Structural and functional analyses of antibodies specific for modified core N-glycans suggest a role in TH 2 responses.

BACKGROUND: Immune responses to N-glycan structures from allergens and parasites are often associated with pronounced, high affinity IgE reactivities. Cross-reactive carbohydrate determinants (CCDs) are constituted by modified N-glycan core structures and represent the most frequently recognized epitopes in allergic immune responses. Although recently accepted as potentially allergenic epitopes, the biological and clinical relevance as well as structural and functional characteristics of CCD-specific antibodies remain elusive. METHODS: In order to gain structural insights into the recognition of CCDs, two specific antibody fragments were isolated from a leporid immune repertoire library and converted into human/leporid IgE and IgG formats. The antibody formats were assessed by ELISA and surface plasmon resonance, structural and functional analyses were performed by X-ray crystallography, mediator release, and ELIFAB assays. RESULTS: The recombinant IgE exhibited highly specific interactions with different types of CCDs on numerous CCD-carrying glycoproteins. Crystal structures of two CCD-specific antibodies, one of which in complex with a CCD-derived disaccharide emphasize that mechanisms of core glycan epitope recognition are as specific as those governing protein epitope recognition. The rIgE triggered immediate cellular responses via FcεRI cross-linking and mediated facilitated antigen presentation by binding of IgE/antigen complexes to CD23, a process that also could be blocked by IgG of allergic patients. CONCLUSIONS: Our study provides evidence for the relevance of N-glycan recognition in TH 2 responses and corroborates that IgE and IgG antibodies to ubiquitous carbohydrate epitopes can be equivalent to those directed against proteinaceous epitopes with implications for diagnostic and immunotherapeutic concepts.

A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases.

Gene therapy critically relies on vectors that combine high transduction efficiency with a high degree of target specificity and that can be administered through a safe intravenous route. The lack of suitable vectors, especially for gene therapy of brain disorders, represents a major obstacle. Therefore, we applied an in vivo screening system of random ligand libraries displayed on adeno-associated viral capsids to select brain-targeted vectors for the treatment of neurovascular diseases. We identified a capsid variant showing an unprecedented degree of specificity and long-lasting transduction efficiency for brain microvasculature endothelial cells as the primary target of selection. A therapeutic vector based on this selected viral capsid was used to markedly attenuate the severe cerebrovascular pathology of mice with incontinentia pigmenti after a single intravenous injection. Furthermore, the versatility of this selection system will make it possible to select ligands for additional in vivo targets without requiring previous identification of potential target-specific receptors.

Human IgE is efficiently produced in glycosylated and biologically active form in lepidopteran cells.

TH2-biased immunity to parasites and allergens is often associated with increased levels of antigen-specific and high affinity IgE. The role in reacting against minute amounts of target structures and to provoke severe anaphylactic reactions renders IgE a mechanistically outstanding isotype. IgE represents the least abundant serum antibody isotype and exhibits a variety of peculiarities including structure, extensive glycosylation and effector functions. Despite large progress in antibody technologies, however, the recombinant access to isotypes beyond IgG such as IgE still is scarce. The capacity of expression systems has to meet the complex structural conformations and the extensive posttranslational modifications that are indispensable for biological activity. In order to provide alternatives to mammalian expression systems with often low yield and a more complex glycosylation pattern we established the recombinant production of the highly complex IgE isotype in insect cells. Recombinant IgE (rIgE) was efficiently assembled and secreted into the supernatant in yields of >30 mg/L. Purification from serum free medium using different downstream processing methods provided large amounts of rIgE. This exhibited a highly specific interaction with its antigen, therapeutic anti-IgE and its high affinity receptor, the FcεRI. Lectins and glyco-proteomic analyses proved the presence of prototypic insect type N-glycans on the epsilon heavy chain. Mediator release assays demonstrated a biological activity of the rIgE comparable to IgE derived from mammalian cells. In summary the expression in insect cells provides rIgE with variant glycosylation pattern, but retained characteristics and biological activity. Therefore our data contribute to the understanding of functional and structural aspects and potential use of the IgE isotype.

Basophil activation test using recombinant allergens: highly specific diagnostic method complementing routine tests in wasp venom allergy.

