EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Carbohydrate epitopes currently recognized as targets for IgE antibodies.

Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub-Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE-binding glycans recognized up to 2008 were considered to be "classical" cross-reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE-binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose-alpha-1,3-galactose (alpha-gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha-gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross-reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub-Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.

Do germinal centers protect most of us from becoming allergic?

OBJECTIVE: To review the literature and discuss a hypothesis as to why most people do not have allergy. This hypothesis is dependent on the following 3 main components: (1) airborne allergens (eg, from pollen or mites) are weak antigens that induce a B-cell response only in immunologically most reactive subjects (ie, with atopy); (2) a roadblock to production of immunoglobulin E (IgE) is the T helper 2/interleukin 4 requirement for class switch to IgE; (3) activated germinal centers prevent the formation of mature IgE-switched B-cells, creating a second roadblock to IgE production. DATA SOURCES: Transgenic reporter mice and a cross-sectional human cohort. STUDY SELECTIONS: From the mouse studies, we selected the data on histology and tissue-derived cell suspensions published by several groups in 2011 to 2014. From the human cohort, we selected our published microarray data on the levels of allergen-specific IgE and IgG in serum. RESULTS: The immune response to airborne atopic allergens entails both IgE and IgG antibodies rather than just an IgG or IgE response. However, as expected for an immune response without mature germinal centers, the specific IgG levels will be very low, typically in the ng/ml range. CONCLUSION: Control of IgE production is not just through the T helper 2/interleukin 4-mediated class switch. Recent studies suggest that mature germinal centers are likely to provide protection against the development of allergy to airborne allergens, as well. This may explain why allergen exposure does not induce allergen-specific IgE in everyone.

Identification of the amino-terminal fragment of Ara h 1 as a major target of the IgE-binding activity in the basic peanut protein fraction.

BACKGROUND: Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens. OBJECTIVE: To analyse the allergenic composition of the basic peanut protein (BPP) fraction. METHODS: A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and 125 I-labelled anti-IgE. RESULTS: Most IgE reactivity of the BPP fraction was due to the 5-7 kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive. CONCLUSION: The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1.

Further investigations of the IgE response to tetanus and diphtheria following covaccination with acellular rather than cellular Bordetella pertussis.

BACKGROUND: It has previously been shown in an uncontrolled study that the IgE response to vaccine antigens is downregulated by co-vaccination with cellular Bordetella pertussis vaccine. METHODS: In the present study, we compared in a controlled trial the humoral immune response to diphtheria toxoid (D) and tetanus toxoid (T) in relation to co-vaccinated cellular or acellular B pertussis vaccine. IgE, IgG4, and IgG to D and T were analyzed at 2, 7, and 12 months of age in sera of children vaccinated with D and T (DT, N = 68), cellular (DTPw, N = 68), 2- or 5-component acellular B pertussis vaccine (DTPa2, N = 64; DTPa5, N = 65). RESULTS: One month after vaccination, D-IgE was detected in 10% sera of DTPw-vaccinated children, whereas vaccination in the absence of whole-cell pertussis resulted in 50%-60% IgE positivity. Six months after vaccination, the IgE antibody levels were found to be more persistent than the IgG antibodies. These diphtheria findings were mirrored by those for tetanus. Only minor differences between vaccine groups were found with regard to D-IgG and T-IgG. No immediate-type allergic reactions were observed. CONCLUSION: Cellular (but not acellular) B pertussis vaccine downregulates IgE to co-vaccinated antigens in infants. We assume that the absence of immediate-type allergic reactions is due to the high levels of IgG antibodies competing with IgE antibodies.

Unique patterns of glycosylation in immunoglobulin subclass G4-related disease and primary sclerosing cholangitis.

