Jean-Michel Wal

Bovine milk allergenicity

OBJECTIVE To provide updated data on the characteristics (eg, structure, function, stability) of the main milk proteins identified as allergens and on the characterization of their epitopes. DATA SOURCES Basic literature and the most relevant original recent publications on clinical and epidemiologic aspects of milk allergy and the biochemistry and immunochemistry of milk proteins. STUDY SELECTION The expert opinion of the author was used to select the relevant data for the review. RESULTS Most milk proteins, even proteins present at low concentrations, are potential allergens. Epitopes on milk proteins are both conformational and linear epitopes, widely spread throughout the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule, that comprise highly conserved sequences responsible for IgE cross-reactivity with corresponding milk proteins of other mammals, including humans. Those sequential epitopes have also been proposed as good markers of persistent allergy to milk proteins and may be of particular clinical significance. CONCLUSIONS No specific structure or function is associated with allergenicity of milk proteins. Due to the great variability and heterogeneity of the human IgE response, no single allergen or particular structure can account for a major part of milk allergenicity. Furthermore, the available evidence is not sufficient to establish an intake threshold below which allergic reactions are not triggered or to predict reliably the effect of food processing on allergenic potential of milk proteins.

Cow's milk proteins/allergens

OBJECTIVE The primary objective of this review is to provide updated data on the structure and function of the main cows milk proteins (CMPs) identified as allergens and on the characterization of their epitopes. DATA SOURCES The review represents a synthesis of basic literature and most relevant original recent publications on both topics of clinical and epidemiologic aspects of milk allergy and of milk proteins bio- and immunochemistry. STUDY SELECTION The expert opinion of the author was used to select the relevant data for the review. RESULTS Most CMPs are potential allergens, even the proteins present at very low concentration. There are both conformational and linear epitopes, widely spread all along the protein molecules. They may be short fragments, located in hydrophobic parts of the molecule which comprise highly conserved sequences responsible for immunoglobulin E cross-reactivity with corresponding milk proteins of other mammals, including human beings. Those sequential epitopes have also been proposed as good markers of persistent allergy to CMPs. CONCLUSIONS No specific structure nor function is associated with allergenicity of CMPs. Variability and heterogeneity of the human immunoglobulin E response preclude anticipating the allergenic potential of any CMP or fragment thereof, as well as justify the need for being careful before using peptides for desensitization or proposing any milk protein hydrolysate in a diet for highly allergenic children.

Induction of Mucosal Immune Response after Intranasal or Oral Inoculation of Mice with Lactococcus lactis Producing Bovine Beta-Lactoglobulin

ABSTRACT The bovine beta-lactoglobulin (BLG) is a major cows milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS (“generally regarded as safe”) status, as a delivery vehicle. The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression inL. lactis. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 μg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.

Specificity of the Human IgE Response to the Different Purified Caseins in Allergy to Cow’s Milk Proteins

Background: Cow’s milk is one of the most frequent food allergens. Whole casein appears to be highly allergenic. It corresponds to an association of four different proteins, αs1-, αs2-, β- and *-caseins in approximate proportions of 40, 10, 40, and 10%, respectively. Methods: These different components were thus purified and used as immobilized antigens in an original enzyme immunoassay to measure specific serum IgE response in a population of 58 children (median age 11 months) allergic to cow’s milk who were sensitive to whole casein. Results: A great variability was observed in the affinity and specificity of specific IgE responses in milk-allergic patients’ sera. 85% of the patients presented IgE against each of the four caseins. Statistically higher amounts of specific IgE were found to be directed against the most abundant fractions (αs1- and β-casein). Co- and/or cross-sensitization to the different caseins were seen in most of the patients sensitive to whole casein. Conclusion: These results suggest that both distinct and common epitopes may occur on these different caseins. The major site of phosphorylation which is the most conserved domain in three caseins could be involved in the IgE response to casein and in immunocross-reactivity between these proteins.

Two-site enzyme immunometric assays for determination of native and denatured β-lactoglobulin

Two enzyme immunometric assays suitable for measuring native and denatured beta-lactoglobulin (BLg) have been developed. The assays were performed in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduced and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the other milk proteins and tryptic fragments of BLg. The validity of the assays in different media such as cows milk and cows milk products, saline buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-denatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentration of native BLg was associated with an increase in denatured BLg specifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were able to establish a modified sandwich immunoassay permitting very sensitive detection of cows milk in goats milk.

