Immunization with an adeno-associated viral vectored allergy vaccine containing Der p1-Der p2 effectively alleviates an asthmatic phenotype in mice

doi:10.21203/rs.3.rs-4980552/v1

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Immunization with an adeno-associated viral vectored allergy vaccine containing Der p1-Der p2 effectively alleviates an asthmatic phenotype in mice

https://doi.org/10.21203/rs.3.rs-4980552/v1

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Given the rising incidence of allergic asthma, current symptomatic treatments primarily offer relief rather than halt disease progression. Recombinant allergens, designed with reduced immunoglobulin E (IgE) reactivity and the ability to regulate excessive T helper type 2 (Th2) responses, are emerging as promising candidates for more precise, effective, and safer specific immunotherapy (SIT). SIT remains the only clinical approach capable of potentially curing certain allergic diseases by inducing immunological tolerance. In this study, we explored the protective effects of AAV-Dp12S, an adeno-associated viral vector carrying two house dust mite antigens, Der p1 and Der p2, against allergic asthma. Using a murine model of HDM, immunization with this combination vaccine significantly attenuated the HDM-induced asthmatic phenotype. Invasive lung function assessments revealed improvements following AAV-Dp12S treatment, correlating with marked reductions in goblet cell hyperplasia and pulmonary eosinophilia. Moreover, total serum IgE, HDM-specific IgE (sIgE) titers, and pulmonary inducible nitric oxide synthase levels were effectively reduced. The cytokine profiles in bronchoalveolar lavage fluid (BALF) were modulated, as indicated by decreased levels of type 2 cytokines—interleukin (IL)-4, IL-5, and IL-13—and increased levels of interferon-γ (IFN-γ) and IL-10. Additionally, sIgE titers and production were significantly lowered. Overall, these findings demonstrate the potential of AAV-Dp12S as a therapeutic strategy for both tolerance induction and vaccination in the treatment of allergic asthma.

Biological sciences/Immunology/Inflammation/Chronic inflammation

Biological sciences/Microbiology/Vaccines/Dna vaccines

Biological sciences/Immunology/Inflammation

Biological sciences/Immunology/Vaccines

Health sciences/Diseases/Respiratory tract diseases

Allergy vaccine

Allergic asthma

Recombinant allergen

Allergen-specific immunotherapy

Adeno-associated viral vector

Der p1/Der p2.

Atopic asthma affects approximately 300 million individuals globally, with house dust mite (HDM) being the most prevalent allergen, impacting nearly 50% of these patients [1, 2]. Allergic asthma is the most common phenotype of asthma, typically characterized by sensitization to environmental allergens, with a clinical correlation between allergen exposure and symptoms further substantiating the diagnosis. This allergy-driven form of asthma is associated with T helper type 2 (Th2) polarization in about 90% of cases, resulting in antigen-specific T cell responses, production of allergen-specific IgE, and eosinophilic infiltration in the lungs following allergen inhalation [3]. Th2 cytokines, such as IL-4, IL-5, and IL-13, are pivotal in the onset and progression of allergic asthma, while Th1 cytokines like interferon-γ (IFN-γ) and the anti-inflammatory cytokine IL-10 have the potential to counteract the aberrant Th2 responses [4].

The two most prevalent HDM species are Dermatophagoides pteronyssinus and D. farinae [5, 6]. Fecal pellets from these mites are the primary source of allergens [7], with group 1 and group 2 allergens being the most immunodominant. Studies have shown that 95% of HDM-allergic patients possess specific IgE against either or both of these allergen groups [8]. Group 1 allergens, Der p1 and Der f1, are cysteine proteases with over 90% sequence homology, leading to high IgE cross-reactivity among allergic individuals [9]. Der f2 and Der p2 are lipid-binding proteins with 87% sequence homology, as supported by various studies [10, 11]. HDM allergens are a common trigger worldwide [12], and while 39 different HDM allergens have been identified [13], research indicates that group 1 and group 2 allergens are the most clinically significant [14]. Der p1 and Der p2, which are 25.1-kDa and 14.1-kDa proteins respectively, each contain three disulfide bonds [15]. Der p1 is a more complex protein, expressed as a preproenzyme, with only proDer p1 forms being successfully expressed in eukaryotic systems such as plants, insects, mammalian cells, and the methanotrophic yeast Pichia pastoris [16]. This suggests that the pro-peptide region is essential for the expression of Der p1 in eukaryotic systems.

Current therapeutic vaccines for HDM allergy are based on complex biological extracts derived from one or both HDM species [17]. However, these extract-based vaccines present certain drawbacks, including significant compositional variability, the exclusion of key allergens, and the inclusion of irrelevant molecules [18]. In contrast, recombinant allergen-based vaccines offer an appealing alternative, as they can be produced in large quantities as well-defined and standardized molecules. It has been demonstrated that a combination of D. pteronyssinus allergens, Der p1 and Der p2, can accurately diagnose over 95% of HDM-allergic patients [19–21]. Thus, Der p1 and Der p2 are critical components of an effective vaccine for HDM allergy.

Previously, recombinant fusion proteins comprising Der p1 and Der p2 allergens demonstrated partial folding and retained appropriate antigenic properties. However, these chimeric proteins faced significant challenges related to solubility and stability, which limited their utility in immunotherapy and diagnostic applications [16]. In contrast, Chen et al. (2012) reported that Der p1/Der p2 combination vaccines showed significant promise in preclinical trials, suggesting their potential as safe hypoallergenic molecules for both tolerance induction and vaccination strategies to treat HDM allergies [14]. However, similar to native allergen proteins, recombinant allergen proteins possess B cell epitopes capable of binding and cross-linking sIgE on effector cells, which can trigger degranulation and the release of inflammatory mediators.

