Rapid production of recombinant allergens in Nicotiana benthamiana and their impact on diagnosis and therapy.

BACKGROUND: Type I allergies are immunological disorders that afflict a quarter of the world's population. Recombinant allergens have improved the diagnosis of allergic diseases and allow the formulation of new therapeutic approaches. Over 50% of all allergens are of plant origin. OBJECTIVE: We have applied a novel method of overexpressing plant allergens in the tobacco-related species Nicotiana benthamiana. METHOD: This method is based on the use of a chimeric tobacco mosaic virus that harbors a foreign gene sequence and directs its transcription after the infection of the host plant. RESULTS: We have expressed the model allergen Bet v 1, the major birch pollen allergen, and two Hevea brasiliensis latex allergens, the spina-bifida-associated allergens Hev b 1 and Hev b 3, in N. benthamiana using such a viral vector. Bet v 1, Hev b 1 and Hev b 3 produced by this method were recognized by patients' IgE suggesting that the plant-produced allergens were properly folded. Nonpurified Bet v 1 expressed in N. benthamiana leaves had the same immunogenicity as purified Bet v 1 expressed in Escherichia coli or natural Bet v 1 when tested in a murine model of type I allergy. CONCLUSION: We conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts.

Hev b 9, an enolase and a new cross-reactive allergen from hevea latex and molds. Purification, characterization, cloning and expression.

Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho-D-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials.

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.

Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida.

BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy. OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy. METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen. RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3. CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.

Cloning of the patatin-like latex allergen Hev b 7, its expression in the yeast Pichia pastoris and its immunological characterization.

The 43-kD latex allergen Hev b 7 was purified from the latex of Hevea brasiliensis and identified by N-terminal and internal peptide sequences as highly homologous to patatins. Patatins are storage proteins encoded by a multigene family found in plants such as potato and tomato. We have obtained a cDNA clone coding for a cytoplasmic form of Hev b 7. The recombinant protein was expressed in the methylotrophic yeast Pichia pastoris at 10 mg/l culture supernatant. Both natural Hev b 7 and rHev b 7 were recognized by IgE in 11% of the latex-allergic patients. rHev b 7 inhibited binding to its counterpart in natural rubber latex extracts. Purified rHev b 7 used at concentrations of 10 micrograms/ml in skin prick tests produced wheal-and-flare reactions of sizes equal to those produced by nHev b 7. Furthermore, we were able to show that rHev b 7 possessed esterase activity. A plant expression system for the production of larger quantities of recombinant latex allergens as an alternative to the preparation from H. brasiliensis sap is discussed.

Bet v 1, the major birch pollen allergen, and Mal d 1, the major apple allergen, cross-react at the level of allergen-specific T helper cells.

BACKGROUND: Food allergy to apple is frequent in individuals allergic to tree pollen. The major allergens of birch, Bet v 1, and apple, Mal d 1, have been cloned and sequenced and display a high degree of sequence identity, leading to IgE cross-reactivity. OBJECTIVE: We sought to investigate cross-reactivity between Bet v 1 and Mal d 1 at the level of allergen-specific T lymphocytes. METHODS: PBMCs of 13 patients allergic to birch pollen with oral allergy syndrome to apple were stimulated with rBet v 1 and rMal d 1, respectively, thereby establishing allergen-specific T-cell lines and T-cell clones. rMal d 1-specific T-cell cultures were tested for reactivity with rBet v 1, and rBet v 1-specific T cells were analyzed for reactivity with apple allergen. Cytokine production patterns in response to specific stimulation were evaluated. A selection of cross-reacting T-cell clones was mapped for epitope specificity by the use of overlapping Bet v 1- derived peptides. RESULTS: Nineteen Mal d 1-specific T-cell clones were produced, 79% of which cross-reacted with Bet v 1. Eight of 18 Bet v 1-specific T-cell clones cross-reacted with Mal d 1. Six peptides representing cross-reactive T-cell epitopes could be identified. The respective fragments from birch and apple displayed approximately 50% amino acid identity. Seventy percent of the cross-reactive T-cell clones revealed a T(H2)-like cytokine production pattern. CONCLUSION: The results indicate that cross-reactivity between apple and birch pollen leading to the clinical oral allergy syndrome occurs not only at the serologic, but also at the cellular level.

Identification and cloning of prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris.

Avocado, the fruit of the tropical tree Persea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients. Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined. We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5'-rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids. We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium. The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity. IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases.

cDNA cloning of the 43-kDa latex allergen Hev b 7 with sequence similarity to patatins and its expression in the yeast Pichia pastoris.

