled study in susceptible volunteers with induced influenza B virus infection [51], in whom pooled data from subjects treated with 75 or 150 mg twice daily demonstrated a therapeutic and antiviral effect. In three large treatment trials in children and adults with influenza A or B illness, oseltamivir was clinically less efficacious in all studies and produced a lesser antiviral effect in patients with influenza B infection [52 54]. Oseltamivir IC 50 values for influenza B virus NAs are commonly ~10-fold higher than for influenza A [45 47,49] and might be >70-fold higher depending on the assay method [51,54]. The differences in susceptibility by enzyme inhibition assay might explain the observed differences in virological and clinical responses. It remains to be determined whether higher oseltamivir dose regimens would be more effective than the approved regimens in influenza B illness. The frequency of isolation of viruses with reduced susceptibility after oseltamivir treatment of otherwise healthy individuals with influenza is generally low but with some reports of much higher incidences. Cumulative data from Roche-sponsored clinical trials, involving almost 2,000 oseltamivir-treated patients, indicate that the incidence of reduced susceptibility to oseltamivir is 0.32% in adults and 4.1% in children (if low-level mutants detected by genotyping alone in mixed virus populations are included, then the corresponding values are 0.4% and 5.4%, respectively) (Table 2). The course of influenza disease is generally more protracted in children than in adults, with longer periods of viral shedding and higher viral titres. This might explain why the rates of resistance to oseltamivir are somewhat higher in children. However, for children, all early studies used a dose of 2 mg/kg, including a study reported by Whitley et al.[11] and a Roche study in Japanese children [18]. The incidence of resistance was 10/183 in the former study (nine of these children were infected with an H3N2 virus and were aged 6 years [mean: 3.9 years, unpublished data]) and 7/43 in the latter study (three of these children were 1 year old [18]). It was the findings from these two studies, and the observation that the incidence of resistance was higher the younger the child, that prompted Roche to further examine the kinetics of the drug in young children [55]. It became apparent that, for children below the age of ~5 years, the drug was cleared more rapidly the younger the child and the exposure given by 2 mg/kg became increasingly inadequate. Hence, a different dosing regimen was recommended based on the weight of the child [18], and has been accepted everywhere except in Japan (see footnote to Table 2). This provides a drug exposure for the very International Medical Press

7 Susceptibility and resistance to oseltamivir Table 2. Incidence of resistance and associated mutations in all Roche-sponsored studies of oseltamivir in the treatment of naturally acquired influenza infection Population Oseltamivir dose* Incidence Mutation (n) Adolescents, adults and elderly Otherwise healthy adults (aged 75 mg twice daily 2/211 R292K (3) years) 150 mg twice daily 2/207 E119V (1) Otherwise healthy adolescents 75 mg twice daily 0/496 and adults (aged 13 years old) At risk adults (aged 13 years) 75 mg twice daily 0/61 At risk elderly (aged 65 years) 75 mg twice daily 0/34 At risk elderly 75 mg twice daily 0/7 At risk elderly (nursing home 75 mg twice daily 0/3 residents) Otherwise healthy Japanese 75 mg twice daily 0/88 adults (aged 16 years) Children Otherwise healthy children 3 mg/kg twice daily 0/5 (aged 1 12 years) Otherwise healthy children 2 mg/kg twice daily 10/183 R292K (7) (aged 1 12 years) E119V (2) H274Y (1) At risk children (asthmatics, 2 mg/kg twice daily 0/60 aged 6 12 years) At risk children (asthmatics; 30, 45 or 60 mg twice daily 2/26 R292K (1) aged 6 12 years) (depending on weight) SASG del (1) At risk children (asthmatics; 30, 45 or 60 mg twice daily 0/17 aged years) (depending on weight) Otherwise healthy Japanese 2 mg/kg twice daily 7/43 H274Y (7) children (aged 12 years) Households with children and adults Otherwise healthy adults 75 mg twice daily 0/121 Otherwise healthy children 30, 45 or 60 mg twice daily 0/147 (aged 1 years) Potential phenotypic resistance (reduced susceptibility) was indicated by viruses with an 50% inhibitory concentration in the neuraminidase inhibition assay >2 SD above the mean for pretreatment viruses of the same subtype in the study or fourfold higher than the corresponding pre-treatment virus from the same patient. Resistance was confirmed by a corresponding genotypic change in neuraminidase. Incidence