A mucosal vaccine prevents eosinophilic allergic airway inflammation by modulating immune responses to allergens in a murine model of airway disease

Abstract

Allergic sensitization and viral infections are risk factors for asthma development and progression. Sublingual vaccination with MV130, a whole heat-inactivated polybacterial preparation, protects against viral infections, but its impact on allergic sensitization and asthma development remains unknown. Here we show MV130 prevents house dust mite (HDM)-induced local type 2 immune responses and associated eosinophilic airway inflammation, conferring protection up to 9 weeks after vaccination. MV130 reduces pathophysiological and clinical asthma features in an in vivo experimental mouse model of HDM-induced allergic eosinophilic asthma, restoring normal airway functionality. MV130 impairs allergen-specific IgE sensitization and systemic type 2 inflammation endorsing type 1 and IL-10 responses. In human DCs, MV130 induces a transcriptomic and metabolic reprogramming, and restores non-pathological immune responses to allergens in healthy and asthmatic donors. Additionally, the adoptive transfer of MV130-stimulated BMDCs was sufficient to reproduce the protective features of the vaccine administration in vivo. Collectively, we show MV130 reduces allergic sensitization and eosinophilic asthma. Our findings support the exploration of mucosal interventions aimed at reducing the risk of allergen-induced asthma development.

Fig. 1. MV130 blocks the key pathophysiological asthma features in an experimental model of HDM-induced eosinophilic allergic airway inflammation.

a Scheme of employed models. b Serum total IgE, HDM-specific IgE and IgG1 levels (PBS n = 19, MV130 n = 5, HDM n = 19, HDM + MV130 n = 18 mice; 4 independent experiments). c Left, representative hematoxylin and eosin (H&E) staining in formalin-fixed lung sections. Scale bars: 20 μm. Right, quantification of peribronchial inflammatory infiltrates in 1000 μm² of lung sections (n = 10 bronchioles/mouse for 2 mice per group). d Quantification of total cell numbers in BALF (PBS n = 13, MV130 n = 6, HDM n = 18, HDM + MV130 n = 19 mice; 4 independent experiments). e Quantification of different cell types in BALF (PBS n = 13, MV130 n = 6, HDM n = 16, HDM + MV130 n = 17 mice; 4 independent experiments). f Left, representative α-smooth muscle actin (α-SMA) staining of formalin-fixed lung sections. Scale bars: 20 μm. Right, quantification of α-SMA staining area (μm²) relative to total bronchiole area (n = 10 bronchioles/mouse of 2-3 mice per group). g Enhanced pause (Penh) measurement at increasing doses of methacholine as determined by whole-body plethysmography (WBP)^#.HDM vs MV130, *HDM vs HDM + MV130, and ⁺HDM vs PBS comparisons (PBS n = 3, MV130 n = 4, HDM n = 4, HDM + MV130 n = 5 mice per experiment; a representative example of 2 independent experiments is shown). h Left, representative periodic acid Schiff (PAS) staining of formalin-fixed lung sections. Scale bars: 20 μm. Right, quantification of the percentage of μm² of PAS positive staining relative to μm² of bronchiole (n = 10 bronchioles/mouse of 2 mice per group). i Invasive lung function measurement of resistance index (RI) to increase doses of methacholine^#.HDM vs MV130, *HDM vs HDM + MV130, and ⁺HDM vs PBS comparisons (PBS n = 5, MV130 n = 4, HDM n = 5, HDM + MV130 n = 3 mice; 3 independent experiments). Values are mean ± SEM. Statistical significance was determined using unpaired One-way ANOVA (c and e (macrophages, B and T cells)), unpaired Kruskal–Wallis (b, d, e, f and h), two-side unpaired Student’s t test (e (ILC2s)) or unpaired Two-way ANOVA (g and i): *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Exact p values and source data are available in the Source Data file.

