The Rise and Fall of IgE

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Dissertation for the Degree of Doctor of Philosophy in Molecular Immunologypresented at Uppsala University in 2002

Abstract

Vernersson, M., 2002. The rise and fall of IgE. Acta Universitatis Upsaliensis.Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science andTechnology. 742. 61 pp. Uppsala. ISBN 91-554-5388-0.

Immunoglobulin E (IgE) occurs exclusively in mammals and is one of fiveimmunoglobulin (Ig) classes found in man. Unlike other isotypes, IgE is best knownfor its pathological effects, whereas its physiological role remains somewhat elusive. To trace the emergence of IgE and other post-switch isotypes we have studied Igexpression in two monotreme species, the duck-billed platypus (Ornithorhynchusanatinus) and the short-beaked echidna (Tachyglossus aculeatus), leading to the cloningof IgE, two IgG isotypes in platypus and echidna IgE. The presence of IgE and theconservation of the overall structure in all extant mammalian lineages indicates an earlyappearance in mammalian evolution and a selective advantage of structural maintenance.Furthermore, both of the two highly divergent platypus γ chains have three constantdomains. Hence, the major evolutionary changes that gave rise to the IgE and IgGisotypes of present day mammals occurred before the separation of monotremes from themarsupial and placental lineages, estimated to have occurred 150-170 million years ago.As the central mediator in atopic allergy, IgE is a prime target in the development ofpreventive treatments. This thesis describes an active immunization strategy that has thepotential to reduce IgE to a clinically significant extent. The active vaccine componentis a chimeric IgE molecule, Cε2-Cε3-Cε4. The receptor-binding target domain, Cε3, isderived from the recipient species, whereas the flanking domains, acting both asstructural support and to break T-cell tolerance, are derived from an evolutionarilydistant mammal. Vaccination of ovalbumin-sensitized rats resulted in a substantialreduction in total IgE in three out of four strains, accompanied by a significant reductionin skin-reactivity upon allergen challenge. No cross-linking activity was observed andthe response to vaccination was reversible with time. The apparent safety and efficacy ofthe vaccine suggest that active immunization against IgE has the potential to become atherapeutic method for humans.Furthermore, the cloning and expression of the pig (Sus scrufa) ε chain will facilitatethe development of sensitive and specific assays for pig IgE, thus increasing thepossibilities of using the pig model in future studies of IgE-mediated reactions.

Key words: Immunoglobulin, IgE, evolution, atopic allergy, vaccine

Molly Vernersson, Department of Cell and Molecular Biology, Immunology Program,Uppsala University, Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden.

© Molly Vernersson 2002ISSN 1104-232XISBN 91-554-5388-0Printed in Sweden by Eklundshofs Grafiska, Uppsala 2002

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Problems worthyof attack

prove their worthby hitting back

Grooks by Piet Hein

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To my Family

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List of Publications

This thesis is based on the following publications and manuscripts, which are referred toin the text by their roman numerals.

I . Vernersson M, Aveskogh M, Munday B and Hellman L. Evidence for an early appearance of modern post-switch immunoglobulinisotypes in mammalian evolution (II); cloning of IgE, IgG1 and IgG2 from amonotreme, the duck-billed platypus, Ornithorhynchus anatinus.Eur J Immunol. 2002; 32:2145-2155.

II. Vernersson M, Aveskogh M and Hellman L.Cloning of IgE from the echidna (Tachyglossus aculeatus) and a comparativeanalysis of ε chains from all three extant mammalian lineages.Manuscript. 2002.

III. Vernersson M, Pejler G, Kristersson T, Alving K and Hellman L.Cloning, structural analysis, and expression of the pig IgE ε chain.Immunogenetics. 1997; 46:461-468.

IV. Vernersson M, Ledin A, Johansson J and Hellman L.Generation of Therapeutic Antibody Responses against IgE through Vaccination.FASEB J. 2002; 16:875-877.FASEB J. express article 10.1096/fj.01-0879fje, 2002.

Reprints were made with permission from the publishers.© Springer-Verlag GmbH & Co. KG© WILEY-VCH Verlag GmbH 2002

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Contents

Abstract .......................................................................................... 2List of Publications.................................................................. 6Contents .......................................................................................... 7Abbreviations and definitions ........................................... 8Introduction ................................................................................. 9Immunoglobulins ..............................................................................10Immunoglobulin features ..............................................................................10Immunoglobulins in evolution......................................................11What about Mammals ..................................................................................13The rise of IgE............................................................................................14IgE features .......................................................................................15Switching to IgE.................................................................................16The IgE Network ................................................................................16FcεRI........................................................................................................17FcεRII/CD23..............................................................................................17Regulation of and by IgE ..............................................................................18Why do we have IgE? ..........................................................................19IgE and parasitic defense................................................................................19Other functions? .........................................................................................20IgE deficiency .............................................................................................21Atopic allergy ..................................................................................21The allergic response....................................................................................22Why are allergies increasing? .........................................................................25Current therapeutic strategies .................................................26Hyposensitization........................................................................................27Allergen independent treatments .............................................28Passive immunization against IgE ..................................................................28Active Immunization against IgE....................................................................29Present study .............................................................................32Aim .........................................................................................................32Results and discussion...................................................................32Cloning and phylogenetic analysis of monotreme ε- and γ chains (I and II).............32Comparative analysis of mammalian ε chains (Paper II)......................................34Cloning, structural analysis and expression of Pig IgE (Paper III) .........................37Active immunization against IgE (paper IV) .....................................................39Loose Ends .........................................................................................45Concluding remarks........................................................................47Acknowledgements..................................................................49References....................................................................................51

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Abbreviations and Definitions

APC antigen presenting cellBCR B cell receptorC constantCD23 the low affinity receptor for IgECDR complementarity-determining regionCH1, -2, -3 and -4 constant heavy chain domain 1, -2, -3 and -4, respectively.Cε1, -2, -3 and -4 constant ε chain domain 1, -2, -3 and -4, respectively.ε chain the heavy chain of IgEELISA enzyme-linked immunosorbent assayeutherians placental mammalsFcεRI the high affinity receptor for IgEFcεRII the low affinity receptor for IgE, also called CD23FcεRIα the IgE binding alpha chain of the high affinity receptorγ chain the heavy chain of IgGGST-C2C3 a fusion protein consisting of the Cε2-Cε3 region of rat IgE

coupled to glutathione-S-transferase of S. japonicumH heavyIFN interferonIg immunoglobulinIL interleukinκ kappa (light chain isotype)λ lambda (light chain isotype)L lightMAb monoclonal antibodymetatherians marsupial mammalsMHC major histocompatibility complexmIg membrane bound immunoglobulinmonotremes a small group of egg-laying mammalsOMO recombinant protein: OpossumCε2-MouseCε3-OpossumCε4OOO recombinant protein: OpossumCε2-Cε3-Cε4ORO recombinant protein: OpossumCε2-RatCε3-OpossumCε4OROO recombinant protein: OpossumCε2-Rat/OpossumCε3-

OpossumCε4Ova ovalbuminPCR polymerase chain reactionprototherians monotremesTCR T cell receptorTh cell T helper celltherian mammals placental and marsupial mammalsV variable

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Introduction

We are constantly exposed to a great variety of potentially pathogenic microbes, such asbacteria, viruses, protozoa and multicellular parasites. Survival in this rather hostileenvironment requires effective and versatile means of protection. The first lines ofdefense are physical and chemical barriers, such as the skin and lysozyme in saliva,which passively, but effectively, prevent entry of unwanted intruders. However, if thesebarriers fail, the cells and molecules that constitute our immune system take a moreoffensive course of action to destroy the intruder.

Our immune system consists of a wide range of distinct cell types and molecules, eachwith important roles. Some components are present and armed before the infection, likephagocytic cells and complement proteins in blood, which recognize and respond tonon-self molecular patterns commonly displayed by microbes. These cells andmolecules are responsible for innate immunity, characterized by rapid responses that areessentially identical from infection to infection with a given microbe.

Other components develop after the infection and adapt to it; namely, the lymphocytesand their products, which constitute adaptive immunity. The hallmarks of adaptiveimmunity are specific recognition of a virtually infinite variety of distinct non-selfstructures, antigens, and the ability to “remember” and respond more vigorously uponrepeated exposure to any given microbe. The lymphocytes fall into two majorpopulations, B cells and T cells, which specifically recognize antigens through theirmembrane-bound antigen receptors, the B cell receptor (BCR) and the T cell receptor(TCR). The immunoglobulins (Igs) are the soluble forms of the BCR and they operateas adaptors in the immune system by linking the specific recognition of microbes to thecells and molecules that ultimately destroy the intruder, like the phagocytic cells andcomplement proteins of innate immunity. The various components of the immunesystem work in concert to eliminate the intruder using the cells and molecules that aremost efficient against the microbe in question.

However, sometimes the immune system reacts unnecessarily vigorously againstotherwise harmless environmental agents, allergens. These overreactions are termedhypersensitivity reactions and fall into four subgroups, types I – IV, depending on themechanism behind the reaction. The type I hypersensitivity reactions are mediated byallergen-specific IgE and are responsible for the clinical manifestations of atopicallergies, such as hay fever, food allergies, eczema, asthma and anaphylaxis. More than20% of the general population suffers from allergic diseases in one form or another andthe alarming increase in their prevalence over the past decades have placed them amongthe most common human diseases in developing countries.

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Although IgE is best known for its detrimental effects, causing allergic disease canhardly be the primary function of IgE. When did IgE appear as a separate isotype, andwhy? What is the selective advantage of having IgE? IgE may have evolved as aprotective mechanism against certain parasitic infections, but its overall contribution tohost defense remains a subject of controversy. Do we still need IgE? Is it possible tovaccinate against IgE? This thesis features IgE, focusing on some of the unresolvedquestions, opening with an evolutionary perspective and ending with the development ofan anti-IgE vaccine as a potential therapeutic strategy against atopic allergy.

Immunoglobulins

Immunoglobulins, or antibodies, are heterodimeric glycoproteins produced by B cells.They share several characteristics with the TCR, including antigen recognition andvarious mechanisms that generate diversity, as well as structural features. However,immunoglobulins recognize native antigens, such as proteins, polysaccarides and lipids,whereas the TCR recognizes peptide fragments presented together with the majorhistocompatibility complex (MHC) class I and II molecules on the surface of infectedand antigen-presenting cells (APC). Furthermore, in contrast to the TCR,immunoglobulins occur in a bifunctional secreted form. In addition to antigen binding,secreted immunoglobulins exhibit one or more effector functions accounted for bydistinct structures spatially separated from the antigen-binding sites.

Immunoglobulin featuresImmunoglobulins are usually composed of two identical heavy (H) chains and twoidentical light (L) chains held together by disulfide bonds and stable non-covalentinteractions. Both H chains and L chains consist of a variable (V) region responsible forantigen binding and a constant (C) region which determines the isotype. Mammalsexpress five major immunoglobulin classes; IgM, IgD, IgA, IgG, and IgE, which aredefined by their H-chain C-region genes; µ, δ, α, γ, and ε. The H-chain C-region genesoccur in a tandem array in the H-chain locus and share V-region genes. The L chain alsooccurs in two different isotypes, kappa (κ) and lambda (λ), but they have separate lociand, accordingly, combine with separate sets of V-region genes.

Both L chains and H chains are folded into repeated globular motifs, immunoglobulindomains, each consisting of about 110 amino acids and composed of two layers of anti-parallel β-pleated sheets that are joined together by a disulphide bond. The L-chainisotypes are composed of two such Ig domains, one V domain and one C domain. Themammalian H-chain isotypes are composed of a V domain and, depending on theisotype, three (α and γ) or four (µ and ε) C domains.

In germline configuration, the genes encoding the V regions are composed of multiplecopies of variable (V), diversity (D) and joining (J) segments (H-chain) or V and Jsegments (L-chain). The enormous V region variability is, in part, generated by the

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random rearrangement of these segments into a functional V region by a genetic somaticevent called V(D)J recombination. The H chain V region (VH) and L chain V region (VL)are juxtaposed in the protein and together they form the antigen-binding site. Thenumerous potential VH and VL combinations generate an amazing diversity of the Igrepertoire, which enables recognition of virtually all foreign and potentially pathogenicstructures.

Initially, the VDJ exon encoding the VH is spliced to the Cµ exon, forming a µ chain,which subsequently assembles with a successfully rearranged L chain. Hence, IgM is theinitial isotype expressed during B-cell development. The antigen specificity of a givenB-cell clone is constant: that is, the Ig V regions are retained. However, through amechanism called isotype switching the constant region, Cµ, can be replaced by Cα,C γ or Cε, resulting in expression of IgA, IgG or IgE isotypes that have the sameantigen specificity as the original IgM.

