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Evaluation of the influenza vaccine protection in the house dust mite-induced chronic allergic asthma mice model and the evaluation of squalene oil in water emulsion as an adjuvant candidate

Respiratory Research volume 26, Article number: 132 (2025) Cite this article

Abstract

Background

Despite the importance of influenza vaccination in asthma patients, the efficacy of this vaccine in asthma has not been well elucidated. We aimed to compare the efficacy of an influenza vaccine of the asthmatic and control mice. We also evaluated the efficacy of AddaVax™ as an adjuvant candidate, which is equivalent to the MF59 influenza vaccine adjuvant in the elderly.

Method

House dust mite extracts were intranasally injected into six-week-old female BALB/c mice to induce chronic allergic asthma. Antibody responses after split-inactivated A/Puerto Rico/8/34 H1N1 influenza vaccination with or without AddaVax™ adjuvant were measured using ELISA. Homologous viral protection was determined by measuring the survival rate, lung inflammation level, and lung virus titer after challenge with the human influenza virus strain A/Puerto Rico/8/1934 H1N1. Antigen-specific T cell responses were determined using flow cytometry.

Result

The chronic asthma mice immunized with split-inactivated A/Puerto Rico/8/34 H1N1 influenza vaccine showed significant weight loss and higher lung viral load after homologous influenza infection than naïve vaccinated mice. Antigen-specific IgG, IgG1, and IgG2a production did not differ between the naïve and asthma mice. However, serum HI titer was lower in asthma-vaccinated mice after infection. The application of AddaVax™ to a vaccine for mice with asthma enhanced the efficacy of homologous antiviral protection but elicited eosinophil infiltration in the lungs after homologous influenza virus infection.

Conclusion

The immune response after split inactivated A/PR8 vaccine differed between asthma and naïve mice, particularly in terms of antibody activity and T cell populations. This study enhances our understanding of how asthma status may influence the effectiveness of influenza vaccine and offers insights into the AddaVax™-induced eosinophilic inflammation, guiding the development of virus vaccine strategies for both healthy individuals and asthma patients.

Background

Asthma is a chronic airway inflammatory disease that affects 300 million people worldwide [1]. They have altered immune status and structural remodeling in the airway due to allergen-induced chronic inflammation [2, 3]. Malfunction of the airway and exacerbation of asthma increase the risk of developing severe complications of influenza virus infection. During the 2009 influenza A H1N1pandemic (pH1N1), asthma was one of the most common underlying medical conditions among patients hospitalized for pH1N1 infections in the U.S. and worldwide [4]. However, despite the importance of influenza vaccination in patients with asthma, there has been limited research on the effectiveness of vaccines and the detailed T cell function against viral infection in asthma.

Allergic asthma is the most common type of asthma [5]. It exhibits pathological features of airway remodeling, including airway thickening, narrowing, edema, and mucous plugging [6, 7]. It is immunologically characterized by allergen-specific immunoglobulin E (IgE) production and eosinophil-dominant T-helper type 2 (Th2)-biased inflammation [8,9,10]. In a previous study, we demonstrated an increased percentage of exhausted T cells after chronic allergen exposure, with decreased influenza virus clearance in an asthma mouse model [11]. T cell exhaustion refers to the gradual regression of T cell function and number owing to the persistence of low-grade chronic inflammation [12, 13]. These findings suggest that asthma can cause immune dysfunction owing to prolonged inflammation, which may raise concerns about the reduced response to vaccination.

Influenza virus is a highly mutable ssRNA virus, with new strains emerging every year. The success of annual influenza vaccine protection is highly dependent on the accuracy of the prediction of prevalent viruses during the upcoming flu season [14]. Therefore, a robust and effective vaccine that covers both homologous and heterologous strains is needed to reliably increase the success rate of influenza vaccines, which is even more important for people with asthma.

In this study, we evaluated whether asthma status affects protective vaccine responses with homologous and heterologous protection, using a house dust mite (HDM)-induced chronic asthma mouse model. HDM is an important indoor allergen in humans. The HDM mouse model closely resembles the features of human allergic asthma, such as airway hyper-responsiveness and remodeling, increased HDM-specific serum IgE levels, eosinophilic bronchitis, and increased levels of inflammatory cytokines, including interleukin (IL)-4 and IL-13. It is widely used in asthma research to investigate underlying mechanisms and explore treatment options [15,16,17]. Furthermore, we assessed the effects of AddaVax™, which is equivalent to the MF59 adjuvant, on the vaccine efficacy in asthmatic mice. MF59 is a safe and effective squalene oil-in-water vaccine adjuvant used as an influenza vaccine for immunocompromised individuals, such as the elderly, children, and pregnant women [18]. Our study contributes to the understanding of vaccine immunity in asthma, and offers insights into potential developmental strategies.