Fig. 2. MV130 inhibits the HDM-induced recruitment of inflammatory eosinophils (iEos) without affecting homoeostatic resident eosinophils (rEos).

a Representative flow cytometry dot plots of Siglec-F and CD125 expression on live CD45⁺ gated mouse isolated lung cells. b Histograms of CD101 (in red) and CD62L (in green) expression on inflammatory eosinophils (iEos) and resident eosinophils (rEos). Fluorescence minus one (FMO) of each fluorophore is shown in yellow. c Percentage of lung iEos and rEos relative to CD45⁺ live lung cells (n = 8 mice per group; 2 independent experiments). d Percentage of eosinophil major basic protein (MBP) positive staining in lung sections (n = 15 fields/lung/mouse for 1 mouse per group). e Representative MBP (left) and Congo Red (right) stained mouse lung sections of the peribronchial area (1 independent experiment). Scale bars: 50 μm. Black arrows point to the eosinophils. f Representative MBP (left) and Congo Red (right) stained mouse lung sections of the lung parenchyma (1 independent experiment). Scale bars: 50 μm. Black arrows point to the eosinophils. g Left, representative eosinophil peroxidase (EPX) positive stained mouse lung sections of peribronchial area. Right, percentage of EPX positive staining in lung sections (n = 13 fields/lung/mouse for 1 mouse from each group). h Left, quantification of EPX activity in BALF (PBS n = 3, HDM n = 4, HDM + MV130 n = 5 mice; 1 independent experiment). Values are mean ± SEM. Statistical significance was determined using unpaired One-way ANOVA (c, g and h) and unpaired Kruskal–Wallis test (d): *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Exact p values and source data are available in the Source Data file.

Fig. 3. MV130 restores non-pathological innate and adaptive immune responses against HDM.

Left, representative flow cytometry dot plots of intracellular staining of IFN-γ- (a), IL-5- (b) or IL-10-producing (c) lung T cells, respectively, relative to CD3⁺ lung T cells. Right, percentage of IFN-γ-, IL-5- or IL-10-producing lung T cells, respectively, relative to CD3⁺ lung T cells (n = 4 mice per group of one representative experiment out of 2). d Correlation between the percentage of iEos and the % of IL-5– (left) and IFN-γ- (right) producing lung T cells, respectively. “r” indicates Pearson correlation coefficient. e Left, representative flow cytometry dot plots of CD4⁺CD25⁺FOXP3⁺ lung Treg cells. Right, percentage of CD4⁺CD25⁺FOXP3⁺ Treg cells relative to live cells in the lungs (n = 4 mice per group of one representative experiment out of 2). f Percentage of CD23⁺ resident and migratory DCs in mediastinal lymph nodes (MLNs) (PBS n = 6, HDM n = 6, HDM + MV130 n = 10 mice; 2 independent experiments). g Percentage of total ILC2s, and IL-5-, IL-13- and IL-13/IL-5-producing ILC2s of CD45⁺ live lung cells (PBS n = 6, HDM n = 11, HDM + MV130 n = 11 mice; 2 independent experiments). h Left, levels of serum albumin in BALF 24 h after last HDM challenge (PBS n = 7, HDM n = 10, HDM + MV130 n = 11 mice; 2 independent experiments). Right, lactate dehydrogenase (LDH) levels in BALF 72 h after last HDM challenge (PBS n = 12, HDM n = 14, HDM + MV130 n = 13 mice; 2 independent experiments). Values are mean ± SEM. Statistical significance was determined using two-side unpaired Student’s t test (a), unpaired One-way ANOVA (b, e, and h), unpaired Kruskal–Wallis test (c and g), two-side Pearson test (d) and unpaired Two-way-ANOVA (f): *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Exact p values and source data are available in the Source Data file.

