BALB/c mice (6–8 weeks old, female) were immunized intraperitoneally with 1000 PNU of A. vulgaris natural pollen extract (Burly, Almaty, Kazakhstan) emulsified in complete Freund’s adjuvant, followed by three booster injections at two-week intervals with the same antigen dose in incomplete Freund’s adjuvant. Three days after the final booster, spleens were harvested and splenocytes were fused with SP2/0 myeloma cells using polyethylene glycol (PEG 1500, Roche) according to standard hybridoma technology, as previously described (20). Hybridomas secreting allergen-specific IgG₁ antibodies were screened by ELISA using plates coated with either A. vulgaris pollen extract or the recombinant major allergen Art v 1. Positive clones were subsequently subcloned by limiting dilution to ensure monoclonality.

To assess the binding specificity of mAb, direct ELISA was performed using either recombinant Art v 1 protein or A. vulgaris pollen extract as the coating antigen. The recombinant Art v 1 (rArt v 1) protein was obtained as previously described (21). Antigens were diluted to a final concentration of 100 PNU/mL (A. vulgaris pollen extract) or 1 µg/mL (rArt v 1 protein) in carbonate buffer (pH 9.5), and 100 µL of the solution was added to each well of a high-binding polystyrene microplate (KHB, China). Plates were incubated overnight at 4 °C. Following removal of the coating solution by vacuum, wells were washed once with ELISA washing buffer (0.1% Tween-20, Serva, Germany, in 0.9% NaCl, Merck, Germany) and subsequently blocked with blocking buffer (0.1 M phosphate buffer containing 0.9% NaCl and 0.5% hydrolyzed casein) for 2 h at room temperature (RT). After blocking, plates were dried at RT for 48 h and stored until use. Hybridoma culture supernatants or purified mAb were serially diluted in ELISA dilution buffer (0.1 M phosphate buffer containing 0.9% NaCl and 0.1% hydrolyzed casein). Aliquots of 100 µL were added to each well and incubated for 30 min at 37 °C. Wells were then washed three times with washing buffer, followed by incubation with horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG antibodies (Cat# AS302-HRP, Xema, Finland) for 30 min at 37 °C. After five washes, TMB substrate solution (Cat# R055, Xema) was added to each well and incubated for 15 min at RT. The reaction was terminated by the addition of 5% sulfuric acid, and absorbance was measured at 450 nm using a HiPo microplate reader (Biosan, Latvia). Only clones demonstrating binding to both the rArt v 1 protein and natural pollen extract were selected for further characterization.

To assess the ability of mAb to inhibit the interaction between human IgE and A. vulgaris allergens, a reverse binding ELISA was performed using ready-to-use components from a specific IgE detection kit (#K200S, Xema). The assay was conducted following a modified protocol. At the first step, 100 µL of serum containing high levels (Class III) of mugwort-specific IgE from a single allergic patient (nArt v 1-specific IgE [w231] level: 5.21 kU/L, determined by immunofluorescence on a Phadia 250 analyzer, Sweden) was added to microplate wells pre-coated with anti-human IgE monoclonal antibodies. The plate was incubated for 60 min at 25 °C with continuous shaking on a platform shaker (ELMI, Latvia). After three washes with ELISA washing buffer (0.1% Tween-20 in 0.9% NaCl), 50 µL of biotinylated natural mugwort allergen was added to each well along with 50 µL of one of the following: ELISA buffer (negative control) or purified anti-mugwort pollen mAb. The plate was incubated for another 60 min at 25 °C under the same shaking conditions, followed by three additional washes. In the third step, 100 µL of streptavidin–polymerized HRP conjugate (working dilution) was added to each well and incubated for 30 min at 25 °C without shaking. After five final washes, 100 µL of TMB substrate (Cat# R055, Xema) was added and incubated for 15 min at RT. The enzymatic reaction was stopped by adding 5% sulfuric acid, and optical density (OD) was measured at 450 nm using a HiPo microplate reader (Biosan, Latvia). All samples were assayed in triplicate. The percentage of inhibition of IgE-allergen binding was calculated using the following formula:

Ninety-six-well microplates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 100 µL per well of a capture antibody specific for mouse IgE (ELISA MAX™ Standard Set Mouse IgE, BioLegend) and incubated overnight at 2–8 °C. The next day, the plates were blocked with 200 µL of blocking buffer per well and incubated at RT for 1 h on a PST-60HL thermal shaker (BIOSAN, Latvia). After blocking, wells were washed four times with washing buffer. Mouse serum samples - obtained from either A. vulgaris-sensitized mice (as previously described (18, 19)) - were diluted 1:5 in ELISA Assay Diluent. Subsequently, 100 µL of each diluted serum sample was added to the wells and incubated for 1.5–2 h at RT with shaking. After four additional washes, 50 µL of biotinylated natural mugwort allergen or rArt v 1 protein was added to each well, along with 50 µL of either ELISA buffer (negative control) or purified anti-mugwort pollen mAb. The remaining steps, including the detection procedure and calculation of the percentage inhibition of IgE-allergen binding, were performed as described previously for the human IgE assay. For subsequent in vivo studies, only those mAb demonstrating the strongest and most consistent inhibition of mouse IgE binding to both A. vulgaris extract and rArt v 1 were selected. Among these, the clone XA19 showed the most potent allergen-blocking activity and was subsequently sequenced (GenBank accession numbers BankIt2947155 Seq1 PV483362 and BankIt2947155 Seq2 PV483363).

The gene sequence for the synthetic construct Art v 1 major Artemisia pollen allergen was retrieved from the NCBI GenBank database (accession number: PQ223694.1). The Open Reading Frame (ORF) was identified using the NCBI ORF Finder tool, yielding the amino acid sequence:

MGSSHHHHHHSSGLVPRGSHMAGSKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHKREAGKESCFCYFDCSKSPPGATPAPPGAAPPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPSTH. To obtain the mature protein sequence for structural modelling of Art v 1, the N-terminal His-tag & linker sequence (MGSSHHHHHHSSGLVPRGSH) were removed. The resulting sequence was used for subsequent protein modelling analyses. To determine the molecular interactions driving the binding of XA19 to Art v 1, structural models of XA19 fab region and of Art v 1 were generated using AlphaFold3 and docking of the structural models was performed using the AlphaFold webserver. An all-atom molecular dynamic simulation of the XA19– Art v 1 complex was conducted using GROMACS [v2022.6 GPU], with the production run extending over 100 ns. The system was propagated using the leap-frog integrator with a 2 fs timestep, totalling 50 million steps. Coordinate and velocity outputs to the.trr file were suppressed to reduce file size, while compressed coordinates were saved every 10 ps. Energies and log files were also recorded at 10 ps intervals. All bonds involving hydrogen atoms were constrained using the LINCS algorithm to enable the use of a 2 fs timestep. Electrostatic interactions were treated using the Particle Mesh Ewald (PME) method with a 1.2 nm cutoff and van der Waals interactions employed a force-switch scheme between 1.0 and 1.2 nm. Temperature was maintained at 298 K using a velocity-rescaling thermostat, and isotropic pressure coupling at 1.0 bar was applied using the C-rescale barostat with a compressibility of 4.5 x 10–⁵ bar^-1. Periodic boundary conditions were applied in all three spatial dimensions, and the simulation continued from the equilibrated NPT ensemble without reinitialization of velocities. Detailed residue-residue interaction analysis was carried out for both the variable light (VL) and variable heavy (VH) chains of the Fab in complex with Art v 1.

mAbs were purified from hybridoma culture supernatants by affinity chromatography using Protein G-agarose Resin 4 (Agarose Bead Technology, Spain), following the manufacturer’s protocol. Briefly, clarified supernatants were filtered through a 0.22 µm membrane and loaded onto a pre-equilibrated Protein G column. After binding, the column was washed extensively with phosphate-buffered saline (PBS, pH 7.4) to remove unbound proteins. Bound antibodies were eluted using 0.1 M glycine-HCl buffer (pH 2.7) and immediately neutralized with 1 M Tris-HCl (pH 9.0). Eluted fractions were analyzed by absorbance at 280 nm, and those containing IgG were pooled, dialyzed against PBS, and stored at –20 °C until use.