data are cumulative from all studies, irrespective of the doses used. *Recommended doses for treatment of influenza are 75 mg twice daily in adults and children weighing >40 kg, and 30, 45 and 60 mg twice daily in children weighing 15, and >23 40 kg, respectively. Both of these children had a degree of immunosupression being treated with relatively high dose steroids. Unit dosing in this study was based on the age of the children. young that is more closely matched to that in adults with a dose of 75 mg twice daily. High resistance rates (9/50 patients [18%]) were also observed in an independent trial in Japanese children, and a novel NA mutation (N294S) was noted in an H3N2 virus from one patient [56]. The dose of oseltamivir used was 2 mg/kg (see above), and the incidence of resistance observed (9/50) was very similar to that in the Roche trial of Japanese children with H1N1 infections that used the same dose. Moreover, the age range was markedly skewed towards those whose exposure to the drug was likely to be inadequate when using the 2 mg/kg dose; the oldest child in whom resistant virus was selected was 3 years old, five of the patients did not get the full 5 days of treatment, and the study included at least eight children aged <1 year who were treated off-label. When studies using only the currently recommended doses of oseltamivir were analysed, the overall incidence of resistance was 2/900 (0.2%) in adults and adolescents and 2/190 (1.1%) in children. In these trials, observed mutations were R292K, E119V and a unique SASG del NA mutation, all in H3N2 viruses (Table 2). A general observation from clinical studies is that oseltamivir-resistant virus occurred only transiently in Antiviral Therapy 12:4 Pt B 609

8 FY Aoki et al. virus excreted by patients in clinical trials [17,56]. Sequential viral samples were analysed for NA sequence for 17 patients who excreted a resistant virus population or subpopulation at some point during one of four clinical trials. Most of these patients were children in the early trial by Whitley et al. [11], which used the 2 mg/kg dose. Resistant virus did not emerge until study day 4 or 6, and had cleared by day 8 in adults and day 10 in children. In a further study, resistant viruses were detected in 2/54 experimentally infected adults, 60 and 84 h after the initiation of oseltamivir treatment [57]. In each case, the onset of resistance was associated with a transient spike in viral shedding that rapidly subsided. In all of these instances, clearance of resistant virus was most probably achieved by the immune system, as with placebo. In seasonal influenza, the clinical course of patients excreting oseltamivir-resistant virus is generally indistinguishable from that in patients infected with wild-type virus. For example, nine children in whom resistant influenza virus emerged on days 4 and 6 after starting treatment continued to recover normally [16]. There appears to be no evidence for enhanced pathology in humans excreting resistant viruses and patient symptom scores continue to decrease after the emergence of resistant virus [16]. The subtype specificity and predominance of the resistance mutations arising during clinical studies with oseltamivir were generally consistent with those predicted by the in vitro studies. During clinical studies, as observed in vitro, R292K was the predominant NA mutation and its occurrence was restricted to the influenza N2 subtype, whereas H274Y was observed only in the influenza N1 subtype. One exception to the predictive pattern of in vitro studies was the occurrence of E119V in influenza N2 in clinical trials; this mutation had previously only been observed in an N9 reassortant virus in vitro (N2 and N9, however, are structurally very similar [22]). Recently, Hatakeyama et al. reported that 1/74 (1.4%) immunocompetent children (median age: 3 years) infected with influenza B in the season and treated with oseltamivir carried a G402S mutation in the NA of a post-treatment virus sample [58]. This conferred a 3.9-fold decrease in the sensitivity of the NA to oseltamivir carboxylate. They also identified variants with reduced sensitivity to oseltamivir in 6/422 (1.4%) pre-treatment influenza B samples. In three cases, the variant was I222T and the other three were D198N. These variants/mutations have been previously reported in virus from untreated [48] and immunocompromised [61] patients. Consistent with these previous findings, the decrease in sensitivity to oseltamivir carboxylate was approximately sevenfold and fourfold, respectively. The extent of susceptibility change that leads to loss of oseltamivir effectiveness