Improved Immune Responses in Young and Aged Mice with Adjuvanted Vaccines against H1N1 Influenza Infection

Abstract

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.

Keywords: H1N1; T helper 1; adjuvant; elderly; influenza; vaccine.

Figure 1

Adjuvanted sH1N1 vaccines induce antigen-specific IgG1 and IgG2c antibodies in young mice and hemagglutination inhibition (HAI) titers in young and aged CB6F1 mice. (A) Scheme of immunization procedure: CB6F1 mice were immunized i.m. either once or twice, 3 weeks apart, and antibody analysis was determined on sera collected 3 weeks following immunization. All mice were challenged with 100LD₅₀ A/H1N1/California/4/2009 at day 54, and their physical condition was monitored over 14 days. Data for young mice were color-coded as blue; aged mice were color-coded as red for entire figure. (B) Sera from saline, sH1N1, sH1N1 + MF59-like, sH1N1 + SE, and sH1N1 + GLA-SE groups were analyzed for H1-specific IgG1 and IgG2c endpoint titers. Results are represented as the mean endpoint titer (log10) ± SEM. ** indicates p-value <0.01; *** indicates p-value <0.001; **** indicates p-value <0.0001. (C) Sera harvested from mice after a prime (day 20) or boost (day 42) immunization were analyzed for HAI titers. An HAI titer of five was assigned to responses below the assay detection limit. p-Values are denoted as follows: * indicates <0.05; ** indicates <0.01; *** indicates < 0.001; **** indicates < 0.0001.

Figure 2

Enhanced body weight and survival in young and aged CB6F1 mice with adjuvanted sH1N1 vaccines after boost immunization. Following immunization with adjuvanted and unadjuvanted sH1N1 vaccines (as shown in Figure 1A) and challenge with 100LD₅₀ A/H1N1/California/4/2009, (A) the body weight and (B) survival were monitored every day over the course of 14 days. Young mice were color-coded as blue; aged mice were color-coded as red. p-Values are denoted as follows: ** indicates < 0.01; *** indicates < 0.001; **** indicates < 0.0001. ^$ compares p value between saline vs. sH1N1 groups; ^& compares p value between saline vs. MF59-like groups; ^# compares p value between saline vs. SE groups; * compares p value between saline vs. GLA-SE groups.

Figure 3

MF59-like and GLA-SE-based sH1N1 vaccine promotes the generation of cytokine-producing T helper cells, GC B cells, and long-lived bone marrow plasma cells in young CB6F1 mice. (A) Scheme of slightly modified immunization procedure: CB6F1 mice were immunized i.m. twice, 4 weeks apart and (B) the immunogenicity of splenic cytokine-producing CD4⁺ T cells, (C) the percentage of CD4⁺CXCR5⁺PD-1⁺ Tfh cells in inguinal LNs, and (D) the number of B220⁺CD95⁺GL7⁺ GC B cells in spleen and in inguinal LNs were determined 1 week after the boost immunization with adjuvanted or unadjuvanted sH1N1 vaccines as indicated (E) The number of H1-specific BMPCs were determined 4 weeks after a boost immunization. Data from young mice were color-coded as blue; data from aged mice were color-coded as red. * indicates p value < 0.05; ** indicates p value < 0.01. Error bars indicate mean ± SEM. GC, germinal center; LN, lymph node; Tfh, follicular T helper cells; BMPC, bone marrow plasma cells. Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparison test except 3E, which used one-way ANOVA and Bonferroni’s test.

Figure 4

BMDCs from aged mice produce lower levels of cytokine upon GLA-SE stimulation and lung homogenates from aged mice have severely impaired cytokine-producing ability. (A) BMDCs and (B) lung homogenates from young and aged CB6F1 mice were stimulated with 10-fold dilutions of GLA-SE (2, 20, 200, 2, and 20 µg/ml) or SE (0.00008, 0.0008, 0.008, 0.08, and 0.8%) overnight, followed by harvesting the supernatants the next day for cytokine analysis. Open triangles indicate the concentration from highest (left) to the lowest (right). 10 ng/ml lipid A 506 was used as positive control. Cells from young mice were color-coded as blue; cells from aged mice were color-coded as red. Results are represented as the mean ± SEM. p-values are denoted as follows: * indicates < 0.05; ** indicates < 0.01; *** indicates < 0.001; **** indicates < 0.0001. BMDC, bone marrow dendritic cells.

Figure 4

BMDCs from aged mice produce lower levels of cytokine upon GLA-SE stimulation and lung homogenates from aged mice have severely impaired cytokine-producing ability. (A) BMDCs and (B) lung homogenates from young and aged CB6F1 mice were stimulated with 10-fold dilutions of GLA-SE (2, 20, 200, 2, and 20 µg/ml) or SE (0.00008, 0.0008, 0.008, 0.08, and 0.8%) overnight, followed by harvesting the supernatants the next day for cytokine analysis. Open triangles indicate the concentration from highest (left) to the lowest (right). 10 ng/ml lipid A 506 was used as positive control. Cells from young mice were color-coded as blue; cells from aged mice were color-coded as red. Results are represented as the mean ± SEM. p-values are denoted as follows: * indicates < 0.05; ** indicates < 0.01; *** indicates < 0.001; **** indicates < 0.0001. BMDC, bone marrow dendritic cells.

Figure 5

GLA-SE adjuvanted sH1N1 vaccine decreases viral load and prevents prolonged lung inflammation in young CB6F1 mice. Young CB6F1 mice (age of 6–8 weeks old) were immunized once with saline, sH1N1, sH1N1 + SE, or sH1N1 + GLA-SE, and sera were harvested 3 weeks after prime. All mice were challenged with 100LD₅₀ A/H1N1/California/4/2009 at day 28 and BALF and lung homogenates were harvested at day 3 and day 6 post-infection. (A) Hemagglutination inhibition (HAI) titer against A/H1N1/California/4/2009 was determined. (B) The relative expression of H1 was determined by real time Q-PCR. The expression of H1 of each sample was normalized to GAPDH (graph on log10 scale). (C) BALF from all infected mice were harvested at day 3 and day 6 postinfection, and the levels of IL-6, IFN-γ TNF-α, IL-12p70, IL-4, and IL-5 cytokines were determined by Luminex. (D) The percentage of AMs was determined by flow cytometry and were normalized to uninfected, naïve mice (as 100%). AMs in the lung homogenates were defined as CD11b⁻CD11c⁺Siglec-F⁺NK1.1⁻ population. (E) The intracellular expression of TLR7 by AMs was determined by flow cytometry and was normalized to uninfected, naïve mice (as 100%). p Values are denoted as follows: * indicates <0.05; ** indicates <0.01; *** indicates <0.001; **** indicates <0.0001. Results are represented as the mean ± SEM. BALF, bronchoalveolar lavage fluid; AM, alveolar macrophages.