Discussion
The hemagglutinin molecule of H1N1 influenza possesses several important antigenic sites, which are targeted by hemagglutination inhibiting (HI) antibodies (15, 16). These epitopes are named Sa, Sb, Ca1, Ca2, and Cb (4). They are subjected to antibody-mediated immune pressure and are therefore variable (16). Strain(S)-specific sites are located at the top of the hemagglutinin head and display high levels of variation, whereas cross-reactive(C)-sites display less variability, most likely due to their more sideward (less exposed) location on the hemagglutinin. The antigenic sites are targeted by different species in a different manner, a phenomenon called immunodominance (17). HA-reactive antibodies of humans are preferentially affected by both sites Sb and Sa, whereas those of ferrets are mainly affected by site Sa (17). Therefore, similarities exist between both species regarding epitope Sa. HA antigenic drift occurs in response to increasing population immunity induced by infection or vaccination, which acts as a strong evolutionary pressure (18). The influenza season 2019-2020 has been the third consecutive season featuring prominent circulation of A(H1N1)pdm09 viruses in Germany: In 2017-2018, shortly after B/Yamagata influenza viruses had passed their peak, A(H1N1)pdm09 influenza viruses surged, effectively constituting a second influenza wave within that season. In 2018-2019 A(H1N1)pdm09 viruses circulated in parallel with A(H3N2) viruses. In 2019-2020, a similar pattern of A(H1N1)pdm09 and A(H3N2) viruses circulating in parallel was observed, which was followed by the circulation of B/ Victoria influenza viruses. Circulation of A(H1N1)pdm09 viruses over three successive seasons thus far may have induced higher-than-usual population immunity levels, thus augmenting evolutionary pressure on A(H1N1)pdm09 viruses. The emergence of antigenic variants displaying substantially reduced reactivity towards antisera against vaccine viruses would be a logical consequence of this pressure. The data reveal a clear intra-seasonal antigenic drift within the influenza A(H1N1)pdm09 subtype, which is associated with the N156K substitution in the Sa antigenic site of the hemagglutinin. Importantly, this kind of antigenic drift has been predicted by Guarnaccia et al. (2013), based on the results of sequential passaging studies in ferrets: HA-N156K emerged during serial passaging of A(H1N1)pdm09 virus in suboptimally vaccinated ferrets, where the N156 HA variant outgrew wildtype virus and showed altered receptor binding preferences (18). Igarashi et al. (2010) predicted similar evolutionary trends, based onin silico analyses of the previous seasonal, and historical A(H1N1) viruses (5).
A(H1N1)pdm09 viruses have thus far been highly immunogenic and in most years, vaccine effectiveness against A(H1N1)pdm09 viruses exceeded that observed for A(H3N2) and influenza B viruses (7). Patient data analysis did not indicate a reduction of vaccine effectiveness, probably for the following reasons: (i) there is still cross reactivity between these viruses and the vaccine virus at lower level; ii) there is probably still a high level of population immunity camouflaging effects of cross-reactivity at lower level. However, time to medical consultation medians differed by 1 day between N156K and non-N156K viruses. Although N156K is not located in the receptor binding site, it is positioned at the interface of three antibody binding sites (19-21). It has been postulated to increase receptor binding avidity, thereby supporting extensive replication (18). The shorter time to medical consultation observed in patients infected with H156K HA viruses may be an indicator of this presumed extensive replication pattern. The proportion of patients receiving antiviral therapy was higher among those infected with HA-N156K viruses, which could be related to more severe clinical courses of infection with these variants.
To date, A(H1N1)pdm09 viruses are sensitive to neuraminidase inhibitors (https://influenza.rki.de/Wochenberichte.aspx). Among the N156K viruses, only one displayed a mutation associated with mild drug resistance, similar to that observed in Australia (14).
The investigations reflect influenza A(H1N1)pdm09 virus hemagglutinin antigenic drift, following ten years of circulation of these viruses in the human population. These drift variants, characterized by an asparagine to lysine exchange at position 156 of the HA gene, emerged in different genetic clades during the same season, indicating convergent evolution. This HA-N156K substitution arose under conditions favoring increased immune selection pressure on A(H1N1)pdm09 viruses, given three consecutive years of prominent circulation, which likely augmented population immunity. HA-N156K viruses still react with antiserum directed against the vaccine virus A/Brisbane/02/2018 but at substantially reduced levels; ultimately, this may result in a shorter duration of vaccine-induced immunity, which could considerably impact vaccine effectiveness and have important public health implications. These findings were taken into consideration when the decision was made on the 2020 vaccine composition: A(H1N1)pdm09 HA-N156K viruses were included in the 2020 influenza vaccines recommended for the Southern hemisphere and in 2020/21 influenza vaccines for the Northern hemisphere. In Germany, as well as in other countries, the 2019-2020 influenza season was cut short by COVID-19 targeted non-pharmaceutical interventions (NPIs) (22). Similarly, 2020/21 influenza activity was historically low. Thus, it remains to be seen whether these NPIs have led to the extinction of this first A(H1N1)pdm09 HA antigenic drift variant as an unintended benefit. Taken together with animal model data predicting this same amino acid exchange as the basis for immune escape (18), our findings indicate that even if HA-N156K variants have been extinguished for now, they may reemerge in future influenza seasons. This highlights the major importance of laboratory-based virological surveillance in order to monitor viral evolution, which gives rise to novel pathogen variants, thereby carrying important public health implications.