Design of the recombinant influenza neuraminidase antigen is crucial for protective efficacy

Abstract

Supplementing influenza vaccines with recombinant neuraminidase (rNA) remains a promising approach for improving the suboptimal efficacy. However, correlations among rNA designs, properties, and protection have not been systematically investigated. Here, we performed a comparative analysis of several rNAs produced from different construct designs using the baculovirus/insect cell system. The rNAs were designed with different tetramerization motifs and NA domains from a recent H1N1 vaccine strain (A/Brisbane/02/2018) and were analyzed for enzymatic properties, antigenicity, thermal and size stability, and protection in mice. We found that rNAs containing the NA head-domain versus the full-ectodomain possess distinct enzymatic properties and that the molecular size stability is tetramerization domain-dependent, whereas protection is more contingent on the combination of the tetramerization and NA domains. Following single-dose immunizations, a rNA possessing the full-ectodomain, non-native enzymatic activity, and the tetramerization motif from the human vasodilator-stimulated phosphoprotein provided substantially higher protection than a rNA possessing the head-domain, native activity and the same tetramerization motif. In contrast, these two rNAs provided comparable protection when the tetramerization motif was exchanged with the one from the tetrabrachion protein. These findings demonstrate that the rNA design is crucial for the protective efficacy and should be thoroughly evaluated for vaccine development, as the unpredictable nature of the heterologous domain combination can result in rNAs with similar key attributes but vastly differ in protection. IMPORTANCEFor several decades it has been proposed that influenza vaccines could be supplemented with recombinant neuraminidase (rNA) to improve the efficacy. However, some key questions for manufacturing stable and immunogenic rNA remain to be answered. We show here that the tetramerization motifs and NA domains included in the rNA construct design can have a profound impact on the biochemical, immunological and protective properties. We also show that the single-dose immunization regimen is more informative for assessing the rNA immune response and protective efficacy, which is surprisingly more dependent on the specific combination of NA and tetramerization domains than common attributes for evaluating NA. Our findings may help to optimize the design of rNAs that can be used to improve or develop influenza vaccines.