Allergen mimotopes for 3-dimensional epitope search and induction of antibodies inhibiting human IgE.

There is no definite information available on the structural characteristics of IgE binding epitopes on allergenic molecules, although it is widely accepted that most of them are conformational. In the current study we aimed to characterize the IgE epitope of Bet v 1, the major birch pollen allergen, by the application of phage display peptide libraries. We purified IgE specific for Bet v 1 from allergic patients' sera to select mimotopes representing artificial IgE epitopes by biopanning of phage libraries. By linear alignment, it was not possible to attribute mimotope sequences to the primary structure of Bet v 1. We developed a computer-aided, 3-dimensional coarse-grained epitope search. The 3-dimensional search, followed by statistical analysis, revealed an exposed area on the Bet v 1 molecule (located between residues 9-22 and 104-123) as the IgE binding structure. The IgE epitope was located at a 30 A distance from a previously described IgG epitope and the respective mimotope, designated Bet mim E. Such mimotopes could potentially be used for the induction of IgG capable of interfering with the IgE/allergen interaction. To test this hypothesis, we immunized BALB/c mice with the phage-displayed Bet mim E. Immunizations resulted in the induction of Bet v 1-specific IgG, which was able to block the IgE binding to Bet v 1 in vitro. Based on these observations, we propose that immunotherapy with IgE mimotopes generated by biopannings result in formation of blocking IgG. We conclude that mimotope immunotherapy may represent a new and promising concept for treatment of type I allergic disease.     

T cell epitope-containing hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1, induce blocking antibodies

Allergen-specific immunotherapy represents one of the few curative approaches toward type I allergy. Up to 25% of allergic patients are sensitized against the major birch pollen allergen, Bet v 1. By genetic engineering we produced two recombinant (r) Bet v 1 fragments comprising aa 1-74 and aa 75-160 of Bet v 1, which, due to a loss of their native-like fold, failed to bind IgE Abs and had reduced allergenic activity. Here we show that both fragments covering the full Bet v 1 sequence induced human lymphoproliferative responses similar to rBet v 1 wild type. The C-terminal rBet v 1 fragment induced higher lymphoproliferative responses than the N-terminal fragment and represented a Th1-stimulating segment with high IFN-gamma production, whereas the N-terminal fragment induced higher IL-4, IL-5, and IL-13 secretion. Immunization of mice and rabbits with rBet v 1 fragments induced IgG Abs, which cross-reacted with complete Bet v 1 and Bet v 1-related plant allergens and strongly inhibited the IgE binding of allergic patients to these allergens. Thus, our results demonstrate that hypoallergenic T cell epitope-containing rBet v 1 fragments, despite lacking IgE epitopes, can induce Abs in vivo that prevent the IgE binding of allergic patients to the wild-type allergen. The overall demonstration of the immunogenic features of the hypoallergenic rBet v 1 fragments will now enable clinical studies for safer and more efficient specific immunotherapy.     

Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.

Type I allergies are immunological disorders that afflict a quarter of the world's population. Improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. We describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. We expressed our model allergen Bet v 1, the major birch pollen allergen, in the tobacco-related species Nicotiana benthamiana using a tobacco mosaic virus vector. Two weeks postinoculation, plants infected with recombinant viral RNA containing the Bet v 1 coding sequence accumulated the allergen to levels of 200 microg/g leaf material. Total nonpurified protein extracts from plants were used for immunological characterizations. IgE immunoblots and ELISA (enzyme-linked immunoassay) inhibition assays showed comparable IgE binding properties for tobacco recombinant (r) Bet v 1 and natural (n) Bet v 1, suggesting that the B cell epitopes were preserved when the allergen was expressed in N. benthamiana plants. Using a murine model of type I allergy, mice immunized with crude leaf extracts containing Bet v 1 with purified rBet v 1 produced in E. coli or with birch pollen extract generated comparable allergen-specific IgE and IgG1 antibody responses and positive type I skin test reactions. These results demonstrate that nonpurified Bet v 1 overexpressed in N. benthamina has the same immunogenicity as purified Bet v 1 produced in E. coli or nBet v 1. We therefore conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts. In addition, there may be a broad utility of this system for the development of new and low-cost vaccination strategies against allergy.     

