Peramivir and laninamivir susceptibility of circulating influenza A and B viruses

Influenza viruses collected from regions of Asia, Africa and Oceania between 2009 and 2012 were tested for their susceptibility to two new neuraminidase inhibitors, peramivir and laninamivir. All viruses tested had normal laninamivir inhibition. However, 3·2% (19/599) of A(H1N1)pdm09 viruses had highly reduced peramivir inhibition (due to H275Y NA mutation) and <1% (6/1238) of influenza B viruses had reduced or highly reduced peramivir inhibition, with single occurrence of variants containing I221T, A245T, K360E, A395E, D432G and a combined G145R+Y142H mutation. These data demonstrate that despite an increase in H275Y variants in 2011, there was no marked change in the frequency of peramivir- or laninamivir-resistant variants following the market release of the drugs in Japan in 2010.


Introduction
Currently, the neuraminidase inhibitors (NAIs) are the only class of antivirals that are effective against circulating influenza A and B viruses due to widespread resistance against the older adamantane class of drugs. 1 Two NAIs, orally administered oseltamivir (Tamiflu â ) and inhaled zanamivir (Relenza â ), have been licensed in many countries since 1999. In 2010, two new NAIs, peramivir (Rapiacta â ) and laninamivir (Inavir â ), were licensed in Japan. 2 Peramivir has also been licensed in South Korea 3 and most recently in China, as a response to concerns of A(H7N9) avian influenza. 4 Peramivir is delivered intravenously and therefore is well suited to treating severely ill patients who may be unable to use oral or inhaled formulations. 5 Laninamivir octanoate, a prodrug of laninamivir, is administered by inhalation (in a powdered form) and is quickly converted into its active form in the lungs. 6 However, compared with oseltamivir and zanamivir, which are administered twice daily for 5 days, laninamivir is long-acting, requiring only a single administration for retention of the drug for at least five days. 7 During 2011-2012, laninamivir was the largest selling NA inhibitor in Japan. 8 As with any new antiviral agents that are released to the market, it is important to monitor circulating influenza strains for the development of resistance to both peramivir and laninamivir. [9][10][11] This is particularly pertinent given the widespread resistance to the NAI oseltamivir that occurred in seasonal A(H1N1) viruses in 2008. 12,13 Here, we evaluate the peramivir and laninamivir susceptibility of influenza A and B viruses circulating in parts of Asia, Africa and Oceania between 2009 and 2012, a time frame that spans pre-and post-market launch of these drugs in Japan and South Korea.
In line with the recommendations implemented by the WHO GISRS Expert Committee for Antiviral Resistance in Influenza, virus susceptibility was classified based on fold differences compared with the respective NA inhibitor median IC 50 of the matching subtype/type. 15

