Stéphane Hazebrouck

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.

Trypsin resistance of the major peanut allergen Ara h 6 and allergenicity of the digestion products are abolished after selective disruption of disulfide bonds

SCOPE 2S-albumins Ara h 2 and Ara h 6 are the most widely recognized and potent allergens for peanut-allergic patients. These allergens are particularly resistant to proteolysis and the digestion products generally retain significant allergenicity. Five disulfide bridges (DB) stabilize Ara h 6 overall structure and their influence on the trypsin resistance and on the allergenicity of the digestion products was investigated. METHODS AND RESULTS Progressive disruption of each DB was performed by site-directed mutagenesis. Successful refolding of Ara h 6 variants was confirmed by circular dichroism. Trypsin resistance, IgE-binding capacity and allergenic potency, as assessed by in vitro mediator release assay with sera from peanut-allergic patients, was not affected by the deletion of the C-terminal DB at Cys(84) -Cys(124) . Additional disruption of DB at Cys(14) -Cys(71) or at Cys(73) -Cys(115) rendered Arg(16/20) or Arg(114) susceptible to trypsinolysis, respectively, but affected principally the IgE-binding capacity of Ara h 6. DB disruption at Cys(26) -Cys(58) or at Cys(59) -Cys(107) led to an extensive proteolytic degradation and a complete loss of allergenic potency of the digestion products. CONCLUSION Selective disruption of the DB stabilizing the protease-resistant core of Ara h 6 eliminated the IgE-binding capacity of the trypsin-degradation products and their ability to trigger mast cell degranulation.

Allergic Sensitization to Bovine β-Lactoglobulin: Comparison between Germ-Free and Conventional BALB/c Mice

Background: The ‘hygiene hypothesis’ suggests that high hygienic standards met in western countries lead to a lack of microbial exposure, thus promoting the development of atopy by preventing the proper maturation of the immune system. Germ-free animals are deprived of the immune stimulation that occurs during postnatal gut colonization by commensal bacteria. Germ-free mice could thereby provide an attractive model for studying the impact of gut microbiota on the development of Th2-mediated disorders such as allergy. Methods: Germ-free and conventional BALB/c mice were sensitized to β-lactoglobulin (BLG), a major cow’s milk allergen, by means of intraperitoneal injections in the presence of incomplete Freund’s adjuvant. Time courses of serum and fecal BLG-specific antibody responses were monitored and cytokine production was assayed in BLG-reactivated splenocytes. Results: Serum BLG-specific IgG1 and IgE concentrations were significantly higher in germ-free mice during the primary immune response and IgE production persisted longer in germ-free mice. Furthermore, secretion of BLG-specific IgA was evidenced only in feces from germ-free mice while, in contrast, fecal IgG1 concentrations were at least 3-fold higher in conventional mice than in germ-free mice. Production of IL-5, IL-10 and IFN-γ was 3-fold enhanced in BLG-reactivated splenocytes from germ-free mice. Conclusion: The absence of gut microbiota significantly affects the BLG-specific immune response in BALB/c mice, thus suggesting that this model might be of interest for further studies exploring the influence of gut colonization by different bacterial strains on the development of an allergic-type sensitization.

Influence of the route of administration on immunomodulatory properties of bovine β-lactoglobulin-producing Lactobacillus casei.

Because of their intrinsic immunomodulatory properties, some lactic acid bacteria were reported to modulate allergic immune responses in mice and humans. We recently developed recombinant strains of Lactobacillus casei that produce beta-lactoglobulin (BLG), a major cows milk allergen. Here, we investigated immunomodulatory potency of intranasal and oral administrations of recombinant lactobacilli on a subsequent sensitization of mice to BLG. Intranasal administration of the BLG-producing Lb. casei stimulated serum BLG-specific IgG2a and IgG1 responses, and fecal IgA response as well, but did not inhibit BLG-specific IgE production. In contrast, oral administration led to a significant inhibition of BLG-specific IgE production while IgG1 and IgG2a responses were not stimulated. After both oral and intranasal administrations, production of IL-17 cytokine by BLG-reactivated splenocytes was similarly enhanced, thus confirming the adjuvant effect of the Lb. casei strain. However, a mixed Th1/Th2 cell response was evidenced in BLG-reactivated splenocytes from mice intranasally pretreated, with enhanced secretions of Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-4 and IL-5) whereas only production of Th1 cytokines, but not Th2 cytokines, was enhanced in BLG-reactivated splenocytes from mice orally pretreated. Our results show that the mode of administration of live bacteria may be critical for their immunomodulatory effects.

Goat's milk allergy without cow's milk allergy: suppression of non‐cross‐reactive epitopes on caprine β‐casein

Goats milk (GM) allergy associated with tolerance to cows milk (CM) has been reported in patients without history of CM allergy and in CM‐allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM‐allergic/CM‐tolerant patients recognize caprine β‐casein (βcap) without cross‐reacting with bovine β‐casein (βbov) despite a sequence identity of 91%. In this study, we investigated the non‐cross‐reactive IgE‐binding epitopes of βcap.

Allergenic and immunogenic potential of cow's milk β-lactoglobulin and caseins evidenced without adjuvant in germ-free mice

SCOPE In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ-free (GF) mice exhibit Th2-polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant. METHODS AND RESULTS β-lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward κ- and αS1-caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG-reactivation, IL-4, IL-5, IL-13 and IFN-γ productions by splenocytes were higher in GF mice than in CV mice. Heat-treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat-treatment increased protein immunogenicity and led to the production of anti-BLG and anti-κ-casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL-4, IL-5 and IL-13 in BLG-reactivated splenocytes from CV mice. CONCLUSION Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins.

