Pattern of sensitization to yellow jacket venom and expression of recombinant antigen 5 (Ves v 5) from yellow jacket venom

Abstract

Background

Hymenoptera venom allergy is a significant allergic reaction that affects a substantial proportion of adults. Accurate diagnosis of this allergy using venom extracts is challenging due to molecular cross-reactivity. Pure recombinant allergens offer a promising solution to identify the specific venom responsible for allergic reactions. This study aimed to produce recombinant phospholipase A5 (Ves v 5) from yellow jacket venom and evaluate the pattern of bee venom sensitization in a group of sensitive patients.

Methods and results

A total of seven individuals, including four sensitive and three non-sensitive participants, were recruited for this study. Blood samples were collected, and serum was isolated to assess susceptibility to bee venom and recombinant allergens. Expression of Ves v 5 in Escherichia coli resulted in the production of soluble proteins, which were subsequently purified through affinity chromatography. The functionality of the recombinant allergens was evaluated through enzymatic and biophysical analyses, such as dot blot and SDS‒PAGE tests. The diagnostic relevance of Ves v 5 was further investigated using ELISA-based analyses of sera from yellow jacket venom-sensitized patients. Successful production of soluble Ves v 5 in Escherichia coli was achieved. The recombinant Ves v 5 exhibited distinct biochemical and functional characteristics. Evaluation of IgE reactivity in sera from patients underscored the importance of Ves v 5 in hymenoptera venom allergy.

Conclusions

Our findings suggest that recombinant allergens can serve as an alternative to natural extracts for diagnostic purposes. Furthermore, allergen-specific immunotherapy holds the potential to enhance efficiency and specificity in the treatment of hymenoptera venom allergy.

Introduction

Allergic diseases are immune-mediated conditions that typically occur due to IgE-dependent reaction to allergens. In industrialized countries, allergy incidence rates range from 5 to 30% [1, 2] and in severe cases, allergic disorders can lead to systemic anaphylaxis [3]. Anaphylaxis is a rapid and life-threatening hypersensitivity response of the body to allergens. Foods, medicines, and insects are among the most common causes of anaphylaxis Based on European data, the general population experiences a 0.3–7.5% incidence rate of systemic reactions to Hymenoptera stings, with the rate being 0.3–0.8% in children and 14–43% in beekeepers [4]. While a common localized reaction to a Hymenoptera sting is typically observed, in 3–5% of cases, it can also result in a serious systemic reaction and multiorgan failure [5]. The prevalence of insect-sting allergies varies from 0.4 to 4%, with a fatality rate ranging from 0.09 to 0.45/1,000,000 individuals per year [6, 7].

Hymenoptera-related allergies predominantly caused by the superfamilies Apidae and Vespidae [8]. The most common reactions of Hymenoptera stings are caused by yellow jackets¹ and honeybees². Prominent allergens in yellow jacket venom (YJV) include phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2.0101), and antigen 5 (Ves v 5), which are highly abundant proteins in the venom. Ves v 5 (antigen 5) is a 25-kDa protein derived from Vespula venom and is a potent allergen in the Vespidae family [9]. The diagnosis of Hymenoptera venom allergy relies on the clinical history, skin prick test (SPT), and the detection of specific IgE in the serum sample. The sensitivity and specificity of both in vivo and in vitro diagnostic tests are significantly influenced by the quality and quantity of the source extracts. Therefore, the variability in natural extracts complicates test reliability [10]. Utilizing recombinant allergens provides an alternative approach to address the limitations and complexities associated with natural allergenic extracts [11].

E. coli remains the predominant platform for recombinant protein production [12]. The successful utilization of this prokaryotic system relies on its high protein output, fast growth kinetics, genetic plasticity, and simplicity of media and growth conditions. However, heterologous expression in E. coli frequently leads to overproduction, aggregation and development of inclusion bodies, which leads to protein malfunction and insolubility [13]. Various strategies can be employed to enhance the solubility of the expressed protein, and one of them is targeting the secretion of the target protein to the periplasm. This study aimed to produce recombinant Ves v 5 in E. coli and assess its diagnostic relevance for Hymenoptera venom allergy, focusing specifically on this single allergen.

