The forgotten 2S albumin proteins: Importance, structure, and biotechnological application in agriculture and human health.

2S albumin proteins are a group of important seed storage proteins (SSPs) essential to seeds at early and late developmental stages, by providing amino acids and other nutrients during germination and for seed defense. 2S albumins possess a well-conserved cysteine supporting the stability of temperature, pH, and proteolysis. The 3D structure rich in alpha-helices and positively charged is particularly suited for antibacterial and antifungal activity, which is presented by many 2S albumins. However, the hypervariable region present in 2S albumins induces allergenic reactions. Because of that, 2S albumins have never been recognized for their biotechnological potential. However, the development of servers used for the rational design of antimicrobial molecules has now brought a new application to 2S albumins, acting as a model to design antimicrobial molecules without the toxic or allergenic effects of 2S albumins. Therefore, this review is focused on discussing the importance of 2S albumins to seed development and defense and the biochemical, structural and functional properties of these proteins thought to play a role in their antimicrobial activity. Additionally, the application of 2S albumins to design synthetic antimicrobial peptides is discussed, potentially bringing new functions to these forgotten proteins.

A phase 1 study of heat/phenol-killed, E. coli-encapsulated, recombinant modified peanut proteins Ara h 1, Ara h 2, and Ara h 3 (EMP-123) for the treatment of peanut allergy.

BACKGROUND: Immunotherapy for peanut allergy may be limited by the risk of adverse reactions. OBJECTIVE: To investigate the safety and immunologic effects of a vaccine containing modified peanut proteins. METHODS: This was a phase 1 trial of EMP-123, a rectally administered suspension of recombinant Ara h 1, Ara h 2, and Ara h 3, modified by amino acid substitutions at major IgE-binding epitopes, encapsulated in heat/phenol-killed E. coli. Five healthy adults were treated with 4 weekly escalating doses after which 10 peanut-allergic adults received weekly dose escalations over 10 weeks from 10 mcg to 3063 mcg, followed by three biweekly doses of 3063 mcg. RESULTS: There were no significant adverse effects in the healthy volunteers. Of the 10 peanut-allergic subjects [4 with intermittent asthma, median peanut IgE 33.3 kUA /l (7.2-120.2), and median peanut skin prick test wheal 11.3 mm (6.5-18)]; four experienced no symptoms; one had mild rectal symptoms; and the remaining five experienced adverse reactions preventing completion of dosing. Two were categorized as mild, but the remaining three were more severe, including one moderate reaction and two anaphylactic reactions. Baseline peanut IgE was significantly higher in the five reactive subjects (median 82.4 vs 17.2 kUA /l, P = 0.032), as was baseline anti-Ara h 2 IgE (43.3 versus 8.3, P = 0.036). Peanut skin test titration and basophil activation (at a single dilution) were significantly reduced after treatment, but no significant changes were detected for total IgE, peanut IgE, or peanut IgG4. CONCLUSIONS: Rectal administration of EMP-123 resulted in frequent adverse reactions, including severe allergic reactions in 20%.

An unfolded variant of the major peanut allergen Ara h 2 with decreased anaphylactic potential.

BACKGROUND: Peanut allergy causes severe type 1 hypersensitivity reactions and conventional immunotherapy against peanut allergy is associated with a high risk of anaphylaxis. OBJECTIVE: Our current study reports proof of concept experiments on the safety of a stably denatured variant of the major peanut allergen Ara h 2 for immunotherapy. We determined the impact of structure loss of Ara h 2 on its IgE binding and basophil degranulation capacity, T cell reactivity as well as anaphylactic potential. METHODS: The secondary structure of untreated and reduced/alkylated Ara h 2 variants was determined by circular dichroism spectroscopy. We addressed human patient IgE binding to Ara h 2 by ELISA and Western blot experiments. RBL-SX38 cells were used to test the degranulation induced by untreated and reduced/alkylated Ara h 2. We assessed the anaphylactic potential of Ara h 2 variants by challenge of sensitized BALB/c mice. T cell reactivity was investigated using human Ara h 2-specific T cell lines and splenocytes isolated from sensitized mice. RESULTS: Reduction/alkylation of Ara h 2 caused a decrease in IgE binding capacity, basophil degranulation and anaphylactic potential in vivo. However, the human T cell response to reduced/alkylated and untreated Ara h 2 was comparable. Mouse splenocytes showed higher metabolic activity upon stimulation with reduced/alkylated Ara h 2 and released similar IL-4, IL-13 and IFNγ levels upon treatment with either Ara h 2 variant. CONCLUSIONS AND CLINICAL RELEVANCE: Reduced/alkylated Ara h 2 might be a safer alternative than native Ara h 2 for immunotherapeutic treatment of peanut allergic patients.

Ara h 2 peptides containing dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic.

BACKGROUND: Peanut allergy is a life-threatening condition; there is currently no cure. Although whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy. OBJECTIVE: This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4(+) T cells but do not bind IgE as candidates for safe peanut-specific immunotherapy. METHODS: Multiple CD4(+) T-cell lines specific for the major peanut allergen Ara h 2 were generated from PBMCs of 16 HLA-diverse subjects with peanut allergy by using 5,6-carboxyfluorescein diacetate succinimidylester-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA restriction of core epitope recognition was investigated by using anti-HLA blocking antibodies and HLA genotyping. Serum-IgE peptide-binding was assessed by dot-blot. RESULTS: Five dominant CD4(+) T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, HLA-DP, and HLA-DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants containing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE, and could directly target T(H)2-type T cells in peripheral blood of subjects with allergy. CONCLUSION: Short CD4(+) T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell-targeted peanut-specific immunotherapy for an HLA-diverse population.