T cell epitope peptides, typically short and lacking conformational B cell epitopes, do not cross-link cell-bound IgEs and, therefore, do not activate mast cells (MCs) and basophils. The significant efficacy, shorter treatment durations, and minimal non-systemic adverse events associated with T cell epitope immunotherapy make it an attractive therapeutic option [22]. A recent breakthrough involved the creation of a hypoallergenic hybrid molecule containing T-cell epitopes from Der p1, Der p2, and Der p23 allergens, which holds promise for allergen immunotherapy (AIT) in patients co-sensitized to D. pteronyssinus major allergens [23]. Although T-cell epitope vaccines have not yet achieved sustained clinical efficacy, they have the potential to induce long-lasting immunity [24].

Adeno-associated virus (AAV) is a remarkable vector, belonging to the Depend parvovirus genus within the Parvoviridae family, with at least 12 naturally occurring serotypes that differ in their tissue tropism [25, 26]. This characteristic is exploited for the targeted delivery of AAV gene therapy vectors to specific tissues. Notably, AAV infection is asymptomatic and can persist throughout an individual's lifetime. Moreover, AAV is an excellent candidate for gene therapy due to its ability to be produced in large quantities, support long-term transgene expression without requiring integration into the host genome, and infect a wide range of cell types [27]. Unlike adenovirus vectors, which elicit strong innate immune responses leading to inflammation and efficient clearance of the vectors, the immune response to AAV delivery is minimal [22, 28].

The objective of this study was to develop a recombinant vaccine candidate against respiratory allergies associated with common HDM species. To achieve this, the protective effects of AAV-Dp12S were evaluated in a mouse model of HDM-induced asthma. The study examined the impact of AAV-Dp12S immunization on airway hyperresponsiveness (AHR), pulmonary inflammation, and Th1/Th2 immune responses in the airway. We are excited to report that AAV-Dp12S shows potential as a candidate for AIT, offering both tolerance induction and vaccination strategies for treating allergic asthma. If AAV-Dp12S can effectively reduce IgE production, induce allergen-specific blocking antibodies, and restore the balance between Th1/Th2 immune responses, it could represent a significant advancement in the field of allergy treatment.

Construction and Characterization of AAV Vector Carrying Der p1/2 Fusion Antigens

To develop hypoallergenic and more effective combination vaccines targeting the major dust mite allergens Der p1 and Der p2, we designed a mosaic protein that includes the full sequence of Der p1 and the mature Der p2 (Fig. 1A). This design was informed by previous studies indicating that only chimeras containing proDer p1 could be successfully expressed in yeast [16], and that the mature Der p2 was identified as hypoallergenic [16]. The proenzyme form of Der p1, which includes its pro-region, exhibits weak IgE reactivity, making it a promising candidate for use as a hypoallergenic molecule in immunotherapy [29, 30]. To further enhance the hypoallergenic properties, we replaced the 12 cysteine residues (indicated by dotted vertical lines) with serine residues (Fig. 1A-B), a modification expected to reduce potential aggregation of the Der p1-Der p2S mosaic protein (Fig. 1B). The mosaic protein was engineered with a C-terminal His-tag to facilitate purification.

Furthermore, Der p1 and mature Der p2 were linked using a P2A auto-cleavage linker, which enables the post-translational cleavage of the fusion protein into distinct Der p1 and Der p2 peptides (Fig. 1C). Codon usage was optimized to improve expression, and the construct was incorporated into an AAV vector, designated AAV-Dp12S. The mosaic protein was initially expressed in E. coli BL21 cells (Fig. 2A), and its antibody reactivities were evaluated through immunoblotting, using either anti-Der p1 or anti-Der p2 (monoclonal or polyclonal) antibodies. The fusion protein expressed in E. coli appeared as a monomeric protein and showed strong reactivity with both anti-Der p1 and anti-Der p2 antibodies (Fig. 1D, Fig. S1C).

To confirm the expression of the AAV-Dp12S fusion antigen, HEK-293 cells were infected with the AAV-Dp12S vector at equivalent viral particle concentrations, with an AAV-empty vector serving as a negative control (Fig. S1A). A detectable fluorescence signal indicated successful expression of the fusion proteins in HEK-293 cells (Fig. S1B). Subsequent analysis of cell lysates and culture medium via SDS-PAGE and western blotting confirmed efficient expression of the fusion antigen (Fig. 1E). Additionally, overexpression of AAV-Dp12S in HEK-293 cells resulted in a significant increase in mRNA transcripts for both Der p1 and Der p2 compared to the negative control (Fig. S1D-E).

To investigate whether the subcellular localization of Der p1 and Der p2 was affected by the fusion strategy, HEK-293 cells were infected with vectors carrying the Der p1-Der p2 fusion genes. Immunofluorescence assays revealed that both Der p1 and Der p2, whether expressed separately or as a fusion protein, were primarily co-localized in the cytoplasm (Fig. 2). This result indicates that codon optimization and the use of a protein linker did not significantly alter the subcellular distribution patterns of Der p1 and Der p2.

Dp12S Exhibits Lower IgE Reactivity Than Parental Allergens and Allergen Extract in D. pteronyssinus Reactive Sera

We evaluated the IgE reactivity of Dp12S, DpE (HDM extract), rDer p1, and rDer p2 in plasma samples from individuals with allergic rhinitis and asthma using ELISA (Table S1). The hypoallergenic Dp12S demonstrated a significant reduction in IgE reactivity compared to rDer p1 and rDer p2, with reductions of 76% and 57%, respectively (Fig. 3A).

To further assess whether Dp12S exhibited reduced IgE binding activity in comparison to HDM extracts, rDer p1, and rDer p2, we conducted a dot blot analysis using pooled serum from nine highly sensitized patients allergic to HDM. The results revealed that Dp12S exhibited significantly less IgE binding compared to equimolar amounts of rDer p1, rDer p2, and HDM extract (Fig. 3B). Serum from a nonallergic control did not show any reactivity to any of the four allergens tested. These findings indicate that the recombinant Dp12S vaccine has substantially reduced IgE reactivity, making it a potentially safer option for immunotherapy.