BACKGROUND: Skin testing can expose allergic subjects to potential systemic reactions, sensitization against unrelated proteins, and increased risk of future sting reactions. Therefore the continuous improvement of in vitro diagnostic methods is desirable. Recombinant allergens have been shown to improve the sensitivity of specific IgE (sIgE) detection in vitro whilst no data is available regarding their application and reliability in basophil activation test (BAT). Here we aimed to compare the specificity and sensitivity of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 in both specific IgE (sIgE) detection in vitro and basophil activation test. METHODS: sIgE detection by ELISA or ImmunoCAP and BAT towards the panel of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 were performed in 43 wasp venom allergic patients with a history of anaphylactic reaction and sIgE seropositivity, as well as 17 controls defined as subjects with a history of repetitive wasp stings but absence of any allergic symptom. RESULTS: The BAT performed with the recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to the respective sIgE detection in serum. CONCLUSIONS: BAT performed with the recombinant allergens Ves v 5, Ves v 3 and Ves v 1 provides an emerging highly specific in vitro method for the detection of wasp venom allergy, compared to the sIgE detection. Recombinant allergens applied to BAT represent a step forward in developing reliable in vitro tests for specific diagnosis of allergy.

Vitellogenins are new high molecular weight components and allergens (Api m 12 and Ves v 6) of Apis mellifera and Vespula vulgaris venom.

BACKGROUND/OBJECTIVES: Anaphylaxis due to hymenoptera stings is one of the most severe clinical outcomes of IgE-mediated hypersensitivity reactions. Although allergic reactions to hymenoptera stings are often considered as a general model for the underlying principles of allergic disease, venom immunotherapy is still hampered by severe systemic side effects and incomplete protection. The identification and detailed characterization of all allergens of hymenoptera venoms might result in an improvement in this field and promote the detailed understanding of the allergological mechanism. Our aim was the identification and detailed immunochemical and allergological characterization of the low abundant IgE-reactive 200 kDa proteins of Apis mellifera and Vespula vulgaris venom. METHODS/PRINCIPAL FINDINGS: Tandem mass spectrometry-based sequencing of a 200 kDa venom protein yielded peptides that could be assigned to honeybee vitellogenin. The coding regions of the honeybee protein as well as of the homologue from yellow jacket venom were cloned from venom gland cDNA. The newly identified 200 kDa proteins share a sequence identity on protein level of 40% and belong to the family of vitellogenins, present in all oviparous animals, and are the first vitellogenins identified as components of venom. Both vitellogenins could be recombinantly produced as soluble proteins in insect cells and assessed for their specific IgE reactivity. The particular vitellogenins were recognized by approximately 40% of sera of venom-allergic patients even in the absence of cross-reactive carbohydrate determinants. CONCLUSION: With the vitellogenins of Apis mellifera and Vespula vulgaris venom a new homologous pair of venom allergens was identified and becomes available for future applications. Due to their allergenic properties the honeybee and the yellow jacket venom vitellogenin were designated as allergens Api m 12 and Ves v 6, respectively.

Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy.

The generation and use of avian antibodies is of increasing interest in a wide variety of applications within the life sciences. Due to their phylogenetic distance, mechanisms of immune diversification and the way in which they deposit IgY immunoglobulin in the egg yolk, chickens provide a number of advantages compared to mammals as hosts for immunization. These advantages include: the one-step purification of antibodies from egg yolk in large amounts facilitates having a virtually continuous supply; the epitope spectrum of avian antibodies potentially grants access to novel specificities; the broad absence of cross-reactivity with mammalian epitopes avoids assay interference and improves the performance of immunological techniques. The polyclonal nature of IgY antibodies has limited their use since avian hybridoma techniques are not well established. Recombinant IgY, however, can be generated from mammalian monoclonal antibodies which makes it possible to further exploit the advantageous properties of the IgY scaffold. Moreover, cloning and selecting the immune repertoire from avian organisms is highly efficient, yielding antigen-specific antibody fragments. The recombinant approach is well suited to circumvent any limitations of polyclonal antibodies. This review presents comprehensive information on the generation, purification, modification and applications of polyclonal and monoclonal IgY antibodies.

Anti-tumor activity of a B-cell receptor-targeted peptide in a novel disseminated lymphoma xenograft model.