BACKGROUND AND AIM: Immunoglobulin subclass G4-related disease (IgG4-RD) is characterized by an abundance of IgG4 antibodies in the serum and tissue. Glycosylation status of antibodies can impact on immune effector functions and disease pathophysiology. We sought to establish glycosylation patterns in a prospective cohort of patients with IgG4-RD and the relationship with disease activity and response to treatment. METHODS: We assessed IgG Fc-tail and Fab-arm glycosylation status in patients with IgG4-RD (n = 22), disease controls with primary sclerosing cholangitis (PSC) (n = 22), and healthy controls (n = 22). Serum IgG and subclasses were quantified using ELISA. Fc and Fab glycosylation were analyzed by mass spectrometry and lectin affinity chromatography, respectively. Disease activity, organ damage, and response to treatment were assessed using the IgG4 Responder Index. RESULTS: Immunoglobulin G Fab sialylation was increased in IgG4-RD compared with PSC and healthy control (P = 0.01), with a preferential increase in IgG4-specific Fab sialylation, which was independent of IgG4 Fab-arm exchange. There was a reduction in IgG1-specific Fc bisection and hybrid structures in IgG4-RD (P < 0.01), which recovered upon steroid treatment and correlated with disease activity. Overall, IgG Fc galactosylation was reduced in both IgG4-RD and PSC (P < 0.01), with a preferential reduction in IgG1-specific sialylation and enhancement of IgG4-specific bisection in PSC. IgG4 fucosylation and IgG1/2/3 hybrid structures negatively correlated with complement C3 and C4 levels in IgG4-RD (P < 0.01), but not PSC. CONCLUSION: We report the first study showing unique antibody glycosylation status in a prospective cohort of IgG4-RD and PSC patients, which may determine modulation of the immune system and contribute to disease pathophysiology.

The Developmental History of IgE and IgG4 Antibodies in Relation to Atopy, Eosinophilic Esophagitis, and the Modified TH2 Response.

A common reaction from anyone confronted with allergy is the question: what prevents universal allergy? We will discuss recent findings in the mouse system that have provided us with clues on why allergy is not more common. We will also address one crucial aspect of atopic allergy in humans, which is absent in most mouse model systems, an IgG/IgE ratio <10. We consider the typical mouse IgE response to be more closely related to the "modified TH2" response in humans. We will discuss the similarities and differences between the IgE and IgG4 response to allergens and an update on the IgG4 B cell, partly derived from studies on eosinophilic esophagitis and IgG4-related diseases.

Dynamics of inter-heavy chain interactions in human immunoglobulin G (IgG) subclasses studied by kinetic Fab arm exchange.

Interdomain interactions between the CH3 domains of antibody heavy chains are the first step in antibody assembly and are of prime importance for maintaining the native structure of IgG. For human IgG4 it was shown that CH3-CH3 interactions are weak, resulting in the potential for half-molecule exchange ("Fab arm exchange"). Here we systematically investigated non-covalent interchain interactions for CH3 domains in the other human subclasses, including polymorphisms (allotypes), using real-time monitoring of Fab arm exchange with a FRET-based kinetic assay. We identified structural variation between human IgG subclasses and allotypes at three amino acid positions (Lys/Asn-392, Val/Met-397, Lys/Arg-409) to alter the strength of inter-domain interactions by >6 orders of magnitude. Each substitution affected the interactions independent from the other substitutions in terms of affinity, but the enthalpic and entropic contributions were non-additive, suggesting a complex interplay. Allotypic variation in IgG3 resulted in widely different CH3 interaction strengths that were even weaker for IgG3 than for IgG4 in the case of allotype G3m(c3c5*/6,24*), whereas G3m(s*/15*) was equally stable to IgG1. These interactions are sufficiently strong to maintain the structural integrity of IgG1 during its normal life span; for IgG2 and IgG3 the inter-heavy chain disulfide bonds are essential to prevent half-molecule dissociation, whereas the labile hinge disulfide bonds favor half-molecule exchange in vivo for IgG4.

Increased IgG4 responses to multiple food and animal antigens indicate a polyclonal expansion and differentiation of pre-existing B cells in IgG4-related disease.

