BET11 Antibody

What is Bet v 1 and why is it significant in antibody research?

Bet v 1 is the major allergen from birch pollen responsible for triggering allergic reactions in sensitized individuals. It has become a significant focus in antibody research because it serves as a model allergen for understanding the mechanisms of type I allergic reactions and developing immunotherapeutic approaches.

The significance of Bet v 1 in antibody research stems from several factors. First, it allows researchers to investigate differences in antibody responses between allergic and non-allergic individuals. Studies have demonstrated that birch pollen allergic patients (BPA) produce IgE antibodies that primarily recognize conformational epitopes on Bet v 1, whereas immunoglobulin G (IgG) responses in both allergic and non-allergic subjects predominantly target unfolded and sequential epitopes . This distinction is crucial for understanding the pathophysiology of allergic reactions and developing targeted interventions.

Second, Bet v 1 research contributes to our understanding of cross-reactivity with structurally related allergens from the PR-10 protein family, which are found in various foods like apples (Mal d 1), hazelnuts (Cor a 1), and alder pollen (Aln g 1) . This cross-reactivity explains why many birch pollen-allergic individuals also experience oral allergy syndrome when consuming certain fruits and nuts.

Finally, Bet v 1 serves as a platform for developing novel therapeutic approaches, including hypoallergenic derivatives for allergen-specific immunotherapy and engineered antibodies or nanobodies that can block IgE binding to prevent allergic responses .

How can researchers distinguish between conformational and sequential epitopes in Bet v 1 antibody studies?

Distinguishing between conformational and sequential epitopes is methodologically critical in Bet v 1 antibody research. Researchers employ several complementary approaches to make this distinction.

One primary method involves comparing antibody binding to folded recombinant Bet v 1 versus unfolded fragments or peptides. In recent studies, researchers have used ELISA or micro-array analysis to test sera from allergic and non-allergic subjects for reactivity against folded recombinant Bet v 1, two unfolded recombinant Bet v 1 fragments (F1 covering the N-terminal half and F2 covering the C-terminal half), and synthetic peptides spanning the complete Bet v 1 sequence . Antibodies recognizing only the intact folded protein but not the fragments or peptides are considered specific for conformational epitopes, while those binding to fragments or peptides recognize sequential epitopes.

Another approach involves competition ELISAs, where researchers assess whether antibodies binding to unfolded/sequential epitopes can be inhibited by folded Bet v 1. In studies of natural Bet v 1-specific IgG antibodies, researchers found that IgG specific for unfolded/sequential epitopes was not inhibited by folded Bet v 1, suggesting these epitopes represent cryptic determinants not accessible in the native protein conformation .

Thermal or chemical denaturation experiments also help distinguish epitope types. By systematically altering the protein's conformation through heating or chemical treatments and monitoring changes in antibody binding, researchers can determine whether epitope recognition depends on the protein's tertiary structure.

These methodological approaches have revealed that IgE antibodies from birch pollen allergic patients react almost exclusively with conformational epitopes, whereas IgG, IgG1, and IgG4 antibodies from both allergic and non-allergic subjects recognize mainly unfolded and sequential epitopes .

What are the key differences in Bet v 1-specific antibody responses between allergic and non-allergic individuals?

The differences in Bet v 1-specific antibody responses between allergic and non-allergic individuals reveal important insights into the immunological mechanisms of allergy and tolerance. These differences manifest in several distinct ways.

First, the epitope specificity differs significantly between groups. Detailed studies have shown that IgE antibodies from birch pollen allergic patients (BPA) recognize almost exclusively conformational epitopes on Bet v 1. In contrast, IgG antibodies from both allergic patients and non-allergic subjects recognize mainly unfolded and sequential epitopes . This fundamental difference suggests that IgE- and IgG-producing B cells may have different clonal origins.

Second, the antibody isotype distribution varies. While BPA patients produce high levels of Bet v 1-specific IgE, non-allergic individuals predominantly have IgG antibodies without significant IgE production. Within the IgG subclasses, research has examined IgG1 and IgG4 reactivity patterns across different subject groups including BPA, non-birch pollen allergic patients (NBPA), and non-allergic subjects (NA) .