Receptor-targeted therapies have become standard in the treatment of various lymphomas. In view of its unparalleled specificity for the malignant B-cell clone, the B-cell receptor (BCR) on B cell lymphoma cells is a potential therapeutic target. We have used two BCR epitope mimicking peptides binding to the Burkitt's lymphoma cell lines CA46 and SUP-B8. We proved their functionality by demonstrating calcium flux and BCR-mediated endocytosis upon peptide receptor binding. Toxicity experiments in vitro via cross-linking of the BCR with tetramerized epitope mimics lead to apoptosis in both cell lines but was far more effective in SUP-B8 cells. We established a SUP-B8-based disseminated Burkitt's lymphoma model in NOD/SCID mice. Treatment of tumor-bearing mice with tetramerized epitope mimics had significant anti-tumor effects in vivo. We conclude that peptide-mediated, BCR-targeted therapy is a promising approach which may be explored and further developed for application in highly aggressive lymphoma.

Generation and epitope analysis of human monoclonal antibody isotypes with specificity for the Timothy grass major allergen Phl p 5a.

The scarcity of monoclonal human IgE antibodies with specificity for defined allergens is a bottleneck for the molecular characterisation of allergens and their epitopes. Insights into the characteristics of such antibodies may allow for analyses of the molecular basis underlying allergenicity and cross-reactivity, standardisation of allergens as well as improvement of allergy diagnostics and therapeutics. Here we report the generation and application of the first set of authentic human IgG, IgE and IgA antibodies. On the basis of a Phl p 5a specific antibody fragment, a lambda light chain and the IgG1, IgG4, IgE, IgA1, and IgA2 heavy chains, the corresponding human immunoglobulins were constructed and produced in mammalian cells. In parallel, a murine hybridoma line with specificity for Phl p 5a was established, recloned and produced as human chimeric IgE. After purification, immunoreactivity of the antibodies with the allergen was assessed. Applicability in allergy diagnostics was confirmed by establishment of artificial human sera. Functionality of both antibodies was further demonstrated in receptor binding studies and mediator release assays using humanised rat basophil leukaemia cells (RBL-SX38) suggesting the presence of spatially separate epitopes. By using Phl p 5 fusion proteins and recombinant IgE in immunoblotting and mediator release assays we assigned the epitope of the authentic IgE to a looped stretch exclusively present in Phl p 5a. In summary, the Phl p 5-specific antibodies are the first full set of allergy-related antibody isotypes of their kind and represent valuable tools for studies of fundamental mechanisms and structure/function relationships in allergy.

Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells.

Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.

Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity.

BACKGROUND: Hymenoptera venoms are known to cause life-threatening IgE-mediated anaphylactic reactions in allergic individuals. Proper diagnosis of hymenoptera venom allergy using venom extracts is severely affected by molecular cross-reactivities. Although non-glycosylated marker allergens would facilitate the identification of the culprit venom, the major allergen phospholipase A1 (Ves v 1) from yellow jacket venom (YJV) remained unavailable so far. METHODS: Expression of Ves v 1 as wild type and enzymatically inactivated mutant and Ves v 5 in insect cells yielded soluble proteins that were purified via affinity chromatography. Functionality of the recombinant allergens was assessed by enzymatic and biophysical analyses as well as basophil activation tests. Diagnostic relevance was addressed by ELISA-based analyses of sera of YJV-sensitized patients. RESULTS: Both major allergens Ves v 1 and Ves v 5 could be produced in insect cells in secreted soluble form. The recombinant proteins exhibited their particular biochemical and functional characteristics and were capable for activation of human basophils. Assessment of IgE reactivity of sera of YJV-sensitized and double-sensitized patients emphasised the relevance of Ves v 1 in hymenoptera venom allergy. In contrast to the use of singular molecules the combined use of both molecules enabled a reliable assignment of sensitisation to YJV for more than 90% of double-sensitised patients. CONCLUSIONS: The recombinant availability of Ves v 1 from yellow jacket venom will contribute to a more detailed understanding of the molecular and allergological mechanisms of insect venoms and may provide a valuable tool for diagnostic and therapeutic approaches in hymenoptera venom allergy.

Identification, recombinant expression, and characterization of the 100 kDa high molecular weight Hymenoptera venom allergens Api m 5 and Ves v 3.

