pET-prof, a plasmid for high-level expression of recombinant peptides fused to a birch profilin-derived hexadecapeptide tag: a system for the detection and presentation of recombinant antigens.

We have previously identified a birch pollen profilin hexadecapeptide (Bp36/51), which was recognized by a monoclonal antibody (moAb 4A6) with high affinity. Here, we report the construction of a T7 RNA polymerase-driven high-level plasmid expression system, pET-prof, capable of producing proteins and peptides containing the Bp36/51 birch profilin-derived peptide fused to their N-terminus. As examples, the cDNAs coding for two major timothy grass (Phleum pratense) pollen allergens, Phl p 2 and Phl p 6, as well as for an alder (Alnus glutinosa) pollen allergen, Aln g 4, were overexpressed in Escherichia coli as BP36/51-tagged proteins. All three recombinant allergens were readily detected in nitrocellulose-blotted E. coli extracts by the Bp36/51-specific moAb 4A6. We demonstrate comparable IgE recognition of Bp36/51-tagged and untagged recombinant allergens by immunoblotting. A sandwich ELISA was developed using plate-bound moAb 4A6 to immobilize and present Bp36/51-tagged recombinant allergens to IgE antibodies of allergic patients. Using immunoelectronmicroscopy, we demonstrate that even under harsh fixation conditions, tagged allergens can be localized simultaneously in situ by moAb 4A6 and allergen-specific antisera. We suggest the use of the pET-prof system for the high-level expression of Bp36/51-tagged polypeptides that can be rapidly detected in total protein extracts, immunolocalized in situ, immobilized and presented to other antigen-specific antibodies (e.g. IgE), even when they occur in minute concentrations.

Major cat and dog allergens share IgE epitopes.

BACKGROUND: Patients allergic to cats and dogs frequently display IgE reactivity against allergens from different animals, suggesting a cross-sensitization to common allergenic determinants. Although albumins have been recognized as relevant cross-reactive allergens, little is known regarding cross-reactive epitopes of the major cat and dog allergens. OBJECTIVE: In this study, sera from patients allergic to cats and/or dogs were used to investigate the presence of common IgE epitopes among the major cat and dog allergens. METHODS: The IgE reactivity profile of 109 patients who were allergic to allergens from several species of animals was determined with nitrocellulose-blotted cat and dog allergens. Sera from patients who were strongly allergic to the major cat and dog allergens were tested for the presence of cross-reactive IgE antibodies by one-dimensional and two-dimensional immunoblot inhibition experiments and by quantitative measurements obtained with the CAP-FEIA system (Pharmacia). RESULTS: Sixty-eight of 109 patients with animal allergy showed IgE reactivity to cat allergens and dog allergens. Sera from patients with both cat and dog allergy detected allergens of similar molecular weight in nitrocellulose-blotted cat and dog hair/dander extracts. Common, as well as species-restricted, IgE epitopes of the major cat and dog allergens could be demonstrated by IgE inhibition studies. CONCLUSION: Shared IgE epitopes of the major cat and dog allergens may provide an explanation for the clinical observation that allergies to cats and dogs are frequently associated.

Escherichia coli expression and purification of recombinant dog albumin, a cross-reactive animal allergen.

BACKGROUND: Animal hair-dander represents an important source of indoor allergens. Diagnosis and therapy of animal allergy would benefit from the availability of defined recombinant allergens. They may be preferred to animal-derived proteins, particularly for in vivo application in patients. OBJECTIVE: The purpose of this study was to express and purify recombinant dog albumin, an important cross-reactive animal allergen. METHODS: Complementary (c)DNA sequences coding for dog albumin were obtained by reverse transcription and subsequent PCR amplification from dog liver RNA. Dog albumin-encoding cDNA sequences were inserted into phage lambdagt11, and IgE-reactive phage clones were isolated by immunoscreening with serum IgE from a patient with dog allergy. Dog albumin was expressed as IgE-reactive recombinant protein in Escherichia coli and purified by inclusion body preparation, resolubilization, and diethylaminoethyl cellulose chromatography. Cross-reactivity of dog albumin with cat and human albumin was examined by immunoblot, as well as ELISA inhibition experiments. RESULTS: A cDNA sequence coding for complete dog albumin was obtained by reverse transcription and subsequent PCR amplification from dog liver. The cDNA and deduced amino acid sequence of dog albumin was highly homologous to the sequences of albumins from animals to human subjects, thus explaining the extensive cross-reactivities among albumins. Recombinant dog albumin was expressed in E coli and purified. It reacted with serum IgE from patients allergic to dog albumin and a monoclonal anti-human albumin antibody. Immunologic competition experiments performed with serum IgE from patients allergic to dog albumin and a mouse monoclonal antihuman albumin antibody indicated the presence of similar epitopes on dog, cat, and human albumin. CONCLUSION: Recombinant dog albumin may be used for diagnostic purposes to identify patients who are cross-sensitized to many animal species and perhaps for specific immunotherapy of sensitized individuals.

IgE cross-reactivities against albumins in patients allergic to animals.

BACKGROUND: Type I allergic symptoms and severe asthma in particular are frequently caused by animal hair/dander proteins, among which albumins are possible cross-sensitizing allergenic components. METHODS: The significance and degree of IgE-cross-reactivities against various albumins were studied in a representative number (n = 200) of patients allergic to animals with hair/dander extracts, purified albumins from different animals, and a recombinant dog albumin fragment expressed in lysogenic Escherichia coli Y1089 and purified as a beta-galactosidase fusion protein. RESULTS: Despite a high degree of sequence homology among different albumins, a remarkable variability of IgE cross-reactivities was observed, indicating that some patients were sensitized preferentially against certain albumins. Most of the patients allergic to albumins, however, reacted to dog, cat, and horse albumin, which also bound a high percentage of albumin-specific IgE. CONCLUSION: The purified recombinant dog albumin fragment, representing 265 amino acids of the mature protein, bound IgE from all 15 patients allergic to albumin tested suggesting its potential usefulness for diagnosis and perhaps therapy.

Molecular characterization of dog albumin as a cross-reactive allergen.

Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander. In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs. In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat. The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90%. By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones. All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin. The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%). Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence. Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin.

Immunization with purified natural and recombinant allergens induces mouse IgG1 antibodies that recognize similar epitopes as human IgE and inhibit the human IgE-allergen interaction and allergen-induced basophil degranulation.

Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (beta-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.