Method

Induction and confirmation of HDM induced asthma mice model

Six-week-old female BALB/c mice were purchased from Orient Bio and maintained at Jeju National University Animal Facility. The BALB/c mouse model of HDM-induced chronic allergic asthma was used in this study [19]. 25 µg of house dust mite (Dermatophagoides pteronyssinus) extract (Stallergenes Greer) were resuspended in 35 µL of PBS and intranasally delivered to mice 5 days per week. Control or naïve groups were intranasally administered 35 µL of PBS without HDM extract. Mice were challenged up to six weeks. Asthma mice were sacrificed at 6 weeks after induction and their asthma features were evaluated. Experiment was done twice with qualitatively similar outcomes. As an indicator of the allergic response, the levels of immunoglobulin E (IgE) in the sera and the population of eosinophils in the lung and bronchoalveolar lavage fluid (BALF) were measured. The population of inflammatory cells in the lungs and BALF, levels of inflammatory cytokines and chemokines, and hematoxylin and eosin (H&E)-stained histological features of lung tissues were analyzed to evaluate lung inflammation. To investigate the properties of T cells in asthmatic mouse, the expression of the T cell exhaustion marker, thymocyte selection-associated HMG BOX (TOX) and programmed cell death receptor-1 (PD-1) were determined using flow cytometry [20, 21]. HDM-specific IFN-γ and IL-4 cytokine-producing T cell percentages were measured after re-stimulation with the HDM extracts.

Split influenza vaccine and viruses

The human influenza virus strain A/Puerto Rico/8/1934 H1N1 (A/PR8) was obtained from ATCC (VR-95™) and amplified in 9-day old embryonated chicken eggs. The virus was harvested from allantoic fluid. To produce the split vaccine, the allantoic fluid was inactivated with 1% neutralized buffered formalin overnight at 4 °C. The virus was purified by ultracentrifugation for 1 h at 30,000 rpm to obtain an inactivated virus. After incubation with 1% Triton X-100 for 2 h at 4 °C to fragment the virus, the virus was transferred to a dialysis cassette and washed five times with PBS. The split A/PR8 virus vaccine and live A/PR8 virus were maintained at − 80 °C until use.

Vaccine immunization and virus challenge

Mice were divided into six groups (n = 12/group): naïve, naïve A/PR8 vaccine (naïve vac), naïve A/PR8 vaccine AddaVax™ adjuvant (naïve vac AddaVax™), asthma, asthma A/PR8 vaccine (asthma vac), and asthma A/PR8 vaccine AddaVax™ adjuvant (asthma vac AddaVax™). Vaccine groups were intramuscularly vaccinated twice at 4 and 6 weeks after asthma induction at a dose of 1 µg of split A/PR8 vaccine with or without AddaVax™. For immunization and infection, the mice were anesthetized using isoflurane. Sera were collected at 13 days after the prime vaccination and 14 days after the boost vaccination to measure vaccine-specific immunoglobulin levels and hemagglutination inhibitory titers.

Mice were sacrificed at three different time points (n = 4/group): (1) 2 weeks after the boost vaccination (2) 5 days after homologous infection (3) 10 days after homologous infection.

At 2 weeks after the boost vaccination, memory antibody production function of plasma cells (bone marrow) and memory B cells (spleen) were measured. At 2 weeks after the boost vaccination, mice were challenged intranasally with live A/PR8 virus (H1N1, at a dose of 2.5 LD50) to determine homo protection. The LD50 of the A/PR8 viral stock was determined using preliminary mouse viral challenge experiments. 5 days after viral infection, mice were sacrificed to evaluate the pathogenesis of influenza. Viral titers were determined as the 50% egg infectious dose (EID50)/mL. After monitoring the daily body weight change and survival for10 days after infection, mice were sacrificed and T cell populations in lung and spleen cells were measured. Experiment was done twice with qualitatively similar outcomes. The immunization and infection schedules are shown in Supplementary Fig. 1.

Sample collection and preparation

Sera were harvested via centrifugation of blood collected from the caudal vena cava. BALF samples were collected by delivering 1.2 mL PBS through the trachea using a 25-gauge catheter. Lung tissues were obtained separately for histological and cellular/cytokine analysis. For histological analysis, the lung tissues were immediately fixed with 10% formalin. Lung tissues for cellular/chemical analysis were mechanically mashed and filtered using a 100-µm cell strainer. Following centrifugation, the supernatants were stored at − 80 °C until cytokine enzyme-linked immunosorbent assay (ELISA). Following red blood cell lysis, the lung cell pellets were resuspended in 1 mL PBS containing 2% fetal bovine serum (FBS, FACS buffer) for flow cytometry. Lung extracts for virus titration were stored at − 80 °C until use.

Serum antigen-specific ELISA

To measure A/PR8-specific IgG, IgG1, IgG2a, and HDM-specific IgE levels in the serum, the ELISA plate was coated with inactivated PR8 vaccine or HDM protein (400 ng/well) before adding the serum. Serially diluted sera were added to antigen-coated ELISA plates after blocking. Horseradish peroxidase (HRP)-labeled anti-mouse IgG, IgG1, IgG2a, and IgE secondary antibodies (Southern Biotech) were used to detect antigen-specific immunoglobulins in the serum. Tetramethylbenzidine solution was used as the substrate, and the reaction was stopped using sulfuric acid. Optical density was measured at 450 nm.