is uncertain, but, importantly, no appreciable differences in the clinical or viral course of infection were observed between patients infected with wild-type viruses and those with reduced sensitivity when treated with oseltamivir, although numbers were small. It is possible that some of the variant viruses detected before treatment could have been selected by oseltamivir treatment and transmitted to family contacts. Importantly, the researchers observed no appreciable differences in the clinical course of viral infection or the extent of virus shedding (duration and titre) between patients infected with wild-type viruses and those with reduced sensitivity when treated with oseltamivir, although numbers were small. Further studies of treated subjects who are not involved in clinical trials are needed to assess the rate of oseltamivir resistance. Oseltamivir resistance in immunocompromised hosts In immunocompromised individuals infected with influenza, prolonged viral shedding often occurs, meaning there is an increased risk of the emergence of drug-resistant strains [59 61]. In an early case report, Weinstock et al. described a severely immunocompromised individual infected with an H1N1 virus [61]. The infection persisted until eventual death, despite treatment with amantadine, oseltamivir, rimantadine and zanamivir. Isolates were found to be resistant to rimantadine and oseltamivir, but susceptible to zanamivir. Sequence analysis of the NA gene revealed the presence of the H274Y mutation. More recently, Ison et al. (2006) documented influenza viruses resistant to oseltamivir (as assessed by a NA activity inhibition assay) in three severely immunocompromised patients [62]. The first patient, who was infected with influenza B and treated with oseltamivir and ribavirin, experienced progressive influenza illness. The patient did not respond to treatment and eventually died. An isolate with a 10-fold reduction in susceptibility to oseltamivir carboxylate was subsequently recovered from the patient and carried a novel NA D198N mutation along with a HA S285A mutation. The other patients were both infected with influenza A H3N2 virus and treated with oseltamivir and rimantadine without benefit. One patient recovered after treatment with zanamivir plus rimantadine, whereas the other experienced progressive influenza illness despite subsequent rimantadine therapy, and died after a haemorrhagic stroke. Isolates recovered from these patients early in the course of oseltamivir treatment were susceptible to the drug. Isolates carrying the NA E119V mutation were later recovered (>9 and 4 days, International Medical Press

9 Susceptibility and resistance to oseltamivir respectively, after treatment with oseltamivir was initiated). These isolates displayed 100-fold reduced sensitivity to oseltamivir and both carried M2 mutations (S31N for both patients) and HA mutations (R142G, Y195F and I239R for one patient, V226I for the other). In these cases, the resistant variants disappeared with the cessation of oseltamivir treatment, although wild-type virus persisted. Over a period of 12 months in another report, several viral isolates were obtained from a child with severe combined immunodeficiency disease who had been treated for influenza infection with oseltamivir without a clinical response and then with amantadine and zanamivir [60]. After 38 days of oseltamivir treatment, an influenza A variant with NA mutations E59G, E119V and I222V was detected. In virus from this patient, the E119V mutation persisted for 8 months after oseltamivir was discontinued [60]. Overall, these results highlight the need for increased vigilance with respect to identification of emerging drug resistance in immunocompromised patients. Properties of influenza virus with decreased sensitivity to oseltamivir Various ferret models of influenza virus infection have been developed, including models of infection (pathogenicity) and models of transmission. The ferret is an appropriate model of human influenza infection because the sialic acid linkage on the receptors of the mucosal cell surface of the respiratory tract is very similar in both species. Therefore, it is possible to infect ferrets with influenza virus isolates taken directly from patients, and thus eliminate the risk of, or need for, adaptation changes occurring in either the HA or NA gene during cell culture. Using these models, investigators studied the infectivity, pathogenicity and transmissibility of influenza viruses carrying the E119V, H274Y and R292K mutations that have been observed in the clinic [63 66]. Compared with wild-type virus, the infectivity and replicative