Identification of Seasonal Variations of Antibodies against PR-10-Specific Epitopes Can Be Improved Using Peptide-Phage Display.

BACKGROUND: In pollinosis patients, allergen-specific antibody titers show seasonal variations. Little is known about these variations at the epitope level. OBJECTIVES: We aimed at investigating seasonal variations on the level of allergen epitope recognition in patients with Bet v 1-related food allergy using a peptide phage display approach. METHODS: Serum samples collected over 1 year from 4 patients of the placebo arm of the birch-associated soya allergy immunotherapy trial were included. To identify epitopes from Bet v 1-related food allergens, patient sera were used in peptide phage display experiments. In silico analysis of enriched allergen-related motifs was performed. RESULTS: We identified epitope motifs related to Bet v 1 and its homologs in soya and hazelnut (Gly m 4 and Cor a 1, respectively) that were enriched in accordance with birch and hazel pollen exposure. Within several weeks after the birch pollen season peak, the pattern of identified epitope motifs differed considerably among patients. Data for amino acid preferences in homologous Bet v 1 and Cor a 1 epitope motifs identified for one of the investigated patients suggest changes in concentration or specificity of serum antibodies for the Cor a 1 epitope motif. CONCLUSIONS: Peptide phage display data suggest an impact of birch and hazel pollen exposure on the recognition pattern of Bet v 1-like allergen epitopes. Epitope-oriented analyses could provide deeper, personalized details regarding the allergen epitope recognition influenced by pollen exposure beyond the capability of current methods.

Epitope Mapping by HDX-MS Elucidates the Surface Coverage of Antigens Associated with High Blocking Efficiency of Antibodies to Birch Pollen Allergen.

Epitopes of a native pollen allergen protein, birch Bet v1, against four of the noncompeting anti-Bet v1 antibodies individually or in combination, were identified by solution-phase amide backbone H/D exchange (HDX) coupled with high-resolution Q-TOF or Orbitrap mass spectrometry. The HDX results indicates that the four anti-Bet v1 antibodies protected specific regions of Bet v1, explaining the difference in their blocking efficiency of each antibody against Bet v1 binding to polyclonal IgEs in Bet v1 allergic patients. An in-house HDX-MS system was further developed to explore the surface protection of Bet v1 in the presence of all four antibodies with 100% sequence coverage and high redundancy. The data demonstrated that four anti-Bet v1 antibodies were able to simultaneously bind to Bet v1 in solution to provide the most effective blocking for 9 of 10 tested IgE donors in an in vitro antibody-blocking assay. For the first time, we have applied HDX to elucidate the therapeutic advantage of combination antibodies compared with individual antibodies in treating Bet v1 induced allergy.

Challenges related to the immunogenicity of parenteral recombinant proteins: Underlying mechanisms and new approaches to overcome it.

Immune response elicited by therapeutic proteins is an important safety and efficacy issue for regulatory agencies, drug manufacturers, clinicians, and patients. Administration of therapeutic proteins can potentially induce the production of anti-drug antibodies or cell-mediated immune responses. At first, it was speculated that the immunogenicity is related to the non-human origin of these proteins. Later on, it was confirmed that the human proteins may also show immunogenicity. In this review article, we will focus on a number of factors, which play crucial roles in the human protein immunogenicity. These factors are related to the patient's status (or intrinsic properties) and molecular characteristics of the therapeutic protein's (or extrinsic properties). Furthermore, we will discuss available in silico, in vitro, and in vivo methods for the prediction of sequences, which may generate an immune response following parenteral administration of these proteins. In summary, nowadays, it is possible for drug manufacturers to evaluate the risk of immunogenicity of therapeutic proteins and implement a management plan to overcome the problems prior to proceeding to human clinical trials.

A recombinant isoform of the Ole e 7 olive pollen allergen assembled by de novo mass spectrometry retains the allergenic ability of the natural allergen.

