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Background IXIARO® is a Vero cell-derived, inactivated Japanese encephalitis (JE) vaccine licensed mainly in western countries for children and adults traveling to JE endemic areas. Limited immunogenicity and safety data in elderly travelers have been available. Objectives To evaluate safety and immunogenicity of IXIARO in elderly subjects. Methods Open-label, single arm, multi-centered study. Two-hundred subjects with good general health, including adequately controlled chronic conditions, received two doses of IXIARO®, 28 days apart. Protective levels of antibodies were tested 42 days after the second dose. Systemic and local adverse events (AEs) were solicited for 7 days after each dose, unsolicited AEs were collected up to day 70 and in a phone call at month 7. Summary of results Subjects were aged 64–83 years (median 69.0 years). Nineteen percent of subjects had serious or medically attended AEs up to Day 70 (primary endpoint), none of them causally linked to IXIARO. Solicited local AEs were reported by 33.5% (most common: local tenderness) and solicited systemic AEs by 27% (most common: headache) of subjects. The seroprotection rate was 65% with a geometric mean titre (GMT) of 37. Subjects with tick borne encephalitis (TBE) vaccinations in the past 5 years ( N = 29) had a SCR of 90% and GMT of 65. Conclusions IXIARO is generally well tolerated in the elderly, and the safety profile is largely comparable with younger adults. SCR and GMT are lower compared to younger adults, but SCR is in the range reported in elderly for other vaccines e.g. against TBE, hepatitis-A virus (HAV)/hepatitis-B virus (HBV), influenza. The differences in SCR and GMT from younger to elderly adults were in the range of other vaccines. Duration of protection is uncertain in older persons, therefore a booster dose (third dose) should be considered before any further exposure to JE virus.

BACKGROUND Japanese encephalitis remains a serious health concern in Asian countries and has sporadically affected pediatric travelers. In the present study, we monitored the safety profile of the Japanese encephalitis virus vaccine IXIARO (Valneva Austria GmbH, Vienna, Austria) in a pediatric population. METHODS We randomized 1869 children between 2 months and 17 years of age in an age-stratified manner to vaccination with IXIARO or one of the control vaccines, Prevnar (formerly Wyeth Pharmaceuticals Inc., now Pfizer Inc., Kent, United Kingdom) and HAVRIX 720 (GlaxoSmithKline Biologicals, Rixensart, Belgium). Adverse events (AEs) (unsolicited and solicited local and systemic AEs), serious AEs and medically attended AEs were assessed up to day 56 and month 7 after the first dose. RESULTS Incidences of AEs, serious AEs or medically attended AEs did not differ significantly between the groups in any age stratum. AEs were most frequent in children

Japanese encephalitis (JE) is a major public health concern in Asia and poses a small but potentially fatal threat to travelers from nonendemic countries, including children. No JE vaccine for pediatric use has been available in Europe and the United States.Age-stratified cohorts of children between 2 months and 17 years received 2 doses of Vero cell-derived inactivated JE virus vaccine (IXIARO; Valneva Austria GmbH, Vienna, Austria) administered 28 days apart [3 years, 0.25 mL (half adult dose); ≥3 years, 0.5 mL (full adult dose)]. Immunogenicity endpoints were seroconversion rate, 4-fold increase in JE neutralizing antibody titer and geometric mean titer assessed 56 days and 7 months after the first vaccination in 496 subjects of the intent-to-treat population. The immune response to JE virus at both time points was also analyzed according to prevaccination JE virus and dengue virus serostatus.At day 56, seroconversion was attained in ≥99.2% of subjects with age-appropriate dosing, 4-fold increases in titer were reported for 77.4%-100% in various age groups, and geometric mean titers ranged from 176 to 687, with younger children having the strongest immune response. At month 7, seroconversion was maintained in 85.5%-100% of subjects. Pre-existing JE virus immunity did not impact on immune response at day 56; however, it led to a better persistence of protective antibody titers at month 7.IXIARO is highly immunogenic at both doses tested in the pediatric population, leading to protective antibody titers at day 56 in99% of subjects who received the age-appropriate dose.