Plasma cell, memory B cell antigen-specific immunoglobulin ELISA

The A/PR8 coated plates were blocked with 10% fetal bovine serum containing RPMI 1640 medium for 1 h at room temperature to measure the memory B cell response to the influenza virus. Bone marrow and spleen cells were collected at 7 days post-infection, seeded at a density of 2 × 106 cells/mL onto the plates, and then incubated at 37 °C for 1 or 5 days. Anti-mouse IgG, IgG1, and IgG2a antibodies were used to detect influenza-specific antibodies secreted by the antibody-producing B cells.

Hemagglutination inhibitory (HI) assay

Sera samples were incubated overnight at 37 °C with RDE II (DENKA SHEIKEN CO. LTD.) at a 1:3 ratio. The samples were inactivated at 56 °C for 30 min and stored at 4 °C until further use. The samples were initially diluted tenfold, followed by twofold serial dilutions of up to 1280-fold. Serum samples (25 µL) were incubated with 25 µL of 3 hemagglutination units of A/PR8 virus for 30 min at room temperature. Then, 50 µL of 0.5% chicken red blood cells (RBC) was added to each well and incubated for 30 min at room temperature. The endpoint dilution of HI activity in Chicken RBCs was determined.

Cytokine and chemokine ELISA

Cytokines in the BALF and lung extracts were measured using a tumor necrosis factor (TNF)-α, IL-6, IL-12 p40 Mouse Uncoated ELISA Kit (Invitrogen), and interferon (IFN)-γ, IL-4, granzyme B DuoSet ELISA kit (R&D Systems) according to the manufacturer’s protocols.

Flow cytometry

For cell phenotype staining, the Fc receptors of the harvested cells were blocked using anti-CD16/32 (clone 2.4G2) antibody after washing with FACS buffer. Each antibody cocktail was then added to the cells and incubated for 30 min at room temperature in the dark. Intracellular cytokine staining for TOX, IL-4, and IFN-γ was performed using a BD Cytofix/Cytoperm kit. To measure IL-4 and IFN-γ cytokine-producing T cells, cells were incubated for 4 h with 5 µg/mL of antigen (HDM or split A/PR8) after treatment with Golgi-stop. Data were acquired using a BD LSR Fortessa at the Bio-Health Materials Core Facility, Jeju National University, and analyzed using FlowJo software. Table 1 lists the antibodies used in this study and the gating strategy is shown in Supplementary Fig. 2.

Table 1 List of antibodies used in flow cytometry analysis
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Histopathological examination of mice lung

Portions of the left lower lobe lung tissue were fixed in 10% neutralized buffered formalin, processed, and embedded in paraffin. Sections were cut in 4 μm of thickness and stained with H&E. Lung Sections (15–20 photos per group) were scored from 0–4 blindly by three researchers for bronchial lesions and alveolar lesions using an adapted histological scoring system (Table 2) that was originally described by Dubin et al. [22]. To determine eosinophil infiltration in the lungs, modified Congo Red staining was performed [23].

Table 2 Lung inflammation scoring of mice after influenza infection
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Lung virus titration

The lung extracts from the influenza-infected mice groups were serially diluted in PBS from 10−2 to 10−12, and 300 μL of each dilution was inoculated into 9-day old embryonated chicken eggs (three eggs per dilution). After incubation for three days, 50 μL of allantoic fluid was collected and added to a 96 well U-bottom plate. 50 µL of 0.5% RBC was added to each well and incubated for 30 min at room temperature The 50% endpoint for hemagglutinin activity in chicken RBC was determined according to the method of Reed and Muench [24].

Statistical analysis

All results are presented as mean ± standard error of the mean, and statistical significance was analyzed using GraphPad Prism software 10.09 (GraphPad Software Inc.). Significant differences between two groups were analyzed using unpaired t-test. One-way ANOVA or two-way ANOVA was used for the comparison of multiple groups. Statistical significance was set at p < 0.05.

Result

A 6-week HDM-induced asthma mice model showed typical features of chronic allergic asthma

To establish a HDM mouse model, mice were sensitized and challenged intranasally 5 days per week for up to 6 weeks with 25 µg of HDM extract suspended in 35 µl of PBS. Asthma mice successfully represented the features of allergic asthma. They also showed increased serum levels of HDM-specific IgE, TNF-α, IL-12p40, and IL-4 (Fig. 1A–D) in the lung, as well as representative broncho-pathological characteristics, such as inflammatory cell infiltration around the bronchus and blood vessels, and mucous plugging. They also exhibited structural changes such as airway thickening, smooth muscle thickening, and increased alveolar cellularity in lung histology (Fig. 1E, F). The population of infiltrated inflammatory cells was determined using flow cytometry. A significant increase in the number of eosinophils, monocytes, neutrophils, and dendritic cells (DC) was observed in the lungs and BALF of asthma mice. The number of eosinophils, monocytes, neutrophils, and DC in the lungs and BALF increased in asthmatic mice (Fig. 1G, H). Among the inflammatory cells, the population of eosinophils showed the largest increase in the lungs (Fig. 1I). In addition, we analyzed the population of T cell subsets in asthma mice. They showed an increase in the population of effector memory CD4+ T cells (Fig. 2A, B, C) in the lungs. However, the percentages of IFN-γ or IL-4 cytokine-producing CD4+ T cells in the lungs after HDM restimulation were lower than those in the control (Fig. 2A, D, E). Instead, the percentages of exhausted CD4+ and CD8+ T cells in the lungs were significantly increased in asthma mice (Fig. 2A, F, G).

