To overcome the key limitations of traditional allergen immunotherapy (AIT), we integrated three innovative strategies aimed at enhancing safety, efficacy, and patient compliance.

• Firstly, by rationally designing hypoallergenic variants of six major allergens (pollen, dust mites, and seafood derivatives), we mutated the IgE binding epitopes while retaining T-cell reactivity, thereby reducing the risk of anaphylactic shock.
• Secondly, by combining the hypoallergenic units with self-assembling peptides (EFK8), we developed a broad-spectrum fusion protein, enabling multi-allergen targeting and extending the treatment half-life.
• Thirdly, we implemented a micro-needle-based delivery platform for painless and continuous antigen release, improving accessibility and long-term compliance.

These methods collectively addressed the challenges of artificial immunotherapy (AIT) in terms of side effects, complexity, and invasiveness, while inducing a strong immune tolerance.

In this part, we designed and produced recombinant hypoallergenic derivatives derived from six common allergens: Phl p1 and Bet v1 (pollen allergens), Der p1 and Der p2 (dust mite allergens), and Met e1 and Cyp c1 (seafood allergens). Through rational protein design, we eliminated or disrupted key conformational IgE-binding epitopes while strategically preserving T-cell epitopes, and we have named these hypoallergenic derivatives as: mPhl p1, mBet v1, mDer p1, mDer p2, mMet e1, mCyp c1 (Table 1). This approach maintains the immunogenicity required for inducing immune tolerance while significantly reducing the risk of triggering allergic reactions.

Table 1 Design Principles for Hypoallergenic protein Sequences

The plasmids were designed using the pET-32a vector, which features a T7 promoter for high-level expression, a His-tag for simplified purification, and a Trx tag to enhance soluble expression of the target allergen protein. The gene fragments encoding the engineered ​​hypoallergenic proteins​​ were first amplified via ​​PCR​​. The pET-32a vector was then digested with the corresponding ​​restriction enzymes​​ (BamHI and XhoI). Following purification, the target gene fragments and vector were assembled using ​​homologous recombination​​ (Figure 1).

Figure 1 Schematic diagram of the plasmid construction.

​ As shown in Figure 2, PCR products and linearized vector were validated via agarose gel electrophoresis. Target gene fragments (330-720 bp) were confirmed for purity and accuracy through staining and UV visualization. Following gel extraction and purification, six allergen genes (mPhl p1, mBet v1, mDer p1, mDer p2, mCyp c1, mMet e1) were inserted into the linearized pET32a vector using homologous recombination technology, completing recombinant plasmid construction.

Figure 2 Agarose gel electrophoresis results of (A) gene amplification and (B) vector linearization.

The recombinant products were then transformed into E. coli Top 10 competent cells, and positive clones were selected on LB agar plates, respectively. Colony PCR and DNA sequencing verified the correct insertion and integrity of the allergen genes. Selected positive clones were sequenced, and the sequence data confirmed that each plasmid construct contained the correct allergen gene sequence with high fidelity (Figure 3). The validated plasmids were subsequently transformed into E. coli Origami (DE3) competent cells to enhance soluble expression of the recombinant hypoallergenic proteins.

Figure 3 (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

Next, we expanded the cultivation and used IPTG to induce the expression of the hypoallergenic protein derivatives. Our goal was to express and purify the recombinant allergen proteins for subsequent research. After cell lysis and centrifugation, we performed SDS-PAGE to identify the protein expression and purification conditions. The SDS-PAGE analysis results showed that all six hypoallergenic protein derivatives were successfully expressed, and the molecular weights were consistent with the expectations (Figure 4 A). Among them, mPhl p1, mBet v1, and mDer p1 were mainly expressed in the precipitate part, with less background proteins and high purity. These insoluble proteins were then denatured and refolded to obtain soluble proteins. For the three soluble-expressed proteins: mDer p2, mMet e1, and mCyp c1, we used nickel columns to purify them separately. The SDS-PAGE results indicated that we successfully obtained highly pure proteins (Figure 4 B).

Figure 4 SDS-PAGE results of (A) protein expression and (B) purification

To validate whether our engineered hypoallergenic derivatives indeed exhibit reduced allergenicity (IgE binding capacity), we also produced the corresponding native allergens. Notably, since native rDer p1 requires glycosylation and cannot be produced in E. coli, it was commercially purchased from WOLCAVI BIOTECH. For the other five native allergens (rPhl p1, rBet v1, rDer p2, rMet e1, rCyp c1), we introduced reversion mutations via primers, obtained the fragments through PCR, and subsequently constructed the recombinant plasmids using homologous recombination (Figure 5).