Peramivir susceptibility
The peramivir susceptibility of 2548 influenza A and 1238 influenza B viruses was tested, of which 96Á8% (580/599) of A (H1N1)pdm09 and 99Á4% (1231/1238) of influenza B viruses demonstrated normal peramivir inhibition, while all A (H3N2) isolates (n = 1949) exhibited normal peramivir inhibition. The mean (AEstandard deviation) peramivir IC 50 of the influenza B viruses with normal inhibition was 0Á74 AE 0Á33 nM, four-fold higher than the mean IC 50 of the influenza A(H1N1)pdm09 or A(H3N2) viruses (Table 1). In addition, there was no significant difference in the median peramivir IC 50 s of B Victoria compared with B Yamagata lineage viruses exhibiting normal inhibition.
Nineteen A(H1N1)pdm09 viruses (19/599, 3Á2%) had highly reduced peramivir inhibition (Figure 1), with a mean IC 50 value of 31Á3 AE 10Á3 nM, 241-fold above the median peramivir IC 50 of A(H1N1)pdm09 viruses with normal inhibition. Genetic analysis of these viruses revealed that they all contained the H275Y NA substitution (N1 numbering, codon 274 in N2 numbering), a mutation known to confer highly reduced oseltamivir inhibition. 12 Forty-two per cent (8/19) of the H275Y variants detected were from a cluster of cases in Australia in 2011, 16  Six influenza B virus isolates were identified as having reduced or highly reduced peramivir inhibition ( Figure 1, Table 2). The following influenza B residues are numbered based on straight influenza B NA amino acid numbering starting from the first methionine residue, GISAID accession numbers for sequences of the variant viruses are listed in Table 2. B/Malaysia/210/2012 contained two novel NA mutations Y142H and G145R, with the resulting isolate demonstrating a 487-fold increase in peramivir IC 50 ( Table 2). Y142H is located on the surface of the NA active site and could indirectly affect the binding pocket scaffold loop region including G145R (Figure 2). This may explain how G145R together with Y142H have a strong additive inhibitory effect. Other novel substitutions located in a framework residue (D432G) and outside the active site (K360E and A395E) ( Figure 2) were also identified in three influenza B viruses from Thailand and Malaysia with reduced or highly reduced inhibition. B/Bangkok/29/2012, which contained A395E, had a minor five-fold increase in peramivir IC 50 , while B/Malaysia/283/2012 and B/Malaysia/221/2012, which contained K360E and D432G NA mutations, respectively, had 165-and 41-fold increases in peramivir IC 50 ( Table 2). All five of these B variants had normal laninamivir, oseltamivir and zanamivir inhibition, apart from B/ Bangkok/29/2012 (A395E NA mutation) which had a fivefold increase in oseltamivir IC 50 . The final two B strains with reduced or highly reduced peramivir inhibition, B/Waikato/ 21/2011 and B/Wellington/39/2011, have previously been reported to have reduced inhibition to zanamivir and/or oseltamivir. 17 (Table 2). Variant viruses with either an I221T or I221V NA mutation have also been reported in a number of B viruses from USA and China. 18,19 Compared with wild-type viruses, the I221T variant reported here had a much greater increase in peramivir IC 50 (43-fold), than reported for the I221V variants from the USA, which exhibited an eight-fold increase. 19 I221T and A245T are both located near the substrate binding site of the NA (Figure 2). Apart from reductions in peramivir sensitivity, the I221T B variant also demonstrated reduced oseltamivir inhibition 17 , while the A245T mutation was found to affect sensitivity to three of the four NA inhibitors, oseltamivir (20-fold reduction), zanamivir (32-fold reduction) and peramivir (five-fold reduction), even though the residue is not located within the NA active site. The original clinical specimens of many of these isolates were not available to the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, for sequence analysis (details listed in Table 2) as clinical specimens are often discarded by submitting laboratories once virus isolates are cultured. Therefore, we were unable to investigate whether the mutations had arisen during cell culture, as has been the case for some NAI-resistant variants previously reported. 20

Laninamivir susceptibility
All 511 A(H1N1)pdm09, 1950 A(H3N2) viruses and 1238 influenza B had IC 50 values that fell within the normal laninamivir inhibition range. The mean (AEstandard deviation) laninamivir IC 50 values for A(H1N1)pdm09 and A (H3N2) viruses were 0Á27 AE 0Á05 nM and 0Á62 AE 0Á05 nM, respectively. The mean (AEstandard deviation) laninamivir IC 50 for the 1238 influenza B isolates was 3Á26 AE 0Á26 nM, 5-12-fold higher than the mean IC 50 of the influenza A (H1N1)pdm09 and A(H3N2) viruses. Again, no difference was observed between the laninamivir susceptibility of the two B lineages.

Conclusion
Although peramivir and laninamivir are currently only licensed in Japan (and in the case of peramivir, also in South Korea and China), approval in other countries is likely to follow as late-phase clinical trials are completed. Of the viruses analysed, none of the A(H1N1)pdm09, A(H3N2) and influenza B viruses had reduced or highly reduced laninamivir inhibition, while a small number of A(H1N1)pdm09 (3Á2%) and B (0Á5%) viruses had reduced peramivir susceptibility. Of the viruses considered to have normal inhibition, the mean and median values IC 50 for peramivir were lower than those for laninamivir for all of the three influenza types/ subtypes tested. The clinical implications of these differences, and for the H275Y variant viruses with highly reduced peramivir inhibition, are currently unknown and therefore require further study. However, given that the high concentration of peramivir achieved in the blood following intravenous administration 21 , it could be expected that the H275Y and influenza B variants detected here would be inhibited. However, clinical studies have suggested that peramivir is less effective when treating H275Y variant viruses compared with viruses with normal inhibition. 22  This study has found no evidence of widespread emergence of viruses with highly reduced peramivir or laninamivir inhibition since the market launch in 2010, although the increased number of community cases of A(H1N1)pdm09 viruses with H275Y NA mutation in 2011 (which had highly reduced peramivir inhibition) was concerning. However, a limitation of this study is that the majority of the viruses tested were from regions where peramivir or laninamivir has not been approved for use. Therefore, although our data suggest that peramivir-and laninamivir-resistant viruses are not spreading from regions where the drugs are being used, further studies are required to assess the susceptibility of Japanese and South Korean viruses collected from both drugtreated and untreated patients.