Immunization with N2 neuraminidase can protect mice against a heterologous influenza A virus challenge even in the absence of cross-NA inhibiting antibodies

Neuraminidase (NA) inhibition (NAI) titers have been identified as an independent correlate of protection against influenza. Few studies, however, have investigated the breadth of NA-based immune protection. Previously, we have reported that N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017 can be subdivided into four antigenic groups. Here, we (i) immunized mice with recombinant soluble tetrameric NA from H3N2 strains representing those four antigenic groups or (ii) passively transferred N2 NA immune serum into naïve mice to evaluate the breadth of protection against a heterologous HxN2 influenza virus challenge. We show that the breadth of protection goes beyond the breadth of NAI and is associated with the presence of cross-reactive antibodies. Interestingly, in the absence of cross-reactive antibodies, the immunization of DBA/2J mice with heterologous recombinant N2 NA from A/Perth/16/2019 was associated with an early onset of disease upon challenge with HxN2 virus with NA from A/Indiana/08/2011 (H3N2) or with H2N2 A/Singapore/1/1957. This early onset of disease was unidirectional and was not observed when mice that had been immunized with recombinant NA from A/Indiana/08/2011 were challenged with HxN2 virus with NA from A/Perth/16/2009 (H3N2).IMPORTANCEAlthough seasonal influenza vaccines mainly target hemagglutinin (HA), the viral surface protein neuraminidase (NA) can also induce protective immunity. In an earlier work, we profiled neuraminidase inhibition across a broad panel of H3N2 viruses and defined four major NA antigenic groups. Here, we tested whether immunizing mice with NA elicits cross protection against challenge viruses carrying NAs from different antigenic groups. Our data separate the predictive value of NA-binding and NA-inhibiting antibody responses and show that protection can extend beyond measurable NA inhibition when cross-reactive antibodies are present. These findings support the inclusion of standardized NA antigens as vaccine components to enhance the breadth of protection offered by seasonal influenza vaccine.