Allergenicity of peanut component Ara h 2: Contribution of conformational versus linear hydroxyproline-containing epitopes.

BACKGROUND: The 2S-albumin Ara h 2 is the most potent peanut allergen and a good predictor of clinical reactivity in allergic children. Posttranslational hydroxylation of proline residues occurs in DPYSP(OH)S motifs, which are repeated 2 or 3 times in different isoforms. OBJECTIVES: We investigated the effect of proline hydroxylation on IgE binding and the relative contributions of linear and conformational epitopes to Ara h 2 allergenicity. METHODS: Peptides containing DPYSP(OH)S motifs were synthesized. A recombinant variant of Ara h 2 without DPYSP(OH)S motifs was generated by means of deletion mutagenesis. IgE reactivity of 18 French and 5 American patients with peanut allergy toward synthetic peptides and recombinant allergens was assessed by using IgE-binding inhibition assays and degranulation tests of humanized rat basophilic leukemia cells. RESULTS: Hydroxyproline-containing peptides exhibited an IgE-binding activity equivalent to that of the unfolded Ara h 2. In contrast, corresponding peptides without hydroxyprolines displayed a very weak IgE-binding capacity. Despite removal of the DPYSP(OH)S motifs, the deletion variant still displayed Ara h 2 conformational epitopes. The IgE-binding capacity of Ara h 2 was then recapitulated with an equimolar mixture of a hydroxylated peptide and the deletion variant. Hydroxylated peptides of 15 and 27 amino acid residues were also able to trigger cell degranulation. CONCLUSIONS: Sensitization toward linear and conformational epitopes of Ara h 2 is variable among patients with peanut allergy. Optimal IgE binding to linear epitopes of Ara h 2 requires posttranslational hydroxylation of proline residues. The absence of hydroxyprolines could then affect the accuracy of component-resolved diagnostics by using rAra h 2.

Immunomodulatory potential of partially hydrolyzed ß-lactoglobulin and large synthetic peptides.

The immunomodulatory potential of fragments derived from the cow's milk allergen bovine ß-lactoglobulin (BLG) was assessed in a mouse model of oral tolerance (OT) [Adel-Patient, K.; Wavrin, S.; Bernard, H.; Meziti, N.; Ah-Leung, S.; Wal, J. M. Oral tolerance and Treg cells are induced in BALB/c mice after gavage with bovine ß-lactoglobulin. Allergy 2011, 66 (10), 1312-1321]. Native BLG (nBLG) and chemically denatured BLG (lacking S-S bridges, dBLG), products resulting from their hydrolysis using cyanogen bromide (CNBr) and some synthetic peptides, were produced and precisely characterized. CNBr hydrolysates correspond to pools of peptides of various sizes that are still associated by S-S bridges when derived from nBLG. nBLG, dBLG, and CNBr hydrolysate of nBLG efficiently prevented further sensitization. CNBr hydrolysate of dBLG was less efficient, suggesting that the association by S-S bridges of peptides increased their immunomodulatory potential. Conversely, synthetic peptides were inefficient even if covering 50% of the BLG sequence, demonstrating that the immunomodulatory potential requires the presence of all derived fragments of BLG and further supporting the use of partially hydrolyzed milk proteins to favor OT induction in infants with a risk of atopy.

Update on optimized purification and characterization of natural milk allergens.

Highly purified allergens namely cow's milk alpha-lactalbumin (ALA). (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5) and casein (Bos d 8) and goat's milk casein were prepared from the raw milk from a single animal with a known genetic background. Consequently the natural isoforms are limited, constant and characterized. Purification included selective precipitations and chromatographical steps. Characterization of structure and allergenic activity assessment of milk allergens were carried out using physicochemical and immunochemical methods. Taken together data demonstrated the absence of impurities and of contamination by other milk allergens in each preparation. NMR and circular dichroism analyses confirmed the native conformation and proper folding of ALA and BLG and the expected absence of folding of bovine and caprine casein. Enzyme immuno assays confirmed the native conformation of BLG and the purity and immunoreactivity of all the proteins. The allergenic activity, e. g. the IgE binding capacity, of purified proteins was identical as that of those proteins when present in milk. The purified proteins also demonstrated the ability to provoke the degranulation of humanized rat basophilic leukaemia cells. All the data thus confirm the purity, identity, structural conformation and functionality of the prepared milk allergens.

Cytotoxic and allergenic potential of bioactive proteins and peptides.