Background A patch vaccine containing heat-labile toxin (LT) from enterotoxigenic Escherichia coli (ETEC) has demonstrated to be beneficial in reducing the rate and severity of travelers' diarrhea in Latin America. To evaluate the efficacy of this transdermal vaccine system in an area with a different diarrheal pathogen profile, an additional phase 2 study was conducted in European travelers to India. Methods For this multicenter, randomized, double-blinded, placebo-controlled field study 723 subjects were recruited; 603 (299 LT vaccine, 304 placebo) were included in the per-protocol-population (PPP). Results Although the LT patch induced a measurable LT immune response in recipients, it failed to protect against LT ETEC or all-cause diarrhea. In the PPP the incidence rate of diarrhea as per primary endpoint was 6.0% (18 of 299) in the vaccine group and 5.9% (18 of 304) in the placebo group. Additionally, lower than expected rates of LT ETEC diarrheas were observed in India. The vaccine delivery system frequently produced rash and pruritus at the site of application, long term hyperpigmentation persisted in a minority of LT recipients, and also few site reactions were noted in the placebo group. Conclusions The evaluated patch vaccine failed to satisfy mainly with respect to protective efficacy. Noninvasive prophylactic agents against travelers' diarrhea, particularly vaccines against the most frequent pathogens, thus continue to be badly needed.

IC43 is a recombinant outer membrane protein-based vaccine against Pseudomonas aeruginosa (P. aeruginosa) consisting of OprF- and OprI- epitopes (Opr, outer membrane protein; OprF/I, OprF/OprI hybrid vaccine) with an N-terminal His 6 tag (Met-Ala-(His)6-OprF190-342-OprI21-83).The study aimed to confirm the optimal dose of IC43 in adults with regard to immunogenicity, safety, and tolerability after vaccination with three different dosages of IC43, compared with placebo, and to investigate a potential immune-enhancing effect of the adjuvant, aluminum hydroxide. Subjects were randomly allocated in a 1:1:1:1:1 ratio to one of five treatment groups: 50, 100, or 200 µg IC43 with adjuvant, 100 µg IC43 without adjuvant, or placebo (0.9% sodium chloride) and two intramuscular injections were given in the deltoid region 7 d apart.The primary immunogenicity analysis of OprF/I-specific IgG antibody titers on day 14 demonstrated statistically significant differences among treatment groups (P0.0001), with a significantly higher immune response detected in each IC43 treatment group compared with placebo. From day 0 to day 14, a ≥4-fold increase in OprF/I-specific immunoglobulin G (IgG) antibody titers were observed in90% of subjects in all IC43 treatment groups in the per-protocol (PP) and intention-to-treat (ITT) populations; a ≥50-fold titer increase was observed in 42.6% subjects including all IC43 treatment groups. On day 90, OprF/I-specific IgGs started to decline in all IC43 treatment groups but remained significantly higher until 6 mo compared with placebo. Assessment of functional antibody induction by opsonophagocytic assay (OPA) followed a similar pattern compared with OprF/I-specific IgG kinetics. At day 14, a ≥2-fold increase in OPA titer was observed in 54.5% subjects within all IC43 treatment groups. An increase in antibody avidity index was observed after the second vaccination. At day 14,96% of subjects in each IC43 treatment group had detectable OprF/I-specific IgG antibodies. Anti-histidine IgG antibody titers peaked on day 14 and were reduced on day 90 in all IC43 treatment groups. OprF/I-specific IgG secreted by antibody-secreting cell (ASC) was detected in all IC43 groups by B-cell ELIspot after the second vaccination and up to 6 mo. All vaccinations were safe and well tolerated up to the maximum cumulative dosage of 400 µg IC43.IC43 doses equal to or greater than 50 µg were sufficient to induce a plateau of IgG antibody responses in healthy volunteers. Higher doses, whether adjuvanted or non-adjuvanted, were not more effective.In this phase I, randomized, placebo-controlled, observer-blinded, multicenter clinical trial, 163 healthy volunteers (18-65 y) were randomly assigned to five treatment groups (1:1:1:1:1). Three groups received IC43 with adjuvant: 50 µg (n=32), 100 µg (n=33), or 200 µg (n=33). One group received IC43 100 µg without adjuvant (n=32), and one group received placebo (0.9% sodium chloride) (n=33). Each subject received two intramuscular vaccinations, separated by a 7-d interval (days 0 and 7) (Fig. 1). Humoral immune response was assessed by measurement of outer membrane protein F/I (OprF/I)-specific antibodies determined by enzyme-linked immunosorbent assay (ELISA), anti-histidine antibodies determined by ELISA, and functional antibody activity determined by opsonophagocytic assay (OPA), up to 6 mo post-vaccination. Antibody avidity was measured on days 7 and 14 from samples that had detectable vaccine antibody-specific immunoglobulin G (IgG) antibody titers. At the Austrian site only, the B-cell ELIspot assay was used to determine specific ASC responses. Safety was assessed using adverse event monitoring and clinical laboratory tests. Local and systemic tolerability was recorded in a subject diary for 7 d after each vaccination and by investigators up to 6 mo post-vaccination.

