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.