Co-Delivery of Multiple Toll-Like Receptor Agonists and Avian Influenza Hemagglutinin on Protein Nanoparticles Enhances Vaccine Immunogenicity and Efficacy.

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.