Fig. 1
figure 1

Evaluation of asthma features of HDM-induced asthma mouse model. Sera and lung samples were collected at 6 weeks post HDM sensitization. A HDM-specific IgE levels in the sera were measured using ELISA. B–D Inflammatory cytokine levels in the lungs were measured by ELISA. E, F Lung tissues were stained with H&E, and representative histological analyses of each group at magnification × 200 and × 400 are shown. G, H Frequencies of inflammatory cells in lungs and BALF were measured using flowcytometry. I Percentages of inflammatory cells in lungs were shown as parts to whole. Data were shown in mean ± SEM. For statistical analysis, two-way ANOVA and unpaired t test was performed. *p < 0.05; **p < 0.01; ***p < 0.001 ****p < 0.0001 between the indicated groups (n = 4/group)

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Fig. 2
figure 2

Evaluation of HDM-specific T cells and exhausted T cell populations in asthmatic mouse lungs. Lung samples were collected at 6 weeks post HDM sensitization. Golgi-stop was treated to cells and cells were re-stimulated for 4 h at 37 °C with HDM extract (5 µg/mL). T cell populations in the lungs were measured using flow cytometry. A Gating strategy and representative data were shown. B–G Frequencies of each T cell population were shown. Data were shown in mean ± SEM. For statistical analysis, unpaired t test was performed. *p < 0.05; **p < 0.01; ***p < 0.001 ****p < 0.0001 between the indicated groups (n = 4/group)

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Asthma mice exhibited lower serum HI titer compared to naïve mice after split A/PR8 vaccination, and AddaVax™ adjuvant enhanced antibody response and HI titer against A/PR8 in both naïve and asthma mice

To investigate vaccine efficacy in asthma, mice were immunized twice with a split A/PR8 vaccine, with or without AddaVax™ supplementation. The level of sera IgG, and IgG1 were increased after prime and boost vaccination in both naive and asthma mice. There was no significant difference in the antibody levels between the naive and asthma vac groups (Fig. 3A–F). AddaVax™ adjuvant effectively enhanced the levels of IgG and IgG1, but not IgG2a (Fig. 3A–F). The level of IgG1 was slightly lower in the asthma vac AddaVax™ group than in the naive vac AddaVax™ group after boost vaccination (Fig. 3A, B).

Fig. 3
figure 3

A/PR8-specific antibody production after prime and boost vaccination. Serum samples were collected at 2 weeks after prime and boost vaccination. Sera Ig G, G1, G2a levels were measured using ELISA (A–F). Bone marrow (BM) cells and spleen cells were collected at 2 weeks after boost vaccination and cultured in A/PR8 pre-coated 96 well cell-culture plate for 1 day (BM) and 7 days (spleen). Memory Ig G response in the (G) bone marrow and (H) spleen were measured using ELISA. 2 weeks post boost vaccination serum samples were initially diluted tenfold, followed by twofold serial dilutions of up to 1280-fold and incubated with 3 HA unit of A/PR8 virus. I The endpoint dilution of HI activity in Chicken RBCs was determined. Data were shown in mean ± SEM. For statistical analysis, two-way ANOVA (A–F) and one-way ANOVA (G–I) were performed. ****p < 0.0001 between the indicated groups (n = 4/group)

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We also assessed the antibody-producing capacity of the plasma cells in the bone marrow and memory B cells in the spleen. Split A/PR8 vaccine alone did not significantly enhance antibody-producing cell responses after vaccination. AddaVax™ supplementation significantly increased IgG production in the bone marrow (Fig. 3G), and spleen cells (Fig. 3H). There was no difference in the levels between naïve and asthma mice.

Although the naïve and asthma vac groups exhibited similar levels of A/PR8-specific serum IgG and IgG1, they showed large differences in the serum HI titer. The naïve and asthma vac AddaVax™ groups showed the highest dilution titer, followed by the naïve vac group, whereas the asthma vac group remained undetectable (Fig. 3I).