Materials and methods

Ethics approval and consent to participate

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki (https://www.wma.net/policies-post/wma-declaration-of-helsinki/). The study was approved by the Ethics Committee of Birjand University of Medical Sciences (IR.BUMS.REC.1399.327), and all participants provided written informed consent. A total of seven individuals participated in the study, including four who were sensitive to yellow jacket venom and three who were non-sensitive. Sera from these individuals were evaluated using the immunoblotting test (Euroimmun, Germany). Each participant completed a questionnaire that gathered information on their demographic details, history of other allergic diseases such as rhinitis, atopic dermatitis, and asthma, anaphylactic stimulants, anaphylactic attacks, and anaphylaxis-related symptoms.

Strains, vector and culture media

E. coli BL21 (DE3) cells (Novagen, USA), transformed with construct pET-22b_Ves v 5, were used for protein expression. To ensure optimal expression in E. coli cells, the DNA sequence referring to the mature form of the Ves v 5 peptide (GenBank accession no. M98858.1) was codon-optimized. The construct included the N-terminal pelB leader sequence, which facilitated periplasmic secretion through the Sec translocation machine, and the C-terminal hexahistidine tag. The transformed clones were obtained using Luria Bertani (LB) agar medium (Merck, Germany) containing 100 µg/mL ampicillin. Aliquots containing transformed bacteria were stored in 20% (v/v) glycerol at − 70 °C for long-term use.

Expression of recombinant proteins in E. coli

A single isolated colony of transformed bacteria was inoculated in 10 ml of LB medium containing 100 µg/mL ampicillin and cultivated overnight at 37 °C with shaking. Five milliliters of the overnight culture were transferred into 500 ml of LB broth medium containing 100 µg/mL ampicillin and incubated at 37 °C until they reached the exponential phase (OD600 nm of 0.6–0.8). The expression of Ves v 5 was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM overnight at 16 °C.

To extract the periplasmic E. coli fractions, osmotic shock was employed. The pelleted bacteria were resuspended in 10 ml of hypertonic buffer (30 mM Tris, 20% (w/v) sucrose, pH 8.0). EDTA was added to a final concentration of 1 mM. The suspension was incubated at 4 °C for 30 min with gentle agitation, followed by centrifugation at 8000 g/4°C for 20 min. The resulting supernatant (S1) was collected and kept on ice. Cells were resuspended in a hypotonic solution of 5 mM MgSO4 (9 mL) and incubated for 30 min at 4 °C, followed by an additional centrifugation. The supernatant from the hypotonic solution (S2) was combined with the supernatant from the hypertonic solution (S1), and the mixture was centrifuged to remove debris. The resulting pellet was dialyzed against PBS overnight at 4 °C.

Purification of recombinant protein by IMAC

The periplasmic solution containing soluble Ves v 5 was clarified over a 0.45 μm filter and purified by Ni2 + affinity chromatography as follows. Ni-NTA resin (Qiagen, Germany) was added to a disposable column and equilibrated with wash buffer. Then, the periplasmic solution containing soluble Ves v 5 was passed twice through the preequilibrated Ni-NTA matrix. After the sample was applied to the column, the column was washed twice with 10 volumes of wash buffer containing 20 mM imidazole. This wash step helps remove impurities and unbound proteins that may have nonspecifically bound to the column. Next, the bound proteins, including Ves v 5, were eluted from the column using elution buffer containing 250 mM imidazole. Finally, the eluted proteins were collected in five fractions, each containing 500 µL of elution buffer.

Quantification of protein

SDS‒PAGE was performed to assess the size and purity of the protein obtained in this study. The Mini-Protean^® Tetra Cell System (Bio-Rad, Sao Paulo, Brazil) was employed for gel electrophoresis. Following electrophoresis, the gels were stained with Coomassie Brilliant Blue G-250 to visualize the protein bands. The protein concentration of the samples was quantified using the Pierce™ BCA Protein Assay Kit with a bovine serum albumin (Thermo Fisher Scientific, US) standard.

Immunoblot and ELISA

The purified recombinant Ves V 5 allergen, along with bovine serum albumin (BSA) and anti-IgE (kpl, USA) as negative and positive controls, respectively, were spotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) allowed to air dry. To block any unreacted protein-binding sites, the membrane was immersed in a 2% skim milk solution for 2 h. Subsequently, the membrane was washed three times with PBS-T (PBS containing 0.1% Tween 20), with each wash lasting 3 min. Next, the membranes were incubated with a 1/2 diluted solution of positive and negative sera for two hours at room temperature, followed by three washes with PBST buffer. Then, an anti-human IgE antibody coupled with biotin was added to the membranes and incubated for 45 min with constant shaking at room temperature. Next, the membranes were incubated with streptavidin-alkaline phosphatase for 20 min, followed by a final round of washing. The chromogenic substrate BCIP/NBT (bromochloroindolyl phosphate/Nitro Blue Tetrazolium) was added to the membrane for color development, and the reaction was allowed to proceed for 10–20 min. The membrane was then washed with ddH2O.

