Synergistic effect of non-neutralizing antibodies and interferon-γ for cross-protection against influenza.

Current influenza vaccines do not typically confer cross-protection against antigenically mismatched strains. To develop vaccines conferring broader cross-protection, recent evidence indicates the crucial role of both cross-reactive antibodies and viral-specific CD4+ T cells; however, the precise mechanism of cross-protection is unclear. Furthermore, adjuvants that can efficiently induce cross-protective CD4+ T cells have not been identified. Here we show that CpG oligodeoxynucleotides combined with aluminum salts work as adjuvants for influenza vaccine and confer strong cross-protection in mice. Both cross-reactive antibodies and viral-specific CD4+ T cells contributed to cross-protection synergistically, with each individually ineffective. Furthermore, we found that downregulated expression of Fcγ receptor IIb on alveolar macrophages due to IFN-γ secreted by viral-specific CD4+ T cells improves the activity of cross-reactive antibodies. Our findings inform the development of optimal adjuvants for vaccines and how influenza vaccines confer broader cross-protection.

Nasal-subcutaneous prime-boost regimen for inactivated whole-virus influenza vaccine efficiently protects mice against both upper and lower respiratory tract infections.

Although influenza vaccines are effective for reducing viral transmission and the severity of clinical symptoms, influenza viruses still induce considerable morbidity and mortality worldwide. Seasonal influenza viruses infect the upper respiratory tract initially but then often induce severe pulmonary complications in the lower respiratory tract. Therefore, influenza vaccines that prevent viral infection at both the upper and lower respiratory tracts are highly anticipated. Here, we examined whether using different vaccination routes for priming and boosting achieved protection in both regions of the respiratory tract. To this end, we used inactivated whole-virion influenza vaccines to immunize mice either subcutaneously or intranasally for both priming and boosting. Regardless of the route used for boosting, the levels of virus-specific IgG in plasma were higher in mice primed subcutaneously than those in control mice, which received PBS only. In addition, intranasal priming followed by subcutaneous boosting induced higher levels of virus-specific IgG in plasma than those in control mice. The levels of virus-specific nasal IgA were higher in mice that were primed intranasally than in control mice or in mice primed subcutaneously. Furthermore, intranasal priming but not subcutaneous priming provided protection against viral challenge in the upper respiratory tract. In addition, when coupled with subcutaneous boosting, both subcutaneous and intranasal priming protected against viral challenge in the lower respiratory tract. These results indicate that intranasal priming followed by subcutaneous boosting induces both virus-specific IgG in plasma and IgA in nasal washes and protects against virus challenge in both the upper and lower respiratory tracts. Our results will help to develop novel vaccines against influenza viruses and other respiratory viruses.

Lipid Nanoparticle Acts as a Potential Adjuvant for Influenza Split Vaccine without Inducing Inflammatory Responses.

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.

Murine Cross-Reactive Nonneutralizing Polyclonal IgG1 Antibodies Induced by Influenza Vaccine Inhibit the Cross-Protective Effect of IgG2 against Heterologous Virus in Mice.

Annual vaccination against influenza viruses is the most reliable and efficient way to prevent and control annual epidemics and protect from severe influenza disease. However, current split influenza vaccines are generally not effective against antigenically mismatched (heterologous) strains. To broaden the protective spectrum of influenza vaccines, adjuvants that can induce cross-reactive antibodies with cross-protection via Fc-mediated effector functions are urgently sought. Although IgG2 antibodies are generally more efficient than IgG1 antibodies in Fc-mediated effector functions, it is not yet clear which IgG isotypes show superior cross-protection against heterologous strains. It also remains unclear whether these IgG isotypes interfere with each other's protective effects. Here, we found that influenza split vaccine adjuvanted with aluminum salts, which predominantly induce cross-reactive IgG1, did not confer cross-protection against heterologous virus challenge in mice. In contrast, split vaccine adjuvanted with CpG oligodeoxynucleotides, which predominantly induce cross-reactive IgG2, showed cross-protection through the interaction of cross-reactive nonneutralizing IgG2 and alveolar macrophages, indicating the importance of cross-reactive nonneutralizing IgG2 for cross-protection. Furthermore, by using serum samples from immunized mice and isolated polyclonal antibodies, we show that vaccine-induced cross-reactive nonneutralizing IgG1 suppress the cross-protective effects of IgG2 by competitively inhibiting the binding of IgG2 to virus. Thus, we demonstrate the new concept that cross-reactive IgG1 may interfere with the potential for cross-protection of influenza vaccine. We propose that adjuvants that selectively induce virus-specific IgG2 in mice, such as CpG oligodeoxynucleotides, are optimal for heterologous protection.IMPORTANCE Current influenza vaccines are generally effective against highly similar virus strains by inducing neutralizing antibodies. However, these antibodies fail to neutralize antigenically mismatched (heterologous) strains and therefore provide limited protection against them. Efforts are being made to develop vaccines with cross-protective ability that would protect broadly against heterologous strains, because the mismatch between predicted and epidemic strains cannot always be avoided, resulting in low vaccine efficacy. Here, we show that nonneutralizing IgG2 antibodies induced by an optimal adjuvant play a crucial role in cross-protection against heterologous virus challenge in mice. Furthermore, nonneutralizing polyclonal IgG1 suppressed the cross-protective effects of nonneutralizing polyclonal IgG2 by competitively blocking the binding of IgG2 to its antigen. These data shed new light on the importance of IgG isotypes and the selection of appropriate adjuvants for the development of universal influenza vaccines. Furthermore, our findings are applicable to the rational design of vaccines against other pathogens.

Peptides with the multibasic cleavage site of the hemagglutinin from highly pathogenic influenza viruses act as cell-penetrating via binding to heparan sulfate and neuropilins.

Cell-penetrating peptides (CPPs) show promise as an attractive delivery vehicle for therapeutic molecules-including nucleic acids, peptides, proteins, and even particulates-into several cell types. It is important to identify new CPPs and select the optimal CPP for each application, because CPPs differ in their internalized efficiency and internalization mechanisms. Here, we identified new CPPs derived from the peptides with the hemagglutinin cleavage site (pHACS) of highly pathogenic influenza viruses. We compared the potential of peptides from the pHACS of four subtypes of influenza A virus (H1, H3, H5, and H7) and an influenza B virus (H1-pHACS, H3-pHACS, H5-pHACS, H7-pHACS, and B-pHACS, respectively) to serve as CPPs. H5-pHACS and H7-pHACS, but not the other peptides, bound to mouse dendritic cells and human epithelial cells and were internalized efficiently into these cells. H5-pHACS and H7-pHACS required glycosaminoglycans, especially heparan sulfate and neuropilins, to bind to the cells. In addition, we designed a mutant H7-pHACS with superior cell-binding capability by changing a single amino acid. Furthermore, when conjugated with antigen, H5-pHACS and H7-pHACS induced antigen-specific antibody responses, demonstrating the usefulness of this antigen-delivery vehicle. Our results will improve our understanding of the mechanisms of CPPs and facilitate the development of novel drug-delivery vehicles designed to improve therapeutic efficacy.

Lipid Nanoparticles Potentiate CpG-Oligodeoxynucleotide-Based Vaccine for Influenza Virus.

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.