BACKGROUND: IgG4-related disease (IgG4-RD) is a systemic fibroinflammatory condition, characterised by an elevated serum IgG4 concentration and abundant IgG4-positive plasma cells in the involved organs. An important question is whether the elevated IgG4 response is causal or a reflection of immune-regulatory mechanisms of the disease. OBJECTIVES: To investigate if the IgG4 response in IgG4-RD represents a generalised polyclonal amplification by examining the response to common environmental antigens. METHODS: Serum from 24 patients with IgG4-RD (14 treatment-naive, 10 treatment-experienced), 9 patients with primary sclerosing cholangitis and an elevated serum IgG4 (PSC-high IgG4), and 18 healthy controls were tested against egg white and yolk, milk, banana, cat, peanut, rice and wheat antigens by radioimmunoassay. RESULTS: We demonstrated an elevated polyclonal IgG4 response to multiple antigens in patients with IgG4-RD and in PSC-high IgG4, compared with healthy controls. There was a strong correlation between serum IgG4 and antigen-specific responses. Responses to antigens were higher in treatment-naive compared with treatment-experienced patients with IgG4-RD. Serum electrophoresis and immunofixation demonstrated polyclonality. CONCLUSIONS: This is the first study to show enhanced levels of polyclonal IgG4 to multiple antigens in IgG4-RD. This supports that elevated IgG4 levels reflect an aberrant immunological regulation of the overall IgG4 response, but does not exclude that causality of disease could be antigen-driven.

The enigma of IgE+ B-cell memory in human subjects.

Our understanding of the origin and fate of the IgE-switched B cell has been markedly improved by studies in mouse models. The immediate precursor of the IgE-switched B cell is either a relatively naive nonswitched B cell or a mature IgG-switched B cell. These 2 routes are referred to as the direct and indirect pathways, respectively. IgE responses derived from each pathway differ significantly, largely reflecting the difference in time spent in a germinal center and thus time for clonal expansion, somatic hypermutation, affinity maturation, and acquisition of a memory phenotype. The clinical and therapeutic implications for IgE responses in human subjects are still a matter of debate, largely because the immunization procedures used in the animal models are significantly different from classical atopic sensitization to allergens from pollen and mites. On the basis of the limited information available, it seems likely that these atopic IgE responses are characterized by a relatively low IgG/IgE ratio, low B-cell memory, and modest affinity maturation, which fits well with the direct switching pathway. It is still unresolved how the IgE response evolves to cover a wide epitope repertoire involving many epitopes per allergen, as well as many different allergens from a single allergen source.

Nanomolar to sub-picomolar affinity measurements of antibody-antigen interactions and protein multimerizations: fluorescence-assisted high-performance liquid chromatography.

Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.

Species-specific determinants in the IgG CH3 domain enable Fab-arm exchange by affecting the noncovalent CH3-CH3 interaction strength.

A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3-CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.

Consensus statement on the pathology of IgG4-related disease.

IgG4-related disease is a newly recognized fibro-inflammatory condition characterized by several features: a tendency to form tumefactive lesions in multiple sites; a characteristic histopathological appearance; and-often but not always-elevated serum IgG4 concentrations. An international symposium on IgG4-related disease was held in Boston, MA, on 4-7 October 2011. The organizing committee comprising 35 IgG4-related disease experts from Japan, Korea, Hong Kong, the United Kingdom, Germany, Italy, Holland, Canada, and the United States, including the clinicians, pathologists, radiologists, and basic scientists. This group represents broad subspecialty expertise in pathology, rheumatology, gastroenterology, allergy, immunology, nephrology, pulmonary medicine, oncology, ophthalmology, and surgery. The histopathology of IgG4-related disease was a specific focus of the international symposium. The primary purpose of this statement is to provide practicing pathologists with a set of guidelines for the diagnosis of IgG4-related disease. The diagnosis of IgG4-related disease rests on the combined presence of the characteristic histopathological appearance and increased numbers of IgG4⁺ plasma cells. The critical histopathological features are a dense lymphoplasmacytic infiltrate, a storiform pattern of fibrosis, and obliterative phlebitis. We propose a terminology scheme for the diagnosis of IgG4-related disease that is based primarily on the morphological appearance on biopsy. Tissue IgG4 counts and IgG4:IgG ratios are secondary in importance. The guidelines proposed in this statement do not supplant careful clinicopathological correlation and sound clinical judgment. As the spectrum of this disease continues to expand, we advocate the use of strict criteria for accepting newly proposed entities or sites as components of the IgG4-related disease spectrum.