Together, the immunoglobulins mediate the effector functions of humoral immunity,the principal defense against extracellular microbes. However, each isotype engagesdistinct effector mechanisms for disposing of the antigen, including interaction withisotype specific receptors on various immune cells, activation of complement andtransfer across the placenta or through epithelial layers. In short, mammalian IgMserves as the predominant BCR and it is the principal antibody of primary immuneresponses. IgA is the primary isotype in mucosal immunity, providing a frontlinedefense at epithelial surfaces. IgG is the predominant serum antibody and the role ofmast cell activation, and thus of mediating anaphylactic reactions, falls mainly on IgE.

Immunoglobulins in Evolution

The immunoglobulins and all other components of adaptive immunity (TCR, MHC andrecombination enzymes) seem to have emerged at the appearance of the jawedvertebrates, gnatosomes [1-3]. Since these components appear to be absent ininvertebrates as well as agnathans, the adaptive immune system has been proposed tohave appeared in a relatively short period of evolutionary time, possibly in a single stepcatalyzed by horizontal transfer of recombination genes from prokaryotes into thevertebrate genome. This putative event has been described as a “Big Bang” in molecularevolution [2, 3]. Since the time of this putative “Big Bang”, the complexity of theadaptive immune system has increased gradually throughout vertebrate evolution (Fig.1). IgM, the only isotype that occurs in all investigated gnatosome vertebrates, appearsto be the first and most ancient isotype. Accordingly, IgM is the major serum isotypefound in elasmobranches (sharks and rays) and bony fish [4]. Fish B cells apparently donot undergo isotype switching [4]. However, depending on the species, an IgD-like Ig,IgNAR, IgNARC, IgW or IgR may also be expressed, either through differentialsplicing (the IgD-like Ig) or by their presence on loci separate from that of IgM,reviewed in [4].

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Gnatosomateancestor

Bony fish

Amphibians

Birds

Mammals

?

?

IgEIgM

IgYIgM IgA

IgM IgY IgX

IgM

IgD

IgM

IgD IgAIgG

Figure 1. Scheme for the evolutionary development of immunoglobulins. Putative geneduplication events that gave rise to new isotypes are indicated by dashed arrows. So far, thereis no evidence of IgD in amphibians or in birds, but the number of investigated species i slimited.

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Amphibians (Xenopus) express two non-IgM post-switch isotypes, IgY and IgX [5].The IgY isotype has a wide distribution in non-mammalian vertebrates, includingamphibians; axolotl [6] and Xenopus [7], reptiles; lizard [8] and turtle [9] and birds;chicken [10] and duck [11]. In contrast, the IgX isotype appears to be restricted toXenopus, but the number of species investigated so far is limited. The two Xenopuspost-switch isotypes may have originated from independent duplications of the Cµ gene,and based on membrane exon analysis, IgY appeared before IgX [12]. The latterhypothesis is further supported by the presence of IgY, but not IgX, in the moreprimitive axolotl [6] and indications of an IgY-like isotype in lungfish [6, 13, 14].Hence, IgY is believed to be the first post-switch isotype to appear in evolution and themechanism of isotype switching may have emerged with the tetrapods, or possibly inthe putative ancestors of tetrapods, the lungfish or the coelacanth (Latimeria) [6, 12].

Although Xenopus IgX appears to have a distribution and effector functions similar tothose of the IgA isotype expressed by birds and mammals, it is probably not the directhomologue of mammalian IgA [15]. Hence, in addition to IgM and IgY, birds expressIgA [16]. Like IgM and IgY, avian IgA contains four constant domains, whereasmammalian IgA has only three. Sequence comparisons suggest a common progenitorCα gene for avian (chicken) and mammalian IgA and that the hinge region found inmammalian IgA has replaced the Cα2 domain in the putatively more primitive avianIgA [16].

What about mammals?Most studies of immunoglobulin expression in mammals have focused on placental(eutherian) mammals, and all investigated species express IgM, IgA, IgG and IgE. Sofar, IgD has been found only in rodents and primates. Some Ig classes; namely, IgG andIgA, are often expressed from more than one H-chain gene; that is, several subclasses ofIgG and IgA occur in many species. For example, humans express four IgG isotypes(IgG1 through IgG4) and two IgA isotypes (IgA1 and IgA2). Only one IgG isotypeoccurs in rabbits [17], but at least ten out of thirteen different Cα genes are expressed invivo [18, 19]. Conversely, only one Cα gene occurs in the pig [20], whereas eight C γgenes have been detected in genomic Southern blots and at least five of these areexpressed [21].

Apart from the eutherians, there are two other extant mammalian lineages, themarsupials (metatheria) and the monotremes (prototheria). The evolutionary separationof the marsupials from the placental mammals is generally dated back to approximately130 to 175 million years ago [22-24], whereas the major radiation of the placentalmammals probably occurred 60 to 120 million years ago [24].

Immunoglobulin expression has only been studied in a few of the approximately 270existing marsupial species. The cloning of IgG, IgE and IgA from the gray short-tailedopossum (Monodelphis domestica) revealed that all of the eutherian post-switch

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isotypes occur also in marsupials [25]. Furthermore, as in placental mammals, multiplesubclasses of IgG are reportedly present in several marsupial species, as reviewed in[26].

The monotremes, which are represented by only three species; namely, the duck-billedplatypus and two echidna species, have been regarded as "reptile-like" or "primitive"mammals simply because they lay eggs. However, they do possess several of the majormammalian features. Histological studies show that the spleen, thymus and gut-associated lymphoid tissues in both the platypus and the echidna are well developed andcomparable in histological structure to those in therian mammals (eutherians andmarsupials) [27, 28]. However, in sites where lymph nodes would be expected in therianmammals, monotremes were found to have lymphoid nodules that resemble the jugularbodies of the amphibians, which indicates that monotremes have a somewhat moreprimitive immune system [27, 28]. Early studies of immunoglobulin expression in theechidna established that the major immunoglobulin classes were very similar to humanIgM and IgG [29]. This has been confirmed recently by the cloning of echidna IgM andIgG1 [22, 30].

The evolutionary relationship between marsupials and monotremes is not fully resolved.The Marsupionata hypothesis, supported mainly by mitochondrial data, suggests thatmarsupials and monotremes are sister lineages [31-34]. In opposition, the Theriahypothesis proposes that marsupials and eutherians are sister lineages and that themonotremes constitute the most divergent lineage. The Theria hypothesis initiallyemerged from morphological data and has more recently gained additional support frompaleontological data [35, 36] and a number of nuclear gene sequences [22, 23, 37-40].Studies based on IgM and protamine sequences, both of which support the Theriahypothesis, estimate the time of divergence of the monotremes from the othermammalian lineages at around 150 to170 million years ago [22, 23].

The Rise of IgESo far, IgE as well as IgG have been found exclusively in mammals. However, the IgYisotype found in amphibians, reptiles and birds (see above) shares characteristics withboth IgE and IgG, and based on several observations, a common phylogenic history forthe IgY isotypes and mammalian IgE and IgG has been suggested (Fig. 1) [6, 12, 41,42].

In structure, IgY is more similar to IgE. Both IgY and IgE have four constant domains,whereas the IgG isotype has only three. Sequence comparisons among the differentdomains of IgY and IgG suggest that the equivalent of the Cυ2 domain is absent inmammalian IgG [41, 42]. However, the Cυ2 domain shares features with themammalian hinge, suggesting that a truncated Cυ2 domain formed the hinge in whatevolved into mammalian IgG [41, 42]. Two additional cysteine residues, putativelyinvolved in the formation of a second intra-domain disulfide-bridge, are present in the

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Cυ1 domain of IgY. These additional cysteine residues also occur in most of thecharacterized ε chains, excluding those of possum and rodents, as well as in some γchains, including those of rabbit and pig [17, 21, 42].

In analogy with IgG in mammals, IgY is the major serum antibody in birds andamphibians, and hence is the major isotype involved in the defense against systemicinfections. However, IgY can also mediate anaphylactic reactions [43], which usually,but not exclusively, is ascribed to IgE in mammals. In some mammalian speciesanaphylactic reactions can be mediated by IgG, e.g., in the guinea pig [44] and to alesser extent in mice [45]. Nevertheless, it may have been of selective advantage toseparate the two functions of IgY into two separate isotypes, IgG and IgE, and therebyrestrict potentially dangerous reactions to an isotype that normally occurs in lowconcentrations.

IgE Features (or what makes IgE so special?)IgE is the least abundant of the human immunoglobulin classes and was accordingly thelast to be discovered in the late 1960s [46-48]. The concentration of IgE in normalhuman sera is between 10 and 400 ng/ml, whereas IgG is present at concentrations thatare thirty thousand times higher (9.5-12.5 mg/ml) [49]. Furthermore, at least inhumans, the turnover rate of IgE is much more rapid than that of IgG. The half-life ofIgE in the circulation is estimated at 2-2.5 days, whereas serum IgG has a half-life ofabout 3 weeks [49].

As previously mentioned, mammalian species often express multiple subclasses of theother post-switch isotypes, IgG and IgA. In contrast, to my knowledge, only onefunctional ε gene occurs in all investigated species. However, several alternativelyspliced ε chain mRNA transcripts have been identified in humans [50-57] and a few,non-homologous to the human isoforms, in mice [58]. A few of the human splicevariants seem to be translated and give rise to stable protein products in vivo [53],including the so-called tailpiece isoform, IgEtp [56, 59, 60]. In IgEtp, the two carboxy-teminal amino acids of the classical secreted IgE are replaced by eight novel aminoacids, including a carboxy-terminal cysteine residue. In a previous study, Diaz-Sanchezand colleagues reported a positive correlation between the amount of IgEtp mRNA andhigh total IgE levels, as observed in atopy or parasitic infections [59]. However, recentstudies by the same group did not enable assignment of any unique characteristics toIgEtp and no correlation between serum IgE levels and IgEtp expression was observed[60]. Hence, the contribution of the splice variants to the characteristics of human IgEappears to be limited. Furthermore, most of the alternatively spliced transcripts appearto be by-products of the splicing machinery, encoding non-functional protein productsthat are retained within the cells and degraded [55].

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No reports of alternative splicing of IgE are available for any mammalian species otherthan humans and mice and efforts to identify splice variants of rat IgE were fruitless[61]. Hence, in most mammalian species, IgE probably occur in the two classical formsonly, that is, the secreted and the membrane bound form of IgE.

Finally, IgE is shaped like no other isotype. Unlike IgM and IgA, the secreted form ofIgE is monomeric. Unlike IgA and IgG, the other post-switch isotypes, IgE has nohinge and is therefore less flexible. However, unlike IgM, the other isotype with fourconstant domains, IgE adopts a bent structure in solution [62].

Switching to IgE

The production of IgE antibodies is initiated when an antigen is recognized by the mIgon the B cell surface. However, to further B-cell proliferation and differentiation intoIgE-secreting plasma cells, two additional signals are required [63]. One is theCD40/CD40L interaction and the other is interleukin-4 (IL-4) and/or IL-13, which directrecombination to the Cε gene, resulting in ε-chain transcription.

A subset of T helper (Th) cells, Th2 cells, can supply both of these signals [63]. Hence,the antigen is internalized and processed into peptide that become associated with MHCclass II molecules and transported to the B-cell surface. Specific T cells that carry TCRswith antigenic epitopes recognize and bind to the processed antigen. This recognitionresults in a transient expression of CD40L on the T cell. CD40L/CD40 interactionupregulates B7-1/B7-2 (CD80/CD86) expression by the B cell and stimulates B-cellproliferation. Binding of B7 to CD28 on the T cell activates the T cell, which starts toproduce IL-4 and other cytokines and thus triggers the differentiation of B cells into IgE-secreting plasma cells. However, this is a very simplified scenario. The Th2 subset maywell be the main source of IL-4, but these cells also require IL-4 to become polarizedinto Th2 cells, a hen and egg situation. What is the original source of IL-4? Mast cells,basophils and eosinophils, the effector cells of IgE, all produce type 2 cytokines,including IL-4, as reviewed in [64]. In addition, both mast cells and basophils expressthe CD40L and can induce IgE synthesis [65]. Nevertheless, IgE synthesis involvesTh2-type cytokines and IgE responses against a given antigen are regulatedpredominantly by Th2-polarized T cells [66]. In contrast, interferon-γ (IFNγ) producedby Th1 polarized cells inhibit both Th2 responses and IgE synthesis.