Background Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 20% children and 9% adults worldwide. AD patients are often sensitized against a broad variety of allergens and more than 90% of them suffer from skin superinfections with Staphylococcus aureus.

3055Background: Casitas-B-lineage lymphoma protein – b (cbl-b), an E3 ubiquitin ligase is an important intracellular checkpoint limiting activation of lymphocytes and NK cells. Silencing of cbl-b e...

Zusammenfassung: Typ I Allergie ist eine Immunglobulin E (IgE)-mediierte Uberempfindlichkeitsreaktion, die mehr als 25 % der Bevolkerung betrifft. Die serologische Diagnose beruht auf IgE Antikorper Nachweis und wird zur Zeit mit Allergenextrakten durchgefuhrt. Allergenextrakte werden durch Extraktion aus Allergenquellen (z. B. Birkenpollen) gewonnen und enthalten eine Vielzahl allergener und nicht-allergener Molekule, deren Konzentration im Extrakt nur schwierig zu standardisieren ist. Allergenextrakte sind in der Allergiediagnostik geeignet, um die Sensibilisierung eines Patienten gegen eine Allergenquelle festzustellen, sie konnen aber keine Information liefern, gegen welche Komponenten (= einzelne Allergene) des Extraktes der Patient allergisch reagiert. Fortschritte in der rekombinanten DNS-Technologie haben es ermoglicht, die krankheitsauslosenden Molekule in den haufigsten Allergenquellen zu charakterisieren und als rekombinante Allergene herzustellen. Die erweiterte Diagnostik basierend auf rekombinanten Allergenen erlaubt es dem Allergologen, das individuelle IgE-Reaktivitatsprofil eines Patienten gegen die einzelnen Komponenten in einem Extrakt zu erstellen. Diese Informationen ermoglichen Kreuzsensibilisierungen gegen Allergenquellen, die immunologisch verwandte Allergene besitzen, nachzuweisen, therapeutische Strategien zu optimieren und auch den Verlauf der Erkrankung wahrend der spezifischen Immuntherapie mit herkommlichen Allergenextrakten zu kontrollieren. Basierend auf der „Komponenten-spezifische Diagnostik” mit rekombinanten Allergenen kann in Zukunft auch die spezifische Immuntherapie mit genau jenen rekombinanten Allergenen durchgefuhrt werden, gegen die der Patient tatsachlich sensibilisiert ist. Hypoallergene Derivate mit reduzierten allergenen Eigenschaften befinden sich bereits in klinischer Erprobung. Summary: Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25 % of the population. Currently, in vitro diagnosis of allergy is performed with allergen extracts, containing a variety of allergenic and non-allergenic molecules, and their allergen content is often cumbersome to standardize. While it is possible to use crude allergen extract-based diagnostic tests to define allergen containing sources, they cannot identify the disease-eliciting molecules. With the introduction of molecularbiology techniques into the field of allergen characterization, the identities of many important allergens have been revealed and increasing numbers of recombinant allergens have become available, leading to new forms of component-resolved diagnosis (CRD) based on recombinant allergens. Using recombinant allergens in in vitro diagnostic devices, a patient`s individual IgE reactivity profile can be established. The presence of IgE to cross-reactive allergen components can thus be determined and used to predict clinically relevant sensitization to allergen sources which contain immunologically related allergens. Moreover, component-resolved diagnosis can assist the clinician in choosing optimal therapeutic strategies among available treatment forms (e.g. allergen extract-based immunotherapy) and to monitor the success of the applied therapeutic measures. Based on CRD, component-resolved forms of immunotherapy are currently being developed using recombinant allergens with reduced allergenic activity.