To determine the reactivity of human IgE antibodies with the recombinant protein, an ELISA was performed. In brief, ELISA microtiter plates were coated overnight at 4 °C with 100 µl of r-Ves V 5 at a concentration of 10 µg/mL in PBS (pH 7.5), along with positive and negative controls. Diluted sera (pooled sera from 4 allergic patients) were added to each well, and the wells were incubated with 100 µl of HRP (horseradish peroxidase)-labeled anti-human IgE. Finally, the color was developed by adding TMB (3,3’,5,5’-tetramethylbenzidine) and finally sulfuric acid was added as stop solution, and the absorbance of the wells was measured at 450 nm using an ELISA reader (Bio Tek Epoch). The measurement of absorbance values was repeated twice.

Result

Protein expression and purification with Ni-affinity chromatography

To express rVes v 5 in bacteria, the pET22b plasmid (Novagen) was used to construct vectors targeting Ves v 5 to the E. coli periplasm. The vectors, containing the pelB secretion signal, were transformed into BL21 (DE3) cells. In this strain, the chromosomal copy of the T7 RNA polymerase gene is regulated by the lacUV5 promoter. The effective transformation of BL21 (DE3) cells with the vector was verified, subsequently inducing the expression of the target protein with 0.1 mM IPTG at 16 °C (Fig. 1.A). Upon induction, T7 RNA polymerase is expressed, resulting in the transcription of the Ves v 5 gene under the control of the T7 promoter. The resulting polypeptide was transported to the E. coli periplasm via the Sec-dependent transport pathway facilitated by the N-terminal pelB secretion signal [14]. The signal peptide was cleaved, and the protein underwent folding in the periplasm with the assistance of chaperones and disulfide bond isomerases. A hexahistidine tag at the C-terminus facilitated protein purification. Purification by Ni-NTA affinity chromatography involved applying the periplasmic solution to pre-equilibrated Ni-NTA (Qiagen, Germany) resin. and washed with 20 mM imidazole to remove impurities. Ves v 5 was eluted with 250 mM imidazole in five 500 µL fractions. This process yielded purified Ves v 5 for further analysis.

Molecular weight determination

Based on the BCA assay, the protein concentration of the purified pelB component was determined to be approximately 360 µg/mL. Additionally, SDS‒PAGE examination confirmed the anticipated molecular weight of Ves v 5, a single band at about 25 kDa (Fig. 1.B).

Fig. 1

Analysis of Ves v 5 expression in BL21 (DE3) using SDS‒PAGE 12.5%. A: Lanes 1 and 2: bacterial protein before and after induction with IPTG, respectively; Lane 3: bacterial pellet after induction with IPTG; Lane 4: pre-stained protein marker. B: Lane 1: protein marker; lanes 2–6: 5 eluates from the Ni-NTA column

Confirmation test for determining sensitive sera

The collected sera were validated for their sensitivity to various allergens derived from bees and wasps, utilizing a commercial blotting kit provided by Euroimmun (Germany). The results validated the reactivity of sera from patients suspected of having allergies to bees and wasps when tested by this blotting kit.

IgE reactivity analysis

The immunological function of the recombinant Ves v 5 was assessed via immunoblotting and ELISA techniques. Dot blot assays confirmed IgE reactivity of purified recombinant Ves v 5 using sera from hymenoptera venom-sensitized patients, with no binding observed for the negative control (BSA) (Fig. 2). Direct ELISA further confirmed IgE binding, as the recombinant protein showed higher absorbance compared to the negative control (Fig. 3). Inhibition ELISA revealed reduced IgE binding in sera pretreated with recombinant Ves v 5, though the reduction was not dose-dependent (Fig. 4). These results confirm the biological activity of recombinant Ves v 5.

