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Vaccination is a critical and reliable strategy for controlling the spread of influenza viruses in populations. Conventional seasonal split vaccines (SVs) for influenza evoke weaker immune responses than other types of vaccines, such as inactivated whole-virion vaccines, although SVs are highly safe compared to other types. Here, we assessed the potential of the lipid nanoparticle (LNP) we developed as an adjuvant for conventional influenza SV as an antigen in mice. The LNP did not induce the production of cytokines such as interleukin-6 (IL-6) and IL-12 p40 by dendritic cells or the expression of co-stimulatory molecules on these cells in vitro. In contrast, an SV adjuvanted with LNP improved SV-specific IgG1 and IgG2 responses and the Th1 response compared to the SV alone in mice. In addition, SV adjuvanted with an LNP gave superior protection against the influenza virus challenge over the SV alone and was as effective as SV adjuvanted with aluminum salts in mice. The LNP did not provoke inflammatory responses such as inflammatory cytokine production and inflammatory immune cell infiltration in mice, whereas aluminum salts induced inflammatory responses. These results suggest the potential of the LNP as an adjuvant without inflammatory responses for influenza SVs. Our strategy should be useful for developing influenza vaccines with enhanced efficacy and safety.

Current influenza vaccines are generally effective against highly similar (homologous) strains, but their effectiveness decreases markedly against antigenically mismatched (heterologous) strains. One way of developing a universal influenza vaccine with a broader spectrum of protection is to use appropriate vaccine adjuvants to improve a vaccine's effectiveness and change its immune properties. Oligodeoxynucleotides (ODNs) with unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG ODNs), which are Toll-like-receptor 9 (TLR9) agonists, are among the most promising adjuvants and are already being used in humans. However, the development of novel delivery vehicles to improve adjuvant effects in vivo is highly desirable. Here, we assessed the potential of lipid nanoparticles (LNPs) as CpG ODN delivery vehicles in mice to augment the vaccine adjuvant effects of CpG ODN and enhance the protective spectrum of conventional influenza split vaccine (SV). In vitro, compared with CpG ODN, LNPs containing CpG ODNs (LNP-CpGs) induced significantly greater production of cytokines such as IL-12 p40 and IFN-α by mouse dendritic cells (DCs) and significantly greater expression of the co-stimulatory molecules CD80 and CD86 on DCs. In addition, after subcutaneous administration in mice, compared with CpG ODN, LNP-CpGs enhanced the expression of CD80 and CD86 on plasmacytoid DCs in draining lymph nodes. LNP-CpGs given with SV from H1N1 influenza A virus improved T-cell responses and gave a stronger not only SV-specific but also heterologous-virus-strain-specific IgG2c response than CpG ODN. Furthermore, immunization with SV plus LNP-CpGs protected against not only homologous strain challenge but also heterologous and heterosubtypic strain challenge, whereas immunization with SV plus CpG ODNs protected against homologous strain challenge only. We therefore demonstrated that LNP-CpGs improved the adjuvant effects of CpG ODN and broadened the protective spectrum of SV against influenza virus. We expect that this strategy will be useful in developing adjuvant delivery vehicles and universal influenza vaccines.