The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software. Thirteen out of the 457 de novo sequenced peptides obtained allowed assembling its full-length amino acid sequence. Then, Ole e 7-encoding cDNA was synthesized and cloned in pPICZαA vector for its expression in Pichia pastoris yeast. The analyses by Circular Dichroism, and WB, ELISA and cell-based tests using sera and blood from olive pollen-sensitized patients showed that rOle e 7 mostly retained the structural, allergenic and antigenic properties of the natural allergen. In summary, rOle e 7 allergen assembled by de novo peptide sequencing by MS behaved immunologically similar to the natural allergen scarcely isolated from pollen. SIGNIFICANCE: Olive pollen is an important cause of allergy. The non-specific lipid binding protein Ole e 7 is a major allergen with a high incidence and a phenotype associated to severe clinical symptoms. Despite its relevance, its cloning and recombinant expression has been unable by classical techniques. Here, we have inferred the primary amino acid sequence of Ole e 7 by mass-spectrometry. We separated Ole e 7 isolated from pollen by 2DE. After in-gel digestion with trypsin and a direct analysis by nLC-MS/MS in an LTQ-Orbitrap Velos, we got the complete de novo sequenced peptides repertoire that allowed the assembling of the primary sequence of Ole e 7. After its protein expression, purification to homogeneity, and structural and immunological characterization using sera from olive pollen allergic patients and cell-based assays, we observed that the recombinant allergen retained the antigenic and allergenic properties of the natural allergen. Collectively, we show that the recombinant protein assembled by proteomics would be suitable for a better in vitro diagnosis of olive pollen allergic patients.

Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Possible effect of landscape design on IgE recognition profiles of two generations revealed with micro-arrayed allergens.

The aim of this study was to investigate possible effects of landscape design on the IgE sensitization profile toward inhalant allergens in patients with respiratory allergy from Uzbekistan where green areas have been changed during the last two decades by a State program. Sera from two different generations of Uzbek (n=58) and, for control purposes, from two generations of Austrian (n=58) patients were analyzed for IgE reactivity to 112 different micro-arrayed allergen molecules by ImmunoCAP ISAC technology. Changes in molecular IgE sensitization profiles to pollen allergens in the young vs the middle-aged Uzbek population were associated with replanting, whereas those in the Vienna populations reflected natural changes in plant growth. Our data indicate that anthropologic as well as natural changes in the biome may have effects on IgE sensitization profiles already from one to another generation.

TepiTool: A Pipeline for Computational Prediction of T Cell Epitope Candidates.

Computational prediction of T cell epitope candidates is currently being used in several applications including vaccine discovery studies, development of diagnostics, and removal of unwanted immune responses against protein therapeutics. There have been continuous improvements in the performance of MHC binding prediction tools, but their general adoption by immunologists has been slow due to the lack of user-friendly interfaces and guidelines. Current tools only provide minimal advice on what alleles to include, what lengths to consider, how to deal with homologous peptides, and what cutoffs should be considered relevant. This protocol provides step-by-step instructions with necessary recommendations for prediction of the best T cell epitope candidates with the newly developed online tool called TepiTool. TepiTool, which is part of the Immune Epitope Database (IEDB), provides some of the top MHC binding prediction algorithms for number of species including humans, chimpanzees, bovines, gorillas, macaques, mice, and pigs. The TepiTool is freely accessible at http://tools.iedb.org/tepitool/. © 2016 by John Wiley & Sons, Inc.

The conformational IgE epitope profile of soya bean allergen Gly m 4.

BACKGROUND: Birch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. OBJECTIVE: To identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. METHODS: Sera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. RESULTS: All subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kUA /L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. CONCLUSION AND CLINICAL RELEVANCE: Serum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.

Immunoregulatory T cell epitope peptides: the new frontier in allergy therapy.