This review article deals with the assessment of cytotoxic and allergenic potential of bioactive proteins and peptides. It is evident that 'novel' foods or nutraceuticals containing bioactive proteins and peptides must fulfill their proposed "health claim". Furthermore, there is a need to assess their potential to exert adverse effects before they can be made widely available to consumers. A brief overview of compounds (i.e. proteins and peptides of animal and plant origin) and mechanisms involved in cytotoxic and allergenic (adverse) reactions is given along with some recent results obtained from ongoing studies. There are numerous proteins and peptides of plant and animal origin that are known to exhibit cytotoxic effects. There is evidence that many cytotoxic compounds described in the literature exclusively affect malignant cells leading to the assumption that a cancer protective effect could exist for such bioactive proteins and peptides. All the constituents that are responsible for the allergenicity of foods (as well as of pollens) are proteinaceous in nature. Some protein breakdown products, i.e. peptide fragments, may conserve part of the allergenicity of the native protein and thus can also be considered as allergens. The molecular basis of IgE recognition underlying cow's milk protein allergy is described. Some results from studies on volunteers fed caseinophosphopeptides or potentially hypotensive milk protein hydrolysates illustrate the major difference between allergenicity and immunogenicity. The data presented on the relationship between the structure of food proteins and peptides and their allergenicity shows the difficulty in trying to assess the "non-allergenicity" of products derived from an allergenic source, even if the process used involved extensive hydrolysis of the native protein(s). A 'weight of evidence approach' for assessing the potential allergenicity of a novel protein with no history of prior allergenicity is also presented with regard to the current EU Regulations.

Intranasal coadministration of live lactococci producing interleukin-12 and a major cow's milk allergen inhibits allergic reaction in mice.

The Th1/Th2 balance deregulation toward a Th2 immune response plays a central role in allergy. We previously demonstrated that administration of recombinant Lactococcus lactis strains expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, partially prevents mice from sensitization. In the present study, we aimed to improve this preventive effect by coadministration of L. lactis BLG and a second recombinant L. lactis strain producing biologically active interleukin-12 (IL-12). This L. lactis strain producing IL-12 was previously used to enhance the Th1 immune response in a tumoral murine model (L. G. Bermúdez-Humarán et al., J. Immunol. 175:7297-7302, 2005). A comparison of the administration of either BLG alone or BLG in the presence of IL-12 was conducted. A BLG-specific primary Th1 immune response was observed only after intranasal coadministration of both L. lactis BLG and IL-12-producing L. lactis, as demonstrated by the induction of serum-specific immunoglobulin G2a (IgG2a) concomitant with gamma interferon secretion by splenocytes, confirming the adjuvanticity of IL-12-producing L. lactis. Immunized mice were further sensitized by intraperitoneal administration of purified BLG, and the allergic reaction was elicited by intranasal challenge with purified BLG. Mice pretreated with BLG in either the presence or the absence of IL-12 were rendered completely tolerant to further allergic sensitization and elicitation. Pretreatment with either L. lactis BLG or L. lactis BLG and IL-12-producing L. lactis induces specific anti-BLG IgG2a production in serum and bronchoalveolar lavage (BAL) fluid. Although specific serum IgE was not affected by these pretreatments, the levels of eosinophilia and IL-5 secretion in BAL fluid were significantly reduced after BLG challenge in the groups pretreated with L. lactis BLG and L. lactis BLG-IL-12-producing L. lactis, demonstrating a decreased allergic reaction. Our data demonstrate for the first time (i) the induction of a protective Th1 response by the association of L. lactis BLG and IL-12-producing L. lactis which inhibits the elicitation of the allergic reaction to BLG in mice and (ii) the efficiency of intranasal administration of BLG for the induction of tolerance.

Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells.

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenes inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of noninvasive L. lactis strains as a DNA delivery vehicle. We constructed two Escherichia coli-L. lactis shuttle plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine beta-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis strain MG1363(pLIG:BLG1) was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in Caco-2 human colon carcinoma cells after 3 h of coincubation with (i) purified pLIG:BLG1, (ii) MG1363(pLIG:BLG1), (iii) a mix of MG1363(pLIG) and purified pLIG:BLG1, and (iv) MG1363. Both BLG cDNA and BLG expression were detected only in Caco-2 cells coincubated with MG1363(pLIG:BLG1). There was a decrease in the BLG cDNA level in Caco-2 cells between 24 and 48 h after coincubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after coincubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.

