Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current "one-shot" universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA stalk-reactive antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) was

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... nt for an attached substrate but not for unattached small-molecule cleavage of sialic acid. This finding suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab′)2 fragments that occupied reduced area without loss of avidity or disrupted HA/NA interactions showed significantly reduced NI activity. Notably, HA stalk-binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of protection mediated by HA stalk-reactive antibodies. IMPORTANCE This study reports a new mechanism of protection mediated by influenza hemagglutinin stalk-reactive antibodies, i.e., inhibition of neuraminidase activity by steric hindrance, blocking access of neuraminidase to sialic acids when it abuts hemagglutinin on whole virions.