PBMCs from Allergic Individuals Stimulated with Dp12S Show High Levels of Regulatory and Th1 Cytokines

We next examined the cytokine profiles of peripheral blood mononuclear cells (PBMCs) from HDM-allergic and nonallergic donors, particularly focusing on the secretion of IFN-γ, a cytokine predominantly produced by Th1 cells that suppresses IgE production and promotes the generation of protective IgG antibodies. Using ELISA, we compared the IFN-γ levels in PBMC supernatants from allergic individuals stimulated or unstimulated with DpE. The results indicated a reduction in baseline levels of IFN-γ in these subjects (Fig. 3C). However, upon stimulation with Dp12S, there was a notable increase in baseline levels of IFN-γ, IL-2, and IL-10 cytokines, both in stimulated and unstimulated conditions (Fig. 3C).

Moreover, the baseline levels of Th2 cytokines, such as IL-4, IL-5, and IL-13, were reduced in PBMC supernatants from allergic individuals stimulated with Dp12S (Fig. 3D). Conversely, there was a trend towards increased levels of these Th2 cytokines when PBMCs were stimulated or unstimulated with DpE (Fig. 3D). Additionally, upon Dp12S stimulation, a significant increase in IL-10, IL-2, and IFN-γ levels was observed in PBMCs from allergic subjects compared to those from healthy donors (Fig. 3D). These findings suggest that Dp12S induces a lower Th2 cytokine response than HDM extract in the PBMCs of allergic patients, highlighting its potential as a more effective and safer therapeutic option.

AAV-Dp12S Immunization Reduces AHR and Eosinophilia and Induces Macrophage Proliferation in HDM-Induced Asthmatic Mice

We administered AAV-Dp12S or AAV-GFP to HDM-sensitized mice prior to the HDM challenge (Fig. 4A). The results showed that HDM/AAV-Dp12S mice, but not HDM/AAV-GFP mice, exhibited significantly lower Penh values in response to 50 mg/ml aerosolized methacholine (MCh) compared to HDM-only mice (Fig. 4B). AAV-Dp12S immunization led to a marked reduction in AHR at both 100 and 200 mg/ml MCh, in contrast to AAV-GFP immunization and the asthma control group (Fig. 4B). These findings demonstrate that AAV-Dp12S immunization effectively inhibits AHR.

Next, we conducted a flow cytometric analysis to examine the cellular composition within the lungs (Fig. 4C-F). In saline-treated control mice, pulmonary macrophages were the predominant cell type (Fig. 4D). However, in the Asthma-Control and HDM/AAV-GFP groups, there was a significant infiltration of eosinophilic granulocytes (68%) and lymphocytes (22%), while the percentage of macrophages was notably reduced (Fig. 4D-E). In contrast, in HDM/AAV-Dp12S-treated mice, eosinophils were reduced to 22.5%, resulting in a substantial increase in the proportions of lymphocytes and macrophages (45% and 28%, respectively). Notably, the reduction in eosinophilia following HDM/AAV-Dp12S treatment did not lead to airway neutrophilia (Fig. 4F). Additionally, we observed a similar reduction in total cells, eosinophils, and macrophages in the bronchoalveolar lavage fluids (BALFs) of AAV-Dp12S-treated mice compared to AAV-GFP-treated mice (Fig. 4G). The percentage of eosinophils in HDM/AAV-Dp12S mice was significantly lower than in the other experimental groups (Fig. 4H). Interestingly, in the BALFs of HDM/AAV-Dp12S mice, macrophages were the dominant cell type, similar to those observed in saline-treated mice (Fig. 4H). These results collectively suggest that AAV-Dp12S has potent anti-inflammatory effects by inhibiting the infiltration of inflammatory cells.

AAV-Dp12S Treatment Reduces IgE Production in Mice Allergic to *D. pteronyssinus

Allergies and asthma are strongly associated with elevated IgE titers, particularly in affected tissues [31]. Therefore, we assessed the levels of IgE. Before the HDM challenge, all groups exhibited comparable serum IgE levels (Fig. 4I). Following the challenge, both the HDM and HDM/AAV-GFP groups showed increased IgE levels, while HDM/AAV-Dp12S mice had significantly lower serum IgE levels compared to the HDM and HDM/AAV-GFP groups (Fig. 4I), indicating that AAV-Dp12S effectively suppresses IgE production.

We also analyzed anti-HDM IgE levels in the BALF. In contrast to the saline-treated mice, where IgE was almost undetectable, the Asthma-Control group exhibited robust levels of HDM-specific IgE in the BALF (Fig. 4J). However, HDM/AAV-Dp12S-treated animals showed significantly reduced IgE titers, whereas HDM/AAV-GFP-treated mice had IgE levels comparable to those in the Asthma-Control group (Fig. 4J).

Additionally, we assessed the titers of HDM-specific IgG1 and IgG2a as indicators of a potential Th1 immune response shift. Unlike the IgE response, sIgG1 was significantly increased following AAV-Dp12S treatment compared to the control and asthma groups (Fig. 4J). A trend toward increased sIgG2a levels was also observed when compared to the control group (Fig. 4F). These findings suggest that AAV-Dp12S not only suppresses IgE production but also induces the production of blocking antibodies, thereby contributing to a shift towards a Th1 immune response and offering protection against the allergen.

AAV-Dp12S Immunization Ameliorates Pulmonary Inflammation and Mucus Overproduction

A common symptom in asthmatic patients is the marked hyperplasia and activation of mucus-producing goblet cells, which is associated with increased mortality[32]. To determine if the immunological improvements observed with AAV-Dp12S immunization would also result in a decrease in goblet cell numbers, we examined lung tissue three days after the final airway challenge. Lung sections from the right lobes were stained with H&E and PAS. The peribronchial and perivascular inflammation in HDM/AAV-Dp12S mice was significantly reduced compared to HDM or HDM/AAV-GFP mice (Fig. 5A, B). This reduction in inflammation was consistent with a notable decrease in mucus-secreting goblet cells following AAV-Dp12S immunization (Fig. 5C, D). In saline-treated mice, PAS-positive cells, indicative of mucus-producing goblet cells, were completely absent, whereas the Asthma-Control group showed pronounced goblet cell hyperplasia. In contrast, HDM/AAV-Dp12S-immunized mice displayed only a minimal presence of PAS-positive cells. No significant change in goblet cell hyperplasia was observed in AAV-GFP-treated mice (Fig. 5C, D).