Background Cross-linking of mast cell–bound IgE releases proinflammatory mediators, cytokines, and proteolytic enzymes and is a key event in allergic inflammation. Objective We sought to study the effect of proteases released on effector cell activation on receptor-bound IgE and their possible role in the regulation of allergic inflammation. Methods Using molar ratios of purified recombinant tryptase and human IgE, we studied whether tryptase can cleave IgE. Similar experiments were performed with mast cell lysates in the presence or absence of protease inhibitors. IgE cleavage products were detected in supernatants of allergen cross-linked, cultivated mast cells and in tissue fluids collected from patients' skin after IgE-mediated degranulation. The effects of protamine, an inhibitor of heparin-dependent proteases on IgE-mediated allergic in vivo skin inflammation in human subjects were studied. Results We show that β-tryptase, a major protease released during mast cell activation, cleaves IgE. IgE degradation products were detected in tryptase-containing tissue fluids collected from sites of allergic inflammation. The biologic significance of this mechanism is demonstrated by in vivo experiments showing that protease inhibition enhances allergic skin inflammation. Conclusion We suggest that IgE cleavage by effector cell proteases is a natural mechanism for controlling allergic inflammation.

Background: The 2 EF-hand calcium-binding allergen from timothy grass pollen, Phl p 7, contains the majority of relevant IgE epitopes among calcium-binding allergens occurring in pollen species of different plants. Objective: To describe the ultrastructural localization of Phl p 7 allergen in timothy grass pollen and its homologues in a broad spectrum of allergologically relevant pollens from grasses (timothy grass, rye grass), trees (birch, alder, olive) and weeds (mugwort, ribwort, ragweed) commonly growing in Europe. Materials and Methods: Mature pollens from 8 different plant species were collected and anhydrously prepared for transmission electron microscopy. In ultrathin sections, allergens were localized using an antibody prepared against a Phl p 7-derived peptide comprising the C-terminal half of the Phl p 7 wild-type molecule in combination with a secondary antibody coupled to 10-nm colloidal gold particles. Results: Phl p 7 and Phl p 7 homologues were detected in pollen from each of the 8 pollen species investigated. The allergens were found in the cytoplasm of the pollen grains (cytoplasmic matrix, mitochondria, nuclei) and in the pollen wall (preferably the exine). Reserve materials were unlabeled. Conclusions: The 2 EF-hand calcium-binding allergen Phl p 7 from timothy grass and its homologues can be localized in all pollen species under investigation. This finding confirms that Phl p 7 is a marker allergen for sensitization of patients to a novel family of 2 EF-hand calcium-binding pollen allergens occurring in a number of important allergenic plants in Europe.

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhoea and colitis and the most common pathogen of health care-associated infections. In the US, CDI causes approximately half a million infections and close to 30,000 deaths. Despite antibiotic treatment of C. difficile associated diarrhoea, the disease is complicated by its recurrence in up to 30% of patients.An open-label, partially randomized, dose-escalation Phase I trial was performed in two parts. Sixty volunteers aged ≥18 to65 years were randomized into five treatment groups to receive three immunizations (Day 0, 7, 21) of VLA84 (20μg with Alum, 75μg with or without Alum, 200μg with or without Alum). Eighty-one volunteers aged ≥65 were randomized into four treatment groups (75μg with or without Alum, 200μg with or without Alum) and received four immunizations (Day 0, 7, 28 and 56). All subjects were followed for safety and immunogenicity for six months.VLA84 was safe and well tolerated. Fifty-one adult volunteers (85%) and 50 elderly (62%) experienced at least one solicited or unsolicited adverse event (AE). Forty-eight adult volunteers (80%) and 40 elderly (49%) experienced related AEs which were mostly mild or moderate. No related serious adverse event and no death occurred. The vaccine induced high antibody titres against Toxin A and Toxin B in both study populations.VLA84 was safe, well tolerated and highly immunogenic in adult volunteers aged ≥18 to65 years and elderly volunteers aged ≥65 years. This study is registered at ClinicalTrials.gov under registration number NCT01296386.

