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bioRxiv · 10.1101/2024.07.09.602673

Triton-X 100-treated virus-based ELLA demonstrates discordant antigenic evolution of influenza B virus haemagglutinin and neuraminidase

Koutsakos, M.·Do, T. T. H.·Wille, M.·Wheatley, A. K.
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Abstract

Neuraminidase (NA)-specific antibodies have been associated with protection against influenza and thus NA is considered a promising target for next-generation vaccines against influenza A (IAV) and B viruses (IBV). NA inhibition (NI) by antibodies is typically assessed using an enzyme-linked lectin assay (ELLA). However, ELLA can be confounded by anti- hemagglutinin (anti-HA) antibodies that block NA by steric hindrance (termed HA interference). While strategies have been employed to overcome HA interference for IAV, similar approaches have not been assessed for IBV. We found HA interference is common in ELLA using IBV, rendering the technique unreliable. Anti-HA antibodies were not completely depleted from sera by HA-expressing cell lines and this approach was of limited utility. In contrast, we find that treatment of virions with Triton-X 100, but not Tween-20 or ether, efficiently separates the HA and NA components and overcomes interference caused by anti-HA antibodies. We also characterise a panel of recombinant IBV NA proteins that further validated the results from Triton-X 100-treated virus-based ELLA. Using these reagents and assays we demonstrate discordant antigenic evolution between IBV NA and HA over the last 80 years. This optimized ELLA protocol will facilitate further in-depth serological surveys of IBV immunity as well as antigenic characterisation of the IBV NA on a larger scale. ImportanceInfluenza B viruses contribute to annual epidemics and may cause severe disease, especially in children. Consequently, several approaches are being explored to improve vaccine efficacy, including the addition of neuraminidase. Antigen selection and assessment of serological responses will require a reliable serological assay to specifically quantify Neuraminidase inhibition. While such assays have been assessed for influenza A viruses, this has not been done of influenza B viruses. Our study identifies a readily applicable strategy to measure inhibitory activity of neuraminidase-specific antibodies against influenza B virus without interference from anti-hemagglutinin antibodies. This will aid broader serological assessment of influenza B virus-specific antibodies and antigenic characterisation of the influenza B virus neuraminidase.

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Koutsakos, M., Do, T. T. H., Wille, M., Wheatley, A. K.. 2024-07-09. Triton-X 100-treated virus-based ELLA demonstrates discordant antigenic evolution of influenza B virus haemagglutinin and neuraminidase. https://doi.org/10.1101/2024.07.09.602673

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