Third, the functional properties of antibodies differ substantially. IgG antibodies from non-allergic subjects have been found to inhibit IgE binding to Bet v 1 only poorly in competition studies, suggesting they do not effectively block allergic responses. Surprisingly, some natural Bet v 1-specific IgG antibodies could even enhance Bet v 1-specific basophil activation , indicating a potential pro-allergenic role under certain circumstances.

Finally, cross-reactivity patterns with related allergens differ between the groups. IgG reactivity to Bet v 1 peptides did not correlate with IgG reactivity to the corresponding Mal d 1 (apple allergen) peptides, suggesting that IgG responses to Bet v 1 are not primarily the result of sensitization to PR10 allergen-containing foods .

These differences have significant implications for understanding the immunological basis of allergy versus tolerance and for developing targeted therapeutic approaches.

How are basophil activation tests used to evaluate the inhibitory potential of Bet v 1-specific antibodies?

Basophil activation tests represent a crucial functional assay for evaluating the inhibitory potential of Bet v 1-specific antibodies in allergy research. The methodology involves several carefully controlled steps to ensure reliable and reproducible results.

The most commonly used system employs rat basophilic leukemia (RBL) cells expressing the human high-affinity IgE receptor (FcεRI). These cells are first sensitized with serum from allergic patients containing Bet v 1-specific IgE antibodies. In a typical protocol, RBL cells (4 × 10^5/well) are seeded in sterile, transparent, flat-bottomed 96-well cell culture plates and incubated with allergic patient's sera (1:10 diluted in medium) overnight at 37°C . After washing to remove unbound IgE, the cells are ready for the allergen challenge phase.

Before challenging the sensitized cells, preliminary titration experiments are performed to identify the allergen concentration range that causes β-hexosaminidase release between background level and maximal response for each individual serum . This critical step ensures the assay operates within its linear range, allowing accurate assessment of inhibition.

To evaluate the inhibitory potential of Bet v 1-specific antibodies or nanobodies, the allergen is pre-incubated with the potential inhibitor before adding to the IgE-sensitized cells. For example, in experiments with the nanobody Nb32 and its trimeric form Nb32ILZ, allergens (0.2 pM – 125 pM) were pre-incubated with nanobodies (0.25 μM – 6.25 μM) for two hours at room temperature before challenging the sensitized cells . The inclusion of appropriate controls is essential:

• Spontaneous release controls: IgE-loaded cells incubated with buffer only

• Non-specific activation controls: Non-sensitized cells incubated with allergen

• Total release controls: Cells lysed with 1% TritonX100 to determine 100% release

After allergen challenge, β-hexosaminidase release is measured using a fluorogenic substrate (4-methylumbelliferyl N-acetyl-β-D-galactosaminide), and results are expressed as percentages of total mediator content . The degree of inhibition is calculated by comparing release in the presence of the inhibitor versus allergen alone.

This methodology has revealed that while monomeric nanobodies like Nb32 only partially inhibit Bet v 1-induced basophil degranulation despite high-affinity binding, trimeric forms like Nb32ILZ can decrease basophil degranulation by up to approximately 90% , demonstrating their superior inhibitory potential.

How do trimeric Bet v 1-specific nanobodies compare to monomeric forms in suppressing allergic responses?

Trimeric Bet v 1-specific nanobodies represent a significant advancement over monomeric forms in suppressing allergic responses, with multiple studies demonstrating their enhanced efficacy through several mechanisms.

The fundamental difference lies in their structure-function relationship. Monomeric nanobodies (like Nb32) consist of a single variable domain that recognizes a specific epitope on Bet v 1. While these can bind with high affinity, they cover only a limited surface area of the allergen. In contrast, trimeric nanobodies (like Nb32ILZ) are engineered by adding an isoleucine zipper domain to the monomeric nanobody, enabling post-translational trimerization . This trimerization significantly enhances both the avidity and effective size of the nanobody complex, allowing for more comprehensive epitope coverage.

In competitive binding assays, Nb32ILZ showed enhanced capacity to inhibit polyclonal IgE binding to Bet v 1 and related allergens from the PR-10 family, including alder (Aln g 1), hazelnut (Cor a 1), and apple (Mal d 1) allergens . This cross-protective effect extends the potential therapeutic applications beyond birch pollen allergy.