Asthma mice showed reduced vaccine protection against homologous A/PR8 virus challenge, which was effectively compensated for using the AddaVax™ adjuvant

To evaluate the efficacy of the split A/PR8 vaccine and AddaVax™ adjuvanted vaccine in asthma mice, mice were challenged with 2.5 LD50 of A/PR8 H1N1 at 2 weeks after boost vaccination. Naïve mice showed good protection after vaccination with or without AddaVax™, with no weight loss (Fig. 4A), 100% survival (Fig. 4B), and significantly reduced the viral titer in the lungs at 5 days post-infection compared to that in the naïve infection (inf.) group (Fig. 4C). The asthma mice also showed better protection after A/PR8 split vaccination with 100% survival (Fig. 4B) and reduced viral titer (Fig. 4C) in the lungs than the asthma infected mice. However, the asthma vac inf. group showed significant weight loss (Fig. 4A) and higher virus titer (Fig. 4C) compared with the naïve vac inf. groups, whereas the asthma vac AddaVax™ inf. group was fully protected, with no weight loss (Fig. 4A), and a low viral titer, which was even lower than that of the naïve vac AddaVax™ inf. group (Fig. 4C).

Fig. 4
figure 4

Determination of homologous protective efficacy of the A/PR8 split vaccine with or without AddaVax™ in naïve and asthma mice. A Body weight changes were monitored for 10 days after infection with A/PR8 influenza virus (2.5 LD50). B Survival rate of the asthma mice after infection with A/PR8 virus. C Lungs were collected at day 5 post infection and viral titers were determined in EID50/mL. All data were shown in mean ± SEM. For statistical analysis, two-way ANOVA was performed for body weight change. ****p < 0.0001 between asthma vaccine group and naïve vaccine group. One-way ANOVA was performed for virus titer. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 between the indicated groups (n = 4/group)

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AddaVax™ augmented eosinophil infiltration after homologous virus challenge at the site of infection and increased A/PR8 specific IgE level in lung of asthma mice

To further determine the protective effect of the vaccine against A/PR8 virus challenge in asthma mice, inflammatory cytokine levels, inflammatory cell populations in the airway, and histopathology of the lung tissues were determined at 5 days post-infection. In lung, naïve vac inf. and vac AddaVax™ inf. groups showed decreased levels of IL-6 and granzyme B compared to those in the naïve inf group. Asthma inf. group showed higher level of TNF-α, IL-6, and IFN-γ than in the naïve inf. group. Asthma vac AddaVax™ inf. group showed decreased levels of TNF-α, IL-6, IFN-γ, and granzyme B compared with the in those asthma group. Asthma vac inf. group showed decreased level of TNF-α, and IL-6. IL-4 level was higher in the asthma group than in the control group. In addition, it showed higher levels of IL-6, IFN-γ, IL-4, and granzyme B than asthma vac AddaVax™ inf. group (Fig. 5A). In BALF, the levels of TNF-α, IL-6, and IFN-γ among the groups exhibited a pattern consistent with that observed in the lungs. The level of IL-4 increased in naïve vac AddaVax™ inf. group compared to the naïve inf. and asthma vac AddaVax™ inf. group. Asthma vac inf. group showed the highest levels of granzyme B among the groups (Fig. 5B).

Fig. 5
figure 5

Inflammatory cytokine levels and cell infiltration in the lungs and BALF after A/PR8 challenge. Lung samples were collected at 5 days post infection. Inflammatory cytokine levels in the lung (A) and BALF (B) were measured by ELISA. Inflammatory cell populations in the lung (C) and BALF (D) were measured by Flow cytometry. All data were shown in mean ± SEM. For statistical analysis, one-way ANOVA was performed. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 between the indicated groups (n = 4/group)

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The percentage of eosinophils, neutrophils, monocytes, alveolar macrophages, and dendritic cells in the lungs (Fig. 5C), and BALF (Fig. 5D) was measured using flow cytometry. Both the naïve vac inf. and the naive vac AddaVax™ inf. groups exhibited significantly decreased percentages of monocytes and neutrophils in the lungs compared to those in the naive infection group (Fig. 5C). In BALF, naïve vac inf. group exhibited high levels of monocytes and neutrophils, whereas the naïve vac AddaVax™ inf. group exhibited a significant decrease in these cell populations (Fig. 5D). In asthma mice, however, the vac inf. group did not show a significant decrease in the populations of monocytes and neutrophils in the lung and BALF, whereas the vac AddaVax™ inf. group showed a substantial decrease in monocyte populations (Fig. 5C, D). The tendency of inflammatory cell populations was similar in the cell number graphs (Sup. 3).

We also evaluated the histopathology of lung tissues using H&E staining. Representative photos were selected based on the mean inflammation score (Fig. 6A, B). The lungs of the naïve inf. group showed moderate infiltration of inflammatory cells around the bronchioles and blood vessels, with some debris of cells inside the lumen (bronchiole score, 1.8). Cellularity in the alveolar space moderately increased (alveolar score, 2.1). The severity of inflammation in naïve vac inf. group was considerably lower than that of the naïve inf. group (bronchiole score 0.5, alveolar score 0.9). The severity of inflammation in naïve vac AddaVax™ inf. group was increased in both bronchiole and alveolar, showing similar score (bronchiole score 1.8, alveolar score 2.7) to naïve inf. group. The asthma inf. group showed severe inflammatory cell infiltration in the bronchiole region and narrowing of the lumen by mucus and cell debris (bronchiole score, 2.6). They also had significantly increased alveolar cellularity and obliteration of the alveolar space (alveolar score 3.2). Inflammation scores of the asthma vac inf. group (bronchiole score 2.4, alveolar score 2.7) were less severe than those of the asthma infection group, but showed higher bronchiole score than asthma group. Asthma vac AddaVax™ inf. group showed lower inflammation scores (bronchiole score 2, alveolar score 2.2) than asthma inf. group, similar score to asthma group.