ability of the R292K and H274Y mutant viruses in this model were reduced by 2 4 log units (100 10,000 fold) [63,66]. This conclusion was supported by studies in mouse models, in which oseltamivir- and zanamivir-derived R292K mutations were associated with 10,000- [27] and 500-fold [67] reductions in infectivity, respectively. Additionally, in ferrets infected with mutant virus, inflammatory and febrile responses were significantly reduced, indicating that the pathogenicity of the mutant viruses is compromised compared with wild-type virus. These results led the authors to conclude that the R292K and H274Y substitutions in the NA enzyme of influenza virus compromise viral fitness to such a degree that the mutations are unlikely to be of clinical significance [63,66]. Another study in ferrets demonstrated that in addition to the reduced infectivity and pathogenicity exhibited by the R292K virus, this virus was also compromised in terms of ability to infect contacts under conditions in which the wild-type virus was readily transmitted [65]. In a similar study, although the H274Y mutant virus was transmissible to all recipient ferrets and had a similar lung viral titre to wild type, it required a 100-fold higher dose than wild-type virus to infect index ferrets and was transmitted more slowly. However, the infectivity and transmissibility of the E119V mutant virus was not reduced compared with wild-type virus [64]. In contrast to the latter finding, other studies have demonstrated a reduction in infectivity and pathogenicity of the E119V virus of at least 2 3 log units (100 1,000-fold) compared with the wildtype virus [18]. A recent study used recombinant H274Y and N294S mutant viruses generated by reverse genetics in the mouse-adapted, highly virulent influenza A H1N1/WSN/33 background to infect mice [68]. In contrast to the results seen in ferrets with other strains, the recombinant H274Y virus was shown to be as virulent as wild-type virus in mice on the basis of clinical signs, median lethal doses and viral lung titres. On the other hand, the N294S mutant had lower replicative capacities and virulence than the wild-type virus. Overall, however, the weight of evidence suggests that influenza viruses carrying any oseltamivir-resistant mutation in the NA enzyme have decreased fitness and transmissibility. The R292K mutant is probably the most compromised, with the E119V mutant being only marginally less fit than wild-type, perhaps depending on the general genetic background of the NA in which the mutation is selected. The H274Y and N294S mutations [69] appear intermediate. These findings are in contrast to those observed with rimantadine-resistant viruses, which have similar virulence and transmissibility to wild-type virus in ferret models [70]. The data overall suggest that such isolates are unlikely to be of major clinical significance in immunocompetent persons with seasonal influenza, which might in part be because of the usually self-limiting course of illness. Considerations for concomitant therapeutic and prophylactic use of oseltamivir in seasonal influenza No evidence for resistant virus emerged in a clinical study that assessed the efficacy of oseltamivir in treatment and prophylactic use in 277 households [12]. As such, assessment of the transmissibility and pathogenicity of mutant viruses from index cases to close contacts was not possible. This contrasts with a similar study conducted with rimantadine, in which resistant virus emerged in treated patients and was transmitted to, and caused disease in, contacts [71]. Antiviral Therapy 12:4 Pt B 611

10 FY Aoki et al. It is likely that oseltamivir will be used concomitantly for treatment of the index case and for post-exposure prophylaxis within a group of contacts. As a general principle, when the same drug is used concomitantly for treatment and prophylaxis and treated patients shed wild-type virus, then the subjects receiving prophylaxis will be protected from infection. If the treated patients shed mutant, drug-resistant virus, there is potential for the patients receiving prophylaxis to be infected with resistant virus. The consequences of this should theoretically be no worse than had the subjects not been receiving prophylaxis: they would still be protected from wild-type viruses from other sources. Thus, in a closed population taking the drug as both treatment and prophylaxis, there is benefit for the group and, in the worst case, no disadvantage for an individual. When specifically considering viral resistance to oseltamivir carboxylate, several observations can be factored into these general considerations. First, the