The trimeric nanobody also demonstrated efficacy in preventing facilitated antigen binding (FAB) to human B cells expressing CD23. This inhibition is particularly significant because FAB reduction is an important mechanism for decreasing allergen-specific T cell responses. By diminishing the facilitated presentation of Bet v 1 to T cells, Nb32ILZ may reduce downstream allergic inflammation more effectively than monomeric forms .

Importantly, the inhibitory effects of Nb32ILZ on natural allergen extracts were comparable to those observed with recombinant allergens, suggesting its potential effectiveness in real-world allergen exposure scenarios .

What mechanisms explain the clonal origins of IgE versus IgG responses to Bet v 1?

The differing clonal origins of IgE versus IgG responses to Bet v 1 represent a fundamental immunological question with significant implications for allergy treatment. Several mechanisms have been proposed to explain these distinct antibody responses, based on epitope recognition patterns and functional studies.

The most compelling evidence for separate clonal origins comes from epitope specificity studies. Research has demonstrated that IgE antibodies from birch pollen allergic patients recognize almost exclusively conformational epitopes on Bet v 1, requiring the intact tertiary structure of the protein. In stark contrast, IgG antibodies from both allergic and non-allergic subjects predominantly recognize unfolded and sequential epitopes . This fundamental difference in epitope recognition suggests that the B cells producing these antibodies originate from distinct clonal lineages and undergo different selection processes.

The differential recognition may result from several immunological mechanisms. First, the initial antigen presentation and processing pathway may differ for IgE- versus IgG-producing B cell precursors. Antigen-presenting cells might process and present Bet v 1 differently to T cells that subsequently help either IgE- or IgG-producing B cells. Second, the microenvironmental conditions during B cell activation, including cytokine milieu and T cell help, likely differ between allergic and non-allergic responses, driving class switching toward either IgE or IgG.

Competition studies provide additional insight into the separate nature of these responses. IgG antibodies specific for unfolded/sequential Bet v 1 epitopes are not inhibited by folded Bet v 1, suggesting these epitopes represent cryptic determinants not accessible in the native conformation . This finding indicates that IgG antibodies recognize epitopes that are normally hidden in the properly folded protein, becoming exposed only during protein processing or denaturation.

Furthermore, the inability of natural Bet v 1-specific IgG antibodies to efficiently inhibit IgE binding to Bet v 1 in competition assays suggests these antibodies target different regions of the protein. This limited cross-inhibition provides additional evidence for distinct B cell clonal origins rather than a shared precursor population that undergoes different class switching.

Understanding these different clonal origins has important implications for allergen-specific immunotherapy (AIT), as the treatment may need to specifically modulate the IgE-producing B cell lineage or induce protective IgG antibodies that effectively compete with IgE for allergen binding.

How can high-affinity Bet v 1-specific antibodies be isolated and characterized from immunized subjects?

The isolation and characterization of high-affinity Bet v 1-specific antibodies from immunized subjects involves sophisticated molecular and immunological techniques that yield valuable reagents for research and potential therapeutic applications.

One powerful approach for isolating these antibodies utilizes phage display technology to create combinatorial antibody libraries. In a representative study, researchers constructed a phage-displayed single-chain fragment (ScFv) library from the blood of a subject immunized with hypoallergenic recombinant fragments of Bet v 1 . This methodology involves several critical steps:

• RNA isolation from peripheral blood mononuclear cells (PBMCs) of the immunized subject

• cDNA synthesis focusing on immunoglobulin heavy and light chain variable regions

• PCR amplification of variable regions using specialized primer sets

• Assembly of ScFv constructs linking variable heavy and light chains with a flexible peptide linker

• Cloning into phage display vectors and transformation into bacteria

• Selection (panning) of phage-displayed ScFvs against immobilized Bet v 1

• Elution and amplification of binding phages through multiple rounds of selection

This process enriches for high-affinity binders, which are then individually characterized through various binding assays.

For characterizing the isolated antibodies, researchers employ multiple complementary techniques. Affinity measurements using surface plasmon resonance (SPR) or bio-layer interferometry provide quantitative binding parameters including association (kon) and dissociation (koff) rate constants and the equilibrium dissociation constant (KD). Competition ELISAs help determine epitope specificity by assessing whether the isolated antibodies compete with other antibodies or with patients' IgE for binding to Bet v 1 .

