IgE cross-inhibition between Ara h 1 and Ara h 2 is explained by complex formation of both major peanut allergens.

BACKGROUND: Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities. OBJECTIVE: We investigated the unexpected cross-reactivity between peanut major allergens. METHODS: Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n = 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes. RESULTS: Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions. CONCLUSION: True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.

Chemically modified peanut extract shows increased safety while maintaining immunogenicity.

BACKGROUND: Peanuts are most responsible for food-induced anaphylaxis in adults in developed countries. An effective and safe immunotherapy is urgently needed. The aim of this study was to investigate the immunogenicity, allergenicity, and immunotherapeutic efficacy of a well-characterized chemically modified peanut extract (MPE) adsorbed to Al(OH)3 . METHODS: Peanut extract (PE) was modified by reduction and alkylation. Using sera of peanut-allergic patients, competitive IgE-binding assays and mediator release assays were performed. The immunogenicity of MPE was evaluated by measuring activation of human PE-specific T-cell lines and the induction of PE-specific IgG in mice. The safety and efficacy of MPE adsorbed to Al(OH)3 was tested in two mouse models by measuring allergic manifestations upon peanut challenge in peanut-allergic mice. RESULTS: Compared to PE, the IgE-binding and capacity to induce allergic symptoms of MPE were lower in all patients. PE and MPE displayed similar immunogenicity in vivo and in vitro. In mice sensitized to PE, the threshold for anaphylaxis (drop in BT) upon subcutaneous challenge with PE was 0.01 mg, while at 0.3 mg MPE no allergic reaction occurred. Anaphylaxis was not observed when PE and MPE were fully adsorbed to Al(OH)3 . Both PE and MPE + Al(OH)3 showed to be efficacious in a model for immunotherapy. CONCLUSION: In our studies, an Al(OH)3 adsorbed MPE showed reduced allergenicity compared to unmodified PE, while the efficacy of immunotherapy is maintained. The preclinical data presented in this study supports further development of modified peanut allergens for IT.

Cryo electron microscopy reconstructions of the Leviviridae unveil the densest icosahedral RNA packing possible.

We solved the structures of the single-stranded RNA bacteriophages Qbeta, PP7 and AP205 by cryo-electron microscopy. On the outside, the symmetrized electron density maps resemble the previously described cryo-electron microscopy structure of MS2. RNA density is present inside the capsids, suggesting that the genomic RNA of Qbeta, PP7 and AP205, analogous to MS2, contains many coat protein-binding sites in addition to the hairpin on which assembly and packaging are initiated. All four bacteriophages harbour the same overall arrangement of the RNA, which is a unique combination of both triangles and pentagons. This combination has not been found in other icosahedral viruses, in which the RNA structures are either triangular or pentagonal. Strikingly, the unique RNA packing of the Leviviridae appears to deploy the most efficient method of RNA storage by obeying icosahedral symmetry.