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Abstract Most seasonal and pandemic influenza vaccines are derived from inactivated or attenuated virus propagated in chicken eggs, while more advanced delivery technologies, such as the use of recombinant proteins and adjuvants, are under‐utilized. In this study, the E2 protein nanoparticle (NP) platform is engineered to synthesize vaccines that simultaneously co‐deliver influenza hemagglutinin (H5) antigen, TLR5 agonist flagellin (FliCc), and TLR9 agonist CpG 1826 (CpG) all on one particle (termed H5‐FliCc‐CpG‐E2), with uniform molecular orientation significant for immunomodulation. Antigen‐bound NP formulations elicit higher IgG antibody responses and broader homosubtypic cross‐reactivity against different H5 variants than unconjugated antigen alone. IgG1/IgG2c skewing is modulated by adjuvant type and NP attachment. Conjugation of flagellin to the NP causes significant IgG1 (Th2) skewing while attachment of CpG yields significant IgG2c (Th1) skewing, and simultaneous conjugation of both flagellin and CpG results in a balanced IgG1/IgG2c (Th2/Th1) response. Animals immunized with E2‐based NP vaccines and subsequently challenged with H5N1 influenza show 100% survival, and only animals that receive adjuvanted NP formulations are also protected against morbidity. This investigation highlights that NP‐based delivery of antigen and multiple adjuvants can be designed to effectively modulate the strength, breadth toward variants, and bias of an immune response against influenza viruses. 

Abstract The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed ‘IVAX-1’) appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.