Fig. 4. MV130 abrogates systemic T2 immune responses in HDM-sensitised mice.

a Schematic representation of the model employed to assess in vitro responses of splenocytes from the different groups after HDM stimulation. b Cytokine production after 3 days of stimulation of splenocytes with HDM from the different groups (PBS n = 15, MV130 n = 8, HDM n = 12, HDM + MV130 n = 10 mice; 4 independent experiments). c Ratios of cytokine production (HDM n = 12, HDM + MV130 n = 10 mice; 4 independent experiments). Values are mean ± SEM. Statistical significance was determined using unpaired One-way ANOVA (b), unpaired Kruskal–Wallis (b, IFN-ɣ) and two-side unpaired Student’s t test (c): *p < 0.05, **p < 0.01 and ***p < 0.001. Exact p values and source data are available in the Source Data file.

Fig. 5. MV130 prophylactic treatment prevents allergic sensitization and HDM-induced eosinophilic airway inflammation, conferring protection up to 9 weeks after treatment discontinuation.

a Schematic representation of the prophylactic treatment with MV130 or vehicle, resting period (1, 2, 4 or 9 weeks) and following HDM-induced asthma model. Two control groups without sensitization were included, MV130 group receiving PBS in the sensitization and challenge and MV130 during immunization and a PBS group receiving only PBS; i.n., intranasal. b Serum total IgE, HDM-specific IgG1 levels (n = 3–9 mice from each group; 2 independent experiments). c Quantification of total cell numbers of different populations in bronchoalveolar lavage fluid (BALF) as determined by flow cytometry (n = 3–9 mice from each group; 2 independent experiments). d Quantification of total cell numbers of eosinophils, e B cells and f Th2 cells in BALF (n = 3–9 mice from each group; 2 independent experiments). Values are mean ± SEM. Statistical significance was determined using unpaired Two-way ANOVA (b) and unpaired One-way ANOVA (c–f) tests: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Exact p values and source data are available in the Source Data file.

Fig. 6. MV130 impairs HDM-induced T2 allergic profile by generating human DCs with the capacity to polarize Th1 and IL-10 immune responses.

a Scheme of the employed protocol for HmoDCs generation from healthy and HDM-allergic asthmatic donors and in vitro stimulation. b Cytokine production in cell-free supernatants after stimulation of HmoDCs with vehicle (Ctrl-), MV130, HDM or HDM/MV130 for 24 h (Ctrl- n = 17, MV130 n = 6, HDM n = 19, HDM + MV130 n = 19 healthy donors and Ctrl- n = 6, MV130 n = 6, HDM n = 6, HDM + MV130 n = 6 asthmatic donors). c Scheme of the employed protocol for HmoDCs and allogeneic naïve T cells co-cultures. d Left, cytokines produced by allogeneic naïve CD4⁺ T cells primed by HmoDCs in the presence of vehicle (Ctrl-), MV130, HDM or HDM/MV130 after 3 days (Ctrl- n = 9, MV130 n = 4, HDM n = 9, HDM + MV130 n = 9 healthy donors). Right, ratios of indicated cytokines were determined for HDM and HDM/MV130 stimulations (HDM n = 7, HDM + MV130 n = 7 healthy donors). e Left, cytokines produced by allogeneic naïve CD4⁺ T cells primed by HmoDCs in the presence of vehicle (Ctrl-), MV130, HDM or HDM/MV130 (n = 6 asthmatic donors). Right, ratios of indicated cytokines were determined for HDM and HDM/MV130 stimulations (n = 6 asthmatic donors). Values are mean ± SEM. Statistical significance was determined using two-side paired Wilcoxon or Student’s t test (b, d, e) depending on the normal distribution of the compared samples analysed by D’Agostino-Pearson normality test: *p < 0.05, **p < 0.01 and ****p < 0.0001. Exact p values and source data are available in the Source Data file.

