Background: Cow’s milk allergy (CMA) is an important problem worldwide and the development of an in vivo system to study new immunotherapeutic strategies is of interest. Intolerance to soybean formula has been described in CMA patients, but it is not fully understood. In this work, we used a food allergy model in BALB/c mice to study the cross-reactivity between cow’s milk protein (CMP) and soy proteins (SP). Methods: Mice were orally sensitized with cholera toxin and CMP, and then challenged with CMP or SP to induce allergy. Elicited symptoms, plasma histamine, humoral and cellular immune response were analyzed. Th1- and Th2-associated cytokines and transcription factors were assessed at mucosal sites and in splenocytes. Cutaneous tests were also performed. Results: We found that the immediate symptoms elicited in CMP-sensitized mice orally challenged with SP were consistent with a plasma histamine increase. The serum levels of CMP-specific IgE and IgG1 antibodies were increased. These antibodies also recognized soy proteins. Splenocytes and mesenteric lymph node cells incubated with CMP or SP secreted IL-5 and IL-13. mRNA expression of Th2-associated genes (IL-5, IL-13, and GATA-3) was upregulated in mucosal samples. In addition, sensitized animals exhibited positive cutaneous tests after the injection of CMP or SP. Conclusions: We demonstrate that CMP-sensitized mice, without previous exposure to soy proteins, elicited hypersensitivity signs immediately after the oral administration of SP, suggesting that the immunochemical cross-reactivity might be clinically relevant. This model may provide an approach to further characterize cross-allergenicity phenomena and develop new immunotherapeutic treatments for allergic patients.
PurposeSoy-based formulas are widely used as dairy substitutes to treat milk allergy patients. However, reactions to soy have been reported in a small proportion of patients with IgE-mediated milk allergies. The aim of this work was to explore whether P34, a mayor soybean allergen, is involved in this cross-reactivity.MethodsIn vitro recognition of P34 was evaluated by immunoblotting, competitive ELISA and basophil activation tests (BAT) using sera from allergic patients. In vivo cross-reactivity was examined using an IgE-mediated milk allergy mouse model.ResultsP34 was recognized by IgE antibodies from the sera of milk allergic patients, casein-specific monoclonal antibodies, and sera from milk-allergic mice. Spleen cells from sensitized mice incubated with milk, soy or P34 secreted IL-5 and IL-13, while IFN-γ remained unchanged. In addition, the cutaneous test was positive with cow's milk proteins (CMP) and P34 in the milk allergy mouse model. Moreover, milk-sensitized mice developed immediate symptoms following sublingual exposure to P34.ConclusionsOur results demonstrate that P34 shares epitopes with bovine casein, which is responsible for inducing hypersensitivity symptoms in milk allergic mice. This is the first report of the in vivo cross-allergenicity of P34.
BackgroundCross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity.MethodsCow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy.ResultsBoth α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice.ConclusionsGly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.
Reactions to soy have been reported in a proportion of patients with IgE-mediated cow's milk allergy (CMA). In this work, we analyzed if Gly m Bd 28K/P28, one of the major soybean allergens, is a cross-reactive allergen with cow milk proteins (CMP). We showed that P28 was recognized by IgE sera from CMA patients and activated human peripheral basophils degranulation. Moreover, IgE sera of mice exclusively sensitized to CMP recognized P28. Splenocytes from sensitized animals secreted IL-5 and IL-13 when incubated with CMP or soy proteins, but only IL-13 when treated with P28. In addition, a skin test was strongly positive for CMP and weakly positive for P28. Remarkably, milk-sensitized mice showed hypersensitivity symptoms following sublingual challenge with P28 or CMP. With the use of bioinformatics' tools seven putative cross-reactive epitopes were identified. In conclusion, using in vitro and in vivo tests we demonstrated that P28 is a novel cross-reactive allergen with CMP.
reduction of cow's milk proteins using latex peptidases,
Exposure to cow's milk constitutes one of the most common causes of food allergy. In addition, exposure to soy proteins has become relevant in a restricted proportion of milk allergic pediatric patients treated with soy formulae as a dairy substitute, because of the cross-allergenicity described between soy and milk proteins. We have previously identified several cross-reactive allergens between milk and soy that may explain this intolerance. The purpose of the present work was to identify epitopes in the purified αS1-casein and the recombinant soy allergen Gly m 5.0101 (Gly m 5) using an α-casein-specific monoclonal antibody (1D5 mAb) through two different approaches for epitope mapping, to understand cross-reactivity between milk and soy. The 1D5 mAb was immobilized onto magnetic beads, incubated with the peptide mixture previously obtained by enzymatic digestion of the allergens, and the captured peptides were identified by MALDI-TOF MS analysis. On a second approach, the peptide mixture was resolved by RP-HPLC and immunodominant peptides were identified by dot blot with the mAb. Finally, recognized peptides were sequenced by MALDI-TOF MS. This novel MS based approach led us to identify and characterize four peptides on α-casein and three peptides on Gly m 5 with a common core motif. Information obtained from these cross-reactive epitopes allows us to gain valuable insight into the molecular mechanisms of cross-reactivity, to further develop new and more effective vaccines for food allergy.
Plants are economical and sustainable factories for the production of recombinant proteins. Currently, numerous proteins produced using different plant-based systems with applications as cosmetic and tissue culture ingredients, research and diagnostic reagents, and industrial enzymes are marketed worldwide. In this study, we aimed to demonstrate the usefulness of a plant-based system to synthesize a single-chain antibody (scFv)-elastin-like polypeptide (ELP) fusion to be applied as an affinity precipitation reagent of the difficult to produce recombinant proteins. We used the human tissue transglutaminase (TG2), the main celiac disease autoantigen, as a proof of concept. We cloned a TG2-specific scFv and fused it to a short hydrophobic ELP tag. The anti-TG2-scFv-ELP was produced in Nicotiana benthamiana and was efficiently recovered by an inverse transition cycling procedure improved by coaggregation with bacteria-made free ELP. Finally, the scFv-ELP was used to purify both plant-synthesized human TG2 and also Caco-2-TG2. In conclusion, this study showed for the first time the usefulness of a plant-based expression system to produce an antibody-ELP fusion designed for the purification of low-yield proteins. K E Y W O R D S affinity precipitation, downstream processing, elastin like polypeptides, molecular farming, single-chain variable antibody fragments 1 | INTRODUCTION Many recombinant proteins produced in plants are currently in the market, such as tissue culture or cosmetic ingredients, research or diagnostic reagents, and industrial enzymes. Less successful has been the production of biopharmaceuticals, with only one approved product and several vaccines in advanced stages of clinical trials (Fischer & Buyel, 2020). Plant-based platforms can compete across different markets due to specific advantages like inexpensive and massively scalable transgenic plant-based systems, or the rapid scaleup of transient leaf expression systems (Fischer & Buyel, 2020). The bottlenecks of molecular farming include the low product yield, the
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