Immune Netw.  2017 Oct;17(5):326-342. 10.4110/in.2017.17.5.326.

Distinct Effects of Monophosphoryl Lipid A, Oligodeoxynucleotide CpG, and Combination Adjuvants on Modulating Innate and Adaptive Immune Responses to Influenza Vaccination

• Ko EJ
• Lee YT
• Lee Y
• Kim KH
• Kang SM

• KMID: 2400638
• DOI: http://doi.org/10.4110/in.2017.17.5.326

Abstract

Monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG are toll-like receptor (TLR) 4 and 9 agonist, respectively. Here, we investigated the effects of MPL, CpG, and combination adjuvants on stimulating in vitro dendritic cells (DCs), in vivo innate and adaptive immune responses, and protective efficacy of influenza vaccination. Combination of MPL and CpG was found to exhibit distinct effects on stimulating DCs in vitro to secrete IL-12p70 and tumor necrosis factor (TNF)-α and proliferate allogeneic CD8 T cells. Prime immunization of mice with inactivated split influenza vaccine in the presence of low dose MPL+CpG adjuvants increased the induction of virus-specific IgG and IgG2a isotype antibodies. MPL and CpG adjuvants contribute to improving the efficacy of prime influenza vaccination against lethal influenza challenge as determined by body weight monitoring, lung function, viral titers, and histology. A combination of MPL and CpG adjuvants was effective in improving vaccine efficacy as well as in reducing inflammatory immune responses locally and in inducing cellular immune responses upon lethal influenza virus challenge. This study demonstrates unique adjuvant effects of MPL, CpG, and combination adjuvants on modulating innate and adaptive immune responses to influenza prime vaccination.

Keyword

Monophosphoryl lipid A; Oligonucleotide CpG; Adjuvant; Influenza vaccine

MeSH Terms

Animals
Antibodies
Body Weight
Dendritic Cells
Immunity, Cellular
Immunization
Immunoglobulin G
In Vitro Techniques
Influenza Vaccines
Influenza, Human*
Lipid A*
Lung
Mice
Orthomyxoviridae
T-Lymphocytes
Toll-Like Receptors
Tumor Necrosis Factor-alpha
Vaccination*
Antibodies
Immunoglobulin G
Influenza Vaccines
Lipid A
Toll-Like Receptors
Tumor Necrosis Factor-alpha

Figure

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  Figure 1 In vitro activation of bone marrow-derived DCs by adjuvant stimulation. DCs were enriched from mouse bone marrow cells by treatment with mGM-CSF. (A-C) Cytokine levels secreted into the culture supernatants of DCs treated with different concentrations of MPL and CpG were measured by ELISA. For statistical analysis, Two-way ANOVA and Bonferroni post-multiple comparison tests were performed. (D-F) The immature DCs were cultured with MPL (0.2 μg/ml), CpG (1 μg/ml), or MPL (0.2 μg/ml)+CpG (1 μg/ml) for 2 days. Expression levels of DC activation markers were determined by flow cytometry. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison tests were performed. mGM-CSF, mouse granulocyte-macrophage colony stimulating factor. *p<0.033; **p<0.002; ***p<0.001 between the indicated groups.

• 

  Figure 2 In vitro proliferation and activation of T cells by adjuvant-activated DCs. DCs enriched from bone marrow cells were pre-activated by MPL (0.2 μg/ml), CpG (1 μg/ml), or MPL (0.2 μg/ml)+CpG (1 μg/ml) for 2 days. Allogeneic lymphocytes were harvested from spleens of C57BL/6 mice. CFSE-labeled lymphocytes and pre-activated DCs were co-cultured for 5 days. T cell proliferation and cytokine producing cells were determined by flow cytometry. (A) Proliferated CD4+ T cells. (B) Proliferated CD8+ T cells. (C) IFN-γ producing CD4+ T cells. (D) IFN-γ producing CD8+ T cells. (E) IL-4 producing CD4+ T cells. (F) IL-4 producing CD8+ T cells. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison tests were performed. ns, not significant between the indicated groups. *p<0.033; **p<0.002; ***p<0.001.