Analysis of epitope-specific immune responses induced by vaccination with structurally folded and unfolded recombinant Bet v 1 allergen derivatives in man

Previously, we have constructed recombinant derivatives of the major birch pollen allergen, Bet v 1, with a more than 100-fold reduced ability to induce IgE-mediated allergic reactions. These derivatives differed from each other because the two recombinant Bet v 1 fragments represented unfolded molecules whereas the recombinant trimer resembled most of the structural fold of the Bet v 1 allergen. In this study, we analyzed the Ab (IgE, IgG subclass, IgA, IgM) response to Bet v 1, recombinant and synthetic Bet v 1-derived peptides in birch pollen allergic patients who had been vaccinated with the derivatives or adjuvant alone. Furthermore, we studied the induction of IgE-mediated skin responses in these patients using Bet v 1 and Bet v 1 fragments. Both types of vaccines induced a comparable IgG1 and IgG4 response against new sequential epitopes which overlap with the conformational IgE epitopes of Bet v 1. This response was 4- to 5-fold higher than that induced by immunotherapy with birch pollen extract. Trimer more than fragments induced also IgE responses against new epitopes and a transient increase in skin sensitivity to the fragments at the beginning of therapy. However, skin reactions to Bet v 1 tended to decrease one year after treatment in both actively treated groups. We demonstrate that vaccination with folded and unfolded recombinant allergen derivatives induces IgG Abs against new epitopes. These data may be important for the development of therapeutic as well as prophylactic vaccines based on recombinant allergens.     

Allergy vaccine engineering: epitope modulation of recombinant Bet v 1 reduces IgE binding but retains protein folding pattern for induction of protective blocking-antibody responses

Human type 1 immediate allergic response symptoms are caused by mediator release from basophils and mast cells. This event is triggered by allergens aggregating preformed IgE Abs bound to the high-affinity receptor (FcepsilonRI) on these cells. Thus, the allergen/IgE interaction is crucial for the cascade leading to the allergic and anaphylactic response. Two genetically engineered forms of the white birch pollen major allergen Bet v 1 with point mutations directed at molecular surfaces have been characterized. Four and nine point mutations led to a significant reduction of the binding to human serum IgE, suggesting a mutation-induced distortion of IgE-binding B cell epitopes. In addition, the mutated allergens showed a decrease in anaphylactic potential, because histamine release from human basophils was significantly reduced. Retained alpha-carbon backbone folding pattern of the mutated allergens was indicated by x-ray diffraction analysis and circular dichroism spectroscopy. The rBet v 1 mutants were able to induce proliferation of T cell lines derived from birch pollen allergic patients. The stimulation indices were similar to the indices of nonmutated rBet v 1 and natural Bet v 1 purified from birch pollen. The ability of anti-rBet v 1 mutant specific mouse IgG serum to block binding of human serum IgE to rBet v 1 demonstrates that the engineered rBet v 1 mutants are able to induce Abs reactive with nonmodified Bet v 1. rBet v 1 mutants may constitute vaccine candidates with improved efficacy/safety profiles for safer allergy vaccination.     

Birch pollen-associated peanut allergies in children

Panallergens show structural similarities, and they are responsible for many cross-reactions between pollen and plant food sources. The aim of the present study was to investigate IgE reactivity to peanut allergen components in children with birch pollen allergy. Patients experienced symptoms of allergic asthma, allergic rhinitis, and urticaria, and they underwent a complete diagnostic evaluation, including skin prick test (SPT), specific IgE (sIgE) to birch pollen allergen (t3), peanut allergen (f13). In addition, measurement of sIgE to the major birch allergen components, Betula verrucosa (Bet v1, Bet v2), and to peanut allergen components, Arachis hypogaea (genuine componens: Ara h1, Ara h2, Ara h3, and cross-reactive Ara h8) was performed, by using a microarray technique (component resolved diagnosis, CRD). SPT to birch extract was positive in all children, and SPT to peanut extract was positive in 51 % of them. sIgE to both allergens was increased in 39 % of children, 55 % of them had increased sIgE (t3), and one child had increased sIgE (f13). CRD results confirmed that some children were sensitized to Bet v1 only, and some children to genuine Ara h only. Bet v1/Ara h8 cross-reactivity was found in 16 % of children. Results of the present study reveal that SPT, sIgE, and CRD may detect sensitization and co-sensitization with birch and peanut allergens/allergen components, and CRD may help to differentiate sensitization to genuine peanut components from sensitization to peanut cross-reactive component in birch-sensitive children. Diagnostic approach has to be individualized for each patient.     

