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Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. The traditional thiobarbituric acid (TBA) method to quantify NA inhibiting antibodies is cumbersome and not suitable for routine serology. An enzyme-linked lectin assay (ELLA) described by Lambre et al. (1990) is a practical alternative method for measuring NA inhibition (NI) titers. This report describes optimization of the ELLA for measuring NI titers in human sera against influenza A viruses, using H6N1 and H6N2 viruses as antigens. The optimized ELLA is subtype-specific and reproducible. While the titers measured by ELLA are somewhat greater than those measured by a miniaturized TBA method, seroconversion rates are the same, suggesting similarity in assay sensitivity under these optimized conditions. The ELLA described in this report provides a practical format for routine evaluation of human antibody responses to NA.

A(H1N1)pdm09 influenza A viruses predominated in the 2013–2014 USA influenza season, and although most of these viruses remain sensitive to Food and Drug Administration-approved neuraminidase (NA) inhibitors, alternative therapies are needed. Here we show that monoclonal antibody CD6, selected for binding to the NA of the prototypic A(H1N1)pdm09 virus, A/California/07/2009, protects mice against lethal virus challenge. The crystal structure of NA in complex with CD6 Fab reveals a unique epitope, where the heavy-chain complementarity determining regions (HCDRs) 1 and 2 bind one NA monomer, the light-chain CDR2 binds the neighbouring monomer, whereas HCDR3 interacts with both monomers. This 30-amino-acid epitope spans the lateral face of an NA dimer and is conserved among circulating A(H1N1)pdm09 viruses. These results suggest that the large, lateral CD6 epitope may be an effective target of antibodies selected for development as therapeutic agents against circulating H1N1 influenza viruses.

The predominance of A(H3N2) virus in recent influenza seasons has resulted in rigorous investigation on hemagglutinin, but little attention has been paid to the potential role of neuraminidase (NA). Here we show that since 2016, the S245N/S247T (introducing an N-linked glycosylation site at residue 245) and P468H mutations contributed to antigenic drift of the NA of circulating A(H3N2) viruses, compared with earlier viruses represented by the A/Hong Kong/4801/2014 vaccine strain. As a result, some human monoclonal antibodies, including those that have broad-reactivity with NA of the reference 1957 A(H2N2) and 1968 A(H3N2) pandemic viruses as well as contemporary seasonal A(H3N2) strains, lost binding to NA. This antigenic drift also reduced NA antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the NA245 glycosylation site is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift may impact protection against influenza virus infection, highlighting the importance of including NA antigenicity for consideration in optimizing influenza vaccines.

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