The IgE Network

Although the IgE isotype constitutes only a minuscule fraction of the total antibodycontent in human sera, its actions are potent due to interaction with, and the actions of,two structurally unrelated receptors: the high affinity receptor, FcεRI, and the lowaffinity receptor, FcεRII, also known as CD23. FcεRI engages IgE with anexceptionally high affinity, Ka~1010 M-1, which is 1000-fold higher than that of CD23.

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FcεεεεRI

FcεRI belongs to the immunoglobulin superfamily and is highly homologous to theFcγ receptors. It is expressed primarily by mast cells in tissues and basophils in blood,on which it has a tetrameric structure composed of four transmembrane peptides; an α-chain, a β-chain and two disulfide linked γ-chains (αβγ2) [67]. The α-chain containstwo extracellular Ig domains that are responsible for IgE binding, whereas the β- and γ-chains are involved in receptor assembly and signal transduction [67, 68]. High“constitutive” expression of FcεRI is restricted to mast cells and basophils [69].However, in humans, low levels of FcεRI expression have been detected also on othercell types, including eosinophils, neutrophils and platelets, and in a in putative trimericαγ2 structure also on monocytes, Langerhans cells and dendritic cells, as reviewed in[68]. The functional significance of FcεRI on some of these cell types is uncertain. Forinstance, the level of FcεRI expression on eosinophils is very low, only about 0.5% ofthat on basophils, and apparently IgE does not mediate eosinophil effector functions[70]. Considering the central role of the β-chain in receptor activation, the importance ofthe putative trimeric form (αγ2) is also questionable [68]. Nevertheless, its presence onprofessional APCs is suggestive of a role in antigen uptake and presentation [71]. Inrodents, FcεRI is expressed exclusively on mast cells and basophils and has anobligatory αβγ2 structure [68].

The crystal structure of the human IgE-Fc fragment bound to FcεRIα has been solvedand it reveals that one dimeric IgE-Fc molecule binds asymmetrically to two separatesites on one FcεRIα, each site involving one Cε3 domain [72]. Twelve amino acidsfrom one of the Cε3 domains are involved in the interaction with receptor site 1,whereas ten amino acids from the other Cε3 domain interact with site 2. The residues inthe respective Cε3 domains that are involved in binding to site 1 and site 2 overlap, butare not identical. Hence, explaining the 1:1 stoichiometry of the interaction [72].Furthermore, binding to FcεRI appears to induce a conformal change in the Fc region ofIgE, indicating a conformal flexibility of this region and suggesting that the highaffinity of the IgE/FcεRI interaction might be achieved by an induced fit [73, 74]. Inaddition, although binding interactions occur exclusively between the Cε3 domains ofthe IgE and FcεRIα, the conformal change also involves the Cε2 domain and itspresence markedly reduces the dissociation rate of the IgE/FcεRIα complex [74, 75].The dissociation rate of the IgE/FcεRI complex is extraordinary slow, about two weeks[76]. In comparison, the off rate of the interaction between IgG and its high affinityreceptor is only a few minutes [76]. In addition, the Cε4 domain appears to beimportant for dimerization and proper folding of the Cε3 domain [77-79].

FcεεεεRII/CD23

In contrast to most immunoglobulin receptors, CD23 does not belong to theimmunoglobulin superfamily, but is member of the C-type (calcium-dependent) lectinsuperfamily [67]. The CD23 molecule consists of an extracellular C-terminal lectindomain, a stalk region, a transmembrane region and a cytoplasmic N-terminus, which

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self-associate and form trimers by coiling around each other at the stalk region [67]. Thelectin domain comprises the binding site for IgE and the interaction is thought toinvolve two lectin “heads” [67, 80]. The binding site on human IgE for CD23 has beenmapped to the Cε3 domain and appears to overlap with, but is distinct from, that ofFcεRI [80, 81].

Differential splicing of CD23 results in two different isoforms; CD23a and CD23b,which differ only by a few amino acid residues in the cytoplasmic N-terminus [80].Both isoforms are found on B cells: CD23a is constitutively expressed and appears to berestricted to B cells, whereas CD23b is induced in particular by IL-4 and is expressed ona variety of cells, including monocytes, macrophages, eosinophils, platelets, naturalkiller cells, T cells, follicular dendritic cells (FDC) and Langerhans cells, as reviewed in[67, 82, 83]. The CD23a isoform is associated with endocytosis of IgE-coated particlesby B cells and IgE-dependent antigen presentation to T cells [84], whereas the CD23bisoform on macrophages, eosinophils and platelets mediates IgE-dependent cytotoxicityand promotes phagocytosis of soluble IgE complexes [82].

To complicate things further, proteolytic cleavage in the stalk region of CD23 generatesa series of soluble fragments, some of which appear to have cytokine-like properties[80, 84]. In addition to IgE, CD23 also interacts with CD21 on B cells and FDCs, aswell as other members of the integrin family, suggesting a role for CD23 in IgE-independent inflammatory mechanisms [85].

Regulation of and by IgEIL-4 is the spider in the IgE network. It promotes the development of Th2-typeresponses, enhances FcεRI expression, induces the expression of CD23 and mostimportantly, together with IL-13, it directs the switch towards IgE synthesis [67, 68].However, once induced, IgE appears to regulate its own responses via interactions withits receptors.

To begin with, IgE can modulate the surface expression of both FcεRI and CD23.Several studies, both in vitro and in vivo, have demonstrated that IgE enhances FcεRIexpression on mast cells and basophils, both in humans [86-88] and in mice [89, 90].The FcεRI-expression level in peritoneal mast cells from IgE-deficient (IgE-/-) mice isfourfold to fivefold lower than that in wild-type mice [89]. Similarly, B cells derivedfrom IgE-/- mice express fourfold to sixfold less CD23 than those of wild-type animals,as reviewed in [91]. Accordingly, intravenous administration of IgE enhances surfaceexpression of both receptors in the IgE-/- mice [89, 91].

The enhanced FcεRI-surface expression is mediated by IgE/FcεRI interaction andappears to be relatively insensitive to protein synthesis inhibitors [88, 89]. Thissuggests that IgE acts by slowing depletion, thus prolonging the survival of preformedFcεRI on the cell surface, rather than by inducing FcεRI synthesis [88, 89].

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Furthermore, IgE-mediated upregulation of FcεRI on mast cells appears to enhancemast-cell-effector functions by increasing both the sensitivity to antigen challenge andthe intensity of the response [86, 89]. In addition, binding of monomeric IgE to FcεRI,even in the absence of antigen, promotes mast cell survival by preventing growth-factor-deprivation-induced apoptosis [92, 93].

In contrast, CD23 appears to be the most important receptor affecting IgE production.Interaction with CD23 may either inhibit or promote IgE responses, depending on theexpressing cell type, the expression level and the form of CD23. Binding of IgE/antigencomplexes to CD23 on IL-4 stimulated B cells, which express high levels of CD23,inhibits IgE production [94, 95]. Under other conditions, possibly involving lowerdegrees of receptor cross-linking, IgE/antigen complexes enhance antigen-specific IgEresponses via CD23 engagement on B cells, reviewed in [95]. Furthermore, IgE bindingto membrane-bound CD23 inhibits proteolytic cleavage and the release of soluble CD23products, some of which promote IgE synthesis (larger trimeric fragments) possibly viaCD21, whereas others appear to inhibit IgE production [96].

In conclusion, by engaging FcεRI IgE enhances its own effects, whereas by engagingCD23 IgE can keep the responses under control.

Why do we have IgE?

The mere existence of a complex and powerful biological system such as the IgEnetwork implies a strong selective pressure to establish and maintain it. However, IgEis best known for its pathological effects in allergic diseases and the beneficial role ofIgE in host defense remains somewhat uncertain. Nevertheless, IgE and its receptors arebelieved to have evolved as a defense mechanism against parasitic infections, inparticular against parasitic worms, i.e., helminth infections [67].

IgE and parasitic defenseImmune responses against helminth infections, such as Schistosoma mansoni and S.haematobium, are characterized by elevated serum IgE concentrations and eosinophilia[97]. A protective role for IgE was demonstrated by passive transfer of parasite-specificIgE in a rat model of schistosomiasis [97]. Furthermore, several humanseroepidemiological studies have shown that the presence of parasite-specific IgEcorrelates with protective immunity against shistosomiasis [98-100] and other helminthinfections [101]. In human schistosomiasis, antibody-dependent, cell-mediatedcytotoxicity involving eosinophils and IgE appears to be the main mechanism forkilling parasite larvae [97]. Hence, eosinophils may have evolved as a defense againstparasites (such as parasitic worms) that are too large for phagocytic cells. In thiscontext, the principle function of the IgE-mediated mast cell reaction may be to directeosinophils to the site of parasite infection and to enhance their anti-parasitic functions.

20

However, the importance of IgE in parasitic infections remains a controversial issue,mainly due to contrasting findings in murine models of S. mansoni. In one study, IgE-deficient mice developed two-fold greater S. mansoni worm burdens than did wild-typemice [102], whereas no difference in worm burden or egg load was observed in another,similar study [103]. In a third study, neither experimental increase of IgE productionthrough IL-4 stimulation nor passive transfer of parasite specific IgE was associatedwith any significant changes in worm burden or egg load, indicating that IgE has norelevant function in primary murine S. mansoni infections [104]. Similarly,schistosome infections develop normally in FcεRI deficient mice, with similar parasiterecovery and egg load as in wild-type mice, suggesting a limited role for the IgE/FcεRIsignaling pathway in protection against S. mansoni infection [105].

Then again, whereas Th2-type responses and eosinophils appear to be essentialcomponents for parasite expulsion in humans, Th1-type responses and macrophagesappear to play a more important role in mice, at least in schistosomiasis [97],suggesting that, in this context, IgE may be more important in humans than in mice.

Other functions?IgE appears to function as an enhancer of immune responses against antigens that arepresent at low concentrations. This has been demonstrated in vivo in a series ofexperiments in mice, as reviewed in [95]. Intravenous administration of small proteinantigens together with antigen-specific IgE can induce antibody responses that are morethan 1000-fold higher than those induced by antigen alone [95]. The response toIgE/antigen is strictly antigen specific, but not isotype specific, as it involvesupregulation of IgM-, IgG1-, IgG2a- as well as IgE responses. The proposed mechanismbehind IgE-mediated enhancement is endocytosis of IgE/antigen complexes via CD23 byB cells and presentation of antigen peptides to antigen-specific T cells [95]. Arequirement for CD23 in IgE-mediated upregulation was demonstrated by a completelack of enhancement in mice pretreated with anti-CD23 antibodies as well as in CD23deficient mice [106, 107]. The ability of IgE to augment responses under suboptimalconditions is suggestive of a physiological function in early responses [95]. This,together with the comparatively high turnover rate of IgE in serum, raises questionsregarding the positive value of IgE. Does IgE function as a “doorkeeper”, scanning theantigen repertoire of the environment and preparing the individual for potentiallyharmful pathogens? [108]. A rapid turnover may serve to adequately prepare theindividual to deal with its present environment.

Furthermore, surgery and other forms of tissue injury, such as burns and heart attacks,characteristically evoke a transient rise in serum IgE levels [109-113]. Serum IgE levelsappear to rise shortly after surgery, peak by day five and then decline again [110, 113].In contrast, serum IgG as well IgA and IgM fall rapidly after surgical intervention, butby postoperative day 7 they have returned to initial levels [110, 113]. In addition to IgE,an early increase in serum IL-6, a cytokine involved in acute phase protein induction, is

21

also observed after surgical intervention [109]. Hence, the behavior of IgE in situationsinvolving tissue injury led Szczeklik and colleagues to suggest a possible role for IgEin acute-phase responses [111].

Finally, individuals with high IgE levels, such as atopics, apparently have a mildhaemostatic imbalance, which means that their blood takes longer than normal to clot[114, 115]. This imbalance is reflected by a slightly prolonged bleeding time, depressedplatelet aggregation and a delayed generation of thrombin, the final enzyme in thecoagulation cascade. These phenomena could prolong the generation of a clot in acritically obstructed coronary artery and thus protect individuals with high IgE levelsfrom sudden cardiac arrest associated with coronary thrombosis (blocking of vessels thatprovide the heart muscle with blood) [114, 115].