To assess the expression levels of Der p1 and Der p2 in various organs, including the heart, lung, liver, and kidney, we performed qRT-PCR on samples from different treatment groups. As expected, both Der p1 and Der p2 genes were highly expressed in the lungs and showed a slight increase in the heart during AAV-Dp12S immunization. However, there were no noticeable changes in expression levels in the liver and kidney (Fig. S2A, B). Consistent with these findings, quantification of mRNA for Gob5, a mucus-related gene, and inflammatory markers (TNFα, IL-1β, IL-6) in lung sections revealed elevated levels in the Asthma-Control and HDM/AAV-GFP groups compared to the significantly lower levels in HDM/AAV-Dp12S-treated animals (Fig. 5E). Moreover, the levels of Th2 cytokines (IL-4, IL-5, IL-13) were markedly elevated in the Asthma-Control and HDM/AAV-GFP groups but were sharply reduced to near control levels in the lungs of AAV-Dp12S-treated mice (Fig. S2C, D). Interestingly, IL-10, an anti-inflammatory cytokine, was significantly elevated in both the Asthma-Control and HDM/AAV-GFP groups (Fig. S2C, D). In the heart, only IL-5 and IL-10 exhibited a similar profile as in the lungs, while IL-4 and IL-13 levels remained comparable. Overall, these results suggest that AAV-Dp12S substantially reduces airway inflammation and mucus overproduction in response to allergen challenge.

AAV-Dp12S Immunization Modulates Th2 Cytokines and Induces IL-10 and IFN-γ

To further investigate whether AAV-Dp12S immunization modulates Th1/Th2 immune responses, we analyzed cytokine levels in BALF from Dp12S-treated mice compared to the sham-treated group. The results showed significantly reduced levels of Th2 cytokines IL-4, IL-5, and IL-13, while levels of IL-10 and IFN-γ were increased (Fig. 6B). To corroborate these findings, splenocytes from the mice were restimulated for 48 hours with HDM, Dp12S, or their parental allergens (Der p1/p2, Fig. 6A). These stimuli were compared with non-stimulated cells, as shown in Fig. 6C-G. Induction of an asthmatic phenotype in the Asthma-Control group was evidenced by high levels of the classical Th2 cytokines IL-5 and IL-13 (Fig. 6B). Treatment with Dp12S, however, effectively reduced the secretion of IL-4, IL-5, and IL-13, with significantly lower levels observed in both non-stimulated cells and those stimulated with HDM and rDp12S (Fig. 6C-E). Conversely, IL-10 levels increased compared to all other groups, including when splenocytes were stimulated with rDer p1, HDM, and Dp12S (Fig. 6F). Similar significant increases were observed for IFN-γ when cells were stimulated with HDM and rDp12S, in comparison to both the Asthma-Control and HDM/AAV-GFP groups (Fig. 6G). Collectively, these findings suggest that AAV-Dp12S enhances Th1 and anti-inflammatory cytokine responses while decreasing Th2 cytokines in the airway, indicating its potential to modulate immune responses in favor of reducing allergic inflammation.

AAV-Dp12S Immunization Suppresses iNOS Expression and Improves Lung Function

Nitric Oxide (NO) plays a significant role in the pathophysiology of asthma, with inducible Nitric Oxide Synthase (iNOS) being a key enzyme in its production [33]. To determine the impact of AAV-Dp12S immunization on iNOS expression, we analyzed iNOS transcript levels. As expected, HDM-challenged mice, whether treated with AAV or not, exhibited the highest iNOS transcript accumulation compared to healthy control mice (Fig. 7A). In contrast, AAV-Dp12S treatment significantly decreased iNOS expression in comparison to the Asthma-Control and HDM/AAV-GFP groups (Fig. 5D–F). The reduced iNOS levels observed in HDM/AAV-Dp12S mice compared to HDM and HDM/AAV-GFP mice further confirmed that AAV-Dp12S effectively inhibits iNOS expression (Fig. 7B).

Having established the beneficial effects of HDM/AAV-Dp12S on various immunological and histological parameters of the asthma-like phenotype, we next sought to determine whether these improvements translated into better lung function. To assess this, anesthetized mice were tracheotomized, and the increase in airway resistance (Rn), tissue damping (G), and tissue elastance (H) in response to methacholine was measured using the forced oscillation technique, with escalating doses of methacholine administered as an aerosol (Fig. 7C-H). The area under the curve (AUC) for Rn, tissue damping, and elastance in the sensitized groups was significantly different from that of the saline group. Notably, mice in the Asthma-Control and HDM/AAV-GFP groups exhibited a significantly higher increase in these parameters than HDM/AAV-Dp12S mice, even at low doses of methacholine (Fig. 7C-E). These results indicate that AAV-Dp12S treatment leads to an overall improvement in lung function in HDM-induced asthmatic mice.

The development of allergy vaccine candidates will likely depend on well-defined recombinant allergens. Recombinant HDM allergens offer the advantage of being produced at defined concentrations with consistent quality, enabling the creation of vaccines that retain immunogenicity while reducing allergenic activity [34]. This approach, which has proven effective for birch and grass pollen allergens [35, 36], as well as for recombinant hypoallergenic mite allergens [14], shows promise as a clinically viable option for HDM allergy vaccines. Based on IgE reactivity data, group 1 and group 2 allergens from HDM are essential components that must be included in HDM allergy vaccines [37–39]. Previous attempts to create engineered hybrid molecules combining Der p1 and Der p2 in yeast encountered challenges, such as the instability of the chimera and its tendency to form aggregates when only the 80 amino acid residue proenzyme sequence of Der p1 was included [16].