A well-established murine model was used to reproduce allergic airway inflammation through intraperitoneal (i.p.) sensitization followed by aerosol and intranasal allergen challenge, as previously described (18, 19). Briefly, specific pathogen-free (SPF) male BALB/c mice aged 8–12 weeks (n = 5 per group; 15 mice in total) were randomly divided into three groups ( Figure 1 ). Mice in the sensitized groups (mAb treatment and positive control) received two i.p. injections of A. vulgaris pollen extract (Burly, Kazakhstan) at 14-day intervals (days 0 and 14). Each injection contained 1000 PNU in 200 µL PBS, adsorbed onto aluminum hydroxide (1 mg Al³⁺ per mouse). Negative control mice (n = 5) received an equal volume (200 µL) of PBS with alum. On day 21, all mice underwent allergen challenge performed three times at 48-h intervals (on days 21, 23, and 25). Each challenge included: inhalation exposure to A. vulgaris pollen extract aerosol (1000 PNU per group), delivered using a previously described whole-body exposure method (19), and intranasal administration of A. vulgaris extract (200 PNU in 20 µL PBS) or PBS (negative control) under light ketamine-xylazine anesthesia. Anesthesia was induced via i.p. injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) in sterile phosphate-buffered saline. To assess the therapeutic potential of mAbs, mice in the pretreatment group received 20 µg of the XA19 mAb (in 20 µL PBS; approximately 1 mg/kg per dose) via intranasal administration under anesthesia, one h prior to each allergen challenge. Clinical monitoring was performed during the third nebulization challenge to assess the severity of allergic rhinitis, with a focus on nasal rubbing. On day 27, airway responsiveness to methacholine or PBS was assessed. On the final day (day 28), mice underwent: an ear swelling test, blood sampling for measurement of total and allergen-specific IgE levels, necropsy for lung tissue collection, followed by cytokine quantification and histopathological analysis to assess allergic inflammation.

Total and allergen-specific IgE levels were measured using enzyme-linked immunosorbent assay (ELISA). Total IgE was quantified using the ELISA MAX™ Standard Set Mouse IgE kit (BioLegend) according to the manufacturer’s instructions, with results expressed in µg/mL.

To quantify allergen-specific IgE, 96-well microplates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 100 µL per well of anti-mouse IgE capture antibody (from the same ELISA kit) and incubated overnight at 2–8 °C. On the following day, plates were blocked with 200 µL/well of blocking buffer and incubated for 1 h at RT on a thermal shaker (PST-60HL, BIOSAN), followed by four washes with wash buffer. Mouse serum samples were diluted 1:10 in assay diluent and added to the wells (100 µL/well), followed by incubation for 2 h at RT with shaking. After four washes, 100 µL of biotinylated A. vulgaris allergen extract were added to the respective wells and incubated for 1 h at RT with shaking. Biotinylation of allergen extracts was performed using the EZ-Link™ NHS-Biotin kit (Thermo Fisher Scientific, Rockford, IL, USA), which labels primary amines while preserving allergenic epitopes. Plates were washed five times before the addition of 100 µL/well of TMB substrate solution (BioLegend, USA). After a 15 min incubation at RT, the enzymatic reaction was stopped by adding 100 µL of 2.5 M H₂SO₄. OD was measured at 450 nm using a Chromate 4300 microplate reader (Awareness Technologies, USA).

To assess local allergic hypersensitivity, mice were intradermally injected into the right auricle with 10 µL of the A. vulgaris pollen extract (100 PNU per mouse). Mice in the negative control group received 10 µL of PBS instead. After 1.5–2 h, auricular thickness was measured using an electronic digital micrometer (MCC-25 DSWQ0-100II, China). The degree of ear swelling was calculated as the difference in thickness (in mm) between the allergen-injected right ear and the PBS-injected left ear.