IgE deficiencyOne way to increase our understanding of the physiological role of IgE is to study theclinical characteristics of IgE deficiency. One study linked IgE deficiency with anincreased prevalence of additional Ig deficiencies, autoimmune disease and non-allergicreactive airway disease [116]. IgE deficiency has also been associated with recurrent orchronic sinopulmonary infection, indicating a role for IgE in mucosal immunity [117].Lack of IgE could lead to impaired mucosal defense and thereby increase susceptibilityto respiratory tract infections [117]. However, in studies such as these it is alwaysdifficult to establish cause and effect. Why do these individuals have low IgE? Incontrast to the human situation, animal models, i.e., mice knock-out models, allow usto study the effect of a single deficiency. Mice with a targeted deletion of IgE, IgE-/-mice, show no apparent signs of disease, the animals do not die prematurely andlymphocyte development is normal [45, 95]. Hence, life is possible in the completeabsence of IgE, at least for laboratory mice. More importantly, IgE deficiency may alsooccur in humans with an otherwise normal immunologic profile without evidence ofillness [118].

Atopic Allergy

That allergies are widespread and that they may cause serious problems is commonknowledge for anybody who has arranged a birthday party for kids recently. Furthermore,all of us have friends or family members that are allergic to one thing or another. Infact, at least 20% of the Swedish population suffers from one or several of the clinicalmanifestations of atopic allergy, ranging from hay fever to asthma, eczema and, in theworst case, systemic anaphylactic reactions. Asthma is one of the most commonchronic childhood diseases, at least in the industrialized part of the world, and thousandsof people die from asthma annually. Furthermore, asthma is a major cause ofhospitalization and school/work absenteeism. Although hay fever and eczema are notlife-threatening conditions, the quality of life for, and the activity of, affected individuals

22

can be seriously reduced. Taken together, atopic diseases constitute one of the majorproblems of modern day medicine.

Atopic allergies, or hypersensitivity type 1 reactions, are caused by adverse IgE-antibodyresponses against otherwise harmless environmental agents, allergens. Curiously, ourallergic or atopic friends are often allergic to the same things. For instance, birchpollen, cats, nuts and dust mites are common allergen sources. What makes these thingsmore allergenic (better inducers of IgE responses) than others? In other words: Whatmakes an allergen an allergen? Some physical characteristics appear to be more commonamong allergens than among other proteins. Most allergens are soluble proteins andmany are exceptionally heat stable [119]. They are often glycosylated and severalallergens have enzymatic activities, as reviewed in [119]. A neat example of the latter,is Der p 1, the major house-dust-mite allergen, which has proteolytic activity and hasbeen shown to cleave CD23 and promote IgE production [119]. However, none of theabove-mentioned characteristics are unique to allergens, and whether a protein isallergenic appears to have no single structural cause. Furthermore, although none of uscan avoid exposure to birch pollen we are not all allergic. Hence, other factors inaddition to the nature of the allergen predispose to allergy. Nevertheless, the detrimentalrole of IgE is well established and the mechanism behind the allergic response is fairlywell understood.

The allergic responseAllergic responses involve two separate phases, a sensitization phase and an effectorphase (Fig. 2). During the sensitization phase allergen-specific IgE antibodies areproduced and become localized to the effector cells of allergic responses, mast cells andbasophils. The effector phase involves the activation of these cells, which results in therelease of mediators and the recruitment of other inflammatory cells that cause theclinical symptoms of atopic allergy. Both phases begin with the presence of an allergen.

The sensitization phaseAn allergen may enter the body via inhalation, ingestion, injection or through the skin.APCs encounter the allergen at, or close to, epithelial surfaces (skin, airways,intestines) and different cell types function as APCs depending on the site of allergenencounter. Macrophages, activated B cells and dendritic cells act in tissues, such as theairways, whereas Langerhans' cells are the most important APCs in the skin [71]. TheAPCs then migrate to a draining lymph node where they present allergen-derivedpeptides to naïve T cells and induce T-cell activation. Depending on the localconditions, which include the cytokine environment, the type of antigen, the antigendose and the type of APCs, Th cells polarize to a greater or lesser extent into either aTh1- or Th2-like phenotype. The phenotype is defined by the cytokine repertoire andhighly polarized Th1 cells produce mainly IL-2 and IFNγ, whereas Th2 cells produceIL-3, IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. As previously mentioned, polarization toand activation of the Th2 phenotype promotes the development of IgE responses. The

23

key cytokine that directs naïve Th cells to become Th1 effector cells is IL-12 [71]. Theloss of IL-12 responsiveness appears to be an important factor in the polarization toTh2 cells [64]. IL-4 inhibits the expression of an IL-12 receptor subunit, IL-12R β2,and thus directs the loss of IL-12 responsiveness and drives polarization to the Th2phenotype [120, 121]. In contrast, IFNγ promotes IL-12 responsiveness andconsequently, in the presence of IL-12, also commitment to the Th1 pathway. The mainsource of IL-12 is activated macrophages, but other APCs, such as dendritic cells, alsoproduce IL-12 [71]. The capacity of dendritic cells to induce either Th1 or Th2 responsesmay be due to their IL-12 production at the time, which in turn may be influenced bythe pathogen (allergen) [71].

The cytokine environment in the draining lymph node also influences the switchingpattern of allergen-specific B cells. If IL-4 and IL-13 as well as allergen-specific Th2cells are present, i.e., if T cell help is available, a substantial fraction of the allergen-specific B cells will switch to IgE production and mature into IgE-secreting plasmacells. The secreted allergen-specific IgE antibodies leave the lymph nodes via the bloodstream and become localized to mast cells and basophils by binding to FcεRI expressedon the surface of the respective cell-types. In addition, there appears to be an ongoinglocal production of allergen-specific IgE by resident B cells in the nasal and lungmucosa of hay fever and asthmatic patients, respectively [122]. An individual that hasallergen-specific IgE bound to his/her mast cells in the tissues (basophils in the blood)is sensitized to the allergen in question and subsequent encounters with the allergeninitiate the effector phase.

The effector phaseWhen a multivalent allergen associates with receptor-bound IgE on the surface of mastcells and basophils, it cross-links the receptor molecules on the cell membrane. Thiscross-linking initiates signal transduction events and causes an influx of Ca2+ ions intothe target cell that triggers degranulation and the release of preformed chemicalmediators, such as histamine, proteolytic enzymes and heparin. The preformed mediatorsgive rise to an immediate response, usually within 20 minutes. In addition, thedegranulating cell releases de novo synthesized lipid mediators (leukotrienes andprostaglandins) and cytokines (IL-5 and IL-8) that recruit other inflammatory cells(eosinophils, neutrophils and lymphocytes) to the reaction site. The influx ofinflammatory cells causes the late phase of the allergic response, which occurs within 6-24 hours after allergen exposure. Together, the released mediators are responsible for thephysiological effects that characterize allergic reactions, such as increased vascularpermeability, vasodilation, itching, edema, cell infiltration, smooth muscle contractionand hyper production of mucus.

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APC

TCR

Th2

Bcell

MHCII

IL-4

CD40L

CD40

IL-4

IL-13

Th0

Bcell

BCR

Plasma

cell IgE

Mast

cell

Epithelial barrier

Allergen

Effector phase

Sensitization phase

Allergen

FcεR

I

In tissues

Eosinophil

In blood

Basophil

Clinical effect:

Hay fever

Asthma

Eczema

Infiltration &

activation

IL-5

Degranulation

Release of mediators

e.g. Histamine

IL-5

Figure 2.The allergic response. Schem

e of molecular and cellular events behind the developm

ent (sensitization phase) and the expression(effector phase) of allergen induced, IgE

-mediated responses, w

hich lead to the clinical presentations of atopic allergy.

25

Why are allergies increasing?Before trying to find answers to the above question one must first try to establishwhether allergies really are increasing. According to several epidemiological studies,conducted in different parts of the world, the prevalence of allergic diseases has increaseddramatically (twofold to fourfold) during recent decades, as reviewed in [123, 124].However, one might argue that this only reflects that we are better at diagnosis todaythan we were 30 years ago. True or not, several studies indicate that allergic diseaseshave continued to increase even during the last decade, ruling out improved diagnosis asan important parameter. For instance, one survey of Australian schoolchildren recentlyreported an increase in diagnosed asthma (including non-atopic asthma) from 30,5% in1992 to a staggering 38.6% in 1997 [125]. Similar increases in atopic diseases havebeen reported among schoolchildren in Aberdeen (Scotland) [126]. Between 1989 and1994, the prevalence of diagnosed asthma increased from 10.2% to 19.6%, that ofeczema from 12.0% to 17.7% and that of hay fever from 11.9 to 12.7% [126]. Inconclusion, allergies really are increasing and they are reaching epidemical proportions,but why?

The tendency to develop atopic diseases appears to be due to both genetic andenvironmental factors. Atopic diseases often cluster within families and seem to have astrong, but heterogeneous genetic component. More than 20 genes appear to beinvolved and polymorphisms in several candidate genes, including the IL-4 promoter,the IL-4 receptor and the FcεRI β-chain, have been associated with asthma and atopicdisease, as reviewed in [127, 128]. Even so, genetic changes alone cannot account for afourfold increase in one generation. However, a genetic predisposition in combinationwith certain so-called environmental factors that have emerged or become morepronounced during recent decades might well suffice.

Several studies have linked the rise in atopy with increased living standards, a“westernized” lifestyle and a reduced frequency of childhood infections, as reviewed in[123, 124, 129]. For instance, studies conducted in 1991-92, roughly one year after theunification of Germany, revealed that airway hyper-responsiveness and atopicsensitization were less prevalent among children living in Leipzig (former EastGermany) than among children living in Munich (former West Germany) [130]. In1995-96, after five years of integration with Western society, the prevalence of atopicsensitization in schoolchildren living in Leipzig had increased from 19.3% (in 1991-92)to 26.7% [130]. In 1991-92, Leipzig was far more polluted than Munich, indicating thatalthough air pollution is known to aggravate already existing asthma, pollution ingeneral cannot be responsible for the increased prevalence of asthma and other atopicdiseases [131]. Nevertheless, certain pollutants; namely, diesel exhaust particles (DEP),appear to induce allergic sensitization and thus may, in part, explain why allergicdiseases often are more common in cities than in rural areas within a given country,e.g., in Sweden, as reviewed in [129, 132]. Moreover, several epidemiological studies

26

have shown an inverse association between the risk of atopy and family size, birth orderand nursery school attendance, all factors that will increase the risk of infections in earlychildhood, reviewed in [123, 124, 129]. The apparent inverse relationship betweenchildhood infections and atopic diseases is the basis for the Hygiene hypothesis, whichstates that infections during early life, bacterial and/or viral, redirect the maturingimmune system toward Th1 responses and thereby prevent the Th2-polarized allergicresponses [132]. Hence, a reduction in the overall microbial burden, as observed inmodern “Western” societies, may result in unrestrained Th2 responses and an increase inallergic diseases.

Current Therapeutic Strategies

The best way to avoid allergy is simply to avoid the allergen. However, this is onlyfeasible with certain allergens, such as food allergens, and it requires that the allergen isknown. A lot of research is therefore devoted to identifying allergens and developingdiagnostic tests. However, many of the most common allergens are difficult to avoid,e.g., inhalant allergens, demonstrating the need for treatments.

Most of the currently available treatments for atopic allergies involve symptomrelieving drugs which target the effects of the mediators rather than preventing theallergic response. For instance, the actions of histamines and leukotrienes are preventedby histamine H1 receptor antagonists, i.e., anti-histamines (Clarityn and Teldanex) andleukotriene receptor antagonists (Monteleukast), respectively. However, a specificantagonist arrests the effect of only one in the array of mediators involved in the allergicresponse. The antihistamines, are effective in rhinitis and against itch in atopicdermatitis (eczema), but have limited effect in the management of asthma [133]. Thecorticosteroids (Pulmicort, Becotide and Prednisolone) have a more general anti-inflammatory effect and, at present, they constitute the most effective treatment in bothasthma and rhinitis [133]. However, high doses or long-term treatment withcorticosteroids, even topical corticosteroids, may have systemic side effects [133, 134]and lead to permanent insufficiency of the adrenal cortex. Furthermore, althoughcorticosteroids efficiently abolish the inflammation characteristic of the late phaseresponse, they have no effect on the immediate response. Fast relief from theimmediate response in asthma is achieved with β2-stimulators (Bricanyl and Ventolin)that act on β2-receptors and relax bronchial muscles. Sodium chromoglycates (Lomudal)prevent both the immediate and late phase responses by stabilizing mast cells andpreventing degranulation. The side effects of sodium chromoglycates are minor, but theyhave no effect at all in about 1/3 of all patients with allergic asthma. Finally,potentially fatal systemic reactions, anaphylactic shock, are alleviated by promptadministration of adrenaline [134].