Enzyme immunoassay for a urinary metabolite of 4-hydroxynonenal as a marker of lipid peroxidation.

Free radical reactions are involved in the pathogenesis of numerous diseases, so there is a real need to develop biomarkers that reflect these reactions in vivo. 4-Hydroxy-2-nonenal (HNE) is a major product of the lipid peroxidation process that is a consequence of free radical reactions. We present here the development and validation of an enzyme immunoassay (EIA) of the major urinary metabolite of HNE, namely 1,4-dihydroxynonane-mercapturic acid (DHN-MA). EIA allowed direct measurement of DHN-MA in rat urine with good sensitivity (0.02 ng/ml) and precision (intraassay CV = 5.7%). Recovery was complete (99-102%). Cross-reactivity was very low with 1,4-dihydroxynonene and with different mercapturic acids except with one other HNE urinary metabolite. Good correlation (EIA = 0.79 x LC/MS + 14.03, r = 0.877, p < 10(-8)) was obtained between EIA and liquid chromatography/mass spectrometry (LC/MS) quantitation when analyzing urine samples of rats with different oxidative status, due to treatment with either BrCCl(3) or trinitrobenzene sulfonic acid, which are known to induce hepatic lipid peroxidation or colon inflammation, respectively.

Expression in Escherichia coli and disulfide bridge mapping of PSC33, an allergenic 2S albumin from peanut.

In this work, we describe the expression, purification, and disulfide mapping of the named 'peanut seed cDNA 33' (PSC33) peanut allergen. A variant of PSC33 (with N(63), E(64), Q(69) instead of D(63), Q(64), E(69)) has been identified in peanut by proteomic analysis of a highly IgE immunoreactive purification fraction. It is 92% homologous to Ara h 6. We raised monoclonal antibodies against PSC33 and amplified it by PCR from peanut leaf genomic DNA. PSC33 was intron-less and the two NEQ and DQE variants of PSC33 were equally amplified. Since expression of the natural PSC33 (DQE) gene was very low in Escherichia coli even with supplementation of rare codon tRNAs, a synthetic gene optimized for expression in E. coli of PSC33 (DQE) was introduced into a pET9-c vector. A high production of protein occurred in the inclusion bodies that was submitted to refolding using an additive-introduced stepwise dialysis protocol which consists in the gradual removal of the denaturing agent guanidine-HCl with controlled introduction of oxidized and reduced glutathione and l-arginine as a chemical chaperone. After reverse phase HPLC purification, 1mg of pure refolded protein (as assayed by MALDI-TOF mass spectrometry, mouse IgG immunoreactivity and circular dichroism) were obtained with every 100ml of bacterial culture. Trypsin and CNBr hydrolysis of the protein combined with MALDI-TOF mass spectrometry allowed us to assign disulfide bridges and show that the native and refolded proteins were identical. The four disulfides of canonical 2S albumins were conserved and the two supplementary cysteines of PSC33 were paired together.

Epitopic characterization of native bovine beta-lactoglobulin.

Two monoclonal antibodies (mAbs) (mAb 97 and mAb 117) selected from a panel of 52 mAbs directed against beta-lactoglobulin (BLG) have previously been used to develop a two-site enzyme immunometric assay (EIA) specific for the native form of the protein [J. Immunol. Methods 220 (1998) 25]. In the present work, the conformational epitopes recognized by these two mAbs and by the 50 others have been studied. Firstly, an epitope map was drawn using a surface plasmon resonance (SPR) biosensor: the epitopes were organized in a circle of 11 overlapping and 1 nonoverlapping antigenic regions. Secondly, 55 site-directed BLGA mutants were prepared and tested by ELISA and competitive immunoassay to localize these 12 antigenic regions on the protein molecule. Among them, 20 mutants showed a 10- to 7500-fold decrease in relative affinity for the mAbs of one or several neighbouring regions: their circular dichroism (CD) spectra were identical to the spectrum of wild-type (WT) BLGA. At least one mutant was found for each of the 11 overlapping antigenic regions which circled the molecule and for the nonoverlapping one which was localized near the entrance of the calyx. The two mAbs initially chosen were each directed towards very conformation-dependent epitopes and were thus suitable for monitoring native BLG in food products and manufacturing processes. Other mAb pairs could be used to follow the fate of specific regions of the molecule during denaturation or proteolytic digestion.