Following the third nebulization challenge with either allergen or PBS (n = 5 per group), mice were clinically observed for 5 min to assess nasal rubbing frequency, as previously described (22). Observations began with the negative control group (PBS exposure). After each allergen administration, the chamber was thoroughly washed and dried before proceeding with the next group to prevent cross-contamination. Clinical assessments were performed by staff members blinded to the group assignments and study protocol.

Airway hyperresponsiveness (AHR) was evaluated using a whole-body plethysmography (WBP-M) system (Shanghai TOW Intelligent Technology Co. Ltd., Shanghai, China), following the protocol described by Hamelmann et al. (23). Mice were individually placed into the plethysmography chambers and exposed to aerosolized methacholine (25 mg/mL) or phosphate-buffered saline (PBS, negative control) for 5 min. Airway resistance was assessed by measuring the enhanced pause (Penh), a dimensionless parameter indicative of bronchoconstriction. Data acquisition and Penh calculation were performed using ResMass software version 1.4.2 (TOW, China).

Following necropsy, the left lung of each mouse was harvested and processed for cytokine analysis. Tissue samples were placed in 1 mL of Dulbecco’s Modified Eagle Medium (DMEM) and homogenized using a TissueLyser II instrument (QIAGEN, Germany) at 300 oscillations per min for 60 s. The resulting homogenates were centrifuged at 5000 × g for 15 min at 4 °C, and the supernatants were collected and stored at −70 °C until analysis. Proinflammatory cytokine levels, including IL-4, IL-5, IL-13, IL-17, TNF-α, and IFN-γ, were measured in the lung homogenate supernatants using ELISA MAX™ Deluxe Set Mouse kits (BioLegend, San Diego, CA, USA), following the manufacturer’s instructions. Cytokine concentrations were calculated based on standard curves and expressed in pg/mL.

Following euthanasia, mouse nasal turbinates and lungs were excised, rinsed in distilled water, and fixed in 10% neutral-buffered formalin for 7–10 days. After fixation, tissues underwent a standard dehydration protocol consisting of sequential immersion in four changes of 100% isopropyl alcohol, followed by two changes of xylene. Nasal turbinates were decalcified in 20% EDTA-Na solution for 3 days prior to paraffin embedding. Tissues were then infiltrated with four changes of paraffin and embedded into histological blocks. Serial sections of 5 µm thickness were cut using a microprocessor-controlled rotary microtome (MZP-01, KB Tekhnom, Russia). The sections were deparaffinized in two changes of xylene and rehydrated through a graded ethanol series (96%, 80%, and 70%). Staining was performed with hematoxylin (#05-002, BioVitrum, Russia) and eosin (#C0362, DiaPath, Italy), followed by dehydration through ascending concentrations of ethanol (70%, 80%, and 96%) and two final changes of xylene. Coverslips were mounted using Bio Mount synthetic mounting medium (#2813, Bio Optica, Italy). Histological slides were examined using an Mshot MF52-N microscope (China) equipped with an MShot MS23 digital camera. Images were acquired at 100× magnification using the MShot Image Analysis System (China), and selected structures were also examined under oil immersion at 1000× magnification. Calibration was performed using a standardized micrometric scale, and all morphometric measurements were reported in µm. Histopathological changes in the lungs nasal tubinates were assessed using a semiquantitative scoring system described previously (19). The criteria used for scoring are summarized in Table 1 . Histological assessments were performed blinded to treatment groups.

All animal studies were carried out at the certified vivarium facility of the M. Aikimbayev National Scientific Center for Especially Dangerous Infections (NSCEDI), under the Ministry of Health of the Republic of Kazakhstan. SPF BALB/c mice were housed, maintained, and fed in accordance with previously validated procedures (18, 19). Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at NSCEDI (Approval No. 16, dated October 31, 2022), and all procedures complied with national and international standards for the ethical use of animals in research. Humane endpoints were implemented based on IACUC-approved welfare scoring systems to guide timely euthanasia. Prior to terminal sample collection, mice were humanely euthanized under deep anesthesia induced by intraperitoneal administration of ketamine (100 mg/kg) and xylazine (40 mg/kg), followed by cervical dislocation to ensure death.