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Although the current pharmacological treatment of atopic disease often is effective, thedrugs have only a short-term effect (symptoms recur within weeks of withdrawal) andthe adverse side effects of long-term treatment are of great concern.

HyposensitizationWhen treatment with drugs give unsatisfactory results, hyposensitization therapy issometimes applied in an attempt to reduce the patient's sensitivity to the allergen.Hyposensitization therapy involves repeated subcutaneous injections of increasing dosesof allergen over long periods of time (years), a desensitizing strategy that has been usedto treat allergic diseases for nearly 100 years [135]. The exact mechanism behindhyposensitization is unknown, but it may involve immune deviation from Th2- to Th1-polarized responses and/or induction of anergy in allergen-specific T cells, as reviewed in[134, 136]. The highest success rates for this type of therapy are observed in seasonalallergic rhinitis as well as bee-venom and penicillin allergy [134, 136]. A successfultreatment is often accompanied by an increase in the allergen-specific IgG4 to IgE ratioand it has been suggested that IgG4 antibodies prevent sensitization by competing withIgE for the allergen [134, 136]. Furthermore, in successful hyposensitization therapyagainst bee venom allergy, IL-10 secreting cells appear to be critical. IL-10 may have arole both in regulating the IgG4/IgE balance and/or in induction of anergy in allergen-specific T cells, as reviewed in [134, 136]. However, there is no clear-cut correlationbetween any of these findings and clinical improvement in patients. Hence, althoughhyposensitization is sometimes successful in reducing the sensitivity to the injectedallergen, there is no guarantee of improvement. Furthermore, all forms ofimmunotherapy require identification of the allergen and a given patient may besensitized to numerous allergens, which may be difficult to prepare for injection. Mostimportantly, hyposensitization therapy always involves a risk of triggering dangerous,and potentially fatal, anaphylactic reactions.

Consequently, several attempts have been made to minimize the risk of systemicreactions in response to hyposensitization therapy. This may be achieved by modifyingthe allergen in various ways, either chemically with formaldehyde, generating so-calledallergoids, or by recombinant techniques, as reviewed in [136]. The aim of themodifications is to reduce the IgE-binding capacity, but unfortunately theimmunogenicity is often reduced as well and thus also the efficacy of the treatment[134].

Due to the undesirable side effects, drawbacks and sometimes unsatisfactory results ofthe current therapeutic approaches, a lot of research is devoted to finding betteralternatives.

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Allergen-independent treatments

Concomitant with improved understanding of the mechanisms behind atopic allergy,several potential allergen-independent strategies for intervention in the allergic responsehave emerged during the last decade.

As previously mentioned, IL-4 and IL-13 are key factors in inducing IgE synthesis andin addition, IL-4 is directly involved in the differentiation of Th2-type responses. Hence,by blocking the effects of these central cytokines one might prevent the development ofallergic responses. A high-affinity human IL-4 antagonist, obtained by a single aminoacid substitution in the activation domain, has been shown to inhibit both IL-4 and IL-13 induced IgE secretion from human B cells in vitro, as reviewed in [137].Furthermore, an analogous mouse IL-4 antagonist efficiently inhibited IL-4-dependentallergen sensitization in vivo [138].

Other allergen-independent approaches focus on interference with the IgE/FcεRIinteraction and thereby prevent receptor cross-linking and mast-cell degranulation. Forinstance, dimeric human receptor chimeras, consisting of the FcεRI α-chain coupled toeither the entire Fc-region [139] or the CH3 domain only [140] of human IgG1 havebeen developed. Both of these soluble receptor chimeras (receptor antagonists) bind freeIgE with high affinity and inhibit IgE binding to cell-surface-bound FcεRI [139, 140].Other laboratories have focused on the possibility of blocking the receptor with shortpeptides that mimic the receptor-binding properties of IgE, as reviewed in [141].However, to be clinically effective such peptides have to be long-lived, non-immunogenic and bind FcεRI with very high affinity, thus making the process ofdesigning them, at best, difficult.

Due to its central role in atopy and limited involvement in other biological reactions,IgE is considered to be a prime target in the development of anti-allergic treatments.Based on the assumption that a substantial reduction in total and antigen-specific IgElevels will correlate with improved conditions in atopic individuals, two distinctstrategies aiming at IgE depletion have evolved. One approach is passive immunizationwith monoclonal anti-IgE antibodies and the other is active immunization, vaccination,against IgE.

Passive immunization against IgEPassive immunization involves repeated intravenous or subcutaneous injections withmonoclonal antibodies against IgE. To achieve a beneficial pharmacological effect whileavoiding anaphylactic side effects, an antibody that binds specifically to free IgE but notto receptor-bound IgE on the surface of mast cells and basophils is required.Furthermore, to avoid strong immune reactions against the monoclonal antibody (MAb)and thus allow repeated administration, the MAb must not be recognized as foreign.This can be circumvented by so-called humanization, which entails grafting of thecomplementarity determining regions (CDR) from the parent murine MAb into human

29

framework regions, generating reshaped human V regions which then are joined tohuman C regions [142]. These humanized antibodies retain the antigen specificity of theoriginal mouse MAb, but are much less immunogenic.

Hence, different groups of investigators have independently isolated and humanized anumber of high-affinity anti-IgE MAbs [142, 143]. The different research programsjoined forces and one of the most promising MAbs was chosen for further clinicaldevelopment, rhu-MAb-E25. rhu-MAb-E25, also called Omalizumab, is a κ IgG1antibody that binds to the FcεRI-binding region of free IgE, and thus cannot interactwith FcεRI-bound (nor CD23-bound) IgE and cause cross-linking [144, 145]. Inaddition, rhu-MAb-E25 may bind mIgE on the surface of B cells [144, 146, 147].Therefore, the therapeutic effect of rhu-MAb-E25 may be two fold: by both neutralizingfree IgE and down regulating IgE production by B cells [144].

In numerous clinical trials (phase I through phase III), treatment with rhu-MAb-E25 hasresulted in substantial reductions in free-serum-IgE levels that correlate with beneficialeffects in patients with allergic rhinitis [148-151] or asthma [152-156]. The free-serum-IgE levels decrease by ≥90% from pretreatment values within 24h of subcutaneousadministration of rhu-MAb-E25, and continued treatment results in reduced expressionof FcεRI on effector cells and thus reduced responsiveness to antigen stimulation, asreviewed in [145]. rhu-MAb-E25 treatment significantly reduces the severity ofsymptoms in patients with allergic rhinitis as well as their requirements for rescuemedication [149, 151]. In one study, 21 % of the patients that received rhu-MAb-E25considered their symptoms completely controlled, as compared to 2% in the placebogroup [149]. Similar improvements have also been observed in patients with allergicasthma.

Treatment with rhu-MAb-E25 appears to be safe and well tolerated. To my knowledge,only one patient, who was included in a pilot study of aerosolized administration in thelungs, have developed antibody responses against rhu-MAb-E25, as reviewed in [145].Furthermore, no signs of immune-complex-related diseases were reported in any of theabove-mentioned studies, but no long-term studies have been conducted so far. In myopinion, the major disadvantage of passive immunization is the high dose requirement.Subcutaneous dosage of rhu-MAb-E25 is based on body weight and total serum IgElevels as follows: ≥0.016mg/kg/IU/ml. Hence, a patient that weighs 80kg and has atotal serum IgE level of 500ng/ml (200IU) requires a monthly dose of about 250mgrhu-Mab-E-25 [155], which translates to a yearly dose of about 3 g, indicating thattreatment may be very costly.

Active immunization against IgEThe other approach to IgE depletion is active immunization, vaccination, against IgE.Usually vaccination entails injection of, e.g., attenuated microbes with the prospect ofinducing strong anti-microbial immune responses that will protect the recipient from

30

future infection with the microbe in question. However, in this case the target moleculeis not a foreign microbe but a self-protein, IgE. Some years ago, a study was initiatedin the laboratory to explore the possibility of inducing strong and therapeutically usefulantibody responses against self-IgE through vaccination.

Vaccination induces a polyclonal antibody response, but the same requirements thatapply to the MAb, apply also to the polyclonal antibodies. Hence, antibodies generatedin response to vaccination should bind to free IgE with high affinity, but not to FcεRIbound IgE. The crystal structure of the IgE/FcεRI interaction was not available at thetime, but a 76-amino-acid region at the border between the Cε2 and Cε3 domains hadbeen shown to interact with FcεRI and prevent binding of free IgE [157]. Therefore,with the aim of evaluating the vaccine approach in a rat model, a fusion proteinconsisting of the Cε2 and Cε3 domains of rat IgE coupled to glutathione-S-transferase(GST) from Schistosoma japonicum, GST-C2C3, was designed [137, 158-160].

The T cells of a mature immune system are normally self-tolerant, that is, they do notrespond to self-proteins such as IgE. B cells, on the other hand, may be self-reactive,but they will remain in an inactive state in the absence of T-cell help. However, the T-cell tolerance may be circumvented by coupling a foreign-carrier protein, such as GST,to the target self-protein. In theory, B cells with mIg that is specific for the self-protein(the receptor binding region of rat IgE) will internalize and degrade the entire GST-C2C3protein and thus present peptides derived also from GST. Hence, GST-specific T cellscan provide rat-IgE specific B cells with the signals required for proliferation andproduction of anti-rat IgE antibodies.

Vaccination with GST-C2C3 together with Freund’s adjuvant resulted in strong IgGresponses against self-IgE in three of four rat strains [158]. The anti-IgE response wasaccompanied by a significant reduction in total serum IgE, to below the detection limitof the assay (10-20ng/ml). Furthermore, GST-C2C3 vaccination resulted in an almostcomplete inhibition of mast cell reactivity in the skin upon challenge with either across-linking polyclonal anti-IgE antiserum or a specific allergen [158]. These resultswere encouraging indeed.

However, harsh conditions were required to obtain the bacterially expressed GST-C2C3vaccine component in soluble form. Consequently, some of the surface epitopes of theC2C3-derived component in the vaccine might not have retained their nativeconformations, which, in turn, may generate antibody responses with inadequate affinityfor native IgE. Hence, although the initial results were very promising, the solubilityissue and a number of additional questions had to be addressed in order to proceed withthe vaccine strategy in human clinical trials.

If successful, the vaccination strategy has several advantages over that of passiveimmunization. Firstly, vaccination induces a polyclonal response. The risk of immune-

31

complex disease is thus minimal compared to that incurred during long-term treatmentwith high doses of a MAb. Secondly, vaccination is estimated to entail a yearlyadministration dose that is about 10 000 times lower than that of passive immunizationwith a MAb. This will most certainly reduce the cost of treatment significantly.Furthermore, whereas passive immunization might require monthly visits to aphysician, vaccination will probably not necessitate more than four visits a year, a clearbenefit that certainly will affect patient compliance with treatment.

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Present study

AIM

The major aim of this study was to develop an active immunization strategy against IgEas a preventive, allergen-independent treatment for atopic allergy. The central role of IgEin allergic reactions is well established, but its normal physiological function remainssomewhat elusive. Another important objective was thus to increase our understandingof IgE, focusing on structural aspects from an evolutionary perspective.

Results and Discussion

The rise …

Cloning and structural- and phylogenetic analysis of monotreme ε- and γ chains(Paper I and II)To trace the emergence of IgE and other post-switch isotypes in vertebrate evolution wehave studied Ig expression in two monotreme species: the duck-billed platypus(Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus).

Partial platypus IgE and IgG cDNA clones were isolated using a PCR-based cloningstrategy and degenerate primers, as described previously [25]. The clones obtained wereused as probes to screen a platypus-spleen cDNA library. Positive clones were analyzedfurther and the complete nucleotide sequences were determined for one full-length cloneeach of platypus IgE and IgG. Upon further screening using a full-length γ-chain cloneas probe, a novel γ-chain isotype was isolated and sequenced. The two platypus γ-chainisotypes were designated IgG1 and IgG2 in order of “appearance”.

In addition, a partial platypus IgE clone was used as a probe to screen an echidna spleencDNA library. Again, positive clones were selected for further analysis and the completenucleotide sequence of one full-length echidna ε-chain clone was determined.

Sequence comparisons reveal that both echidna and platypus IgE conform to the generalstructure displayed by eutherian and marsupial ε chains (paper I and II). Hence, bothechidna and platypus IgE have four constant domains and all of the generally conservedcysteine residues involved in the various intra- and inter-chain disulfide bridgeformations occur in the monotreme ε chains. Furthermore, the potential N-linkedglycosylation site in the CH3 domain, conserved among all previously characterized εchains, is present also in echidna and platypus IgE (Fig. 3).

