Significance: A noninvasive intranasal (i.n.) influenza vaccine can induce mucosal immune responses in respiratory tracts, preventing infection at the portal of virus entry. However, the absence of appropriate mucosal adjuvants at present hinders the development of such a vaccine. Here, we developed polyethyleneimine-functionalized two-dimensional graphene oxide nanoparticles (GP) that showed high antigen-loading capacities and superior immunoenhancing properties. Robust and broadly reactive immune responses were induced with i.n. immunization with GP-HA nanoparticles, conferring protection against homologous and heterologous viruses. With versatility and flexibility, GP nanoparticles can be easily adapted for constructing mucosal vaccines of different respiratory pathogens.

The intranasal (i.n.) route is an ideal vaccination approach for infectious respiratory diseases like influenza. Polycationic polyethylenimine (PEI) could form nanoscale complexes with negatively charged viral glycoproteins. Here we fabricated PEI-hemagglutinin (HA) and PEI-HA/CpG nanoparticles and investigated their immune responses and protective efficacies with an i.n. vaccination regimen in mice. Our results revealed that the nanoparticles significantly enhanced HA immunogenicity, providing heterologous cross-protection. The conserved HA stalk region induced substantial antibodies in the nanoparticle immunization groups. In contrast to the Th2-biased, IgG1-dominant antibody response generated by PEI-HA nanoparticles, PEI-HA/CpG nanoparticles generated more robust and balanced IgG1/IgG2a antibody responses with augmented neutralization activity and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). PEI-HA/CpG nanoparticles also induced enhanced local and systemic cellular immune responses. These immune responses did not decay over six months of observation postimmunization. PEI and CpG synergized these comprehensive immune responses. Thus, the PEI-HA/CpG nanoparticle is a potential cross-protective influenza vaccine candidate. Polycationic PEI nanoplatforms merit future development into mucosal vaccine systems.

Enhancing influenza vaccine cross-protection is imperative to alleviate the significant public health burden of influenza. Heterologous sequential immunization may synergize diverse vaccine formulations and routes to improve vaccine potency and breadth. Here we investigate the effects of immunization strategies on the generation of cross-protective immune responses in female Balb/c mice, utilizing mRNA lipid nanoparticle (LNP) and protein-based PHC nanoparticle vaccines targeting influenza hemagglutinin. Our findings emphasize the crucial role of priming vaccination in shaping Th bias and immunodominance hierarchies. mRNA LNP prime favors Th1-leaning responses, while PHC prime elicits Th2-skewing responses. We demonstrate that cellular and mucosal immune responses are pivotal correlates of cross-protection against influenza. Notably, intranasal PHC immunization outperforms its intramuscular counterpart in inducing mucosal immunity and conferring cross-protection. Sequential mRNA LNP prime and intranasal PHC boost demonstrate optimal cross-protection against antigenically drifted and shifted influenza strains. Our study offers valuable insights into tailoring immunization strategies to optimize influenza vaccine effectiveness.

Influenza is one of the major threats to public health. Current influenza vaccines cannot provide effective protection against drifted or shifted influenza strains. Researchers have considered two important strategies to develop novel influenza vaccines with improved immunogenicity and broader protective efficacy. One is applying fewer variable viral antigens, such as the haemagglutinin stalk domain. The other is including adjuvants in vaccine formulations. Adjuvants are promising and helpful boosters to promote more rapid and stronger immune responses with a dose-sparing effect. However, few adjuvants are currently licensed for human influenza vaccines, although many potential candidates are in different trials. While many advantages have been observed using adjuvants in influenza vaccine formulations, an improved understanding of the mechanisms underlying viral infection and vaccination-induced immune responses will help to develop new adjuvant candidates. In this review, we summarize the works related to adjuvants in influenza vaccine research that have been used in our studies and other laboratories. The review will provide perspectives for the utilization of adjuvants in developing next-generation and universal influenza vaccines.

MRNA-based vaccine technology has been significantly developed and enhanced, particularly highlighted by the authorization of mRNA vaccines for addressing the COVID-19 pandemic. Various biomaterials are developed in nano-scales and applied as mRNA vaccine delivery platforms. However, how these mRNA nanoplatforms influence immune responses has not been thoroughly studied. Hence, we have reviewed the current understanding of various mRNA vaccine platforms. We discussed the possible pathways through which these platforms moderate the host’s innate immunity and contribute to the development of adaptive immunity. We shed light on their development in reducing biotoxicity and enhancing antigen delivery efficiency. Beyond the built-in adjuvanticity of mRNA vaccines, we propose that supplementary adjuvants may be required to fine-tune and precisely control innate immunity and subsequent adaptive immune responses.