Functional characterization involves testing the antibodies' ability to inhibit IgE binding to Bet v 1 and to block allergen-induced basophil activation. In these assays, researchers pre-incubate Bet v 1 with the isolated antibodies before adding to either an IgE binding assay or to IgE-sensitized basophils, measuring the degree of inhibition compared to controls .

Epitope mapping provides crucial information about the binding sites recognized by the isolated antibodies. Techniques include testing reactivity against Bet v 1 fragments, synthetic peptides spanning the Bet v 1 sequence, and Bet v 1 mutants with specific amino acid substitutions .

These comprehensive characterization studies have revealed that immunization with hypoallergenic Bet v 1 derivatives can induce high-affinity antibodies even in non-allergic subjects, and these antibodies can recognize the folded wild-type allergen , providing valuable insights for immunotherapy approaches.

What techniques are used to evaluate facilitated antigen binding inhibition by Bet v 1-specific antibodies?

Facilitated antigen binding (FAB) inhibition represents a critical mechanism through which Bet v 1-specific antibodies may modulate allergic responses. The evaluation of this inhibition employs sophisticated cell-based assays that model the presentation of allergen-IgE complexes to immune cells.

The cornerstone of FAB inhibition assessment is the B-cell assay utilizing CD23-expressing cells. CD23 (the low-affinity IgE receptor) on B cells can capture allergen-IgE complexes and facilitate allergen presentation to T cells, potentially amplifying allergic responses. Researchers have developed standardized protocols using Epstein-Barr virus-transformed B cell lines (EBV B cells) that constitutively express CD23 on their surface .

The methodology follows several precise steps:

• Confirmation of CD23 expression: Before each experiment, CD23 expression on EBV B cells is verified using flow cytometry with anti-human CD23 PE-labeled antibodies (clone REA1222) .

• Formation of allergen-IgE complexes: Sera from allergic patients containing high Bet v 1-specific IgE titers (>100 kUA/l) are incubated with Bet v 1 at various concentrations (typically 1 ng/ml – 1 μg/ml) at 37°C for 1 hour . This step allows for the formation of allergen-IgE complexes that can subsequently bind to CD23.

• Preincubation with potential inhibitors: To assess inhibitory capacity, Bet v 1 is pre-incubated with potential inhibitors before adding patient sera. These inhibitors may include:

  • Monomeric nanobodies (e.g., Nb32)

  • Trimeric nanobodies (e.g., Nb32ILZ)

  • Bet v 1-specific monoclonal antibodies (e.g., BIP 1)

  • Polyclonal Bet v 1-specific rabbit serum

  • Control antibodies or nanobodies with unrelated specificity

• Binding to CD23-expressing B cells: The allergen-IgE complexes (with or without inhibitors) are incubated with EBV B cells (typically 1×10^5 cells per sample) for one hour on ice .

• Detection and quantification: After washing, bound complexes are detected using FITC-labeled anti-human IgE antibodies and analyzed by flow cytometry. The percentage of inhibition is calculated by comparing the binding in the presence of inhibitors versus allergen-IgE complexes alone .

This methodology has revealed significant differences in the inhibitory capacity of various Bet v 1-specific antibodies and antibody formats. For example, while polyclonal Bet v 1-specific rabbit serum targeting various epitopes fully inhibited IgE binding to CD23 on B cells, bivalent monoclonal antibodies showed strong but not complete reduction. Notably, the trimeric nanobody Nb32ILZ demonstrated superior inhibition compared to its monomeric counterpart Nb32 .

The inhibition of FAB is particularly significant because it represents a mechanism for reducing allergen-specific T cell responses beyond direct blocking of IgE binding to allergens. This dual action mechanism may contribute to the therapeutic potential of Bet v 1-specific antibodies and nanobodies in allergic disease.

How does antibody isotype and subclass distribution affect Bet v 1 recognition and allergy modulation?

The distribution of antibody isotypes and subclasses profoundly influences Bet v 1 recognition patterns and their subsequent capacity to modulate allergic responses. This relationship is complex and multifaceted, with significant implications for understanding allergy pathophysiology and developing targeted therapies.

IgE antibodies represent the primary mediators of allergic responses to Bet v 1. These antibodies bind with high affinity to FcεRI receptors on mast cells and basophils, triggering degranulation upon allergen exposure. Research has demonstrated that Bet v 1-specific IgE antibodies from allergic patients recognize almost exclusively conformational epitopes, requiring the intact tertiary structure of the protein . This restricted epitope recognition pattern suggests a highly specialized B cell response in allergic individuals.