Allergen immunotherapy (AIT) has been practised since 1911 and remains the only therapy proven to modify the natural history of allergic diseases. Although efficacious in carefully selected individuals, the currently licensed whole allergen extracts retain the risk of IgE-mediated adverse events, including anaphylaxis and occasionally death. This together with the need for prolonged treatment regimens results in poor patient adherence. The central role of the T cell in orchestrating the immune response to allergen informs the choice of T cell targeted therapies for down-regulation of aberrant allergic responses. Carefully mapped short synthetic peptides that contain the dominant T cell epitopes of major allergens and bind to a diverse array of HLA class II alleles, can be delivered intradermally into non-inflamed skin to induce sustained clinical and immunological tolerance. The short peptides from allergenic proteins are unable to cross-link IgE and possess minimal inflammatory potential. Systematic progress has been made from in vitro human models of allergen T cell epitope-based peptide anergy in the early 1990s, through proof-of-concept murine allergy models and early human trials with longer peptides, to the current randomized, double-blind, placebo-controlled clinical trials with the potential new class of synthetic short immune-regulatory T cell epitope peptide therapies. Sustained efficacy with few adverse events is being reported for cat, house dust mite and grass pollen allergy after only a short course of treatment. Underlying immunological mechanisms remain to be fully delineated but anergy, deletion, immune deviation and Treg induction all seem contributory to successful outcomes, with changes in IgG4 apparently less important compared to conventional AIT. T cell epitope peptide therapy is promising a safe and effective new class of specific treatment for allergy, enabling wider application even for more severe allergic diseases.

Antibody epitopes on g protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers.

We developed a strategy for creating epitope maps of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs) containing photo-cross-linkers. Using human CXC chemokine receptor 4 (CXCR4) as a model system, we genetically incorporated the photolabile unnatural amino acid p-azido-l-phenylalanine (azF) at various positions within extracellular loop 2 (EC2). We then mapped the interactions of the azF-CXCR4 variants with mAb 12G5 using targeted loss-of-function studies and photo-cross-linking in whole cells in a microplate-based format. We used a novel variation of a whole cell enzyme-linked immunosorbent assay to quantitate cross-linking efficiency. 12G5 cross-linked primarily to residues 184, 178, and 189 in EC2 of CXCR4. Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb epitope footprint with the photo-cross-linked residues clustered around the loss-of-function sites. We also used the targeted photo-cross-linking approach to study the interaction of human CC chemokine receptor 5 (CCR5) with PRO 140, a humanized mAb that inhibits human immunodeficiency virus-1 cellular entry, and 2D7. The mAbs produced distinct cross-linking patterns on EC2 of CCR5. PRO 140 cross-linked primarily to residues 174 and 175 at the amino-terminal end of EC2, and 2D7 cross-linked mainly to residues 170, 176, and 184. These results were mapped to the recent crystal structure of CCR5 in complex with maraviroc, showing cross-linked residues at the tip of the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb epitopes on GPCRs, our targeted photo-cross-linking method is complementary to loss-of-function mutagenesis results and should be especially useful for studying mAbs with discontinuous epitopes.

Modulation of Th1/Th2 immune responses to HIV-1 Tat by new pro-GSH molecules.

We have previously demonstrated that in Ova-immunized mice the increase in intra-macrophage thiol pool induced by pro-GSH molecules modulates the Th1/Th2 balance in favour of a Th1-type immune response. We show now that the same molecules can support a Th1-type over Th2-type immunity against Tat, which is an early HIV-1 regulatory protein and a Th1 polarizing immunomodulator that is increasingly considered in new anti-HIV vaccination strategies. Our results indicate that Tat-immunized mice pre-treated with the C4 (n-butanoyl) derivative of reduced glutathione (GSH-C4) or a pro-drug of N-acetylcysteine (NAC) and beta-mercaptoethylamine (MEA) (I-152), have decreased levels of anti-Tat IgG1 as well as increased levels of anti-Tat IgG2a and IgG2b isotypes suggesting a Th1-type response. Moreover, Th1-(IFN-γ and IL-2) Ag-specific cellular responses were detected by ELISPOT assay in splenocytes of the same animals as well as an increase of IL-12 levels in the plasma. These findings suggest that the Th1 immune response to HIV-1 Tat could be further polarized by these molecules. These results together with those previously reported suggest that pro-GSH molecules could be used to modulate the immune response towards different antigens and may be further exploited for inducing specific Th1 immune responses against other HIV antigens as well as other intracellular pathogens in new Tat-based vaccination protocols.

Mapping of conformational IgE epitopes with peptide-specific monoclonal antibodies reveals simultaneous binding of different IgE antibodies to a surface patch on the major birch pollen allergen, Bet v 1.