Cross-reactivity of pollen and food allergens: soybean Gly m 4 is a member of the Bet v 1 superfamily and closely resembles yellow lupine proteins

In many cases, patients allergic to birch pollen also show allergic reactions after ingestion of certain fruits or vegetables. This observation is explained at the molecular level by cross-reactivity of IgE antibodies induced by sensitization to the major birch pollen allergen Bet v 1 with homologous food allergens. As IgE antibodies recognize conformational epitopes, a precise structural characterization of the allergens involved is necessary to understand cross-reactivity and thus to develop new methods of allergen-specific immunotherapy for allergic patients. Here, we report the three-dimensional solution structure of the soybean allergen Gly m 4, a member of the superfamily of Bet v 1 homologous proteins and a cross-reactant with IgE antibodies originally raised against Bet v 1 as shown by immunoblot inhibition and histamine release assays. Although the overall fold of Gly m 4 is very similar to that of Bet v 1, the three-dimensional structures of these proteins differ in detail. The Gly m 4 local structures that display those differences are also found in proteins from yellow lupine with known physiological function. The three-dimensional structure of Gly m 4 may thus shed some light on the physiological function of this subgroup of PR10 proteins (class 10 of pathogenesis-related proteins) and, in combination with immunological data, allow us to propose surface patches that might represent cross-reactive epitopes.     

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

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

Molecular characterization of human IgG monoclonal antibodies specific for the major birch pollen allergen Bet v 1. Anti-allergen IgG can enhance the anaphylactic reaction

We report the molecular characterization of five human monoclonal antibodies, BAB1-5 (BAB1: IgG(1); BAB4: IgG(2); BAB2, 3, 5: IgG(4)), with specificity for the major birch pollen allergen, Bet v 1. BAB1-5 were obtained after immunotherapy and contained a high degree of somatic mutations indicative of an antigen-driven affinity maturation process. While BAB1 inhibited the binding of patients IgE to Bet v 1, BAB2 increased IgE recognition of Bet v 1, and, even as Escherichia coli-expressed Fab, augmented Bet v 1-induced immediate type skin reactions. The demonstration that IgG antibodies can enhance allergen-induced allergic reactions is likely to explain the unpredictability of specific immunotherapy.     

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.     

Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1

The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 A resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients' IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 A2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients' serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.     

Costimulation blockade inhibits allergic sensitization but does not affect established allergy in a murine model of grass pollen allergy

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments and second structure information of Fag s 1: Fagales allergen from <Emphasis Type="Italic">Fagus sylvatica</Emphasis>

Fagales allergens belonging to the Bet v 1 family account responsible for the majority of spring pollinosis in the temperate climate zones in the Northern hemisphere. Among them, Fag s 1 from beech pollen is an important trigger of Fagales pollen associated allergic reactions. The protein shares high similarity with birch pollen Bet v 1, the best-characterized member of this allergen family. Of note, recent work on Bet v 1 and its homologues found in Fagales pollen demonstrated that not all allergenic members of this family have the capacity to induce allergic sensitization. Fag s 1 was shown to bind pre-existing IgE antibodies most likely primarily directed against other members of this multi-allergen family. Therefore, it is especially interesting to compare the structures of Bet v 1-like pollen allergens, which have the potential to induce allergic sensitization with allergens that are mainly cross-reactive. This in the end will help to identify allergy eliciting molecular pattern on Bet v 1-like allergens. In this work, we report the ¹H, ¹⁵N and ¹³C NMR assignment of beech pollen Fag s 1 as well as the secondary structure information based on backbone chemical shifts.     

Nitration of the egg-allergen ovalbumin enhances protein allergenicity but reduces the risk for oral sensitization in a murine model of food allergy

Background

Nitration of proteins on tyrosine residues, which can occur due to polluted air under “summer smog” conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route.

Methodology/Principal Findings

BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y₁₀₇) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization.

Conclusions/Significance

These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes.     

Crystal structure of the major celery allergen Api g 1: molecular analysis of cross-reactivity

Many patients who have been sensitised to pollen, display allergic symptoms after ingestion of certain plant food such as fresh fruit, vegetables and nuts. The cause is the cross-reactivity between structurally very similar major plant allergens. In particular, allergy to celery is very frequently associated with birch and mugwort pollen sensitization, known as to the birch-mugwort-celery syndrome. The crystal structure of the major celery allergen Api g 1, a homologue of the major birch pollen allergen Bet v 1, has been determined to a resolution of 2.9 A. The structure of Api g 1 is very similar to that of Bet v 1 with major differences occurring in the segment comprised of residues 23-45, preceding the well conserved glycine-rich P-loop, as well as in loops beta3-beta4 and beta5-beta6. In particular, Api g 1 lacks E45, which has been shown to be a crucial residue for antibody recognition in the crystal complex of Bet v 1 with the Fab fragment of a murine monoclonal IgG (BV16) antibody. The absence of E45 and the structural differences in the preceding segment suggest that this region of the Api g 1 surface is probably not responsible for the observed cross-reactivity with Bet v 1. A detailed analysis of the molecular surface in combination with sequence alignment revealed three conserved surface patches which may account for cross-reactivity with Bet v 1. Several residues of Bet v 1 which have been shown by mutagenesis studies to be involved in IgE recognition belong to these conserved surface regions. The structure of Api g 1 and the related epitope analysis provides a molecular basis for a better understanding of allergen cross-reactivity and may lead to the development of hypoallergens which would allow a safer immunotherapy.     