• 

  Figure 3 TLR agonist adjuvant effects on inducing IgG antibodies specific for influenza vaccine antigen. BALB/c mice (n=5) were immunized with sPR8 virus vaccine only or sPR8 virus vaccine in the presence of TLR agonist adjuvants (MPL, CpG, or MPL+CpG). Immune sera were taken 2 weeks after immunization and PR8 virus antigen-specific IgG antibody levels were measured by ELISA. IgG (A), IgG1 (B) and IgG2a (C) levels were shown in OD values at 450 nm. (D) IgG2a/IgG1 ratio was calculated at 102 times sera dilution. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison tests were performed. *p<0.033; **p<0.002; ***p<0.001 between the indicated groups or compared to sPR8 group; †p<0.033 compared to sPR8+MPL.

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  Figure 4 MPL and CpG adjuvant effects on improving protective efficacy of influenza vaccination after lethal virus infection. The immunized mice (n=5) were infected with A/PR8 virus (2×LD50) after 6 weeks of immunization. BW (A) and PenH (B) were measured for 7 days after infection and % changes were calculated based on the day 0. (C) Lung samples were harvested day 7 post infection. Lung virus titers of each immunized mice were measured by using embryonated eggs. EID50 were shown. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison tests were performed. (D) Lung histopathology. Intact lungs were harvested at day 7 post infection, fixed, processed, and stained with hematoxylin and eosin. EID50, 50% egg infection dose; inf., infection. ***p<0.001 compared to naïve infection group; †††p<0.001 compared to sPR8 group.

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  Figure 5 Cytokines and chemokines in lung samples after lethal virus infection of mice. Lung samples were harvested from the immunized mice (n=5) day 7 post A/PR8 virus infection. Cytokine and chemokine levels of each lung samples were measured by ELISA. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison test were performed. *p<0.033; **p<0.002; ***p<0.001 compared to Naïve infection group; †p<0.033; ††p<0.002; †††p<0.001 compared to sPR8 group.

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  Figure 6 Cellular infiltration in lungs after lethal virus infection. The immunized mice (n=5) were infected with A/PR8 virus (2×LD50) after 6 weeks of immunization. Lung samples were harvested day 7 post infection, and cell phenotypes were determined by flow cytometry and calculated by multiplying cell percentages with total cell numbers. (A) Total lung cells. (B) AMs; CD11b−CD11c+F4/80+. (C) Monocytes; CD11b+F4/80+Ly6Chigh. (D) Neutrophils; CD11b+F4/80−Ly6c+. (E) pDCs; CD45+F4/80−CD11c+MHCIIhighB220+. (F) CD103 + DC; CD45+F4/80−CD11c+MHCIIhighCD11b−CD103+. (G) cDC; CD45+F4/80−CD11c+MHCIIhighCD11b+. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison tests were performed. pDC, plasmacytoid dendritic cell; cDC, conventional DC. *p<0.033; **p<0.002 compared to naïve infection group; †p<0.033 compared to sPR8 group.

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  Figure 7 Cytokine producing T cells after immunization and lethal virus infection. The immunized mice were infected with a lethal dose (2×LD50) of A/PR8 virus after 6 weeks of immunization. Lung and BAL samples were harvested day 7 post infection. Intracellular cytokine staining was performed after incubation with MHCI and II-restricted peptides for CD8 and CD4 T cell stimulation as described in the Materials and Methods section. The cytokine producing cell numbers were calculated by multiplying cell percentages with total cell numbers. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison tests were performed. *p<0.033; **p<0.002 compared to naïve infection group; †p<0.033 compared to sPR8 group.

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  Figure 8 Cytokine production of spleen cells from the immunized mice after in vitro antigen stimulation. Spleen cells were harvested from the immunized mice day 7 post infection and then cultured with inactivated A/PR8 virus stimulation. After 3 days culture, cytokine levels in supernatants were determined by ELISA. All results were shown in mean±SEM. For statistical analysis, One-way ANOVA and Tukey's post-multiple comparison tests were performed. nd, not detected or values below detection limit. **p<0.002; ***p<0.001 compared to naïve infection group; ††p<0.002; †††p<0.001 compared to sPR8 group.

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