Allergic inflammation is based on the cross-linking of mast cell and basophil-bound IgE Abs and requires at least two binding sites for IgE on allergens, which are difficult to characterize because they are often conformational in nature. We studied the IgE recognition of birch pollen allergen Bet v 1, a major allergen for >100 million allergic patients. Monoclonal and polyclonal Abs raised against Bet v 1-derived peptides were used to compete with allergic patients' IgE binding to Bet v 1 to search for sequences involved in IgE recognition. Strong inhibitions of patients' IgE binding to Bet v 1 (52-75%) were obtained with mAbs specific for two peptides comprising aa 29-58 (P2) and aa 73-103 (P6) of Bet v 1. As determined by surface plasmon resonance, mAb2 specific for P2 and mAb12 specific for P6 showed high affinity, but only polyclonal rabbit anti-P2 and anti-P6 Abs or a combination of mAbs inhibited allergen-induced basophil degranulation. Thus, P2 and P6 define a surface patch on the Bet v 1 allergen, which allows simultaneous binding of several different IgE Abs required for efficient basophil and mast cell activation. This finding explains the high allergenic activity of the Bet v 1 allergen. The approach of using peptide-specific Abs for the mapping of conformational IgE epitopes on allergens may be generally applicable. It may allow discriminating highly allergenic from less allergenic allergen molecules and facilitate the rational design of active and passive allergen-specific immunotherapy strategies.

Recognition of T cell epitopes unique to Cha o 2, the major allergen in Japanese cypress pollen, in allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen.

BACKGROUND: Pollens from species of the Cupressaceae family are one of the most important causes of respiratory allergies worldwide. Many patients with pollinosis have specific IgE to both allergens from Japanese cedar and Japanese cypress pollen. We set out to identify T cell epitopes in Cha o 2, the second major allergen of Japanese cypress pollen. METHODS: T cell lines (TCL) and T cell clones (TCC) specific to Cha o 2 were generated from allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen. T cell epitopes in Cha o 2 were identified by responses of TCL stimulated with overlapping peptides. Abilities of IL-4/IFN-gamma production by TCC were evaluated using enzyme immunoassay. RESULTS: Using TCL, 11 dominant and subdominant T cell epitopes were identified in Cha o 2. The subsets of TCC were predominantly of T helper 2-type. A T cell epitope p141-160 in Cha o 2 and corresponding peptide in Cry j 2 showed high homology. Although TCC PC.205.159 responded to stimulation with p141-160 in Cha o 2, it did not respond with corresponding peptide in Cry j 2, therefore, the T cell epitope was unique to Cha o 2. CONCLUSIONS: Eleven T cell epitopes that were identified are unique to Cha o 2. Cha o 2 is a putative aeroallergen that can potentially sensitize human T cells. We concluded that generation of T cells specific to Cha o 2 in allergic patients acts as one of the causes of continuous allergic symptoms in April.

Kinetic analysis of a human chorionic gonadotropin-beta epitope-paratope interaction.

Kinetics of protein-protein or ligand-ligate interaction has predominantly been studied by optical spectroscopy (particularly fluorescence) and surface plasmon resonance biosensors. Almost all such studies are based on association kinetics between ligand-ligate and suffer from certain methodological and interpretational limitations. Therefore, kinetic analyses of dissociation data of such interactions become indispensable. In the present investigation, the radiolabeled human chorionic gonadotropin-beta ((125)IhCGbeta) was employed as a probe and nitrocellulose (NC) as a solid support to immobilize monoclonal antibody (MAb) G(1)G(10).1. The NC-G(1)G(10).1-(125)IhCGbeta complex (NC(com)) was prepared and the dissociation of radiolabeled hCGbeta was carried out in the presence of excess unlabeled ligate. From the experimental dissociation data under varying ionic strength, dissociation constants (k(- 1)), association constants (k(+1)) and affinity constants (k(a)) were calculated. The values obtained were utilized in exploring the amino acid residues constituting an epitopic region of hCGbeta involved in interaction with the complementary paratope on MAb G(1)G(10).1. Kinetic data of the present study supported our recently published findings [using single step-solid phase radioimmunoassay (SS-SPRIA)] that the core region of hCGbeta epitope consists of Arg (94,95) and Asp (99) while a Lys (104) and a His (106) are in proximity to the core epitopic region. Based on the results of present investigation, we conclude that dissociation kinetics coupled with SS-SPRIA unequivocally provides considerable insight into the study of ligand-ligate interactions and epitope analysis.