Figure 5 Schematic diagram of the plasmid construction of native allergens.

As shown in Figure 6, PCR products were validated via agarose gel electrophoresis. Target gene fragments were confirmed for purity and accuracy through staining and UV visualization. Notably, due to multiple mutation sites in rCyp c1, its gene fragment (156 bp) was assembled from synthesized oligos and then fused with rCyp-AMP (2847 bp) via overlap PCR, yielding a 2984 bp product. We synthesized the rPhl p1 and rMet e1 fragments in the company. After PCR and agarose gel electrophoresis, the pET32a vector was observed to have a band near 5000 base pairs. Both rPhl p1 (777 base pairs) and rMet e1 (876 base pairs) were of the expected size.After the fragments were recovered through gel cutting, we used homologous recombination to recombine them.

Figure 6 Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the E. coli Top 10 bacteria, and then screen out the positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we successfully obtained the plasmid with correct sequencing (Figure 7). These verified plasmids were subsequently transferred into E. coli Origami (DE3).

Figure 7 (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

Next, we expanded the cultivation scale and used IPTG to induce the expression of the natural allergen proteins. The SDS-PAGE analysis results showed that all five natural allergen proteins were detectably expressed in both the supernatant and the precipitate, and their molecular weights were consistent with the expected values (Figure 8A). Subsequently, we purified them using a nickel column. The SDS-PAGE results indicated that we successfully obtained highly purified proteins (Figure 8B).

Figure 8 SDS-PAGE results of (A) protein expression and (B-C) purification.

To address the issue of multiple allergic reaction sensitivities and simplify the treatment plan, we have designed and manufactured a new type of fusion protein. This protein integrates multiple modified hypoallergenic derivatives that trigger similar immune responses into a single molecular structure. This strategy aims to achieve broad therapeutic effects, potentially avoiding the cumbersome process of multiple separate injections, thereby enhancing treatment efficiency and improving patient compliance. To achieve this, we investigated and selected the EFK8 self-assembling peptide (FEFEFKFK), which undergoes oligomerization driven by hydrophobic stacking of phenylalanine residues and electrostatic interactions of lysine residues. This process forms stable nanostructures that increase molecular size and reduce renal clearance, thereby prolonging the circulation half-life (Song et al., 2023).

We assembled short EFK8 oligonucleotide fragments firstly. Subsequently, we separately amplified single hypoallergenic derivative gene fragments with homologous arms through PCR. Then, we used overlapping extension PCR to splice these fragments together to generate the complete fusion allergen gene sequence. Finally, we performed restriction endonuclease digestion and ligation on the purified fusion gene and linearized vector backbone to obtain the desired recombinant plasmid product. Furthermore, we also constructed a plasmid that integrates the EFK8 peptide and the enhanced green fluorescent protein EGFP, which was used for the subsequent determination of the half-life (Figure 9).

Figure 9 Schematic diagram of the plasmid construction.

As shown in Figure 10, PCR products were validated via agarose gel electrophoresis. Target gene fragments were confirmed for purity and accuracy through staining and UV visualization (Figure 10A-B). Then, we used overlapping extension PCR to splice these fragments together to generate the complete fusion allergen gene sequence (Figure 10C). After the fragments were recovered through gel cutting, we used homologous recombination to recombine them.

Figure 10 Agarose gel electrophoresis results of gene amplification.

Next, we transferred the recombinant plasmid into the E. coli Top 10 bacteria, and then screen out the positive clones on the LB culture medium plate. The results of colony PCR and DNA sequencing indicated that we successfully obtained the plasmid with correct sequencing (Figure 11). These verified plasmids were subsequently transferred into E. coli Origami (DE3).

Figure 11 (A) Transformation, (B) colony PCR and (C) Sequencing results of plasmids.

We expanded the cultivation scale and used IPTG to induce the expression of the natural allergen proteins. The EFK8-mPhl p1-GS-mBet v1 (FPB) and EFK8-mDer p1-GS-mDer p2 (FDD) proteins are both expressed in the precipitate. Subsequently, a denaturation-reconstitution procedure is required to restore their soluble state. The EFK8-mMet e1-GS-mCyp c1 (FMC) protein was expressed in both the supernatant and the precipitate. We purified the soluble FMC protein using a nickel column. The SDS-PAGE results indicated that we were able to successfully purify this protein (Figure 12).