Background: Polcalcins are pollen-specific allergens with two EF-hand calcium-binding sites that exhibit strong cross-reactivity. Our objective was to isolate and express the cDNA coding of the EF-hand calcium-binding allergen from lilac pollen and to study cross-reactivity with other polcalcins from related and nonrelated pollen sources with different specific antibodies and sera from two different populations. Methods: Specific cDNA was amplified by PCR, cloned and expressed in Escherichia coli. Purification was achieved by gel permeation and ion exchange chromatographies. ELISA titration and inhibition assays were performed using the recombinant forms of Syr v 3, Ole e 3, Che a 3 and Phl p 7 with sera from two Spanish regions with different sensitization profiles, as well as Phl p 7- and Ole e 3-specific polyclonal rabbit antisera, and an Ole e 3-specific monoclonal antibody. Results: Syr v 3 displays two EF-hand consensus sites and 8863 Da of theoretical molecular mass. The allergen consists of 80 residues with identities ranging from 66 to 87% with polcalcins included in this study. Syr v 3, Ole e 3, Che a 3 and Phl p 7 showed a similar IgG- and IgE-binding capacity although differences at quantitative level were observed depending on the population of patients’ sera. Conclusion: Syr v 3 is a polcalcin with structural and antigenic similarities to the members of this family. Diagnosis of polcalcin-sensitized patients could be performed whatever polcalcin used, whereas for immunotherapy, primary sensitization to a particular allergenic source should be considered.

PURPOSE OF REVIEW Progress in allergen-specific immunotherapy, the only causative form of allergy treatment, was limited by the lack of defined allergen molecules for vaccine formulation. Today the genetic informations for the most common allergens have been obtained. Here we review recombinant allergen-based technologies for the improvement of diagnosis and therapy of allergy. RECENT FINDINGS Numerous strategies, including the genetic engineering of allergens for reduction of allergenic activity, have been developed to improve allergen-specific immunotherapy. Genetically modified allergen derivatives with reduced allergenic activity, preserved T cell epitope repertoire and retained immunogenicity have been characterized in vitro and in vivo. SUMMARY Based on the review of the recently published data we argue that it is possible to genetically engineer safer and more effective immunotherapy reagents.

The key event of allergic inflammation, allergen-induced crosslinking of mast cell-bound IgE antibodies, is accompanied by release of inflammatory mediators, cytokines, and proteases, in particular beta-tryptase. We provide evidence that protease-mediated cleavage of allergens represents a mechanism that regulates allergen-induced mast cell activation. When used in molar ratios as they occur in vivo, purified beta-tryptase cleaved major grass and birch pollen allergens, resulting in defined peptide fragments as mapped by mass spectrometry. Tryptase-cleaved allergens showed reduced IgE reactivity and allergenic activity. The biological relevance is demonstrated by the fact that lysates from activated human mast cells containing tryptase levels as they occur in vivo cleaved allergens. Additionally, protamine, an inhibitor of heparin-dependent effector cell proteases, augmented allergen-induced release of mediators from effector cells. Protease-mediated allergen cleavage may represent an important mechanism for terminating allergen-induced effector cell activation.

Background Staphylococcus aureus superinfections occur in more than 90% of patients with atopic dermatitis (AD) and aggravate skin inflammation. S aureus toxins lead to tissue damage and augment T-cell–mediated skin inflammation by a superantigen effect. Objective To characterize IgE-reactive proteins from S aureus . Methods A genomic S aureus library was screened with IgE from patients with AD for DNA clones coding for IgE-reactive antigens. One was identified as fibronectin-binding protein (FBP). Recombinant FBP was expressed in Escherichia coli , purified, and tested for specific IgE reactivity in patients with AD. Its allergenic activity was studied in basophil activation experiments and T-cell cultures. The in vivo allergenic activity was investigated by sensitizing mice. Results Using IgE from patients with AD for screening of a genomic S aureus library, an IgE-reactive DNA clone was isolated that coded for FBP. Recombinant FBP was expressed in E coli and purified. It reacted specifically with IgE from patients with AD and exhibited allergenic activity in basophil degranulation assays. FBP showed specific T-cell reactivity requiring antigen presentation and induced the secretion of proinflammatory cytokines from PBMCs. Mice sensitized with FBP mounted FBP-specific IgE responses, showed FBP-specific basophil degranulation as well as FBP-specific T-cell proliferation, and mixed T h 2/T h 1 cytokine secretion. Conclusion Evidence is provided that specific humoral and cellular immune responses to S aureus antigens dependent on antigen presentation represent a novel mechanism for S aureus –induced skin inflammation in AD. Furthermore, FBP may be used for the development of novel diagnostic and therapeutic strategies for S aureus infections.