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Platypus IgG2

CH1 CH2 CH3S HingeVH

L H

CDR1 2 3

Platypus IgG1

CH1 CH2 CH3S HingeVH

L H

CDR1 2 3

Platypus IgE

L H H

CH1 CH2 CH3 CH4S VHCDR1 2 3

Echidna IgE

L H H

CH1 CH2 CH3 CH4S VHCDR1 2 3

Figure 3. Schematic maps of the echidna IgE, and platypus IgE, IgG1 and IgG2 heavy chainclones. The signal sequence (S) is represented in black. The light gray regions in the variableregion (VH) denote the positions of the CDRs. The constant domains are illustrated bystriped boxes and the hinge region (in IgG) is indicated in dark gray. The approximatepositions of potential N-glycosylation sites are indicated by forks, and brackets indicate thepositions of putative intra-chain disulfide bridges. Cysteine residues involved in putativeinter-chain disulfide bridges connecting the two heavy chains and the heavy chain with thelight chain are denoted by H and L, respectively.

Like the previously characterized eutherian and marsupial γ chains, the platypus γ1- andγ2 chains contain three constant domains and a hinge region between the CH1 and CH2domains (Fig. 3). However, the sequence divergence between the two platypus IgGisotypes is remarkably high and they display several structural differences. In platypusIgG1, as in most, if not all, mammalian γ chains, the cysteine residue involved in thedisulfide bridge connecting the heavy chain with the light chain occurs in the C-terminalregion of the CH1 domain. In contrast, in platypus IgG2 this cysteine residue occurs inthe N-terminal region of the same domain, which may be a unique feature amongmammalian γ chains (Fig. 3). Furthermore, one potential inter-chain disulfide-bridge-forming cysteine residue occurs in the hinge region of IgG1, whereas two cysteineresidues are present in the same region of IgG2 (Fig. 3). The two platypus γ chains alsodiffer with respect to the number of potential N-linked glycosylation sites (Fig. 3).However, the lone site in the CH2 domain of IgG1 is conserved between the twoplatypus isotypes and among other γ chains as well. Moreover, this site corresponds tothe generally conserved N-linked glycosylation site in the CH3 domain of mammalianIgE. The sequence divergence between the platypus γ chains is actually greater than that

34

between rodent and human IgG (Fig. 4). Hence, the presence of three constant domainsin both of the two highly divergent platypus γ chains suggests that the deletion of thesecond domain in an ancestral IgG occurred very early in mammalian evolution.

The primary structures of the monotreme ε- and γ chains suggest that all of the majorevolutionary changes that gave rise to the mammalian Ig isotypes occurred before theseparation of the three extant mammalian lineages. However, the results from thephylogenetic analyses are not conclusive with respect to the evolutionary relationshipbetween monotremes and marsupials. Based on the ε-chain sequences, monotremes andmarsupials appear to be sister lineages, which is in agreement with the Marsupionatahypothesis [31-34]. In contrast, the platypus IgG isotypes, together with IgM and IgAsequences support the opposing Theria hypothesis, according to which the monotremesconstitute the most divergent mammalian lineage [22, 23, 35-40] (Fig. 4). Estimatesbased on protamine and IgM sequences indicate that the monotremes separated from theancestors of marsupials and placentals about 150-170 million years ago [22, 23],whereas the separation of early mammalian ancestors from early reptiles may haveoccurred as far back as 310-330 million years ago [24]. Provided that the Theriahypothesis is correct, this leaves us with an evolutionary window for the appearance ofmodern post-switch isotypes of about 150 million years (between 150-170 to 310-330million years ago).

Comparative analysis of mammalian ε chains (Paper II)Including platypus and echidna IgE, fifteen different mammalian ε-chain sequences havebeen isolated so far. Furthermore, each of the three mammalian lineages (placentals,marsupials and monotremes) are now represented by at least two ε-chain sequences,providing us with decent grounds for a detailed comparative analysis of mammalian IgE.

Comparative amino acid sequence analysis reveals that the overall IgE structure isconserved among the various ε chains. All ε chains have four constant domains and,with a few exceptions, the various cysteine residues involved in putative disulfide bridgeformations occur in conserved positions. The most striking deviations occur in therodent ε chains, which lack one of the intra-chain disulphide bridges in the CH1 domainand have an extended C-terminus.

35

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Figure 4. Ig sequence comparisons presented as a phylogenetic tree. A comparativeanalysis of all presently available IgE heavy chains sequences, along with a selection ofmammalian IgG, IgA and IgM sequences as well as some avian and amphibian IgM and IgYheavy chain sequences. The bootstrap tree is based on 1000 independent comparisons and thebootstrap values are added at each branch point. The sequence distance is represented by thelength of the branches according to the time scale (lower left), which corresponds to asequence divergence of 5%.

36

The number and positions of potential N-glycosylation sites differ considerably amongthe various ε chains (Fig. 5). In general, the monotreme and marsupial ε chains containfewer N-glycosylation sites than the eutherian ε chains. Furthermore, no N-glycosylation site occurs in the CH1 domain of monotreme and marsupial ε chains,whereas two or three sites are present in this domain of eutherian IgE. Conversely,some eutherian ε chains (those of primates, pig and horse) lack glycosylation sites inthe CH4 domain. However, one potential N-glycosylation site in the CH3 domainoccurs in all ε chains, suggesting that glycosylation at this position is important forprotein structure and/or solubility. This site actually occurs in the FcεRI binding regionof human IgE, but the involvement of the attached carbohydrate in the interactionappears to be limited [72]. However, glycosylation at this site may be important toensure a proper conformation of the receptor-interacting region [161, 162]. Althoughadditional glycosylation may be of some importance, the differences observed among thevarious ε chains suggest that their positioning is less restricted.

EchidnaPlatypusOpossumPossumMouse

PigHorse

Cat

HumanOrangutanChimpanze

DogCow

SheepRat

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CH2 CH3 CH4

( )

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Figure 5. Variation in glycosylation patterns among mammalian ε chains. The constantregions of the presently available ε chains are indicated as lines below a schematic map ofthe overall ε-chain structure. The approximate positions of potential N-glycosylation sitesin the respective ε chains are indicated by forks.

Interaction between IgE and FcεRI involves both of the CH3 domains, each bindingasymmetrically to two separate sites on one FcεRIα [72]. All together, eighteen aminoacid positions in the CH3 domains are involved in the interaction [72]. Six of theseamino acid residues are identical among all fifteen ε chains and most positions containanalogous amino acid residues (paper II). The putative receptor-binding regions of therodent ε chains are the most divergent, sharing only eight to ten residues with thecorresponding regions of monotreme and primate IgE (paper II). Despite the rather

37

significant differences in the receptor-binding regions, rodent IgE can bind to the humanFcεRI receptor [163]. However, mouse IgE cannot engage human CD23 and human IgEcannot bind to either of the rodent receptors [81, 164]. Inter-specie reactivity has alsobeen reported between dog IgE and primate FcεRI, whereas both human and rhesusmonkey IgE failed to sensitize canine skin [165]. The structural basis for these inter-species interactions and the apparent specie specificity of human IgE is unknown.However, it is tempting to link the latter to three non-analogous substitutions thatoccur exclusively in the receptor-binding region of primate IgE (paper II).

Furthermore, we compared the various ε chains with respect to charge distribution at pH7.0 and isoelectric point (pI) values calculated from their amino acid sequences. Themonotreme and marsupial ε chains all have a considerable negative net charge, resultingin pI values at or below 6.0, whereas the eutherian ε chains have pI values between 6.5and 8.2. Interestingly, there appears to be some correlation between charge distributionand potential N-glycosylation sites (Fig. 5). Domains that lack, or have few, N-glycosylation sites often have a more pronounced negative net charge and vice versa(paper II), thus suggesting that the actual pI values for the various ε chains may bemore similar than what can be discerned from the amino acid sequence alone due to thepresence of sialic acid-containing complex oligosaccharides.

The presence of IgE and the conservation of the overall structure in all extantmammalian lineages indicates an early appearance in mammalian evolution and aselective advantage of structural maintenance. The assumed duplication of an ancestralIgY in the lineage leading to mammals separated the major effector functionscharacteristic of present day IgG and IgE. This separation, which limited the role of themajor plasma Ig isotype (IgG) in mast cell and basophil activation, may have had aselective advantage that could explain the maintenance of both IgG and IgE in all extantmammalian lineages.

Cloning, structural analysis and expression of Pig IgE (Paper III)The pig has been used extensively in biomedical science for many years, especially insurgical and cardiovascular research. Furthermore, due to several similarities with humanphysiology, together with other advantages, the pig is a prime choice as a potentialxenograft donor in human transplantations [166, 167]. Consequently, immunologicalstudies in the pig have gained much attention and the pig has been developed as a largemodel for studies of inflammatory reactions [168-170]. However, relatively few of thereagents required for studies of allergic inflammation are available, including sensitiveand specific assays for total and antigen-specific IgE. To increase our knowledge of IgE,as well as the possibility to use the pig model in studies of IgE-mediated inflammatoryreactions, we have cloned and expressed the ε chain of IgE in the domestic pig (Susscrufa).

38

Two highly conserved regions among the previously characterized ε chains, one in the3’-end of the CH2 domain other in the 5’-end of the CH4 domain, were selected forsynthesis of degenerate oligonucleotide primers (paper III). A partial pig IgE DNAfragment was isolated through PCR amplification using the degenerate primers andsingle stranded cDNA derived from spleen and lymph nodes of Ascaris-sensitized pigsas template. The clone obtained was used as probe to screen a cDNA library constructedfrom mRNA isolated from the spleen and lymph nodes of the same Ascaris-sensitizedanimals. Positive clones were analyzed further and the complete nucleotide sequenceswere determined for one full-length clone of porcine IgE.

Amino acid sequence comparison with the previously characterized ε chains revealed thatthe pig ε chain conforms to the general IgE structure and that it shares the highestdegree of sequence similarity with the sheep ε chain (Fig. 4).

The CH2-CH3-CH4 region of pig IgE was expressed in insect cells using a baculovirusexpression system. In brief, a DNA fragment encoding the AcNPV gp64 signal, whichenables secretion of the target protein, followed by a histidine hexapeptide and the CH2-CH3-CH4 domains of the pig ε chain was obtained through PCR amplifications (paperIII). The final PCR product was ligated into the baculovirus transfer vector pVL1392(PharMingen) and the obtained vector was used as a rescue plasmid in the BaculoGold(PharMingen) expression system. After co-transfection of Sf 9 insect cells and twoadditional rounds of virus amplification, a high-titer recombinant virus stock wasobtained and used to infect High-Five insect cells. Recombinant pig IgE (6xHis-CH2-CH3-CH4) was purified from the conditioned medium on Ni2+-NTA-agarose (QIAGEN)columns.

SDS-PAGE analysis revealed that under reducing conditions the recombinant pig IgEmigrated as a protein with a molecular weight of ~43kD, which corresponds to theestimated size of the 6xHis-CH2-CH3-CH4 protein. However, under non-reducingconditions only a small fraction of the recombinant protein migrated as the expecteddimer. Instead, most of the material migrated as large multimeric complexes. Theaggregation was probably caused by the presence of an unpaired cysteine residue in theN-terminal region of the CH2 domain.

The recombinant pig IgE protein can be used to raise polyclonal and monoclonalantibodies. This will facilitate the development of sensitive and specific assays for pigIgE and thus increase the possibility to use the pig model in future studies of allergicreactions.

At present, rodent models are generally employed in studies of IgE-mediated reactions,owing in part to the availability of the reagents required to monitor the reactions.However, there are several differences between the rodent and human systems that requireconsideration. For instance, rodent FcεRI is expressed exclusively on mast cells and

39

basophils and has an obligatory αβγ2 structure, whereas human FcεRI appears to havea wider distribution including eosinophils and platelets as well as monocytes,Langerhans cells and dendritic cells in a putative trimeric αγ2 structure [68].Furthermore, human mast cells appear to be regulated exclusively by IgE, and no cross-talk between IgE- and IgG mediated responses occurs [171]. In contrast, IgE-deficientmice can display anaphylactic reactions upon antigen challenge, demonstrating that non-IgE pathways for mast cell activation exist in rodents [45]. In addition, in mice, IgE caninteract with inhibitory and activatory Fcγ receptors on the surface of mast cells [172].Hence, although rodent models are useful, extrapolation from rodents to the humansituation may have limitations. So far, the situation in the pig is unknown, but,physiologically, the pig appears to be compatible with humans and the need for morecompatible animal models is apparent.