Figure 12 SDS-PAGE results of (A) protein expression and (B) purification.

Protein concentrations of both purified native allergens and their hypoallergenic derivatives were quantified using the Bradford assay to ensure accurate loading for subsequent IgE-binding affinity assays. A standard curve was constructed using bovine serum albumin (BSA) as a reference, exhibiting excellent linearity (R² = 0.9987) with the regression equation Y = 0.001563x + 0.002397 (Figure 13). All absorbance measurements at 595 nm were performed in triplicate, and sample concentrations were interpolated based on the standard curve (Table 2).

Figure 13 BSA standard curve test by Bradford assay. (A) The absorbance values of the BSA standard samples. (B) Fitting results of the standard curve.

Table 2 Protein quantification results.

Next, we conducted IgE binding affinity analysis, the main purpose of which is to functionally evaluate the allergenic potential of natural and modified hypoallergenic derivatives, and fusion hypoallergenic derivatives. This assessment is crucial because the clinical severity of allergic reactions is largely determined by the affinity of the specific IgE antibodies against the allergen and its target epitope (Fromberg, 2006). High-affinity IgE antibodies can trigger strong activation of mast cells and basophils even in the presence of low concentrations of allergens, leading to significant release of inflammatory mediators and potential anaphylactic shock. In contrast, low-affinity IgE has significantly reduced ability to bind to the FcεRI receptor, thereby reducing the risk of severe allergic reactions (Udoye, Ehlers, & Manz, 2023). Therefore, by quantifying the degree of reduction in IgE binding affinity, it can provide a key indicator for in vitro experiments to determine whether our rational protein design strategy has successfully achieved the reduction of allergenicity.

The standard curve for human IgE quantification was generated using four-parameter logistic (4PL) regression in GraphPad Prism 9.5, demonstrating excellent fit (R² > 0.998), which validated the reliability of subsequent affinity measurements (Figure 14A). The IgE-binding capacities of six purified allergens—both native and hypoallergenic derivatives—were evaluated by ELISA. Coated proteins were incubated with 50 ng/mL human IgE. As shown in Figure 14B, significant reduction in IgE binding (p < 0.0001) was observed in four hypoallergenic derivatives compared to their native counterparts (mBet v1, mDer p2, mMet e1, mCyp c1). Although mPhl p1 and mDer p1 also showed decreased affinity, the reduction was more modest (p>0.05). These results confirmed the effectiveness of the rational design strategy in generating hypoallergenic proteins with markedly reduced IgE reactivity.

Figure 14 IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of natural allergens and hypoallergenic derivatives.

As illustrated in Figure 15A, the IgE standard curve demonstrated excellent linearity (R² = 0.9943), ensuring the reliability of the subsequent binding affinity quantification. Interestingly, comparative analysis revealed that two fusion allergens, ​​EFK8-mPhl p1-GS-mBet v1 (FPB)​​ and ​​EFK8-mDer p1-GS-mDer p2 (FDD)​​, exhibited significantly reduced IgE binding ratios compared to their respective single hypoallergenic constituents (mPhl p1 and mDer p1, respectively). ​​EFK8-mMet e1-GS-mCyp c1 (FMC)​​ also showed low IgE binding affinity, which was comparable to the levels observed for its individual hypoallergenic derivatives (mMet e1 and mCyp c1) (Figure 15B). This consistent reduction across all fusion constructs suggests that the fusion strategy effectively attenuates IgE immunoreactivity beyond the level achieved by individual engineered allergens, likely due to structural masking of key epitopes or altered antigen presentation.

Figure 15 IgE affinity of allergen proteins. (A) IgE standard curve, (B) Comparison of single and fusion hypoallergenic derivatives.

Building upon the IgE-binding affinity results, we further evaluated the cytotoxicity of both single and fusion hypoallergenic derivatives using a CCK-8 assay to assess their biosafety for potential in vivo therapeutic applications. This evaluation is essential to ensure that engineered biologics exhibit minimal adverse effects on cell viability before proceeding to animal studies or clinical use.

After incubating cells with each protein at concentrations of 1 nM, 10 nM, and 100 nM for 24 h and 48 h, significant differences in cytotoxicity profiles were observed. For mBet v1 and mCyp c1, at all tested concentrations and time points, there was almost no significant effect on cell viability, demonstrating extremely high biocompatibility (Figure 16B, H). For mPhl p1 and mMet e1, the toxicity showed a clear time and concentration dependence (Figure 16A, G). At low concentrations and short durations, the impact was minimal, but as the concentrations and durations increased, the toxicity significantly intensified. For mDer p1 and mDer p2, even at lower concentrations and shorter durations, it could cause a significant decrease in cell viability, indicating an inherent toxicity (Figure 16D, E).