Epitope mapping of monoclonal antibody to integrin alphaL beta2 hybrid domain suggests different requirements of affinity states for intercellular adhesion molecules (ICAM)-1 and ICAM-3 binding.

Integrin undergoes different activation states by changing its quaternary conformation. The integrin beta hybrid domain acts as a lever for the transmission of activation signal. The displacement of the hybrid domain can serve to report different integrin activation states. The monoclonal antibody (mAb) MEM148 is a reporter antibody that recognizes Mg/EGTA-activated but not resting integrin alpha(L) beta2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the beta2 hybrid domain. Integrin alpha(L) beta2 binds to its ligands ICAM-1 and ICAM-3 with different affinities. Integrin is proposed to have at least three affinity states, and the position of the hybrid domain differs in each. We made use of the property of mAb MEM148 to analyze and correlate these affinity states in regard to alpha(L) beta2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated alpha(L)beta2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, alpha(L) beta2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that alpha(L)beta2 with intermediate affinity is sufficient to bind ICAM-1 but not ICAM-3, which requires a high affinity state. Furthermore, we showed that the conformation adopted by alpha(L)beta2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation.

Use of molecular simulation for mapping conformational CYP2E1 epitopes.

The identification of the epitopes recognized by autoantibodies against cytochrome P450s (CYPs) associated with drug-induced hepatotoxicity is difficult because of their conformational nature. In the present investigation, we used a novel approach based on the analysis of the whole molecule antigenic capacity following single amino acid substitutions to identify the conformational epitopes on CYP2E1. A molecular model of CYP2E1 was generated based on the CYP2C5 crystal structure, and potential motifs for amino acid exchanges were selected by computer simulation in the surface of alpha helices and beta sheets. Fourteen modified, apparently correctly folded CYP2E1 variants were produced in Escherichia coli and evaluated in immunoprecipitation experiments using sera with anti-CYP2E1 autoreactivity from 10 patients with halothane hepatitis and 12 patients with alcoholic liver disease. Ala substitution of Glu-248 and Lys-251 as well as of Lys-324, Lys-342, Lys-420, and Phe-421 severely decreased or abolished CYP2E1 recognition by the majority of both the halothane hepatitis and alcoholic liver disease sera, whereas the other substitutions had only minor effects. Based on the structural model, these substitutions identified two distinct epitopes on the CYP2E1 surface corresponding to the G-helix and an area formed by juxtaposition of the J' and K'' helices, respectively. The combined use of molecular modeling and single amino acid mutagenesis is thus a useful approach for the characterization of conformational epitopes recognized by autoantibodies.

Common antigenicity between Japanese cedar (Cryptomeria japonica) pollen and Japanese cypress (Chamaecyparis obtusa) pollen, II. Determination of the cross-reacting T-cell epitope of cry j 1 and cha o 1 in mice.

We have previously detected common antigenicity between Cry j 1 and Cha o 1 in B10.S mice. B10.S mice immunized with Cry j 1- or Cha o 1-generated T cells and antibodies reactive to both allergens. In the present study, we investigated the cross-reacting and Cry j 1-specific T-cell epitopes in B10.S mice. Lymph node cells from B10. S mice immunized with Cry j 1 recognized Cry j 1 p111-130, p211-230, and p310-330 as well as Cha o 1 p209-228. The existence of the cross-reacting T-cell epitope in Cry j 1 and Cha o 1 was confirmed by the response of newly established p211-230-specific and Cha o 1 p209-228-specific T-cell lines. The minimum peptide sequence (p213-224) of the cross-reacting T-cell epitope was identical in Cry j 1 and Cha o 1. These findings clearly demonstrate that common antigenicity at the T-cell level between Japanese cedar and cypress pollen allergens was caused by the existence of an identitical-cell epitope in Cry j 1 and Cha o 1.

Epitope mapping: a new method for biological evaluation and immunotoxicology.

The extraordinary capacity of the immune system to recognise and distinguish between molecules that are almost identical can be exploited to generate reagents useful for the identification of specific determinants on drugs, blood-derived products, hormones, receptors or micro-organisms. This manuscript provides a rationale for the use of epitope mapping and a brief outline of its multiple applications.