Intranasal prime-boost immunization with trivalent influenza virus neuraminidase proteins and conserved HCA2 sequences fused to a circularly permuted E. coli heat-labile enterotoxin B subunit

To develop a mucosal influenza vaccine, we used trivalent recombinant neuraminidase (NA) proteins in combination with a circularly permuted TT mutant of the E. coli heat-labile enterotoxin B subunit, LTB(TT), as a mucosal adjuvant. Recombinant tetrameric NA proteins—N1* (with N329/K331T glycan-masking mutations), N2, and BN—were engineered using the tetramerization domains from either Staphylothermus marinus tetrabrachion or human vasodilator-stimulated phosphoprotein, and produced using baculovirus-insect cell expression system. To enhance immune responses, conserved HCA2 sequences with three tandem repeats were genetically fused to LTB(TT), expressed in E. coli, and co-administered intranasally with the trivalent NA proteins. BALB/c mice were immunized using either three- or four-dose prime-boost regimens. In the three-dose regimen, mice receiving 10 μg each of N1*, N2, and BN with 10 μg LTB(TT) or 15 μg LTB(TT)-HCA2 developed higher IgG, NA-inhibiting (NAI), and replication-inhibition antibody titers against H3N2 and influenza B compared to H1N1. Increasing the N1* dose to 15 μg did not enhance responses. Despite eliciting only modest antibody responses against H1N1, but high titers against H3N2 and influenza B viruses, all groups immunized with the trivalent NA formulation combined with LTB(TT)-HCA2 exhibited complete protection and full weight recovery following both H1N1 and H3N2 viral challenges. In the four-dose regimens, using combinations of trivalent NA antigens with LTB(TT), LTB(TT)-HCA2, or LTB(TT)-HCA2 + poly I:C elicited stronger responses to H3N2 and influenza B than to H1N1 viruses. Nonetheless, all immunized groups demonstrated 100% survival and full weight recovery following H1N1 virus challenge. Although the four-dose prime–boost immunization regimen induced cross-subtype serum antibody responses against H7N9, the corresponding mucosal antibody responses were relatively weak and failed to confer protective immunity. These findings support an alternative strategy to influenza vaccine development by targeting the tetrameric ectodomain of NA as a vaccine antigen, and incorporating intranasal delivery, mucosal adjuvants, and conserved HCA2 sequences to advance a potential universal influenza vaccine platform.