Preferential labeling of α-amino N-terminal groups in peptides by biotin: application to the detection of specific anti-peptide antibodies by enzyme immunoassays

Experimental conditions (pH 6.5, 24 h reaction, peptide:biotin ratio 1:5) were defined for preferential incorporation of the biotin molecule in the N-terminal alpha-amino group of peptides. This strategy could be helpful in numerous applications when an entire peptide chain must remain accessible for antibody or receptor binding. We illustrate this advantage in a solid-phase enzyme immunoassay designed to detect antibodies specific for bovine beta-lactoglobulin present in rabbit or human sera. This test involves synthetic peptides biotinylated in different positions and immobilized on a solid phase. The use of biotin/streptavidin interactions permitted more efficient detection of specific anti-peptide antibodies than solid phases prepared using conventional passive-adsorption techniques. The highest levels of antibody binding were measured when biotinylation occurred at the N-terminal extremity of immobilized peptides.

Human IgE binding capacity of tryptic peptides from bovine α-lactalbumin

The specific IgE binding capacity of native bovine α-lactalbumin (α-La), a globular whey protein, and tryptic peptides was investigated using 19 sera from patients with cow’s milk protein allergy. The

Identification of the binding sites of benzyl penicilloyl, the allergenic metabolite of penicillin, on the serum albumin molecule

Tryptic digests of fragment A299 585 of penicilloylated serum albumin obtained from two penicillin‐treated patients or prepared by in vitro conjugation, were analyzed by a tandem immunoaffinity reversed‐phase HLPC. Determinations of benzyl penicilloyl groups (BPO) were performed on the different fractions. Three BPO containing peptides were identified by their amino acid sequence and the bound BPO were located on lysines 432, 541 and 545. Six major BPO binding sites were thus identified on the whole albumin molecule. All of them are lysine residues and correspond to a limited number of definite structures in which lysine and serine residues appear to be closely associated.

Use of Native Lactococci as Vehicles for Delivery of DNA into Mammalian Epithelial Cells

ABSTRACT 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 β-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.

Efficient production and secretion of bovine β-lactoglobulin by Lactobacillus casei

BackgroundLactic acid bacteria (LAB) are attractive tools to deliver therapeutic molecules at the mucosal level. The model LAB Lactococcus lactis has been intensively used to produce and deliver such heterologous proteins. However, compared to recombinant lactococci, lactobacilli offer some advantages such as better survival in the digestive tract and immunomodulatory properties. Here, we compared different strategies to optimize the production of bovine β-lactoglobulin (BLG), a major cows milk allergen, in the probiotic strain Lactobacillus casei BL23.ResultsUsing a nisin-inducible plasmid system, we first showed that L. casei BL23 strain could efficiently secrete a reporter protein, the staphylococcal nuclease (Nuc), with the lactococcal signal peptide SPUsp45 fused to its N-terminus. The fusion of SPUsp45 failed to drive BLG secretion but led to a 10-fold increase of intracellular BLG production. Secretion was significantly improved when the synthetic propeptide LEISSTCDA (hereafter called LEISS) was added to the N-terminus of the mature moiety of BLG. Secretion rate of LEISS-BLG was 6-fold higher than that of BLG alone while intracellular production reached then about 1 mg/L of culture. The highest yield of secretion was obtained by using Nuc as carrier protein. Insertion of Nuc between LEISS and BLG resulted in a 20-fold increase in BLG secretion, up to 27 μg/L of culture. Furthermore, the lactococcal nisRK regulatory genes were integrated into the BL23 chromosome. The nisRK insertion allowed a decrease of BLG synthesis in uninduced cultures while BLG production increased by 50% after nisin induction. Moreover, modification of the induction protocol led to increase the proportion of soluble BLG to around 74% of the total BLG production.ConclusionBLG production and secretion in L. casei were significantly improved by fusions to a propeptide enhancer and a carrier protein. The resulting recombinant strains will be further tested for their ability to modulate the immune response against BLG via mucosal delivery in a cows milk allergy model in mice.