The use of human serum samples in this study was approved by the Local Ethics Committee of S.D. Asfendiyarov Kazakh National Medical University (Approval No. 14 (150), dated April 26, 2024). Written informed consent was obtained from the donor prior to sample collection.

All statistical analyses and data visualizations were performed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). Comparisons of antibody levels, ear swelling measurements, airway responsiveness, lung cytokine profiles, and histopathological scores among experimental groups were conducted using one-way ANOVA followed by Tukey’s multiple comparisons test. A p-value of < 0.05 was considered statistically significant. All graphical data are presented as mean ± standard error of the mean (SEM).

A total of five hybridoma clones (XA15, XA19, XA36, XA37, and XA40) producing IgG₁ antibodies specific to A. vulgaris pollen extract were successfully generated and purified, with final antibody concentrations ranging from 1.0 to 2.5 mg/mL. Of these, only two clones - XA19 and XA15 -recognized both the natural pollen extract and the recombinant major allergen Art v 1 in direct binding ELISA. However, only the culture supernatant of clone XA19 demonstrated IgE-blocking activity in the reverse ELISA, achieving 18% inhibition of human IgE binding to A. vulgaris extract. In murine IgE-blocking ELISA, purified XA19 mAb reduced IgE binding to the natural pollen extract and Art v 1 by 22% and 52%, respectively. Based on its dual specificity and superior IgE-blocking capacity across both human and murine models, clone XA19 was selected for subsequent in vivo studies.

Successful sensitization of mice to A. vulgaris pollen was achieved in both experimental groups (positive control and mAb pretreatment), as evidenced by a significant accumulation of total and allergen-specific IgE, as well as pronounced sensitization according to the ear swelling test compared to the negative control group ( Figure 2 ). Following triple allergen challenge, mice in the positive control group exhibited a marked increase in allergen-specific IgE levels (vs. sensitization), and other laboratory parameters also showed a trend toward elevation. In contrast, in the mAb pretreatment group, although a moderate increase in total and allergen-specific IgE was observed compared to post-sensitization levels, these values remained slightly lower than in the positive control group. Notably, the ear swelling test in the mAb pretreatment group did not follow the same upward trend. Instead, a decrease in this parameter was recorded compared to the sensitization phase and, more significantly, a substantial reduction relative to the positive control group.

At this stage, we evaluated clinical manifestations of rhinitis and bronchial asthma, as well as histopathological alterations in the nasal turbinates and lung tissue, in A. vulgaris-sensitized mice with or without mAb intranasal pretreatment prior to three consecutive allergen challenges. mAb pretreatment markedly reduced both clinical symptoms - reflected by a significant decrease in nasal rubbing episodes - and histopathological signs of rhinitis when compared to the positive control group ( Figures 3a, c ). Histological analysis of nasal turbinates in the mAb pretreatment group revealed well-preserved respiratory epithelium, consisting predominantly of stratified, ciliated columnar epithelial cells ( Figure 3d ). A small number of goblet cells were detected. The olfactory epithelium appeared as pseudostratified columnar epithelium with only minor focal epithelial necrosis. The lamina propria was intact, without signs of inflammatory cell infiltration or thickening. The submucosal glands displayed normal morphology. The mean histopathological score for nasal turbinate inflammation in this group was 0.6 out of 9, with pathological changes detected in only 3 out of 5 mice. Based on the combined clinical signs and histopathological changes in the nasal turbinates, mice in the mAb pretreatment group showed no significant differences compared to the negative control group.

In contrast, all mice (5/5) in the positive control group exhibited pronounced histopathological changes. Goblet cell hyperplasia was observed in focal regions, and the lamina propria displayed structural disruption and signs of inflammatory remodeling ( Figure 3d ). Condensed, pyknotic nuclei characteristic of karyorrhexis were present in both respiratory and olfactory epithelial cells. Large areas of epithelial desquamation were observed, along with individual leukocytes and abundant erythrocytes within the nasal cavity lumen, forming dense cellular debris masses. The mean inflammation score in this group was 3.4 out of 9.

