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Abstract Adjuvants play a central role in enhancing the immunogenicity of otherwise poorly immunogenic vaccine antigens. Combining adjuvants has the potential to enhance vaccine immunogenicity compared with single adjuvants, although the cellular and molecular mechanisms of combination adjuvants are not well understood. Using the influenza virus hemagglutinin H5 antigen, we define the immunological landscape of combining CpG and MPLA (TLR-9 and TLR-4 agonists, respectively) with a squalene nanoemulsion (AddaVax) using immunologic and transcriptomic profiling. Mice immunized and boosted with recombinant H5 in AddaVax, CpG+MPLA, or AddaVax plus CpG+MPLA (IVAX-1) produced comparable levels of neutralizing antibodies and were equally well protected against the H5N1 challenge. However, after challenge with H5N1 virus, H5/IVAX-1–immunized mice had 100- to 300-fold lower virus lung titers than mice receiving H5 in AddaVax or CpG+MPLA separately. Consistent with enhanced viral clearance, unsupervised expression analysis of draining lymph node cells revealed the combination adjuvant IVAX-1 significantly downregulated immune homeostasis genes, and induced higher numbers of antibody-producing plasmablasts than either AddaVax or CpG+MPLA. IVAX-1 was also more effective after single-dose administration than either AddaVax or CpG+MPLA. These data reveal a novel molecular framework for understanding the mechanisms of combination adjuvants, such as IVAX-1, and highlight their potential for the development of more effective vaccines against respiratory viruses. 

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.