Fig. 7. MV130 imprints transcriptomic and metabolic rewiring in human DCs.

a Bar plot showing gene set enrichment analysis (GSEA) the selected significantly enriched gene sets for HALLMARK, KEGG, GOBP and BIOCARTA data bases from 24 h MV130- vs vehicle (Ctrl-)-stimulated HmoDCs Gene Array (n = 4 healthy donors). Enriched gene sets in MV130-stimulated HmoDCs were represented in red and vehicle (Ctrl-)-stimulated HmoDCs were represented in blue. NES (Abs), NES absolute value. All FDR q values < 0.25. b GSEA for HALLMARK_IFN-GAMMA_RESPONSE, BIOCARTA_IL-10_PATHWAY and GOBP_FCƐR_SIGNALING_PATHWAY gene sets showing the genes enriched in the indicated biological pathways differentially enriched in MV130- or vehicle (Ctrl-)-stimulated HmoDCs (n = 4 healthy donors). c GSEA for HALLMARK_GLYCOLISIS gene set significantly enriched in MV130-stimulated HmoDCs from Gene Array results (n = 4 healthy donors). d Scheme of the employed protocol with HmoDCs for in vitro metabolic studies. e Left, real-time analysis of glycolytic Proton Efflux Rate (glycoPER) of HmoDCs after stimulation with HDM, HDM + MV130 or vehicle (Ctrl-) by sequential addition of rotenone/antimycin A (Rot/AA), and 2-deoxyglucose (2-DG). Right, quantification of basal and compensatory glycolysis (n = 4 healthy donors). f GSEA for HALLMARK_OXPHOS gene set significantly enriched in vehicle (Ctrl)-treated HmoDCs from Gene Array results (n = 4 healthy donors). g Fluorescence intensity of HDM- or HDM + MV130-stimulated HmoDCs stained with MitoTracker^TM Red CMXRos (mitochondrial membrane potential) and MitoTracker^TM Green FM (mitochondrial mass) relative to negative control-stimulated cells. MitoTracker Red/MitoTracker Green ratio is also shown (n = 10 healthy donors). Values are mean ± SEM. Statistical significance was determined using two-side paired Student’s t test (e and g): *p < 0.05, and **p < 0.01. Exact p values and source data are available in the Source Data file.

Fig. 8. MV130 reprograming of BMDCs via MyD88-mediated pathways prevents HDM-induced eosinophilic allergic airway inflammation.

a Schematic representation of MV130-BMDCs or vehicle-BMDCs intranasal administration and following HDM-induced asthma model. b Serum total IgE and HDM specific IgE and IgG1 levels (PBS n = 6, Vehicle-BMDCs/HDM n = 6, MV130-BMDCs/HDM n = 7 mice; 2 independent experiments). c Quantification of total cell numbers and d of different cell types in BALF determined by flow cytometry (PBS n = 6, Vehicle-BMDCs/HDM n = 6, MV130-BMDCs/HDM n = 7 mice; 2 independent experiments). e Schematic representation of bone marrow progenitors from wild type and MyD88^−/− mice differentiation to BMDCs and following in vitro stimulation with vehicle (Ctrl-), MV130, or Gefinitib/MV130. f Geometric Mean Fluorescence Intensity (GeoMFI) values for CD40 (Ctrl- n = 3-4, MV130 n = 4-5, Gefinitib/MV130 n = 5 biological replicates; 1 independent experiment). g Cytokine production in cell-free supernatants after in vitro stimulation of wild type and MyD88^−/− BMDCs for 18 h (Ctrl- n = 3-4, MV130 n = 4-5, Gefinitib/MV130 n = 7 biological replicates; 1 independent experiment). h Serum total IgE and HDM specific IgG1 levels (n = 4 mice per group). i Quantification of total cell numbers and j different cell types in BALF determined by flow cytometry (n = 4 mice per group). Values are mean ± SEM. Statistical significance was determined using unpaired One-way ANOVA (b, c and i), unpaired Two-way ANOVA (f and g) and two-side unpaired Student’s t test (d and j): *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Exact p values and source data are available in the Source Data file.