The fall …

Active immunization against IgE (paper IV)

The design and production of a soluble vaccine component against rat IgE

Based on the assumption that a substantial reduction in total and antigen-specific IgElevels will correlate with improved clinical outcomes in atopic individuals, an activeimmunization strategy for IgE depletion was developed in our laboratory [158]. In theprevious studies, a fusion protein consisting of the Cε2-Cε3 region of rat IgE coupledto GST, GST-C2C3, was used as the active component in the vaccine. The initialresults were very promising, but GST-C2C3 could only be obtained in a soluble form ifharsh denaturing conditions were applied [158]. This solubility problem as well asseveral other issues, including the risk of generating cross-linking antibody responses,had to be resolved to proceed with the vaccine strategy in view of human clinical trials.

Initially, we invested a lot of time and effort to obtain the GST-C2C3 vaccinecomponent in a soluble form. However, following several fruitless attempts usingdifferent bacterial expression systems, a yeast system (Pichia pastoris) and abaculovirus expression system we concluded that solubility requires maintenance of theIgE framework, which in turn requires correct folding, dimerization, disulfide bridgeformation and probably also glycosylation [173]. Considering this, the vaccine designitself, a fusion of two radically different proteins with respect to processing, wasprobably a major impediment. GST is an intracellular protein devoid of post-translational modifications, whereas IgE is a glycosylated membrane-bound or secretedprotein. Therefore, it was essential to find an alternative, more suitable, carrier protein.Furthermore, the GST-C2C3 vaccine contained both the Cε2 and Cε3 domains of ratIgE, that is, self-IgE. To limit the risk of generating cross-linking responses we aimedat reducing the self component in the next-generation vaccine.

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Evolutionary studies of IgE in the laboratory had recently led to the cloning of IgE froma marsupial, the opossum (Monodelphis domestica) [25]. Opossum IgE and rat IgE aredistantly related, with a sequence identity of ~43 % on the amino acid level, suggestingthat domains derived from opossum IgE are sufficiently foreign to function as a carrier.However, despite the sequence divergence, the overall structures of opossum and rat IgEappear to be similar [25]. Therefore, domains derived from opossum IgE might alsoserve as structural support for the self-IgE-derived target domain. The Cε2 domain ofIgE is not required for binding to FcεRI, that is, the active conformation of the receptor-binding region occurs in the absence of the Cε2 domain [77]. However, the Cε2 domaincontains both of the cysteine residues involved in the inter-chain disulfide-bridgeformations that occur in the IgE dimer. The Cε4 domains, on the other hand, appear tobe important for the maintenance of the active conformation of the receptor-interactingCε3 domain [77]. Based on these considerations, a sequel vaccine component containingthe third constant domain of the rat ε chain flanked by the second and fourth constantdomain of the opossum ε chain, OpossumCε2-RatCε3-OpossumCε4, ORO, wasdesigned and expressed in a mammalian expression system (Fig. 6).

SAAAA

CH2 CH3 CH4

H H*

Rat OpossumOpossum

Signal seq.cleavage

6xHis

Figure 6. DNA construct of the ORO vaccine component. The signal sequence (S) is derivedfrom human BM40. An asterisk denotes an unpaired cysteine residue that was changed toserine by point mutagenesis.

In brief, the DNA construct of ORO was made from cDNA clones using PCRamplification and subsequent subcloning. For purification purposes a region encoding ahistidine hexapeptide was included in the 5’ primer directed against the region encodingthe N-terminal of the opossum Cε2 domain. DNA fragments corresponding to the OROprotein were cloned into a modified expression vector named pCEP-Pu2, which containsa signal sequence that targets the expressed protein for secretion. Human embryonickidney cells, 293-EBNA (Invitrogen), were transfected with the recombinant plasmidusing Lipofectamine (Gibco BRL) and subsequent selection for the plasmid was effectedby the addition of 0.5µg/ml puromycin (P8833, Sigma). The recombinant ORO proteinwas purified from the conditioned media on Ni-NTA-agarose (QIAGEN) using a batchprocedure which entails protein binding to the adsorbent in solution.

SDS-PAGE analysis revealed that under reducing conditions ORO migrates as a proteinwith a molecular weight of ~48kD. However, under non-reducing conditions most of theprotein migrates at ~97kD, suggesting that recombinant ORO is produced as a disulfide-linked dimer. Furthermore, preliminary Biacore measurements revealed that both ORO

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and its human counterpart (replacing rat Cε3 with human Cε3) bind to the humanFcεRI. Hence, the hybrid proteins appear to fold properly and the receptor-bindingtarget-region occurs in an active and thereby a nearly native conformation. Since B cellsrecognize epitopes of native antigens, a nearly native conformation of the target regionis probably required to evoke an antibody response with sufficiently high affinity fornative IgE, especially considering the high affinity of the competing IgE-FcεRIinteraction (Ka~1010 M-1)

Another objective of the present study was to define the requirements of the targetregion in the vaccine, that is, the receptor-binding region derived from self-IgE. Withthis aim, two additional vaccine components, OROO and OMO, were designed andexpressed (Fig. 7). The purpose of OROO, which contains a part rat, part opossum Cε3domain, was to investigate whether we could trim down the self-component in thevaccine even further without compromising efficacy. The aim of OMO, which containsa mouse Cε3 domain, was to study the efficacy of a vaccine component with an alteredtarget region. The mouse Cε3 domain differs from that of the rat in 21 positions,corresponding to 19% difference in sequence, and can thus be considered as an alteredtarget region. In addition, recombinant OOO (opossum Cε2-Cε4) was produced tofacilitate studies of the response against the carrier protein alone (Fig. 7).

6xHisIgE

CH2

CH3

CH4

6xHis 6xHis6xHis

ORO OROO OMO OOO

Rat MouseOpossum

Figure 7. Schematic structure of the various recombinant proteins.

Vaccination of ovalbumin-sensitized rats

To evaluate the efficacy of the ORO vaccine in a more clinically significant setting, anovalbumin (Ova) sensitized rat model was established through repeated intraperitoneal(i.p.) injections of Ova in PBS prior to vaccination. The study involved four differentrat strains that demonstrate significant, strain-dependant differences in normal serum IgElevels: Brown Norway, Louvain, Wistar and Lewis. The initial IgE levels in the BrownNorway strain were so high (500-110,000 ng/ml) that no additional effect ofsensitization was observed, whereas the Lewis strain had very little IgE (1-16 ng/ml)

42

but did not seem to respond to sensitization with this isotype. However, the initial IgElevels in the Louvain and Wistar strains were 1-27ng/ml and 7-155ng/ml, respectively,and sensitization resulted in an approximately eightfold increase in total serum IgE inboth of these strains. To mimic the deregulated control of IgE expression in atopicpatients, Ova sensitization was continued throughout the vaccination program. Thetreatment program for the Wistar strain is shown in figure 8.

Groups of animals from the four different strains were vaccinated with ORO or treatedwith BSA as negative control. In addition, groups of Wistar rats were vaccinated withOROO, OMO or OOO according to the treatment program (Fig. 8). Blood samples werecollected at several time points (Fig. 8) and comparative IgG anti-IgE titers and totalIgE levels in the sera were determined by ELISA analysis.

-10 -5 0 5 10 2015

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Figure 8. Time line of the treatment program for the Wistar strain. At the initialvaccination, the animals received 250µg recombinant protein (ORO, OROO, OMO or OOO)or BSA in 100µl neutral PBS mixed 50:50 with Freund’s Complete Adjuvant. In week 3 ,100µg recombinant protein or BSA in 100µl PBS mixed 50:50 with Incomplete Freund’sadjuvant (IFA) was administered. At the following two occasions, the animals received 25µgrecombinant protein or BSA in 50µl PBS mixed 50:50 with IFA. At all vaccinations wereadministered i.p.

Antibody responses against IgE were detected in all ORO-vaccinated animals (paper IV).Comparative IgG anti-IgE titers detected in Wistar sera from week 10 of the treatmentprogram are shown in figure 9. The average response in the Wistar strain was ~80% ofthat in the Lewis strain, the strain which generated the most prominent response (paperIV). The average responses against IgE in the Brown Norway and Louvain strains weremuch weaker, about 20-fold and 13-fold lower, respectively, than that in the Lewisstrain (paper IV). Therapeutic effects of the antibody response generated against OROwere observed in three of the vaccinated strains (paper IV). The serum IgE levels werereduced to below 10 ng/ml in all vaccinated Wistar and Lewis rats and in four out ofnine ORO-vaccinated Louvain rats. In comparison with control animals, and based ongeometrical mean values, the reduction in serum IgE in ORO-vaccinated Wistar andLewis rats was 90% and 92%, respectively. The total serum IgE levels determined in theWistar sera at different time points are shown in figure 9. In addition, a significant effectof ORO vaccination was observed in the skin of Ova-sensitized Wistar rats challengedwith the allergen, Ova (paper IV). The apparent mast cell activity was significantlylower in ORO-vaccinated animals compared to BSA-treated control animals, indicating

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that the antibody response generated against ORO also has a clinical effect on mast cellsin the periphery.

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Figure 9. Therapeutic anti-IgE titers and total serum IgE in vaccinated Wistar rats.A) Comparative analyses of anti-IgE antibody titers in 1:5 serial dilutions (in horse sera) ofsera collected in week 10 of the treatment program from BSA-treated and ORO-vaccinatedanimals. B) Total serum IgE concentrations in sera collected throughout the treatmentprogram from the BSA-treated and ORO-vaccinated Wistar rats. The same symbol is used forthe same animal in the anti-IgE (A) and total IgE (B) assays.

In comparison with ORO, vaccination with the modified vaccine components, OROOand OMO, generated considerably lower responses against IgE and no significant effectson total serum IgE levels were observed (paper IV). Hence, the size and the number ofavailable epitopes appear to be of major importance for the efficacy of the vaccine.However, it should be mentioned that recombinant OROO was produced at lower yieldsthan the other proteins and that upon SDS-PAGE analysis under non-reducingconditions the band corresponding to OROO was more smeared and less of the materialmigrated as the dimer (paper IV). It is possible that the design of the dual species Cε3domain caused problems in protein folding and that the reduced efficacy of OROO is dueto a somewhat misfolded target region rather than to the size of the self-component.Nevertheless, the OMO protein appears to be properly folded (paper IV), and consideringthat 81% of the amino acid residues in the rat and mouse Cε3 domains are identical andthat only two of the 18 residues shown to be involved in the receptor interaction differbetween the two species [72] (paper II), the failure of OMO is notable. The outcome ofthese preliminary studies is that, to reach optimal efficacy the target region should be as

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large as possible without running the risk of cross-linking antibody responses, and thespecies-specific sequence should not be altered.

Reversibility of treatment

Given our limited understanding of what future challenges our immune system may faceand the role IgE may play in these challenges, it was important for us to demonstrate areturn to the pre-vaccination state after discontinuation of treatment. The mostprominent anti-IgE response was observed in the Lewis strain and by week 45 of ourstudy, about 11 months after the booster injection, the anti-IgE titers in this strain werereduced by 89-99%. Other modified self-proteins have shown similar reversibilitypatterns [174-176], suggesting that therapeutic antibody responses against a self-proteinare reversible with time and that the antibody response is not potentiated by the presenceof the non-modified self-protein.

Safety of ORO vaccination

Our main concern was the risk of generating potentially cross-linking antibodyresponses against the ORO vaccine. Firstly, antibody responses against the carrier,opossum IgE, might be cross-reactive and thereby potentially cross-linking. Most of thesequence identity between opossum and rat IgE is concentrated around cysteine residuesinvolved in putative disulphide bridge formations and may not be easily available as B-cell epitopes. However, the overall structure of opossum and rat IgE appear to besimilar and therefore, structural epitopes that are difficult to discern from the sequencemay generate cross-linking responses. Secondly, although the self-component issignificantly reduced in the ORO vaccine, the risk of generating potentially cross-linking responses against epitopes outside of the receptor-binding regions in the self-derived Cε3 domain remains. The amino acid regions involved in the FcεRI interactionare spread across the Cε3 domain, but they represent only a fraction of the residues inthis domain [72].

