The 2008-2009 H1N1 influenza virus exhibits reduced susceptibility to antibody inhibition: Implications for the prevalence of oseltamivir resistant variant viruses

Abstract

A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008-2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant.

Figure 1

Comparison of hemagglutination-inhibition activity of antisera against NA 275H and 275Y H1N1 clinical isolates. (A) HI titers were estimated for 20 antisera obtained from individuals immunized with seasonal trivalent vaccine containing A/Brisbane/59/2007-like H1N1 virus. Each of the antisera were tested against 10 susceptible (NA-275H) and 21resistant (NA-275Y) strains of A/Brisbane/59/2007-like H1N1 virus, and the average HI titer for each virus group (susceptible and resistant) calculated for each antisera. The proportion of the antisera samples which inhibited hemagglutination at each progressive antisera dilution based on the average HI titer calculated above was then plotted for the susceptible and resistant virus groups. Error bars represent standard deviation between different virus isolates. (B) HI titers associated with 176 H1 positive antisera identified from 1192 individuals, who had no history of receiving influenza vaccine, were estimated using the A/HK/04633/2008 (NA-275H) and A/HK/17599/2009 (NA-275Y) H1N1 viruses, and plotted as in (A). Statistical significance was determined using the t-test for sera from vaccinated individuals (A) or McNemar test for sera from non-vaccinated individuals (B). Asterisk indicates that the difference in HI reactivity between oseltamivir susceptible and resistant strains is statistically significant (* p<0.05, ** p<0.01).

Figure 2

Phylogenetic trees for the HA (A) and NA (B) genes of H1N1 viruses isolated in Hong Kong during the 2008–2009 influenza seasons. The nucleotide sequences were analyzed with PAUP* software, using the neighbor-joining method with 1000 bootstraps. Sequences of H1N1 viruses characterized in this study are colored blue. Red-hued sequences represent H1N1 influenza vaccine strains recommended by the World Health Organization. Star (*) indicates NA H275Y variant of the H1N1 virus. Genetic lineages of Clade 1, 2A, 2B and 2C H1N1 viruses were based on previous reports (Collins et al., 2009; Hauge et al., 2009).

Figure 3

Testing of the effect of neuraminidase inhibitor treatment on the reactivity of antibodies with oseltamivir susceptible and resistant H1N1 viruses. Hemagglutination inhibition testing was conducted with 20 immunized human sera, with or without pretreatment of viruses with 5uM of the neuraminidase inhibitor, zanamivir. Percentage of sera samples with HI titer > 1:40 represents average from 3 independent experiments. Error bars represent standard deviations between different isolates. Statistical significance was determined using the McNemar test. Asterisk (*) indicates the difference is statistically significant (p value <0.01).

Figure 4

Comparison of hemagglutination-inhibition activity of antisera against NA 275Y and 275H H1N1 recombinant viruses. HA and NA derived from A/Hong Kong/62768/2008, together with six internal genes from the PR8 strain, were used to construct recombinant viruses. One version contains wild type HA and NA (275Y) genes while the other contains wild type HA and NA with residue 275Y mutated to 275H. Recombinant viruses were tested against 20 sera obtained from seasonal flu vaccine immunized individuals, as described in Figure 1A. The experiment was repeated three times and data statistically analyzed using the McNemar test. Error bars represent standard deviation.