Fig. 6
figure 6

Lung histopathological analysis and A/PR8-specific Ig E level in BALF and lung after A/PR8 challenge. The representative histology pictures of lung tissues at magnification × 200 (A) and the inflammation score in lung (B) after A/PR8 influenza virus infection. Lung tissues were collected at 5 days post infection and stained with H&E. The lung inflammation score was blindly quantified (15–20 pictures/group) between 0–4. C Representative histological photos of lung tissues at magnification of × 400 and × 1000, stained with Congo red. D A/PR8-specific IgE levels in BALF and lungs were measured by ELISA. The lung inflammation score graph and IgE level data were shown in the mean ± SEM. For statistical analysis, one-way ANOVA was performed. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 between the indicated groups

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Increased eosinophil infiltration was confirmed by lung histology staining using modified Congo Red staining. An elevated number of eosinophils (red arrowhead) was detected in the peri-bronchiole/peri-vascular region of the lungs in the naïve and asthma vac AddaVax™ inf. groups compared to the other groups (Fig. 6C). Notably the histopathological features of naïve vac AddaVax™ inf. group were similar to allergic inflammation in lung, showing eosinophil infiltration and airway thickening.

We examined A/PR8-specific IgE levels in BALF and lung to determine if the increased eosinophil level is related with allergic response against A/PR8 virus. A/PR8-specific IgE level in BALF were elevated in asthma groups but were only statistically significant in the asthma inf. group. A/PR8-specific IgE level in lung were significantly increased in asthma vac AddaVax™ inf. group (Fig. 6D).

Asthma groups exhibited higher percentage of DN cell populations compare to same treated condition naïve groups after infection

We assessed T cell populations in the lung and spleen at 10 days post infection. Following vaccination, we noted an increase in the percentage of CD4+ T cell populations in CD3+ cell population, whereas the percentage of CD4, CD8 double negative (DN) cell populations decreased in the lung and spleen of naïve and asthma mice (Fig. 7A, B). Also, the percentage of CD4+ T cell population was higher in naïve inf. group compare to the asthma inf. group. In lung, the percentage of CD8+ T cell populations was higher in naïve vac inf. group compare to asthma inf. group. The percentage of DN cell populations was higher in asthma groups compare to same treated condition naïve groups (Fig. 7A). In spleen, the percentage of CD4+ T cell population was lower and the percentage of DN cell populations was higher in asthma groups compare to same treated condition naïve groups. The percentage of CD8+ T cell populations was higher in asthma inf. group than naïve inf. group and asthma vac inf. group compared to the naïve vac inf. group (Fig. 7B).

Fig. 7
figure 7

T cell populations in the lung and spleen at 10 days after infection after A/PR8 re-stimulation. Lung and spleen cells were collected at 10 days after infection. Cells were incubated for 4 h at 37 °C with A/PR8 peptide (5 µg/mL). T cell populations in the lung (A-D) and spleen (E–H) were measured by flow cytometry. All data were shown in mean ± SEM. For statistical analysis, one-way ANOVA was performed. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 between the indicated groups (n = 4/group)

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Discussion

Asthma is a significant disease affecting 300 million people worldwide and increases the risk of hospitalization for the influenza virus [25]. The danger stems not only from lung malfunction and asthma exacerbation [26, 27] but also from the potential alteration in immunity due to chronic inflammation [28]. Our previous study demonstrated T cell exhaustion in asthma condition [11], and Huilei Zhang et al. found effector memory CD8 T cell dysfunction in asthma conditions [29], raising concerns about the efficacy of influenza vaccines.

Previous studies have investigated the impact of asthma on vaccine efficacy using a mouse model; however, this has been controversial [30,31,32]. Here, we demonstrated a clear reduction in the protective efficacy of split A/PR8 vaccination against homologous influenza infections in the asthma condition, whereas the vaccination provided complete protection in normal mice at the same dosage. 6-week HDM exposure increased the Th 2 response and T cell exhaustion.

The viral challenge dose of 2.5 LD50 in this study was effectively mitigated by high antibody levels and HI activity in the naïve vac AddaVax™ group, as evidenced by reduced recruitment of monocytes and neutrophils and low levels of IFN-γ and granzyme B. The naïve vac group exhibited lower antibody levels and HI activity compared to the naïve vac AddaVax™ group. Consequently, activation of cellular immunity appeared necessary for viral clearance, as indicated by the recruitment of monocytes and neutrophils in BALF. The naïve group was unable to withstand the 2.5 LD50 viral challenge.