Fig. 2

Dot blot assay. (1): Negative control (BSA); (2): Recombinant Ves V 5; (3): Native honey bee and (4): Positive control (Anti IgE)

Fig. 3

The absorbance of the recombinant form of Ves V 5 in the enzyme-linked immunosorbent assay (ELISA) experiment

Fig. 4

Results of the IgE binding ELISA inhibition assay with different concentrations of r-Ves V5

Discussion

This research provides an evaluation of recombinant Ves v 5 allergen as a diagnostic marker for yellow jacket venom allergy while addressing the problems associated with natural venom extracts. Yellow jacket venom contains potent allergens that can cause a range of reactions, from localized symptoms to life-threatening anaphylaxis, which can sometimes be fatal even during a first-time reaction [15]. Anaphylaxis resulting from insect stings has been observed in 3% of adults and can be fatal, even during the first reaction [16]. Currently, there are no established parameters to predict which susceptible individuals will experience a systemic response (SSR) in the future. However, certain risk factors, such as mastocytosis and age over 40, have been considered potential indicators of risk. Venom immunotherapy (VIT) is the most effective treatment for individuals who have experienced SSR. One of the possible pathways through which immunotherapy may work is through the induction of environmental tolerance, which occurs through the production of IgG/IgG4 antibodies. These antibodies can inhibit IgE-dependent reactions by binding to high affinity IgE receptors (FcεRI) and low affinity receptors (FcεRII) on basophils, mast cells, and B cells [17]. VIT induces antigen-specific regulatory T cells, stimulates the production of IL-10, suppresses Th2 immunity, and shifts immune responses toward Th1-type responses [18]. B-regulatory cells also play a role in IL-10 production and the development of long-term immune tolerance. Furthermore, VIT reduces the number of effector cells, including mast cells, basophils, intrinsic type 2 lymphocytes, and eosinophils, in target organs [19]. Meta-analysis studies have demonstrated the effectiveness of VIT in preventing SSR incidents and improving quality of life [20]. Natural venom extracts contain a mixture of major and minor allergens. However, obtaining sufficient quantities of natural venom from yellow and red bees poses a challenge. A highly conventional and notably safer approach involves the utilization of recombinant allergens, which have replaced venom extracts. This substitution is significant due to the variability and inconsistency in the composition and allergenic concentration of such extracts. Standardization plays a crucial role in both diagnostic testing and personalized immunotherapy. Several major allergens from honeybees (Apis mellifera), yellow bees (Vespula vulgaris, Dolichovespula maculate, annual polistes), and fire ants (Solenopsis invicta) have been cloned and expressed using various prokaryotic and eukaryotic systems The primary allergens identified in yellow jacket venom (YJV) include phospholipase A1 (Ves v 1), hyaluronidase (Ves v 2.0101), and antigen 5 (Ves v 5) have been well-characterized. It is also worth noting that rVes v 5 has been incorporated into existing diagnostic systems, such as ImmunoCAP (Thermo Fisher Scientific, US) and Polycheck^® Diagnostics, which highlights its utility in clinical diagnostics. Recombinant allergens provide a standardized alternative. n this study, we designed the Ves v 5 plasmid using bioinformatics tools and developed a pET-based expression system to produce recombinant Ves v 5 in the periplasm of Escherichia coli. Our results demonstrate that recombinant allergens can serve as substitutes for crude extracts in the diagnosis and further evaluation of immunotherapy for yellow jacket venom allergy. This aligns with prior research highlighting recombinant allergens’ utility for diagnostics [21, 22]. The findings of the current present study apply to only one major allergen; however, by examining additional recombinant allergens, future studies would significantly contribute greatly to improving diagnosis and enhancing outcomes of immunotherapy. However, our study has certain limitations. The small number of participants may affect the generalizability of our findings. Additionally, technical challenges related to optimizing protein solubility and purification could impact the yield and functionality of the produced allergen. While our results indicate IgE reactivity, future studies should prioritize the assessment of the clinical effectiveness of recombinant Ves v 5 through extensive trials, as well as explore its applicability in personalized immunotherapy strategies.

Conclusion

In the current study, the recombinant form of Ves v 5 was expressed and purified, and its biological activity was confirmed. These findings indicate that recombinant form of this venom can be served as accessible and reliable sources of allergens for diagnostic assessment. Further studies are required because of the limitations of the present study to assess relevance to the clinic. Additionally, Future plans aim to synthesize even more recombinant venom allergens, which will allow for a broader understanding of their utility in diagnostics and therapy.