Next, we compared the cytotoxicity profiles of both individual and fused hypoallergenic derivatives. For the fusion construct EFK8-PT-mPhl p1-GS-mBet v1, we observed that the intrinsic cytotoxicity of mPhl p1 influenced the overall profile of the fusion protein, leading to a detectable reduction in cell viability particularly under high-concentration or prolonged exposure conditions (Figure 16A-C).In contrast, the fusion protein EFK8-PT-mMet e1-GS-mCyp c1 exhibited a different behavior: although mMet e1 alone showed time- and concentration-dependent cytotoxicity, the fusion with mCyp c1—which itself displayed no appreciable toxicity—resulted in a composite molecule that did not induce significant cytotoxicity, even at high doses or after extended incubation (Figure 16D-F). Notably, the fusion protein EFK8-PT-mDer p1-GS-mDer p2 did not exhibit significant cytotoxicity under the tested conditions. This is in clear contrast to its individual components, both mDer p1 and mDer p2, which caused a marked decrease in cell viability, especially at higher concentrations (e.g., 100 nM) and with longer culture periods (48 h) (Figure 16G-I). These results suggest that the fusion strategy can alter the cytotoxic properties of the constituent hypoallergenic derivatives, in some cases mitigating the adverse effects observed with the individual components.

​ The CCK-8 results indicated that most hypoallergenic variants, especially fusion constructs, possess favorable biosafety profiles with minimal impact on cell viability. The marked reduction in cytotoxicity observed for the fusion form of mDer p1, compared to its single allergen counterpart, highlights the potential of fusion strategies not only to broaden immunotherapeutic coverage but also to improve biocompatibility. These findings support the advancement of fusion hypoallergenic derivatives, particularly EFK8-based constructs, toward further evaluation.

Figure 16 The results of the cell proliferation experiment.

To evaluate whether fusion with the self-assembling peptide EFK8 prolongs the half-life of hypoallergenic fusion proteins, we employed EGFP as a model protein incubated in fetal bovine serum at 37 °C for 5 days to simulate in vitro conditions. Fitting the fluorescence decay data to a one-phase exponential model revealed that EFK8-EGFP exhibited a significantly extended half-life (1.820 days) compared to EGFP alone (0.9819 days), nearly doubling its stability (Figure 17). This enhancement is likely attributable to EFK8-mediated oligomerization, which increases molecular size and reduces degradation rate.

Figure 17 Half-life of EGFP and EFK8-EGFP.

To fabricate the dissolvable microneedles (MNs), sodium hyaluronate (50,000 MW) was utilized as the tip-layer material and polyvinyl alcohol (PVA) as the backing layer using a 10 mm × 10 mm mold. The resulting MNs, as shown in the Figure 18A, exhibited integrated structure. Subsequent solubility testing demonstrated that the needle tips dissolved completely within 5 minutes of incubation at 37 °C, indicating their characteristic suitable for efficient drug delivery applications (Figure 18B).

Figure 18 (A) Preparation and (B) Water Solubility Test Soluble Microneedles.

In the above experimental results, we found the significantly reduced IgE binding affinity and cytotoxicity observed in our fusion allergens (EFK8-PT-mPhl p1-GS-mBet v1, EFK8-PT-mDer p1-GS-mDer p2) compared to their monomeric counterparts. We speculated that may be attributed to several synergistic mechanisms facilitated by the fusion strategy and the self-assembling peptide EFK8.

First, fusion-induced ​​steric hindrance​​ and ​​conformational rearrangement​​ likely occlude key IgE-binding epitopes, thereby directly impairing antibody recognition and cross-linking capacity. Secondly, the ​​oligomerization​​ process triggered by EFK8 will increase the molecular size and alter the surface topological structure through hydrophobic and electrostatic interactions, causing conformational changes or masking at specific sites in these fused hypoallergenic derivatives, thereby further leading to the loss of toxic functions. We have predicted the molecular structures of EFK8-PT-mPhl p1-GS-mBet v1 and EFK8-PT-mDer p1-GS-mDer p2, and conducted molecular oligomerization simulations. We found that after oligomerization, some parts of the structures of these hypoallergenic derivatives would be covered. For details, please refer to the Model page.