Mechanism of immunoglobulin G4 Fab-arm exchange.

Immunoglobulin G (IgG) antibodies are symmetrical molecules that may be regarded as covalent dimers of 2 half-molecules, each consisting of a light chain and a heavy chain. Human IgG4 is an unusually dynamic antibody, with half-molecule exchange ("Fab-arm exchange") resulting in asymmetrical, bispecific antibodies with two different antigen binding sites, which contributes to its anti-inflammatory activity. The mechanism of this process is unknown. To elucidate the elementary steps of this intermolecular antibody rearrangement, we developed a quantitative real-time FRET assay to monitor the kinetics of this process. We found that an intrinsic barrier is the relatively slow dissociation of the CH3 domains that noncovalently connect the heavy chains, which becomes rate determining in case disulfide bonds between the heavy chains are reduced or absent. Under redox conditions that mimic the previously estimated in vivo reaction rate, i.e., 1 mM of reduced glutathione, the overall rate is ca. 20 times lower because only a fraction of noncovalent isomers is present (with intra- rather than interheavy chain disulfide bonds), formed in a relatively fast pre-equilibrium from covalent isomers. Interestingly, Fab arms stabilize the covalent isomer: the amount of noncovalent isomers is ca. 3 times higher for Fc fragments of IgG4 (lacking Fab domains) compared to intact IgG4, and the observed rate of exchange is 3 times higher accordingly. Thus, kinetic data obtained from a sensitive and quantitative real-time FRET assay as described here yield accurate data about interdomain interactions such as those between Fab and/or Fc domains. The results imply that in vivo, the reaction is under control of local redox conditions.

When binding is enough: nonactivating antibody formats.

Most therapeutic antibodies currently used in the clinic are based on the human IgG1 format, which is a bivalent molecule that efficiently interacts with the immune system's effector functions. In clinical applications where binding to the target alone is sufficient for therapeutic efficacy; however, engagement of the immune system is not required and may even cause unwanted side-effects. Likewise, bivalent binding to the target may negatively influence the therapeutic efficacy of an antibody. Here we discuss the state of the art for antibody-based therapeutics, designed to be nonactivating (i.e. do not engage the innate immune system's effector functions), in both monovalent and bivalent formats.

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody.

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against α4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus, IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions.

Human IgG4 binds to IgG4 and conformationally altered IgG1 via Fc-Fc interactions.

The Fc fragment of IgG4 can interact with the Fc fragment of another IgG molecule. This interaction is a confounding factor when measuring IgG4 rheumatoid factor levels. Recently, we demonstrated that half-molecules of IgG4 can exchange to form a bispecific Ab. We expected these two phenomena to be related and investigated the physicochemical aspects of IgG4 Fc-Fc interactions. We found that IgG4 is >99% monomeric by size-exclusion chromatography; therefore, IgG4 Fc-Fc interactions in the fluid phase (if any) would be short-lived. However, (125)I-labeled IgG4 does bind to IgG1 and IgG4 coupled to a solid phase. By contrast, IgG1 does not bind to coupled IgG4. Furthermore, conditions that induce partial unfolding/dissociation of the CH3 domains enhance IgG4 Fc binding, suggesting that Fc binding is primarily CH3 mediated. IgG4 slowly associates with both IgG4 and IgG1 coupled to a biosensor chip. Remarkably, subsequent dissociation was much faster for IgG4 than for IgG1. Moreover, after binding of an IgG4 mAb to Sepharose-coupled Ag, we observed additional binding of IgG4 with irrelevant specificity, whereas similar binding was not observed with Ag-bound IgG1. We propose that the IgG4-IgG4 Fc interaction resembles an intermediate of the Fab-arm (half-molecule) exchange reaction that is stabilized because one of the IgG4 molecules is coupled to a solid phase. By contrast, IgG4 Fc recognizes IgG1 only after a conformational change that renders CH3(IgG1) accessible to an interaction with the CH3(IgG4). Such Fc interactions may enhance Ag binding of IgG4 in vivo.