In contrast, IgG antibodies exhibit more diverse epitope recognition patterns. Studies examining IgG, IgG1, and IgG4 reactivity to Bet v 1 have revealed that these antibodies from both allergic and non-allergic subjects predominantly recognize unfolded and sequential epitopes . This finding has important implications for understanding natural tolerance and designing immunotherapy approaches.

Within the IgG class, different subclasses exhibit distinct functional properties:

• IgG1: Generally considered the predominant IgG subclass in allergen responses, IgG1 antibodies can potentially activate complement and engage Fcγ receptors on various immune cells. In Bet v 1 studies, IgG1 antibodies from both allergic and non-allergic subjects have shown reactivity to unfolded and sequential epitopes .

• IgG4: Often associated with successful allergen immunotherapy, IgG4 is functionally monovalent due to Fab-arm exchange, does not activate complement, and has poor binding to most Fcγ receptors. This makes IgG4 potentially blocking without triggering inflammatory responses. IgG4 from both allergic and non-allergic subjects also predominantly recognizes unfolded and sequential Bet v 1 epitopes .

The functional consequences of these isotype and subclass distributions are significant. IgG competition studies have shown that natural Bet v 1-specific IgG antibodies inhibit IgE binding to Bet v 1 only poorly, suggesting limited protective capacity. Surprisingly, some IgG antibodies could even enhance Bet v 1-specific basophil activation , indicating potential pro-allergenic effects under certain circumstances.

These findings challenge the simplistic view that IgG antibodies are uniformly protective in allergic contexts. Instead, they suggest that the specific epitope recognition patterns, subclass distribution, and functional properties of induced antibodies are critical determinants of their modulatory effects on allergic responses. This understanding is essential for developing effective allergen-specific immunotherapy approaches that induce protective rather than potentially enhancing antibody responses.

What are the future directions for Bet v 1 antibody research and therapeutic applications?

The field of Bet v 1 antibody research stands at an exciting frontier with multiple promising avenues for future investigation and therapeutic development. Based on current findings, several key directions are emerging that could significantly advance both our fundamental understanding and clinical applications.

One primary direction involves optimizing antibody and nanobody formats for enhanced therapeutic efficacy. While recent research has demonstrated the superior capacity of trimeric nanobodies like Nb32ILZ to inhibit IgE binding and suppress basophil degranulation , further engineering could yield even more effective molecules. Potential approaches include developing bispecific antibodies targeting multiple non-overlapping epitopes on Bet v 1, creating fusion proteins with immunomodulatory domains, or exploring alternative multimerization strategies beyond trimerization to further enhance avidity and coverage.

A second important direction is expanding cross-reactivity studies to address the broader PR-10 allergen family. Current research indicates promising cross-protection by Nb32ILZ against related allergens from alder, hazelnut, and apple , but comprehensive mapping of cross-reactivity patterns and systematic optimization of cross-protective antibodies could lead to therapies addressing multiple related allergies simultaneously. This approach might be particularly valuable for managing oral allergy syndrome, which affects many birch pollen-allergic individuals.

Deeper mechanistic studies represent another critical frontier. While current research has revealed distinct epitope recognition patterns between IgE and IgG antibodies , the underlying immunological mechanisms driving these differences remain incompletely understood. Investigating the clonal relationship between Bet v 1-specific B cells, their epitope-specific selection, and factors influencing class switching could provide valuable insights for designing more effective immunotherapy approaches.

Translation to in vivo models and eventually clinical studies represents the most challenging but potentially impactful direction. Evaluating the protective capacity of engineered antibodies and nanobodies in animal models of birch pollen allergy, followed by carefully designed human studies, will be essential for establishing their therapeutic potential. These studies should assess not only short-term protection against allergic symptoms but also potential long-term immunomodulatory effects.

Finally, combining antibody-based approaches with established allergen-specific immunotherapy could create synergistic treatment strategies. Engineered antibodies or nanobodies might provide immediate protection while immunotherapy gradually reshapes the underlying immune response, potentially leading to more rapid symptom relief and enhanced long-term efficacy.