Delayed bacterial colonization of the gut alters the host immune response to oral sensitization against cow's milk proteins

SCOPE Cows milk allergy is the most prevalent food allergy in infants whose immune system development is critically stimulated during postnatal gut colonization by commensal bacteria. Allergenic potential of cows milk β-lactoglobulin (BLG) and caseins (CAS) was investigated in germ-free (GF) BALB/c mice and in GF mice conventionalized (CVd) at 6 weeks of age. METHODS AND RESULTS Oral sensitization to cows milk in the presence of cholera toxin led to higher BLG-specific IgE, IgG1, and IgG2a responses in GF mice than in conventional (CV) mice. No significant difference was observed for CAS-specific IgE responses although IgG1 responses to αS1- and κ-caseins were higher in GF mice than in CV mice. CVd mice, orally inoculated with fecal preparations from CV mice, also displayed biased antibody responses compared to CV mice. Secretion of Th2 cytokines by BLG- and CAS-reactivated splenocytes of CVd mice was similar to that of GF mice whereas cytokine production by reactivated cells from mesenteric lymph nodes of CVd mice was equivalent to that of CV mice. CONCLUSION Oral sensitization to BLG and CAS was differentially affected by the absence of gut microbiota and delayed bacterial colonization altered persistently the host immune response to oral sensitization against food antigens.

Specificity of IgE antibodies from patients allergic to goat's milk and tolerant to cow's milk determined with plasmin-derived peptides of bovine and caprine β-caseins

SCOPE Despite a sequence homology of 90% between bovine and caprine β-caseins (CN), IgE antibodies from patients allergic to goats milk (GM), but tolerant to cows milk (CM), recognize caprine β-CN without cross-reacting with bovine β-CN. We investigated this lack of cross-reactivity by evaluating the IgE-reactivity toward peptides isolated from plasmin hydolysates of bovine and caprine β-CN. METHODS AND RESULTS The IgE-binding capacity of plasmin-derived peptides was evaluated with sera from 10 CM-allergic patients and 12 GM-allergic/CM-tolerant patients. In CM-allergic patients, IgE reactivity of caprine fragments (f29-107) and (f108-207), but not (f1-28), was similar to that of the bovine counterparts. In contrast, all bovine fragments were poorly recognized by IgE antibodies from GM-allergic/CM-tolerant patients. The peptide (f29-107) was generally the most immunoreactive fragment of caprine β-CN. By using synthetic peptides, the immunodominant IgE-binding epitope recognized by most GM-allergic/CM-tolerant patients was located in the caprine domain 49-79. CONCLUSION The restricted specificity of the IgE response toward the caprine β-CN in GM-allergic/CM-tolerant patients is mainly directed against the domain 49-79, which differs from its bovine counterpart by only three amino acid substitutions.

Mutational analysis of immunodominant epitopes of caprine β-casein recognized by IgE antibodies from patients allergic to goat’s milk and tolerant to cow’s milk

Background Several cases of allergy to goat’s milk (GM) without allergy to cow’s milk (CM) have been reported. GM-allergy has also been reported in CM-allergic children successfully treated with oral immunotherapy. We previously demonstrated that IgE antibodies from GM-allergic/CM-tolerant patients recognize the caprine b-casein (bcap) without cross-reacting with the bovine b-casein (bbov) despite a high sequence identity (91%). We aimed in the present work to identify the critical amino acids in the non-crossreactive IgE-binding epitopes of bcap. Methods Using site-directed mutagenesis, recombinant bcap was modified by performing residue substitutions with the corresponding amino acids found in bbov. The IgEbinding capacity of the different modified bcap was then evaluated with sera from 9 GM-allergic/CM-tolerant patients and 9 CM-allergic patients. The specificity of murine monoclonal antibodies (mAb) raised against caprine caseins was also analyzed in order to further characterize non-cross-reactive epitopes. The allergenic activity of recombinant bcap was finally assessed by degranulation tests of RBL cells passively sensitized with human IgE antibodies. Results The substitutions A55T/T63P/L75P in the N-terminal part and P148H/S152P in the C-terminal part of bcap induced the greatest decrease of IgE-reactivity of GMallergic/CM-tolerant patients toward the caprine allergen. The threonine 63 was found to be particularly critical, as confirmed by the specificity of mAb SCB1D, whose ability to bind bcap was abolished by the substitution T63P. The recombinant bcap containing the five substitutions was unable to induce the degranulation of RBL cells passively sensitized with IgE from GM-allergic/CM-tolerant patients but was still fully allergenic when testing sera from CM-allergic patients. Conclusion

Specific IgE responses in patients allergic to goat’s milk but tolerant to cow’s milk: involvement of minor differences in primary structure between caprine and bovine caseins

Methods b-Lactoglobulin, whole casein and its four different fractions, i.e. as1-, as2-, band -caseins, were isolated from raw CM and GM. Purified b-caseins were subjected to a mild proteolysis by plasmin which generated 3 peptides, i.e. f(1-28), f(29-107) and f(108-207/9). Synthetic peptides partially recovering the N-terminal f(29107) part of the caprine b-casein were also produced. Immunoreactivity of the purified proteins and peptides was assessed by IgE binding studies using sera from 12 GM-allergic patients tolerant to CM and 10 CM-allergic patients. The capacity of bovine and caprine milk proteins to induce the degranulation of humanized rat mast cells passively sensitized with human specific IgE was also evaluated.