IgE-mediated hypersensitivity to latex proteins present in health care products, particularly in latex gloves, has become an important public health problem in recent years. We purified natural Hev b 7, a 43-kDa patatin-like allergen from the latex of Hevea brasiliensis and determined several internal peptide sequences. A heterologous hybridization probe of a patatin gene of potato, to which these peptides could be aligned best, was used to screen a latex cDNA library. The cDNA encoded an acidic protein of 388 amino acids with a molecular mass of 42.9 kDa. The deduced amino acid sequence had 39-42% identity to patatins from Solanum tuberosum. The purified recombinant Hev b 7 expressed in the yeast Pichia pastoris displayed, similarly to patatins from S. tuberosum, esterase activity. Both natural and recombinant Hev b 7 were recognized by IgE from sera of latex-sensitized allergic individuals. In contrast to patatins from S. tuberosum and Nicotiana tabacum, natural Hev b 7 lacked an N-terminal leader peptide for targeting to the endoplasmatic reticulum and was not glycosylated. These results establish the 43-kDa patatin-like protein as a latex allergen and raise the possibility of different cellular localization and function compared to S. tuberosum patatins.

Genomic characterization of members of the Bet v 1 family: genes coding for allergens and pathogenesis-related proteins share intron positions.

Bet v 1, the major birch pollen allergen, is a member of a multigene family; a number of isoforms and homologous proteins from closely related species (alder, hazel and hornbeam) has been isolated and their cDNAs cloned and characterized. Genomic clones coding for Bet v 1 and homologues from apple and hazel were isolated and sequenced. Some of these clones contained intervening sequences. The exon-intron formation is highly conserved throughout this family of pathogenesis-related proteins in dicot plants and is also found in Aopr1 (Asparagus officinalis), a monocol species. Phylogenetic analysis suggested a possible common origin of the intron position in these homologous proteins at codon 62 in various families of flowering plants, including Fagaceae, Rosaceae and Apiaceae. This conserved 'proto-splice site' may point to a structure/function relationship. A conserved sequence motif (P-loop) was also found in all members of this protein family. Moreover, there is a certain degree of sequence similarity among the proteins derived from various species throughout the dicots and the only monocot examined. This fact is reflected by cross-reactivity from monoclonal and polyclonal antibodies raised against Bet v 1.

Cross-reacting allergens in tree pollen and pollen-related food allergy: implications for diagnosis of specific IgE.

BACKGROUND: A number of recombinant allergens are by now constituents of devices that can be routinely used for the detection of specific IgE. Therefore, the results of diagnostic procedures using conventional allergen extracts can be compared with those employing selected recombinant allergens. METHODS: Thirty-four sera from patients allergic to birch pollen were tested with the standard t3-CAP and rBet v 1a- and rBet v 2-CAP. cDNA was prepared by RT-PCR using primers according to the N terminus of purified allergens. Expression cDNA libraries were screened with IgE from selected patients. RESULTS: Twenty-four patients allergic to birch pollen showed the same RAST class with t3 as with rBet v 1a; 8 patients differed within 1 RAST class. In addition, 3 patients showed RAST class 3 with rBet v 2. Besides Bet v 1 and Bet v 2, 3 allergens from celery and avocado belonging to highly conserved protein families were cloned and sequenced. CONCLUSIONS: rBet v 1a can be expected to represent an excellent tool for the diagnosis of patients allergic to birch pollen in Central, Northern, and Eastern Europe. Still, a much higher number of patients has to be tested. For their high degree of conservation, further protein families have to be identified to explain cross-reactivities of birch pollen allergens other than Bet v 1 and Bet v 2 with, e.g., allergens from vegetable food.

The potential of Betv1 homologues, a nuclear multigene family, as phylogenetic markers in flowering plants.

Betv1 homologues are a ubiquitous group of genes in flowering plants encoding a class of highly conserved defense-related proteins and containing open reading frames from 465 to 480 bp. Betv1-like genes consist of two exons interrupted by an intron of 76-359 bp, with the intron position highly conserved. The pairwise p distance ranged from 0 to 0.583 among flowering plants. Within plant families, the ranges of the p distance were 0-0.403, 0-0.253, and 0.011-0.369, for Apiaceae, Betulaceae, and Fabaceae, respectively. The most striking feature of the betv1 gene phylogeny was that the multiple sequences from each plant family formed a monophyletic group and sequences from each species were generally more similar to each other than those from other species. The almost exclusive paralogous relationships of genes from the same species suggested that the genes of the multigene family underwent strong concerted evolution. Phylogenies of Betulaceae and Fabaceae inferred from betv1 gene trees were generally congruent with those based on morphology and other molecules. Betv1 homologues constitute potential phylogenetic markers at the intrafamilial level or among closely related families in flowering plants.

Cloning and sequencing of Mal d 1, the major allergen from apple (Malus domestica), and its immunological relationship to Bet v 1, the major birch pollen allergen.

The number of tree-pollen-allergic patients showing allergic reactions to apples, various vegetables and nuts is increasing. In this paper the molecular characterization of the major apple allergen, Mal d 1, is reported. The cDNA coding for Mal d 1 was cloned and sequenced. Its open reading frame codes for a protein of 159 amino acids with a predicted molecular mass of 17.7 kDa and a predicted pI of 5.9. Sequence comparison to Bet v 1, the major birch pollen allergen, revealed 64.5% identity on the amino acid level and 55.6% identity on the nucleic acid level. Recombinant Mal d 1 was expressed in the plasmid pMW 175 in E. coli BL 21 (DE3) and its immunological properties were tested. Crossreactivity with Bet v 1 was shown by inhibition assays.