In this study, we aimed to develop a recombinant vaccine candidate for clinical allergen-specific immunotherapy (AIT) trials targeting respiratory allergies associated with common HDM species. We employed codon optimization and cysteine replacement strategies to modify the two major HDM allergens, Der p1 and Der p2. Our efforts successfully produced a hybrid molecule, Dp12S, which demonstrated reduced IgE-binding activity and cross-linking potential while maintaining its T cell stimulatory capacity.

The construction of fusion antigens can be influenced by various factors, including codon usage optimization, signal sequence selection, fusion linker design, and immunization routes. In our study, the subcellular localization of Der p1 and Der p2 was not altered by the fusion strategy. Protein linkers play a crucial role in the design of fusion antigens, as they can enhance folding, stability, bioactivity, and expression levels [40–43]. We utilized the auto-cleavage 2A linker, which proved effective in generating robust systemic and pulmonary cell-mediated immune responses. However, further research is needed to explore whether other linkers might outperform the 2A linker in the construction of Der p1 and Der p2 fusion antigens and to understand the underlying mechanisms.

Given the potential advantages of oral administration, this route should be further investigated for the development of AAV vector-based allergy vaccines. Future studies will focus on evaluating the efficacy of oral and subcutaneous (SC) administration routes in in vivo challenge models, and, if feasible, in non-human primate models. These studies will provide valuable insights into the optimal delivery methods for these vaccines and could pave the way for more effective and convenient treatments for HDM-induced allergies.

The recombinant hypoallergenic hybrid protein Dp12S addresses several previously unresolved challenges in developing a vaccine for HDM allergy. By combining all the sequence elements of Der p1 and primary Der p2, along with all T-cell epitopes within a single molecule, Dp12S can be expressed in large quantities in both E. coli and eukaryotic cells and purified to homogeneity. Importantly, the removal of all cysteines in the construct prevents the formation of aggregates through disulfide bonds, allowing the protein to remain monomeric in solution. This not only enhances the safety profile of the vaccine candidate but also facilitates its large-scale production for clinical trials. Additionally, upon immunization, the mosaic protein induces IgG antibodies that recognize both allergens and inhibit IgE binding to natural allergens, a crucial factor for clinical success in vaccination [35, 36].

In our in vivo study, we demonstrated the hypoallergenic potential of Dp12S in treating pulmonary inflammation induced by an HDM extract-based asthma model. Dp12S treatment altered the total cell counts in bronchoalveolar lavage fluid (BALF) and lung tissue, specifically affecting the numbers of eosinophils, neutrophils, lymphocytes, and macrophages. The reduction in neutrophil and eosinophil levels is associated with improvements in allergic symptoms, as increased levels of these cells are often correlated with disease severity and exacerbation [44]. iNOS and its product, nitric oxide (NO), are known to contribute significantly to tissue damage during airway inflammation [45]. Studies have shown that knocking out all NOS isoforms reduces airway inflammation and decreases Th2 cytokines such as IL-4, IL-5, and IL-13 in asthmatic mice [33]. Therefore, the suppression of iNOS by AAV-Dp12S likely contributes to the inhibition of airway inflammation and Th2 responses.

Our findings also showed that AAV-Dp12S significantly reduced airway hyperresponsiveness (AHR) and improved lung function parameters, including airway resistance (RN), tissue damping (G), and tissue elastance (H). The increase in RN observed in the Asthma-Control group was consistent with the presence of bronchioles occluded by PAS-positive goblet cells. Furthermore, increases in lung elastance (H) and tissue damping (G) are generally associated with peripheral inflammation [46].

Mice treated with Dp12S exhibited reduced IgE levels against both the HDM extract and parental allergens in serum and BALF. In contrast, there was an increase in IgG1 and IgG2a antibodies in Dp12S-treated animals compared to the Asthma-Control, AAV-GFP, and untreated groups. Previous studies involving D. pteronyssinus-derived hypoallergens have reported the induction of high titers of IgG antibodies and the regulation of IgE to lower levels following immunization [47–49], findings that are consistent with our results. The observed reduction in sIgE levels can be attributed to the downregulation of IL-4, IL-5, and IL-13 production in the lungs induced by Dp12S. Allergen-activated Th2 cells secrete these cytokines, which are primarily responsible for promoting IgE production, recruiting eosinophils to the site of inflammation, and stimulating mucus production in the airway epithelium [50–52].

Overall, these findings underscore the potential of AAV-Dp12S as a promising candidate for clinical allergen-specific immunotherapy (AIT) against HDM-induced respiratory allergies. Its ability to modulate immune responses, reduce pulmonary inflammation, and improve lung function highlights its therapeutic efficacy and warrants further investigation in clinical settings.

Splenocyte cultures from allergic mice revealed that stimulation with rDp12S led to higher levels of IL-10 and IFN-γ compared to HDM extract, while it induced lower levels of IL-4, IL-5, and IL-13. These results suggest that rDp12S triggers an immune response distinct from the classical allergic Th2 response, potentially predicting its efficacy in treating allergy [53]. By inducing high levels of IL-10, a cytokine known for its anti-inflammatory and regulatory functions, rDp12S appears to promote T-cell proliferation. IL-10 derived from Th cells plays a crucial role in the success of allergen-specific immunotherapy (AIT), as these cells are central to the regulation of allergic responses [49, 54–56]. The elevated levels of IFN-γ in splenocytes stimulated with rDp12S also contribute to this shift in immune response, which may help inhibit the airway remodeling typically driven by Th2 responses [57]. Unlike the Asthma-Control group, where stimulation with rDp12S led to increased levels of IL-10 and IFN-γ, non-treated cells showed lower levels of these cytokines, highlighting a distinct cytokine production pattern associated with Dp12S, different from that observed for Der p1 [57].