incidence rate for resistance to oseltamivir carboxylate during treatment of influenza in the adult population is very low. Most patients receiving treatment will therefore be shedding only wild-type virus from which the subjects receiving prophylaxis will be protected with an efficacy of about 80 90% [13 15]. Second, the small percentage of patients receiving treatment who shed some resistant virus will shed only wild type for much of their time on treatment, and when resistant virus is shed it is often still in the presence of excess wild-type virus. Finally, as discussed above, animal studies have demonstrated that mutant, oseltamivir-resistant virus genotypes generally have lower infectivity than wild-type virus and are less likely to transmit between animals under conditions where the corresponding wild type can transmit and replicate. Thus, contacts of oseltamivir-treated subjects, whether they are receiving prophylaxis or not, are unlikely to be exposed frequently to resistant virus. Oseltamivir susceptibility and resistance in H5N1 virus Wild-type H5N1 NAs are sensitive to oseltamivir. In a recent study, a group of 55 influenza A (H5N1) viruses isolated from Vietnam, Cambodia, Malaysia, Indonesia and Myanmar between 2004 and 2006 were tested for susceptibility [72]. Almost all of the viruses tested were found to be fully sensitive to oseltamivir by NA inhibition assay, with IC 50 values similar to, or slightly lower than, those of H1N1 viruses. Only two had a significantly higher IC 50 value than the mean of the population as a whole. These carried the NA variants V116A or I117V that gave IC 50 values of 3.6 nm and 5.4 nm, respectively, for oseltamivir (11- and 16-fold higher than wild-type). A further report suggests that sensitivity to oseltamivir has increased over time since the emergence of the 1997 H5N1 isolate in Hong Kong [73]. Sensitivities of the NAs of H5N1 viruses isolated in 2004 and 2005 to oseltamivir were approximately 10-fold higher than those of earlier isolates. This finding suggests that genetic variation in the absence of drug selective pressure has the potential to influence sensitivity to oseltamivir and underscores the need for continuous monitoring. Studies of more recently circulating clade 2 viruses are needed. To date, three confirmed clinical cases of H5N1 infections with reduced susceptibility to oseltamivir have been reported [74,75]. All were infected with clade 1 H5N1 viruses. As expected of an N1 virus, all resistant viruses in these studies were reported to carry the H274Y mutation, although 3/10 viral clones from one patient [75] carried the N294S mutation previously seen only in an N2 virus. Of the three documented cases, one patient received an adequate dose within 48 h of symptom onset [74]; one patient was underdosed with oseltamivir, receiving the prophylactic dose (75 mg once daily) rather than treatment dose (75 mg twice daily) even though she was already exhibiting symptoms of influenza infection [75] and one patient did not receive oseltamivir until day 6 of illness [74]. A further outbreak of H5N1 infection in Egypt in December 2006 resulted in the death of three family members. Moderate resistance to oseltamivir was reported in two virologically confirmed cases [76]. Preliminary reports suggest that the N294S mutation was present in isolates taken from these individuals. As before, it appears that at least two of these patients received oseltamivir late in the course of infection. These limited observations suggest that oseltamivir must be initiated early in the course of the H5N1 infection and at the recommended dose to reduce the risk of resistance. Ongoing surveillance is necessary to determine the prevalence and implications of H5N1 viruses with reduced susceptibility to oseltamivir. Cross-resistance to neuraminidase inhibitors In a recent study, recombinant influenza proteins containing various NA mutations that have previously been found to confer resistance to NA inhibitors were generated and tested for susceptibility to different NA inhibitors [68]. Using NA inhibition assays, three recombinant proteins were analysed for the N1 subtype (E119V, H274Y and N294S) and four were analysed for the N2 subtype (E119V, H274Y, R292K and N294S). The three mutant N1 proteins all conferred a high level of resistance to oseltamivir. The E119V and H274Y N1 mutations also conferred resistance to peramivir (an investigational NA inhibitor) and E119V conferred 2,144-fold resistance to zanamivir. N294S had slightly reduced susceptibility to both peramivir and zanamivir. However, it should be noted that substitutions at codon International Medical Press