The efficacy of vaccine in asthma mice were similar to that in naïve mice when considering survival rates. However, we could find differences in some details between asthma and naïve mice after vaccination. First, serum HI assay showed that HI activity of asthma vac group was substantially lower than naïve vac group, although total serum IgG and IgG1 level was not differ between groups. This is an important finding which might contribute to increased body weight loss and viral titer after challenge in asthma vac inf. group. One possibility is a reduction in antibody affinity, which still bind to influenza antigens but have weaker neutralizing activity. If the chronic HDM antigen exposure depleted the naïve B cell pool in secondary lymphoid organs in asthma mouse, it may lead to the reduction of competition among B cells. This diminished competition can impair the selection of high-affinity memory B cells and plasma cells, ultimately lowering the average antibody affinity. Another possible explanation is a shift in the antibody response away from HA toward internal viral proteins such as nucleoprotein (NP) or matrix protein 1 (M1) by asthma induced inflammation. However, since split-virus vaccines primarily contain HA and NA, with only minimal amounts of NP and M1 (as most internal components and egg proteins are removed during the chemical processing) [33], it is unlikely that asthma-induced inflammation would significantly redirect the antibody response toward these internal proteins, although it remains a possibility worth considering. Lastly, it’s another low possible explanation, that the presence of an expanded population of HDM-induced memory B cells in asthma mice may contribute to the production of antibodies targeting non-neutralizing regions of HA or NA, such as glycan structures rather than receptor-binding sites, which shares a common structure with HDM antigens. This could reduce the proportion of antibodies capable of effectively blocking viral entry, thereby lowering HI titers.. Second, in both lung and spleen, the percentages of DN cell populations were higher in asthma groups compare to same treated condition naïve groups. Possible cell types in this population are double-negative NKT cells and double-negative T cells (DNT) [34, 35]. DNT cells can have memory functions and can expand and activate against pathogens. It is a minor subset of T cells but is associated with multiple disease conditions [36,37,38,39,40]. This study had limitations in distinguishing these cell types. Nevertheless, this result suggests that asthma mice have alternative immune cell populations when responding to viral challenges. Further research investigating the actions of underrepresented immune cell subsets in asthma mice model, including mast cells, NK cells, NK T cells, and basophils, may enhance the understanding the action of asthma immunity against virus and reproducibility of the model.

Despite asthma's pathological feature of a Th 2 skewed immune response, upon exposure to a lethal dose of influenza virus infection, subjects typically elicit an anti-viral response characterized by elevated levels of IFN-γ and granzyme B. IFN-γ is primarily secreted by Th-1 CD4+ T cells [41, 42], cytotoxic CD8+ T cell [43], NK cell [44], and NKT cell [45, 46]. Granzyme B is mainly secreted by NK cell [47, 48], NKT cell [49], cytotoxic CD8 T cell [50, 51] and also mast cell [52]. Furthermore, inflammation was associated with the activation of monocytes and neutrophils rather than eosinophils. Study of Sujin An et al. showed that at the initial stage of infection asthma mice can control viral replication via rapid induction of type III IFN. However, asthma mice become vulnerable to infection after 7 days and intranasal administration of type III IFNs increased the protection [53]. This suggests that asthma mice can also respond normally to the virus, but there are limitations in their immune ability to maintain IFN-mediated immune activation for antiviral responses and consequently exhibit increased susceptibility.

Therefore, the elevated eosinophil levels observed in the vac AddaVax™ groups can be attributed solely to the vaccine's effects. In both naive and asthma mice, AddaVax™ adjuvanted vaccination enhanced serum A/PR8-specific antibody level and HI activity and significantly reduce lung viral titer. However, both of groups exhibited significant eosinophil recruitment in the lung and BALF after infection. It has been reported that split inactivated influenza vaccines can increase eosinophil levels in lung in mice following viral challenge [54]. Furthermore, the addition of AddaVax™ has been observed to enhance this effect [55].

In this study, naïve vac AddaVax™ inf. group showed acute eosinophilic pneumonia and increased IL-4 level in BALF. It did not show the increase in the level of A/PR8-specific IgE in the lung or BALF. Asthma vac AddaVax™ group displayed an elevation in eosinophil levels but not in cytokine levels or observable asthma exacerbation in lung. Allergen sensitivity may be diminished in the chronic asthma stage compared to the acute stage [56, 57]. Elevated A/PR8-specific IgE levels in the lung suggest the development of an allergic response to the A/PR8 virus.

An allergic response to viruses is undesirable and has the potential to develop into a serious disease with repeated influenza vaccination or virus infection. Although there are studies about the anti-bacterial and anti-viral actions of eosinophil [58,59,60], still it is strongly associated with the exacerbation of allergic inflammation [61], and recent reports have highlighted an increase in cases of eosinophilia following COVID-19 vaccination [62,63,64]. Asthma mice also present an unknown risk, as elevated eosinophil levels did not exacerbate lung inflammation but may systemically affect inflammation in other organs. Therefore, more active vaccine research and development is necessary, considering not only antibody levels and protection outcomes but also unidentified risk factors and long-term effects. Furthermore, existing guidelines for influenza vaccination do not recommend the use of live attenuated vaccines or nasal spray formulations for individuals with asthma, due to the potential risk of triggering an asthma attack. Developing a vaccine for asthma patients that enhances cellular response without exacerbating underlying disease poses a novel challenge, particularly in light of the potential for severe seasonal and pandemic respiratory viruses to emerge.