Reduced levels of Th2 cytokines in BALF, accompanied by increased levels of IL-10 and IFN-γ in Dp12S-treated mice, further indicate a shift toward a regulatory and/or Th1-biased immune response. However, it remains unclear which of these two cytokines, IL-10 or IFN-γ, has a more significant influence on the downregulation of IL-4 and IL-5 in this model. Nonetheless, the data suggest that Dp12S has the potential to modulate the immune response away from the Th2 profile typically associated with allergic reactions.

In this study, we also examined the transcript profiles of several cytokines, including IL-4, IL-5, IL-13, IL-10, IL-1β, and IL-6, as well as the inflammatory marker TNFα and the mucus-related gene GOB5. Previous research has shown that IL-1β can prime lung dendritic cells to induce Th2 responses [59–61]. In both humans and mice, elevated levels of these cytokines are associated with a worsening of therapeutic outcomes and exacerbation of asthma [58–60]. Consistent with these findings, our data showed that these genes were up-regulated in the Asthma-Control and AAV-GFP groups, whereas a reduction was observed following stimulation with Dp12S.

To the best of our knowledge, this is the first study to use AAV, one of the most promising in vivo gene delivery tools and a potent vector for eliciting T cell responses[61, 62], as a carrier for D. pteronyssinus antigens to prevent allergic asthma. Our results support the potential of AAV-Dp12S as an alternative immunotherapy for asthma, meriting further exploration. Future studies using Dp12S in a chronic mouse model of allergy will be crucial in demonstrating the long-term benefits of its hypoallergenic and immunogenic properties. These studies could pave the way for novel therapeutic strategies against HDM-induced allergic asthma.

Construction and Characterization of Der p1/2 Mosaic Proteins

Protein sequences of Der p 1.0102, and Der p2.0101 were obtained from the Allergen Nomenclature website (allergen.org) [63, 64]. The fusion gene encoding Der p1/p2 mosaic proteins, designated as Dp12S, codon-harmonized for optimal expression in Escherichia coli (E. coli) and synthesized with an auto-cleave 2A linker by Genomeditech (Shanghai, China). The recombinant mosaic protein consisted of two Der p1 fragments-propeptide (amino acids 19–98) and mature Der p1 (amino acids 99–320)-along with the mature Der p2 (amino acids 18–146). These components were assembled in the following order: Der p1 propeptide, Der p1 mature, and Der p2 mature, with a C-terminal hexahistidine tag for purification. Additionally, codon-optimized sequences were modified by replacing cysteine residues with serine to minimize the potential aggregation behavior of the Der p1/2S protein.

The synthetic genes encoding Der p1, Der p2, and Der p1/2S were cloned into the expression vector GPAAV-CMV-MCS-T2A-eGFP-WPRE using the Hieff Clone™ One Step PCR Cloning Kit (YEASEN). The plasmids (pAAVDp1, pAAVDp2, and pAAVDp12S) were amplified in E. coli strain XLI-Blue and prepared using the Qiagen Plasmid Midi Kit (Qiagen, Cat. No. 12145) according to the manufacturer’s protocol. The DNA sequences of the constructs were confirmed by sequencing (Genomeditech).

The expression vectors for Der p1, Der p2 and Dp12S containing the harmonized sequences were transformed into Escherichia coli BL21(DE3) pLysS (Invitrogen, Carlsbad, CA, USA). One colony was selected for protein expression, which was performed in Luria Bertani (LB) medium, and the induction of expression was performed through the addition of 0.6 mM Isopropyl-b-D-thiogalactopyranoside (IPTG) when OD600 reached 0.6 nm. Then, the expression of recombinant proteins occurred for 6h, 200 rpm at 37°C, bacterial pellets were harvested by centrifugation as described in [23]. Finally, the lysate pellet was resuspended in 6M urea. Purified proteins were run under denaturing and non-denaturing conditions on a 4–15% mini-PROTEAN TGX Stain-Free Protein Gel (Bio-Rad Laboratories, Hercules, Calif), and following blotting.

SDS-PAGE and Western Blot Analysis

To confirm the purity and expression of the recombinant AAV vectors (pAAVDp12 and pAAV-empty), these plasmids were linearized and transfected into HEK293 cells and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

The produced recombinant adeno-associated virus (AAV) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For SDS-PAGE, samples were prepared by incubating them with 6× Laemmli sample buffer (Bio-Rad) containing 10% β-mercaptoethanol, followed by boiling at 100°C for 5 minutes. The denatured proteins were then loaded onto a 15% polyacrylamide gel and electrophoresed at 120 V. The gel was washed with distilled water and stained using Gel Code Blue Protein Safe Stain (Invitrogen).

For Western blot analysis, proteins separated by SDS-PAGE were transferred onto a nitrocellulose membrane by electroblotting. The membrane was blocked with 6% milk in 1× phosphate-buffered saline (PBS) and probed with hybridoma supernatants containing monoclonal antibodies specific for Der p1 (Biorbyt, No. orb14375) or Der p2 (antibodies-online, Cat. No. ABIN7141165). After incubation with a horseradish peroxidase-conjugated secondary antibody, the proteins were visualized using Immobilon chemiluminescent substrate (Beyotime) and detected using the Touch Imaging System (e-BLOT, Shanghai, China).

The expression of inducible Nitric Oxide Synthase (iNOS) in lung tissues was similarly evaluated through Western blot analysis using an anti-iNOS antibody (Abcam, ab49999).

Donors and Sera

Venous blood was collected from non-allergic (n = 8) and allergic (n = 24) individuals using heparin tubes. Plasma samples were evaluated for the presence of specific IgE (sIgE) to D. pteronyssinus using Phadia Diagnostics AB to confirm atopy, defined as detectable sIgE levels ≥ 0.70 kU/L. Additionally, a positive skin prick test (SPT) for D. pteronyssinus extract (positive SPT defined as a mean wheal diameter of 3 mm or larger than the saline control) was used as an inclusion criterion for atopy. Non-allergic donors were included based on the absence of clinical allergy symptoms, a negative SPT reaction, and a lack of detectable sIgE. The reactivity profiles of the donors included in the study are detailed in Table S1. The study was approved by the Ethics Committee on Research of the Faculty of Medicine of the Southwest Jiaotong University of China ([2022] S-15).