Allergens and rhinovirus infections are among the most common elicitors of respiratory diseases. We report the construction of a recombinant combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived VP1, the surface protein which is critically involved in infection of respiratory cells, and a nonallergenic peptide of the major grass pollen allergen Phl p 1. Recombinant hybrid molecules consisting of VP1 and a Phl p 1-derived peptide of 31 aa were expressed in Escherichia coli. The hybrid molecules did not react with IgE Abs from grass pollen allergic patients and lacked allergenic activity when exposed to basophils from allergic patients. Upon immunization of mice and rabbits, the hybrids did not sensitize against Phl p 1 but induced protective IgG Abs that cross-reacted with group 1 allergens from different grass species and blocked allergic patients’ IgE reactivity to Phl p 1 as well as Phl p 1-induced basophil degranulation. Moreover, hybrid-induced IgG Abs inhibited rhinovirus infection of cultured human epithelial cells. The principle of fusing nonallergenic allergen-derived peptides onto viral carrier proteins may be used for the engineering of safe allergy vaccines which also protect against viral infections.

Background Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens. Material and methods Eighty-two grass pollen allergic patients were skin prick tested with serial dilutions of approximately equimolar concentrations of the purified allergens in a double-blind study. Results Phl p 4 and Phl p 13 were identified as major grass pollen allergens according to IgE binding frequency (Phl p 4: 85%; Phl p 13: 56%), but exhibited a five to nine-fold lower allergenic skin reactivity compared to Phl p 1, Phl p 2 or Phl p 5. Conclusion Our results indicate that Phl p 4 and Phl p 13 are not essential components for a therapeutic grass pollen vaccine and underpin the importance of evaluating the in vivo allergenic activity of individual allergens for the formulation of therapeutic vaccines based on purified allergens.

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480 bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

Background: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. Objective: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). Methods: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n=42; Scandinavia, n=8; United States, n=19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. Results: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. Conclusion: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract. (J Allergy Clin Immunol 2003;111:974-9.)

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.

Background: Sensitivity to birch pollen allergens is a common feature among European patients with seasonal pollen allergy. In this in vitro study, we examined the specific serum IgE binding profiles to individual birch pollen allergens in birch-sensitive patients from six European populations. Methods: The study included 242 patients from Finland, Sweden, Austria, France, Switzerland and Italy. All suffered from seasonal rhinoconjunctivitis and/or asthma. Their sera were analyzed for specific IgE reactivity to individual birch pollen allergens (recombinant Bet v 1, Bet v 2 and Bet v 4) and natural birch pollen extract using Pharmacia CAP System™ and immunoblotting. Results: Almost all Finnish, Swedish and Austrian sera contained IgE specific for Bet v 1 (≧98%). Bet v 1-specific IgE antibodies were found in 90% of the French sera, and in 65 and 62% of the sera from Switzerland and Italy, respectively. Few Finnish (2%) and Swedish (12%) patients had IgE to Bet v 2, while Bet v 2 reactivity was more common in the other populations (20–43%). Reactivity to Bet v 4 was rare in all populations (5–11%) except for the Italian patients, in whom 3 of 11 sera were positive (27%). The immunoblot results supported the specific IgE profiles obtained with Pharmacia CAP System showing a broader IgE reactivity profile in patients from central and southern Europe as compared to northern Europe. Conclusion: Component-resolved allergy diagnosis with recombinant allergens reveals that the IgE reactivity profiles to individual birch pollen allergens vary between European populations. This observation may be explained by sensitization to different allergen sources and will have an impact on allergen-specific prevention and therapy strategies.