Insect stings can cause life-threatening IgE-mediated anaphylactic reactions in venom-allergic patients. Although several compounds have already been described as venom allergens, prominent allergen candidates especially in the higher m.w. range have still remained elusive. Tandem mass spectrometry-based sequencing assigned a candidate gene to the most prominent putative high m.w. allergen Api m 5 (allergen C) in honeybee (Apis mellifera) venom and also allowed identification of its homologue Ves v 3 in yellow jacket (Vespula vulgaris) venom. Both proteins exhibit a pronounced sequence identity to human dipeptidyl peptidase IV or CD26. Reactivity of a human IgE mAb verified the presence of these proteins in the venoms. Both proteins were produced in insect cells and characterized for their enzymatic activity as well as their allergenic potential using sera and basophils from insect venom-allergic patients. Both Api m 5 and Ves v 3 were recognized by specific IgE of the majority of patients even in the absence of cross-reactive carbohydrate determinants. Serologic IgE reactivity closely matched activation of human basophils by Api m 5 or Ves v 3, thus underlining their relevance in functional assays. With Api m 5 and Ves v 3, a new pair of homologous allergens becomes available for future clinical applications in diagnosis and therapy that may also contribute to the understanding of molecular mechanisms of insect venoms. Moreover, the patient IgE reactivity together with the cellular activation demonstrates for the first time the relevance of high m.w. allergens in the context of hymenoptera venom allergy.

Dissecting cross-reactivity in hymenoptera venom allergy by circumvention of alpha-1,3-core fucosylation.

Hymenoptera venom allergy is known to cause life-threatening and sometimes fatal IgE-mediated anaphylactic reactions in allergic individuals. About 30-50% of patients with insect venom allergy have IgE antibodies that react with both honeybee and yellow jacket venom. Apart from true double sensitisation, IgE against cross-reactive carbohydrate determinants (CCD) are the most frequent cause of multiple reactivities severely hampering the diagnosis and design of therapeutic strategies by clinically irrelevant test results. In this study we addressed allergenic cross-reactivity using a recombinant approach by employing cell lines with variant capacities of alpha-1,3-core fucosylation. The venom hyaluronidases, supposed major allergens implicated in cross-reactivity phenomena, from honeybee (Api m 2) and yellow jacket (Ves v 2a and its putative isoform Ves v 2b) as well as the human alpha-2HS-glycoprotein as control, were produced in different insect cell lines. In stark contrast to production in Trichoplusia ni (HighFive) cells, alpha-1,3-core fucosylation was absent or immunologically negligible after production in Spodoptera frugiperda (Sf9) cells. Consistently, co-expression of honeybee alpha-1,3-fucosyltransferase in Sf9 cells resulted in the reconstitution of CCD reactivity. Re-evaluation of differentially fucosylated hyaluronidases by screening of individual venom-sensitised sera emphasised the allergenic relevance of Api m 2 beyond its carbohydrate epitopes. In contrast, the vespid hyaluronidases, for which a predominance of Ves v 2b could be shown, exhibited pronounced and primary carbohydrate reactivity rendering their relevance in the context of allergy questionable. These findings show that the use of recombinant molecules devoid of CCDs represents a novel strategy with major implications for diagnostic and therapeutic approaches.

Generation of human monoclonal allergen-specific IgE and IgG antibodies from synthetic antibody libraries.

BACKGROUND: Allergen-specific IgE and IgG antibodies play pivotal roles in the induction and progression of allergic hypersensitivity reactions. Consequently, monoclonal human IgE and IgG4 antibodies with defined specificity for allergens should be useful in allergy research and diagnostic tests. We used combinatorial antibody libraries and subsequent recombinant production to make and assess IgE, IgG1, and IgG4 allergen-specific antibodies. METHODS: We used phage display to select a synthetic single-chain antibody fragment (scFv) library against 3 different allergens, from bee venom, bovine milk, and apple. The scFv obtained were converted into IgG1, IgG4, and IgE antibody formats and assessed for their biochemical properties by ELISA, immunoblotting, and fluorescence-activated cell sorting. RESULTS: Two different antibody formats for each IgG1, IgG4, and IgE antibody were produced in mammalian cells as disulfide-linked and glycosylated Ig, which were usable in allergen-specific ELISA assays and immunoblots. In addition, the recombinant IgE antibodies mediated the binding of allergens to HEK-293 cells transfected with the high-affinity IgE receptor, and this binding was blocked by corresponding IgG antibodies. CONCLUSIONS: The use of synthetic libraries for the generation of allergen-specific recombinant IgE and IgG antibodies should have broad applications in allergological research and diagnosis.

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments.

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.