Reactivity of Human IgE and Culture of Peripheral Blood Mononuclear Cells for Cytokine Determination

The reactivity of human IgE to Dp12S, rDer p1, rDer p2, and DpE allergens was assessed using indirect ELISA and dot blotting techniques. The profile of secreted cytokines and the Dp12S protein-induced reactivity were evaluated in peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from the peripheral blood of allergic (n = 9) and non-allergic (n = 9) individuals as previously described [23, 65]. Cells were incubated in 96-well plates (2 x 10⁵ cells/well) in a humidified atmosphere of 5% CO2 at 37°C, and restimulated with 20 µg/mL DpE, 12.5 µg/mL Dp12S. The 10 µg/mL pokeweed mitogen (PWM) was used as a positive control. Polymyxin B (20 ug/ mL) was used to block lipopolysaccharide (LPS)-related cytokine production. Cultures were performed 120 h and supernatants were collected after this time and stored at -20ºC for further quantification of cytokine concentrations.

AAV Production, Purification, and Titer Quantification

Endotoxin-free recombinant AAV vectors and their auxiliary packaging element plasmids were co-transfected into AAV Pro-293T cells. Six to eight hours post-transfection, the medium was replaced with fresh medium and enhanced buffer, and the supernatant was harvested 72 hours later. The AAV capsid-containing lysates were purified using iodixanol gradients (60%, 40%, 25%, and 15%). The copy numbers of vector genome DNAs were quantified by quantitative PCR using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), as previously described [66].

Asthma Models and Vaccination Strategies

Six-week-old female C57BL/6 mice were purchased and maintained according to local animal care guidelines. The asthma model was established as previously described [67], with modifications. The amount of LPS in the recombinant protein and allergenic extract samples was quantified by the end-point chromogenic LAL assay (QCL-1000 kit; Lonza, Walkersville, MD, USA). When it was needed, LPS was removed from the sample using Pierce High-Capacity Endotoxin Removal Resin (Thermo Fisher Scientific, Waltham, MA, USA). Mice were sensitized via three intraperitoneal (I.P.) injections at weekly intervals with 10 µg of HDM (Grade V, Sigma-Aldrich, St. Louis, MO) mixed with 2 mg of alum (Sigma-Aldrich, St. Louis, MO). One week after the final HDM sensitization, the mice were immunized intratracheally (I.T.) with saline (HDM mice), AAV-GFP (1.0 × 10¹¹ viral particles per mouse; HDM/AAV-GFP mice), or AAV-Dp12S (1.0 × 10¹¹ viral particles per mouse; HDM/Dp12S mice). The AAV particles used for mice are according to previous described [68, 69]. Each mouse received two 50 µL injections into the thigh quadriceps muscles of both hind legs at week 3. Subsequently, the mice were boosted intranasally with the same dose three times. Before immunized by intratracheally or intranasally, mice were anesthetized to minimize pain and distress. Isoflurane gas was used as the anesthetic agent. The mice were placed in an induction chamber pre-filled with 3–5% isoflurane in oxygen at a flow rate of 1–2 liters per minute.

One week after the final immunization, the mice were challenged with aerosolized HDM (2% in saline) for 40 minutes on three consecutive days. One day after the last challenge, the mice were evaluated for airway hyperresponsiveness (AHR) and were euthanized by 100% carbon dioxide (CO2) inhalation as described in [70]. Mice were placed in a clean 14.6-L polyurethane box connected to a CO2 tank (38.5 cm L, 19.5 cm W, 19.5 cm H). The flow rate was 30% displacement volume/minute for mice in the low flow CO2 group and 100% displacement volume/minute for the high flow CO2 group via Western Medica CO2 flow meter (Westlake, OH). The percentages were chosen to represent the lowest acceptable flow rate in the 2020 AVMA euthanasia guidelines and the maximal rate at which CO2 could be displaced [71]. Mice remained in the box until they had stopped breathing for 1 min. Mice were removed from the box and cervically dislocated as a secondary method of euthanasia.

Lymphocytes from the spleen and lung were isolated for subsequent immunological assays.

Analysis of Airway Allergic Inflammation and Lung Function

Lung tissue sections were prepared for the assessment of inflammation using Hematoxylin and Eosin (H&E) staining, and mucus-producing goblet cells were visualized with Periodic Acid-Schiff (PAS) staining. Airway hyperresponsiveness (AHR) and lung function measurements were conducted as previously described [72].

RNA Isolation and Quantitative PCR Analysis

Total RNA from HEK-293 cells and lung tissue was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, RC101–01). The concentration and purity of the extracted RNA were determined using ScanDrop (Analytik Jena AG, Germany), with the 260/280 ratio used as an indicator of purity. The RNA was then converted into cDNA using a reverse transcription kit (Vazyme, R223–01) following the manufacturer’s protocol. Gene expression was quantified by qPCR using a SYBR solution (Vazyme, SQ101) according to the manufacturer’s instructions. The primers used in the study are listed in Supplemental Table 1.

Analysis of Cells from Lung and Bronchoalveolar Lavage Fluid (BALF)

Bronchoalveolar lavage fluids (BALFs) were centrifuged at 2000 g for 10 minutes at 4°C, and the supernatant was stored at -20°C. Erythrocytes in the cellular fraction were lysed using ACK buffer (Lonza, Walkerville, MD, USA). Differential cell types were analyzed using the Diff-Quick kit (Labor und Technik Eberhardt Lehmann, Berlin, Germany) for staining. At least 100 cells were counted according to standard morphological criteria in a blinded fashion.