This study's results are limited to a particular mouse model, with the evaluation focusing on selected cell types only. Also, it has a limitation in fully elucidating the underlying mechanism of vaccine efficacy in asthma condition and identifying the contribution of each factor. To elucidate the mechanisms of asthma immunity against influenza vaccine and virus, further testing, such as antibody affinity assays and monoclonal antibody measurements, would be needed to confirm the mechanism of reduced neutralizing antibody production in split vaccinated asthma mouse. Comprehensive research is required regarding the function of other immune cells under diverse conditions with longitudinal monitoring. Moreover, evaluating various other vaccine formulations will be crucial for advancing vaccine development tailored to asthma patients. Further studies in human subjects are also necessary.

Data availability

All data in the manuscript is available through the responsible corresponding author.

Abbreviations

Th-1:

T helper type 1

Th-2:

T helper type 2

IL:

Interleukin

IgE:

Immunoglobulin E

BALF:

Bronchoalveolar lavage fluid

H&E:

Hematoxylin and eosin

TOX:

Thymocyte selection-associated HMG BOX

PD-1:

Programmed cell death receptor-1

naïve vac:

Naïve A/PR8 vaccine

naïve vac AddaVax™:

Naïve A/PR8 vaccine AddaVax™ adjuvant

asthma vac:

Asthma A/PR8 vaccine

asthma vac AddaVax™:

Asthma A/PR8 vaccine AddaVax™ adjuvant

LD50 :

50% Lethal dose

A/PR8:

Human influenza virus strain A/Puerto Rico/8/1934 H1N1

EID50 :

50% Egg infectious dose

ELISA:

Enzyme-linked immunosorbent assay

PBS:

Phosphate-buffered saline

FBS:

Fetal bovine serum

HRP:

Horseradish peroxidase

TNF:

Tumor necrosis factor

inf.:

Infection

IFN:

Interferon

DC:

Dendritic cell

DN:

Double-negative

DNT:

Double-negative T cells

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Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2023-00211504), the Korea Basic Science Institute (National Research Facilities and Equipment Center) grant funded by the Ministry of Education (2020R1A6C101A188) and the Basic Science Research Program to the Research Institute for Basic Sciences (RIBS) of Jeju National University through the NRF, funded by the Ministry of Education (RS-2019-NR040080).

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Authors and Affiliations

  1. Department of Veterinary Medicine and Veterinary Medical Research Institute, College of Veterinary Medicine, Jeju National University, Jeju, 63243, Republic of Korea

    So Yeon Ahn & Eun-Ju Ko

  2. Interdisciplinary Graduate Program in Advanced Convergence Technology & Science, Jeju National University, Jeju, 63243, Republic of Korea

    Thi Len Ho & Eun-Ju Ko

  3. Bio-Health Materials Core-Facility Center, Jeju National University, Jeju, 63243, Republic of Korea

    Eun-Ju Ko

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Contributions

Conceptualization: S.Y.Ahn, E.J.Ko, Methodology: S.Y.Ahn, E.J.Ko, Validation: S.Y.Ahn, E.J.Ko, Formal analysis: S.Y.Ahn, Investigation: S.Y.Ahn, T.L.Ho, E.J.Ko, Data Curation: S.Y.Ahn, Writing—Original Draft: S.Y.Ahn, E.J.Ko, Writing—Review & Editing: S.Y.Ahn, T.L.Ho, E.J.Ko, Visualization: S.Y.Ahn, E.J.Ko, Supervision: E.J.Ko, Funding acquisition: E.J.Ko.

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All mouse experiments were performed in accordance with the guidelines of Jeju National University approved by the Institutional Animal Care and Use Committee (protocol number 2021-0051).

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Supplementary Information

Additional file 1;Sup.1 Experimental schedule of immunizations and infection

Additional file 2;Sup.2 Gating strategy of immune cell populations with flow cytometry

12931_2025_3209_MOESM3_ESM.pdf

Additional file 3;Sup. 3 Inflammatory cell infiltration in the lungs and BALF after A/PR8 challenge. The number of inflammatory cells in the lung (A) and BALF (B) at 5 days post infection was measured by Flow cytometry. All data were shown in mean ± SEM. For statistical analysis, one-way ANOVA was performed. *p < 0.05; **p < 0.01; ***p < 0.001 between the indicated groups (n = 4/group)

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Ahn, S.Y., Ho, T.L. & Ko, EJ. Evaluation of the influenza vaccine protection in the house dust mite-induced chronic allergic asthma mice model and the evaluation of squalene oil in water emulsion as an adjuvant candidate. Respir Res 26, 132 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-025-03209-6

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