Selection of cryptic B-cell epitopes using informational analysis of protein sequences.

Sub-unit vaccines are synthetic or recombinant peptides representing T- or B-cell epitopes of major protein antigens from a particular pathogen. Epitope selection requires the synthesis of peptides that overlap the protein sequences and screening for the most effective ones. In this study a new method of immunogenic peptide selection based on the analysis of information structure of protein sequences is suggested. The analysis of known B-cell epitope location in the information structure of Aspergillus fumigatus proteins Asp f 2 and Asp f 3 has shown that epitopes are scattered along the sequences of proteins for the exception of sites with Increased Degree Information Coordination (IDIC). Based on these results peptides from different allergens such as Asp f 2, Der p 1, and Fel d 1 were selected and produced in a recombinant form in the context of yeast virus-like particles (VLPs). Immunization of mice with VLPs containing peptides form allergens has induced the production of IgG able to recognize full-length antigens. This result suggests that the analysis of information structure of proteins can be used for the selection of peptides possessing cryptic B-cell epitope activity.

High-affinity IgG to a major A. fumigatus allergen, Asp f 2, retards allergic response.

BACKGROUND: Allergic diseases represent a major health threat to humans. Allergen-specific immunotherapy (SIT) is one of the significant approaches to the treatment of IgE-mediated allergy and its control. The mechanisms involved in SIT-induced responses are complex and still speculative. Immunological events associated with successful SIT include an increase in allergen-specific "blocking" IgG, reduction in cytokine production, and induction of regulatory or suppressor cells. The aim of this study was to estimate the effect of SIT using a single major allergen of A. fumigatus, Asp f 2, or its dominant B-cell epitope, aa254-268, in a murine model of allergic aspergillosis. It is known that A. fumigatus (Af), a ubiquitous fungus, is implicated in the pathogenesis of a number of clinically different allergic diseases. MATERIAL/METHODS: BALB/c and C57BL/6 mice were immunized with Asp f 2, its proteolytic fragments or the recombinant peptide aa254-268 to induce high-affinity IgG to Asp f 2. Allergy to Af was induced by subcutaneous and intranasal immunization of previously SIT-treated animals with an Af crude extract. RESULTS: The results of immunological and lung histological studies demonstrate a simultaneous increase in Asp f 2-specific IgG and amelioration of allergic inflammatory symptoms in mice immunized with Asp f 2 or its peptides before exposure to Af crude allergen. CONCLUSIONS: Thus it was shown that the induction of IgG specific to major allergens or even to their B-cell epitopes induces protection from allergy provoked by natural allergens.

Reactivity of the hydrido/nitrosyl radical MHCl(NO)(CO)(P(i)Pr(3))(2), M = Ru, Os.

The reaction of equimolar NO with the 16 electron molecule RuHCl(CO)L(2) (L = P(i)Pr(3)) proceeds, via a radical adduct RuHCl(CO)(NO) L(2), onward to form RuCl(NO)(CO)L(2) (X-ray structure determination) and RuHCl(HNO)(CO)L(2), in a 1:1 mole ratio. The HNO ligand, bound by N and trans to hydride, is rapidly degraded by excess NO. The osmium complex behaves analogously, but the adduct has a higher formation constant, permitting determination of its IR spectrum; both MHCl(CO)(NO)L(2) radicals are characterized by EPR spectroscopy, and DFT calculations on the Ru system show it to have a "half-bent" Ru-N-O unit with the spin density mainly on nitrogen. DFT (PBE) energies rule out certain possible mechanistic steps for forming the two products. A survey of the literature leads to the hypothesis that NO should generally be considered as a (neutral) Lewis base (2-electron donor) when it binds to a 16 electron complex which is resistant to oxidation or reduction, and that the resulting N-centered radical has a M-N-O angle of approximately 140 degrees, which distinguishes it from NO(-) (bent at <140 degrees ) and from NO(+) (>170 degrees ).

An electron-excessive nitrosyl complex: reactivity of a ligand-centered radical leading to coordinated HNO.

The reaction of RuHCl(CO)L(2) (L = P(i)Pr(3)) with NO initially forms a 1:1 adduct, shown by DFT calculations and EPR spectroscopy (including the RuD isotopomer) to contain a bent ( 90 degree angle Ru-N-O = 143.9 degrees ) nitrosyl where the majority of the spin density is on the nitrosyl nitrogen. This radical adduct transforms further to give equimolar RuCl(NO)(CO)L(2) and RuHCl(HNO)(CO)L(2), the latter with hydride trans to the nitroxyl ligand HN=O. This is the first observation of the synthesis of coordinated HNO from NO itself. DFT calculations lead to the proposal that this H-atom transfer is effected by free NO, and the lifetime of RuHCl(HNO)(CO)L(2) is indeed qualitatively dependent on the presence of free NO.