Lung lymphocytes were prepared as previously described [73]. For flow cytometry (FACS) staining, up to 5 × 10^6 cells were incubated for 20 minutes at room temperature in a 100 µL staining mix containing Fc-block and specific antibodies: SiglecF-AF700, CD45-PerCP-Cy5.5, CD3-APC, CD19-PE-Cy5, CD11c-PE-Cy7, CD11b-eF595/506, Ly6G-PerCP-Cy5.5, and MHC-II-APC-eFluor780, all prepared in FACS buffer. Following staining, cells were washed twice, fixed with 2% paraformaldehyde solution for 20 minutes at room temperature, and analyzed by flow cytometry (Sony, MA900, Japan).

Measurement of Immunoglobulins in Sera and BALF

The concentration of IgE in sera was measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. For the quantification of HDM-specific IgG1, IgG2a, and IgE in the BALF supernatants, the same procedure was followed as detailed in [72].

Fluorescence Microscopy

The fluorescence microscopy protocol was adapted from Zhang et al. (2015). Vero cells were seeded onto glass slides and infected with recombinant pAAVDp12S or the corresponding control vector for 48 hours. The cells were then washed, fixed with 4% paraformaldehyde, permeabilized, and blocked. For inducible Nitric Oxide Synthase (iNOS) immunostaining, lung tissue sections were deparaffinized in xylene for 20 minutes, dehydrated in 100% ethanol for 10 minutes, and washed with PBS for 10 minutes. Endogenous peroxidase activity was inhibited with 0.3% H2O2 for 15 minutes. The specimens were then incubated overnight at 4°C with either anti-Flag antibody (Cat. No. bs-0965R-FITC, 1:50), anti-HA antibody (Cat. No. bsm-0966m-PE, 1:100), or anti-iNOS antibody (Abcam, ab49999) in blocking buffer, followed by development with goat anti-rat IgG. The slides were counterstained with DAPI, and images were captured using confocal microscopy (Olympus, IXplore SpinSR10).

Splenocyte Culture and Cytokine Quantification in Supernatants of Cell Culture

Lymphocytes were isolated from spleens as previously described [23]. Spleens were homogenized, and erythrocytes were lysed. Cells were then counted, and splenocytes (2×10⁵ cells/well) were restimulated with 20 µg/mL of HDM or 12.5 µg/mL of recombinant proteins. Cultures were incubated for 48 or 72 hours, after which the supernatants were collected and stored at -20°C for later cytokine quantification. IL-4, IL-5, IL-13, IFN-γ, and IL-10 were measured using standard ELISA kits according to the manufacturer’s instructions (BD Pharmingen, San Diego, CA, USA).

Data Collection and Statistical Analysis

Flow cytometry data were analyzed using FlowJo software (version 10.8.1; Tree Star, Inc., Ashland, OR). Statistical analysis and graphical presentations were performed using GraphPad Prism software (version 8.01; GraphPad Software Inc., La Jolla, CA). Gel images were analyzed using ImageJ software (NIH, Bethesda, MD). Data are presented as the mean ± standard error of the mean (SEM). Statistical comparisons between groups were conducted using one-way analysis of variance (ANOVA), and Bonferroni post hoc tests were applied when multiple groups were compared. A p-value of < 0.05 was considered statistically significant. Depending on data distribution, one-way ANOVA with Tukey’s or Dunn’s post-tests was used. Results were considered statistically significant at p ≤ 0.05.

AUTHOR CONTRIBUTIONS

LSB and HZS designed the study and supervised the project. QSG and CJZ contributed to the conception and co-supervision of the project. LSB significantly contributed to the experimental work, manuscript writing, and laboratory assays. XAY, YXL, and XJX actively participated in animal experiments, assisted with laboratory assays, and revised the manuscript. YXL, LYY and CJZ analyzed and interpreted the data and contributed to drafting the manuscript. All authors reviewed and approved the final manuscript.

DISCLOSURE STATEMENT

This work was supported by grants from the Sichuan Province Science and Technology Support Program (24NSFSC0429), the Sichuan University Postdoctoral Research and Development Fund (No. 2024SCU12014), a key research project grant from the Chengdu Technology Bureau (2024-YF05-00875-SN), and a grant from the Health Commission of Chengdu (2021021). The remaining authors declare that they have no relevant conflicts of interest.

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No competing interests reported.

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Immunization with an adeno-associated viral vectored allergy vaccine containing Der p1-Der p2 effectively alleviates an asthmatic phenotype in mice

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Version 1

Abstract

Figures

Introduction

Results

Construction and Characterization of AAV Vector Carrying Der p1/2 Fusion Antigens

PBMCs from Allergic Individuals Stimulated with Dp12S Show High Levels of Regulatory and Th1 Cytokines

AAV-Dp12S Immunization Reduces AHR and Eosinophilia and Induces Macrophage Proliferation in HDM-Induced Asthmatic Mice

AAV-Dp12S Treatment Reduces IgE Production in Mice Allergic to *D. pteronyssinus

AAV-Dp12S Immunization Ameliorates Pulmonary Inflammation and Mucus Overproduction

AAV-Dp12S Immunization Modulates Th2 Cytokines and Induces IL-10 and IFN-γ

AAV-Dp12S Immunization Suppresses iNOS Expression and Improves Lung Function

Discussion

Methods

Construction and Characterization of Der p1/2 Mosaic Proteins

SDS-PAGE and Western Blot Analysis

Donors and Sera

Reactivity of Human IgE and Culture of Peripheral Blood Mononuclear Cells for Cytokine Determination

AAV Production, Purification, and Titer Quantification

Asthma Models and Vaccination Strategies

Analysis of Airway Allergic Inflammation and Lung Function

RNA Isolation and Quantitative PCR Analysis

Analysis of Cells from Lung and Bronchoalveolar Lavage Fluid (BALF)

Measurement of Immunoglobulins in Sera and BALF

Fluorescence Microscopy

Splenocyte Culture and Cytokine Quantification in Supernatants of Cell Culture

Data Collection and Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1