Critical role of preconceptional immunization for protective and nonpathological specific immunity in murine neonates

Expression of Th2 immunity against environmental Ags is the hallmark of the allergic phenotype and contrasts with the Th1-like pattern, which is stably expressed in healthy adults throughout life. Epidemiological studies indicate that the prenatal environment plays an important and decisive role in the development of allergy later in life. Since the underlying mechanisms were unclear, an animal model was developed to study the impact of maternal allergy on the development of an allergic immune response in early life. An allergic Th2 response was induced in pregnant mice by sensitization and aerosol allergen exposure. Both, IgG1 and IgG2a, but not IgE, Abs cross the placental barrier. Free allergen also crosses the placental area and was detected in serum and amniotic fluids of neonatal F(1) mice. These F(1) mice demonstrated a suppressed Th1 response, as reflected by lowered frequencies and reduced levels of IFN-gamma production. Development of an IgE response against the same allergen was completely prevented early in life. This effect was mediated by diaplacental transfer of allergen-specific IgG1 Abs. In contrast, allergic sensitization against a different allergen early in life was accelerated in these mice. This effect was mediated by maternal CD4 and OVA-specific Th2 cells induced by allergic sensitization during pregnancy. These data indicate a critical role for maternal T and B cell response in shaping pre- and postnatal maturation of specific immunity to allergens.     

Strain-dependent induction of allergic sensitization caused by peanut allergen DNA immunization in mice

To investigate the potential application of allergen gene immunization in the modulation of food allergy, C3H/HeSn (C3H) mice received i.m. injections of pAra h2 plasmid DNA encoding one of the major peanut allergens, Ara h2. Three weeks following pDNA immunization, serum Ara h2-specific IgG2a, IgG1, but not IgE, were increased significantly in a dose-dependent manner. IgG1 was 30-fold higher in multiply compared with singly immunized mice. Ara h2 or peanut protein injection of immunized mice induced anaphylactic reactions, which were more severe in multiply immunized mice. Heat-inactivated immune serum induced passive cutaneous anaphylaxis, suggesting that anaphylaxis in C3H mice was mediated by IgG1. IgG1 responses were also induced by intradermal injection of pAra h2, and by i.m. injection of pOMC, the plasmid DNA encoding the major egg allergen protein, ovomucoid. To elucidate whether the pDNA immunization-induced anaphylaxis was a strain-dependent phenomenon, AKR/J and BALB/c mice also received multiple i.m. pAra h2 immunizations. Injection of peanut protein into these strains at weeks 3 or 5 following immunization did not induce reactions. Although IgG2a was increased significantly from week 2 in AKR/J mice and from week 4 in BALB/c mice and remained elevated for at least 6 wk, no IgG1 or IgE was detected. These results indicate that the type of immune responses to pDNA immunization in mice is strain dependent. Consequently, models for studying human allergen gene immunization require careful selection of suitable strains. In addition, this suggests that similar interindividual variation is likely in humans.     

A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.     

Modulation of the allergen-induced human IgE response in Hu-SCID mice: inhibitory effect of human recombinant IFN-gamma and allergen-derived lipopeptide

We have previously established a model to study the in vivo human IgE response using humanized SCID mice. Allergic SCID mice were obtained following intraperitoneal injection with mononuclear cells from Dermatophagoides pteronyssinus (Dpt)-sensitive patients, and sensitization by Dpt allergen intraperitoneal injection (immunization) or Dpt aerosol (inhalation). Human serum IgE was measured in allergic SCID mice after administration of human recombinant IFN-gamma or the lipopeptide LP 52-71 (derived from peptide p52-71 from Der p 1, Dpt major allergen, coupled to a lipophilic moiety), during the immunization or the inhalation phase. IFN-gamma inhibited human IgE production when given at the time of immunization, but not during inhalation. This effect was long-lasting as Dpt aerosol, given one month after immunization and IFN-gamma administration, failed to increase IgE levels. Unlike Dpt or p52-71, LP 52-71 failed to induce human IgE production at day 14 and 21 after its injection, but did inhibit the development of the IgE response after a secondary Dpt-challenge. Moreover, LP 52-71 administration 14 days after Dpt inhalation decreased IgE levels, in contrast to peptide 52-71, which increased IgE levels. Thus, taken together these results indicate that the development of the human IgE response in allergic SCID mice can be modulated by modified allergen and a Th1 cytokine.