Collectively, these modifications not only diminish immediate hypersensitivity risks but also mitigate direct cellular toxicity, underscoring the potential of fusion-based designs in developing safer and more effective allergen immunotherapies.

To comprehensively evaluate our engineered EFK8-fusion hypoallergenic derivative, we will conduct a multi-tiered assessment in the future. This includes analyzing their ​​aggregation propensity​​ via biophysical techniques to ensure structural stability, followed by ​​T-cell stimulatory activity assays​​ to confirm preserved immunogenicity for tolerance induction. Furthermore, ​​in vivo immunogenicity studies​​ in murine models will be performed to measure IgG-blocking antibody production and long-term desensitization efficacy. Finally, we aim to develop a ​​microneedle-based biosensor​​ for real-time, minimally invasive monitoring of histamine release in the dermal interstitial fluid, enabling direct pharmacodynamic correlation between treatment and reduction in allergic response.

1. Aggregation Propensity Assessment of EFK8-Fusion Proteins​

To ensure the structural integrity and stability of the novel EFK8 fusion with a hypoallergenic derivative, we will systematically evaluate its aggregation tendency. Specific methods include: quantitative analysis of soluble oligomers via size exclusion chromatography (SEC), determination of hydrodynamic particle size distribution using dynamic light scattering (DLS), and monitoring via exogenous dye binding assays (e.g., bis-fluorescein succinimide). These studies will determine how self-assembled EFK8 peptides influence aggregation kinetics or promote oligomerization, thereby ensuring the stability of subsequent preparations (Grillo et al., 2001).

2. T-cell stimulatory activity assessment

To further validate the T-cell stimulatory activity of our engineered hypoallergenic derivatives, future work will include functional T-cell proliferation and cytokine profiling assays using human peripheral blood mononuclear cells (PBMCs) from sensitized donors (Raaphorst et al., 2025). We plan to utilize standardized methods such as EdU incorporation and dye dilution assays to track proliferation dynamics and measure IL-2, IFN-γ, and IL-10 secretion to evaluate Th1/Tr1 responses. Additionally, we will assess the ability of our constructs to induce antigen-specific T-cell activation in comparison with native allergens, using ELISpot and intracellular staining(van Lier, Brouwer, Rebel, van Noesel, & Aarden, 1989).

3. In Vivo Immunogenicity Assessment​​

In vivo immunogenicity assessment evaluates the ability of engineered hypoallergenic derivatives to stimulate a measurable immune response in a living organism, typically mouse models. This goes beyond simple binding assays to assess complex biological outcomes, such as ​​T-cell activation, cytokine production (e.g., IFN-γ, IL-10), and the generation of allergen-specific IgG blocking antibodies​​. In the future, if permission is granted, this would involve immunizing mice (e.g., BALB/c) with our fusion hypoallergenic derivatives and then analyzing splenocytes or serum. Key readouts include ​​ELISPOT or intracellular cytokine staining for Th1/Tr1 responses​​ and ​​serum IgG titers​​ via ELISA (Thalhamer et al., 2010).

4. Microneedle-Based Histamine Monitoring for Therapeutic Evaluation

To objectively evaluate the in vivo efficacy and pharmacodynamic profile of our novel hypoallergenic therapeutics, we propose developing a dedicated microneedle (MN)-based wearable biosensor for continuous, minimally invasive monitoring of histamine levels in dermal interstitial fluid (ISF). ​​The primary purpose of detecting histamine is to directly quantify mast cell degranulation and allergic response intensity in real-time​​, providing a dynamic and objective measure of therapeutic intervention effectiveness. This approach leverages the core advantages of MN technology, including painless penetration, direct ISF biomarker access, and compatibility with real-time monitoring. We will explore two sensing strategies tailored for histamine detection:

Immunosensor: Utilizing a sandwich ELISA format on MNs tips, where captured histamine is quantified via enzyme-labeled secondary antibodies generating colorimetric/fluorescent signals (Ying et al., 2024).

Electrochemical Sensor: Immobilizing histamine oxidase on MN-integrated electrodes to catalyze histamine, producing hydrogen peroxide, which is then electrochemically oxidized to generate a current signal proportional to histamine concentration (García-Guzmán, Pérez-Ràfols, Cuartero, & Crespo, 2021).

This platform aims to provide direct pharmacological evidence of reduced histamine release following treatment, enabling precise correlation between drug administration and therapeutic response in vivo.

Figure 19 Microneedle based electrochemical sensing (García-Guzmán et al., 2021).

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