The effectiveness of influenza vaccines against circulating A(H1N1)pdm09 viruses 
was modest for several seasons despite the absence of antigenic drift of 
hemagglutinin (HA), the primary vaccine component. Since antibodies against HA 
and neuraminidase (NA) contribute independently to protection against disease, 
antigenic changes in NA may allow A(H1N1)pdm09 viruses to escape from 
vaccine-induced immunity. In this study, analysis of the specificities of human 
NA-specific monoclonal antibodies identified antigenic sites that have changed 
over time. The impact of these differences on in vitro inhibition of enzyme 
activity was not evident for polyclonal antisera until viruses emerged in 2013 
without a predicted glycosylation site at amino acid 386 in NA. Phylogenetic and 
antigenic cartography demonstrated significant antigenic changes that in most 
cases aligned with genetic differences. Typical of NA drift, the antigenic 
difference is observed in one direction, with antibodies against conserved 
antigenic domains in A/California/7/2009 (CA/09) continuing to inhibit NA of 
recent A(H1N1)pdm09 viruses reasonably well. However, ferret CA/09-specific 
antiserum that inhibited the NA of A/Michigan/45/2015 (MI/15) very well in 
vitro, protected mice against lethal MI/15 infection poorly. These data show 
that antiserum against the homologous antigen is most effective and suggest the 
antigenic properties of NA should not be overlooked when selecting viruses for 
vaccine production.IMPORTANCE The effectiveness of seasonal influenza vaccines 
against circulating A(H1N1)pdm09 viruses has been modest in recent years, 
despite the absence of antigenic drift of HA, the primary vaccine component. 
Human monoclonal antibodies identified antigenic sites in NA that changed early 
after the new pandemic virus emerged. The reactivity of ferret antisera 
demonstrated antigenic drift of A(H1N1)pdm09 NA from 2013 onward. Passive 
transfer of serum raised against A/California/7/2009 was less effective than 
ferret serum against the homologous virus in protecting mice against a virus 
with the NA of more recent virus, A/Michigan/45/2015. Given the long-standing 
observation that NA-inhibiting antibodies are associated with resistance against 
disease in humans, these data demonstrate the importance of evaluating NA drift 
and suggest that vaccine effectiveness might be improved by selecting viruses 
for vaccine production that have NAs antigenically similar to those of 
circulating influenza viruses.