No clinical or histopathological signs of rhinitis were observed in mice from the negative control group.

AHR, a defining feature of clinical asthma, was assessed via whole-body plethysmography in response to methacholine challenge ( Figure 3b ). Sensitized mice (positive control group) exposed to allergen challenges with A. vulgaris pollen exhibited pronounced AHR in response to methacholine, compared to the negative control group. In contrast, sensitized mice that received intranasal administration of anti-A. vulgaris mAb one hour prior to each allergen challenge showed significantly reduced AHR. The AHR levels in this group were comparable to those observed in the negative control group.

These functional outcomes were further supported by histopathological analysis of lung tissue ( Figure 3c ). Mice that received mAb intranasal pretreatment displayed significantly reduced pulmonary inflammation compared to the positive control group, with inflammatory levels comparable to those observed in the negative control group.

Histological examination of lung sections from the mAb pretreatment group revealed preserved pleural integrity without thickening or visible signs of inflammation ( Figure 3e ). Bronchial architecture remained intact, although mild goblet cell metaplasia was observed in a minority of bronchi. Low-grade, focal peribronchial lymphocytic infiltrates were detected in 3 out of 5 animals. Alveolar structures were preserved, with normal interalveolar septa, lacking any signs of thickening, inflammation, or hemorrhage. In the positive control group, the pleura also appeared preserved, without thickening or overt inflammation. Bronchial structures remained intact; however, multifocal, marked peribronchial inflammation dominated by macrophages and monocytes was noted in 4 out of 5 mice, along with moderate peribronchial infiltrates. Eosinophils were abundantly present within the inflammatory foci. Goblet cell hyperplasia was prominent in the bronchi of all animals. The alveolar architecture remained preserved, and interalveolar septa showed no signs of thickening, inflammation, or hemorrhage.

No pathological changes were detected in the lung tissues of the negative control group.

Following allergen challenge, mice in the positive control group exhibited a marked increase in the production of key Th2-associated cytokines IL-4 and IL-5 in lung tissue compared to the negative control group ( Figure 4 ). Intranasal pretreatment with anti-A. vulgaris mAb significantly suppressed the expression of IL-4 and IL-5 in sensitized mice, relative to the untreated positive controls. For the remaining cytokines analyzed (IL-13, IL-17, TNF-α, and IFN-γ), a trend toward elevated levels was observed in the positive control group; however, these differences did not reach statistical significance when compared with the negative control group.

Art v 1, the major allergen of A. vulgaris pollen, has a cysteine-rich N-terminal domain that has homology to plant defensins which are small, basic peptides with a cysteine-stabilized alpha-beta (CSαβ) fold that defend plants, animals and insects against pathogens and parasites, through various mechanisms including membrane permeabilization, proteinase and amylase inhibitory activity and inhibition of protein translation (24). Elucidating the structure and mode of binding of XA19 to Art v 1 might enable better understanding of how XA19 mAb is able to inhibit Art v 1 allergenicity. As no high-resolution crystal structure of Art v 1 was available, we used AlphaFold3 to perform a structural prediction of Art v 1. This revealed Art v 1 to have a classic “head and tail” structure ( Figure 5 ), as previously suggested by another modelling study (25). We next used AlphaFold to predict the XA19 Fab antigen binding domain structure and to perform blind docking of it to the predicted Art v 1 structure. GROMACS was then used to perform a molecular dynamic simulation to optimize the docked complex. The modelled complex shows both the heavy and light chains of XA19 bound the head ‘defensin-like’ domain of Art v 1 with binding of XA19 stabilising the Art v 1 structure ( Figure 5 ). Interestingly, the defensin-fold of Art v 1 has been shown to form epitopes recognized by IgE antibodies from allergic patients (26). Hence XA19 may sterically hinder binding of IgE to these same defensin-domain epitopes, as suggested by our competition ELISA data. Further or in the alternative, the binding of XA19 to Art v 1 may induce a conformational change in Art v 1 that in turn induces blocking or masking of key IgE epitopes.