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

Abstract

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.

Keywords: adjuvant; dendritic cell; inflammation; influenza virus; lipid nanoparticle; vaccine.

Figure 1

Cytokine production by mouse-derived dendritic cells (DCs) in response to the lipid nanoparticle (LNP) and LNP-cytosine–phosphate–guanine (LNP-CpG) in vitro. Mouse-derived DCs were treated with the LNP (13.3 or 4.4 μg lipid/mL) or LNP-CpG (13.3 μg lipid with 1.0 μg CpG oligodeoxynucleotide (ODN)/mL or 4.4 μg lipid with 0.33 μg CpG ODN/mL) for 24 h in vitro. (a) Levels of interleukin (IL)-6, IL-12 p40, tumor necrosis factor (TNF)-α and interferon (IFN)-α in the supernatants were measured by using ELISA. (b) Expression levels of CD80 and CD86 on DCs were measured by flow cytometry; percentages of positive DCs are shown. (a,b) n = 5 per group. Data are means ± SD. **** p < 0.0001, as indicated by Tukey’s test.

Figure 2

Split vaccine (SV)-specific antibody responses in vivo after first immunization. Mice were subcutaneously immunized with the SV alone, SV plus LNP, SV plus LNP-CpG, or SV plus alum on day 0. On day 14, levels of SV-specific total IgG, IgG1, IgG2b, and IgG2c in plasma were evaluated by using ELISA. We used 800- (●), 4000- (■), and 20,000- (▲) fold diluted plasma samples. n = 5 per group. Data are means ± SD. Significant differences were only analyzed in the 800-fold-diluted plasma samples. ^††† p < 0.001, and ^†††† p < 0.0001 vs. group immunized with the SV alone; * p < 0.05, ** p < 0.01, and **** p < 0.0001, as indicated by Tukey’s test.

Figure 3

SV-specific antibody responses in vivo after second immunization. Mice were subcutaneously immunized with the SV alone, SV plus LNP, SV plus LNP-CpG, or SV plus alum on days 0 and 21. (a) On day 28, levels of SV-specific total IgG, IgG1, IgG2b, and IgG2c in plasma were evaluated by using ELISA. We used 4000- (●), 20,000- (■), and 100,000- (▲) fold diluted plasma samples. (b) Neutralization titers in plasma samples against Cal7 were evaluated. (a,b) n = 5 per group. Data are means ± SD. (a) Significant differences were only analyzed in the 4000-fold-diluted plasma samples. (a,b) ^† p < 0.05, ^†† p < 0.01, ^††† p < 0.001, and ^†††† p < 0.0001 vs. group immunized with the SV alone; * p < 0.05, ** p < 0.01, and **** p < 0.0001, as indicated by Tukey’s test.

Figure 4

SV-specific T-cell responses in vivo. Mice were subcutaneously immunized with the SV alone, SV plus LNP, SV plus LNP-CpG, or SV plus alum on days 0 and 21. On day 28, splenocytes were cultured in the presence or absence of the SV in vitro. After 1 (for IL-2) or 5 (for IFN-γ and IL-13) days, the levels of IL-2, IFN-γ, and IL-13 were measured by using ELISA. n = 5 per group. Data are means ± SD. ^†††† p < 0.0001 vs. group immunized with the SV alone; **** p < 0.0001, as indicated by Tukey’s test.

Figure 5

Protective effects against influenza virus challenge. Mice were subcutaneously immunized with the SV alone, SV plus LNP, SV plus LNP-CpG, or SV plus alum on day 0. On day 21, mice were challenged with Cal7. (a) Percentages of initial body weights and (b) survival rates were monitored after challenge with Cal7. n = 10. (a) Data are means ± SD.

Figure 6

Hemagglutinin (HA)-specific antibody responses in vivo. Mice were subcutaneously immunized with recombinant HA (rHA) alone, rHA plus LNP, rHA plus LNP-CpG, or rHA plus alum on days 0 and 21. On day 28, the levels of rHA-specific total IgG, IgG1, IgG2b, and IgG2c in plasma were evaluated by using ELISA. We used 160- (●), 800- (■), and 4000- (▲) fold diluted plasma samples. n = 5. Data are means ± SD. Significant differences were only analyzed in the 160-fold-diluted plasma samples. ^† p < 0.05 and ^†††† p < 0.0001 vs. group immunized with rHA alone; ** p < 0.01, *** p < 0.001, and **** p < 0.0001, as indicated by Tukey’s test.

Figure 7

HA-specific T-cell responses in vivo. Mice were subcutaneously immunized with rHA alone, rHA plus LNP, rHA plus LNP-CpG, or rHA plus alum on days 0 and 21. On day 28, splenocytes were cultured in the presence or absence of rHA in vitro. After 1 (for IL-2) or 5 (for IFN-γ and IL-13) days, the levels of IL-2, IFN-γ, and IL-13 were measured by using ELISA. n = 5 per group. Data are means ± SD. ^† p < 0.05 and ^†††† p < 0.0001 vs. group immunized with rHA alone; * p < 0.05, **** p < 0.0001, as indicated by Tukey’s test.

Figure 8

Recruitment of inflammatory immune cells by the LNP and alum in vivo. Mice were treated intraperitoneally with the SV alone, SV plus LNP, or SV plus alum. (a,b) After 20 h, the numbers of macrophages, eosinophils, neutrophils, and inflammatory monocytes in peritoneal lavage fluid were analyzed by flow cytometry. Gating schema for macrophages (Mac), eosinophils (Eosi), neutrophils (Neut), and inflammatory monocytes (iMono) are shown in (a). n = 5 per group. Data are means ± SD. ^†† p < 0.01, ^††† p < 0.001, and ^†††† p < 0.0001 vs. untreated control group; *** p < 0.001 and **** p < 0.0001, as indicated by Tukey’s test.

Figure 9

Production of inflammatory cytokines by the LNP and alum in vivo. Mice were treated intraperitoneally with the SV alone, SV plus LNP, or SV plus alum. After 4 h, the concentrations of IL-6 and granulocyte-colony stimulating factor (G-CSF) in peritoneal lavage fluid were measured by using ELISA. n = 5 per group. Data are means ± SD. ^††† p < 0.001 and ^†††† p < 0.0001 vs. untreated control group; *** p < 0.001 and **** p < 0.0001, as indicated by Tukey’s test.