Background: Calcium-binding plant allergens can be grouped in different families according to the number of calciumbinding domains (EF hands). Objective: We sought to identify pollens containing crossreactive calcium-binding allergens and to investigate structural and immunologic similarities of members belonging to different families of calcium-binding allergens. Methods: By means of multiple sequence alignment and molecular modeling, we searched for structural similarities among pollen allergens with 2 (Phl p 7, timothy grass; Aln g 4, alder), 3 (Bet v 3, birch) and 4 EF hands (Jun o 4, prickly juniper). Purified recombinant Aln g 4 and Jun o 4 were used to determine the prevalence of IgE recognition in 210 patients sensitized to different pollens and to search, by means of ELISA competition, for the presence of cross-reactive epitopes in pollens from 16 unrelated plant species. IgE cross-reactivity among the allergen families was studied with purified rPhl p 7, rAln g 4, rBet v 3, and rJun o 4 and 2 synthetic peptides comprising the N-terminal and C-terminal EF hands of Phl p 7 by means of ELISA competition. Results: Structural similarities were found by using molecular modeling among the allergens with 2, 3, and 4 EF hands. Pollens from 16 unrelated plants contained Aln g 4- and Jun o 4-related epitopes. Twenty-two percent of the patients with multiple pollen sensitization reacted to at least one of the calcium-binding allergens. A hierarchy of IgE cross-reactivity (rPhl p7 > rAln g 4 > rJun o 4 > rBet v 3) could be established that identified rPhl p 7 as the EF-hand allergen containing most IgE epitopes in the population studied. Conclusion: The demonstration that members of different families of calcium-binding plant allergens share similarities suggests that it may be possible to use representative molecules for the diagnosis and therapy of allergies to EF-hand allergens. (J Allergy Clin Immunol 2002;109:314-20.)

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry. As determined by circular dichroism analysis and size exclusion chromatography coupled to mass spectrometry, recombinant mutants showed altered structural fold and lacked calcium-binding capacity, whereas the two synthetic peptides had completely lost their structural fold. Allergic patients’ IgE Ab binding was strongest reduced to the variant containing two mutations in each of the two calcium-binding sites and to the peptides. Basophil histamine release and skin test experiments in allergic patients identified the peptides as the vaccine candidates with lowest allergenic activity. Immunization of rabbits with the peptides induced IgG Abs that blocked allergic patients’ IgE binding to Phl p 7 and inhibited allergen-induced basophil degranulation. Our results indicate that disruption of an allergen’s three-dimensional structure represents a general strategy for the generation of hypoallergenic allergy vaccines, and demonstrate the importance of allergen-specific IgG Abs for the inhibition of immediate allergic symptoms.

Two EF-hand calcium-binding allergens (polcalcins) occur in the pollen of a wide variety of unrelated plants as highly cross-reactive allergenic molecules. We report the expression, purification, immunological characterization, and the 1.75-Å crystal structure of recombinant Che a 3 (rChe a 3), the polcalcin from the weed Chenopodium album. The three-dimensional structure of rChe a 3 resembles an α-helical fold that is essentially identical with that of the two EF-hand allergens from birch pollen, Bet v 4, and timothy grass pollen, Phl p 7. The extensive cross-reactivity between Che a 3 and Phl p 7 is demonstrated by competition experiments with IgE Abs from allergic patients as well as specific Ab probes. Amino acid residues that are conserved for the two EF-hand allergen family were identified in multiple sequence alignments of polcalcins from 15 different plants. Next, the three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 were used to identify conserved amino acids with high surface exposition to visualize surface patches as potential targets for the polyclonal IgE Ab response of allergic patients. The essentially identical three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 explain the extensive cross-reactivity of allergic patients IgE Abs with two EF-hand allergens from unrelated plants. In addition, analyzing the three-dimensional structures of cross-reactive Ags for conserved and surface exposed amino acids may be a first approach to mapping the conformational epitopes on disease-related Ags that are recognized by polyclonal patient Abs.

Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive α-helical contents of the protein but retained its β-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients’ IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.