Redirecting NEU (neuraminidase) antigen to autophagosomes confers enhanced cross-reactive T-cell immunity against heterosubtypic influenza virus infection

NEU (neuraminidase) is a potential cross-reactive antigen for developing broadly protective influenza vaccine, but has suboptimal immunogenicity. We here report that, when NEU antigen was redirected into phagophores, and subsequently autophagosomes, by fusing with MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; NEU-LC3B), it could efficiently activate the autophagosome-lysosome-major histocompatibility complex class II (MHC II) compartment pathway, and thus substantially improve the magnitude, breadth, and polyfunctionality of NEU-specific T cell immunity in mice. Remarkably, we identified several novel NEU-specific T-cell epitopes in response to NEU-LC3B-based immunization. Furthermore, mice immunized with NEU-based constructs were challenged with homologous A/CA/04/09 (H1N1), heterologous within-subtype strain A/Puerto Rico/8/1934 (PR8) (H1N1), and heterosubtypic A/Aichi/2/1968 (H3N2) virus, and the results demonstrated that NEU-LC3B-based vaccine provided a sterilizing immunity to homologous strains and cross-protection against antigenically distinct heterosubtypic challenge. In addition, cell depletion experiment demonstrated that T-cell-mediated immunity contributed to the NEU-LC3B-mediated immune protection. Collectively, this engineered NEU antigen with optimal immunogenicity represents a promising strategy for developing broadly protective influenza vaccines.
Abbreviations: BSA, bovine serum albumin; CQ, chloroquine; ELISpot, enzyme-linked immunosorbent spot; HA, hemagglutinin; ICS, intracellular cytokine staining; IFNG/IFN-γ, interferon gamma; LD50, Median Lethal Doses; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NEU, neuraminidase; NP, nucleoprotein; PBS, phosphate-buffered saline; RAPA, rapamycin; SFCs, spot-forming cells; SV, split vaccine; TCID50, median tissue culture infectious dose; VSV, vesicular stomatitis virus; VSVΔG, VSV vector with the deletion of the G gene.