A skin reactivity assay in high responder Brown Norway rats was used to determine themast-cell activating potential of the antibody responses generated in vaccinated Wistarrats. No skin reactivity was detected upon provocation with sera from Wistar ratsvaccinated with either OOO or ORO, whereas the positive control, a cross-linkingmouse anti-rat IgE MAb, caused extensive mast cell cross-linking and degranulation(paper IV). Crystallization studies of human IgE-Fc Cε3-Cε4 have revealed that FcεRIbinding induces a conformal change in the Cε3 domain [73], which may preventantibodies directed against the Cε3 domain of free IgE from binding to receptor-boundIgE. Hence, conformal changes, together with steric hindrance upon receptor binding,may operate to limit the cross-linking potential of antibody responses generated againstthe entire Cε3 domain. The sequence identity between human and opossum IgE issimilar to that between rat and opossum IgE, which strongly suggests that opossumIgE will be safe to use as a carrier protein in humans as well.

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Loose ends

Here I will focus on the allergy vaccine and an important unresolved issue, that is, theapparent shortcomings of the vaccine in individuals with initially very high IgE levels.

Vaccination with ORO generated detectable antibody responses against IgE in all ratstrains, including the high-responder strain Brown Norway (paper IV). Although this isa significant improvement, since no anti-IgE antibody responses were detected in thisstrain following vaccination with GST-C2C3 [158], the response generated againstORO had no effect on the serum IgE level. Our results indicate that the therapeuticcapacity of the elicited response is dependent on the initial serum IgE concentration.Such high serum IgE levels as those observed in the Brown Norway strain probablyinduce a strong B-cell tolerance in the host that will impede the effects of the vaccine.Since the vaccine is intended as a therapeutic rather than a prophylactic treatment andthat, more often than not, serum IgE levels are elevated in allergic individuals, ongoingstudies are aimed at overcoming this potential limitation of the vaccine.

A successful pretreatment that reduces the IgE level significantly prior to vaccinationmight serve to evade tolerance induction in newly recruited B cells. For humanpurposes, humanized monoclonal anti-IgE antibodies, such as rhu-MAb-E25, mightafford effective pretreatment [142, 143, 145], but such antibodies are not available forrats. In the absence of a rat anti-rat IgE we thought that a non-cross-linking monoclonalmouse IgG1 anti-rat IgE, MAS-314 (Harlan, Loughborough, England) might suffice toput our pretreatment hypothesis to the test.

In brief, Ova-sensitized brown Norway rats were treated with MAS-314 or PBS (controlanimals) weekly for six weeks. At each occasion, 1mg MAS-314 in 500µl PBS wasadministered intramuscularly in the thighs. In addition, a separate group of MAb-treatedanimals were vaccinated with ORO using a similar protocol as in figure 8. The initialdose of ORO was administered two days after the initial MAb treatment. Blood wascollected at several time points throughout the study and analyzed for total serum IgE byELISA.

Four days after the initial MAb treatment (week 1), an almost complete disappearance oftotal IgE was observed in the MAb treated animals, but the reduction was onlytemporary (Fig. 10). By week 3, the IgE levels in the MAb treated animals wereactually elevated in comparison with those in the control animals, and at subsequenttime points the IgE levels were similar in the two groups (Fig. 10). A second ELISAanalysis, to detect antibodies against MAS-314, revealed that a strong response waselicited against the mouse MAb (Fig. 10). This response presumably induced theclearance of subsequent MAb doses and thus prevented further clearance of IgE. Notherapeutic anti-IgE antibody responses were elicited in the ORO-vaccinated MAb-treatedanimals, which was not surprising in view of the brief effect of the MAb treatment.

46

Hence, although mouse and rat IgG1 share ~80% sequence identity over the constantregions, the mouse MAb did not work for pretreatment in our rat model.

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Figure 10. Treatment with MAS 314, a mouse anti-rat IgE MAb. Total serum IgEconcentrations (upper graphs) and antibody titers against MAS 314 (lower graphs) in seracollected throughout the treatment program from PBS-treated (control animals) and MAS314-treated, unvaccinated Brown Norway rats.

A chimeric molecule consisting of the extracellular portion of the human FcεRI α-chain coupled to a truncated human IgG1 heavy chain has been described previously[139]. When expressed in mammalian cells this chimeric molecule was secreted as anantibody-like disulfide-linked homodimer [139]. The chimeric molecule was capable ofbinding free IgE, and since it cannot bind receptor-bound IgE it may, in this respect,function as a non-cross-linking MAb against IgE. Hence, we have designed a rat versionof this chimeric molecule as an alternative to a rat anti-rat IgE monoclonal. Whetherthis chimeric molecule can serve to reduce the IgE level prior to vaccination and if this,in turn, can serve to break the putative B-cell tolerance and, thereby, to enhance thetherapeutic effects of the vaccine, remains to be seen.

Another important issue with regard to the vaccine is the adjuvant problem. In order toapply the vaccine strategy to humans it is essential to find an alternative to Freund’sadjuvant, which is too toxic for human use. At present, aluminum compounds are theonly adjuvants approved in the US, and consequently the only widely used adjuvants forclinical use in humans. However, in a pilot adjuvant study, no antibody responses

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against IgE were detected in Wistar rats vaccinated with ORO together with Al(OH)3

(Johansson et. al, manuscript in preparation). Aluminum adjuvants favor Th2-typeresponses and can induce IgE-antibody responses [177]. Hence, aluminum adjuvantsmay promote IgE-mediated reactions, which seems incompatible with the intended effectof the ORO vaccine. Our results using a water-in-oil formulated, mineral oil-basedadjuvant analogous to Freund’s incomplete adjuvant, Montanide ISA 51 (Seppic, Paris,France) were more encouraging. Vaccination with ORO together with Montanide ISA51 generated therapeutic antibody responses that were at least comparable to thosegenerated with Freund’s adjuvant (Johansson et. al, manuscript in preparation).Montanide ISA 51 has been tested in AIDS, malaria and cancer immunotherapeuticclinical trials and the vaccine formulations were generally well-tolerated [178-180].However, the acceptable level of toxicity is likely to be higher for AIDS, malaria andcancer treatments than for an allergy vaccine. Nevertheless, Montanide ISA 51 is apotential adjuvant candidate for the human version of the ORO vaccine.

Concluding remarks

When did IgE appear?IgE expression appears to be restricted to mammals, but it occurs in all three extantmammalian lineages: placentals, marsupials and monotremes. The conservation of theoverall IgE structure among the available ε chains suggests that IgE was present, inessentially its present form, in a common mammalian ancestor before the evolutionaryseparation of the extant mammalian lineages. According to the Theria hypothesis, themonotremes constitute the most divergent lineage and the estimated time of divergencefrom the therian lineages is about 150-170 million years ago [22, 23]. Hence, anancestral IgE was present at least 150-170 million years ago, but may have appearedalready at, or shortly after, the time of divergence of the early mammalian ancestorsfrom the early reptiles lineages, about 310-330 million years ago [24].

Do we need IgE?Mice with a targeted deletion of the Cε gene, IgE-/- mice, do not suffer from anyapparent disease, they do not die prematurely, lymphocyte development is normal as areserum levels of the other Ig isotypes (IgG, IgA and IgM) [45, 95]. IgE deficiency mayalso occur in humans with an otherwise normal immunologic profile without evidenceof illness [118]. However, laboratory mice, as well as humans in modern Westernsociety, live in an environment that is almost devoid of parasites and the generalmicrobial load is low. Under these circumstances IgE may well be redundant. However,these microbe-depleted environments have emerged relatively recently, especially inrelation to evolutionary time. The presence of IgE and the conservation of the overallIgE structure in all extant mammalian lineages indicate an early appearance in

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mammalian evolution and a selective advantage of structural maintenance. IgE probablyplayed a beneficial role in host defense against parasitic infections during the evolutionof mammalian species and may still do so. Furthermore, IgE functions as an enhancerof an immune response against antigens that occur in low concentrations, as reviewed in[95]. In addition, very low amounts of antigens are required to activate mast cells andbasophils if antigen-specific IgE is present on the cell surface. In an environment wheremicrobes are plentiful, the ability to respond to low amount of potentially fatalmicrobes, to enhance the response and induce mast cell effector functions may becrucial. However, these potentially beneficial effects of IgE might be of limited value inpopulations living in modern Western society, where allergic diseases constitute a farmore important problem than do parasitic infections. This assumption is the basis forthe development of therapeutic strategies aimed at IgE depletion. The clinical studies ofpassive immunization with rhu-MAb-E25 support this assumption in that no adverseside effects of IgE-depletion has been observed in any of the patients. Hence, at least inatopic individuals IgE appears to be dispensable.

Vaccination against IgE – is it possible?The present study suggests that vaccination against IgE is indeed a possible approach toprevent IgE-mediated immune responses. We have successfully produced a solublevaccine component, ORO, which retains the receptor-binding target-region in a native ornearly native conformation. Vaccination resulted in a 90% decrease in serum IgE levelsin sensitized Wistar rats. This is encouraging, since similar reductions in free serum IgEupon passive immunization with rhu-MAb-E25 have led to clinical improvements inatopic patients [148, 149, 154, 155]. In addition, ORO vaccination resulted in asignificant inhibition of mast cell reactivity in the skin upon allergen challenge. Avaccine containing the entire Cε3 domain from self-IgE appears to be safe to use as anantigen. No cross-linking activity was observed in the sera of vaccinated animals and theelicited response was reversible with time. The allergic-rat model has its limitations andthere are still some unresolved questions. However, the apparent efficacy and safety ofORO vaccination, together with the clinical benefits of vaccination over passiveimmunization with a MAb, suggests that the vaccination strategy has potential tobecome a therapeutic method for humans.

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Acknowledgements

I would like to take the opportunity to thank all of you, who in one way or another,have contributed to the completion of this thesis. No one who has not been throughthis can possibly imagine how many people that are affected by the ordeal. In particular,I would like to acknowledge:

Professor Lars Hellman, my supervisor and mentor in the field of immunology, forletting me join the group and the vaccine project, your never-ending ideas, yourencouragement and most of all for your confidence in my work. It has been a privilegeto work for you!

Professor Lars Pilström, for sharing your wisdom of the small (Igs) and big thingsin life, for your encouragement and for leaving me licorice when I couldn’t have the realstuff.

Maria Aveskogh , our outstanding technician, for many shared laughs and for justbeing you. There are bad ways, better ways and Maria’s way to do lab work. Thank youfor taking the time to teach me your way!

Everybody at Resistentia Pharmaceuticals: Markus, Bengt, Christina, Birgitta,Mats, Åsa, Stefan, Anki and Alexis , for your support and for believing in thevaccine enough to make it your bread and butter.

Everybody at ICM and IMBIM, especially Sigrid, Christer, Joakim, Eva, Tonyand Ylva, who made my life easier by taking care of the practical details.

The vaccine girls: Anna, for your positive attitude and for taking the time to read thisand making me feel like Shakespeare with your “suvves”. Jeannette, for being a goodfriend and for sharing the late hours and the agonies of writing.

The rest of ”Hellmans flickor”, Parvin, Camilla, Ulrika, Sara and Maike formaking this place a good one, and coming to work a good thing.

Coauthors and collaborators, especially Gunnar Pejler, for help with the pigmanuscript and Therese Kristersson, for helping me with the pig situation, andIngrid Dahlbom.

The girls that came after, but left before me: Maryam, my first wing-mate, it was apleasure (hire me!), Carolina, for your dimples, Lotta, for your wonderful theoriesof life, Otti, and Frida, for many laughs and for writing the “how to become a PhD”manual, it has been a big help.

The Cod group: Siv, for looking so happy to see me (it’s not only the coffee is it?),Niklas, especially for the cake you pretended to know nothing about, Sirje , Mats,and Anna-Carin.

Anders and Erika, the additional lunch dates, for fruitful discussions…

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Former group members and colleagues: Magnus, Claudia, Tomas and Mårten fortaking care of me when I was a novice.

All my friends outside of this world, no one named, no one forgotten.

My mother-in-law, Margareta Vernersson, also known as “världens bästa farmor”for your help with the kids, I could not have done this without you.

Sonja, my sister, for your support and for being an invaluable friend.

David Eaker (also known as Dad) for linguistic revision.

Mom and Dad, for your endless love and support, for giving me the tools to completethis, for your encouragement and for always believing in me. In short, for being myparents and for doing it so well.

Per, my wonderful husband, for taking also the first part of “i nöd och lust” seriouslyduring the last month. I owe you big time.

Emil and Anna, my beloved children. I will not lie and say that you made this easier,but you are without competition the joys of my life.

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Here is a factthat should help you to fight

a bit longer:

Things that don’t act�ually kill you outright

make you stronger�

Grooks by Piet Hein