CɛmX peptide-carrying HBcAg virus-like particles induced antibodies that down-regulate mIgE-B lymphocytes.

Type-I hypersensitivity reactions play a critical role in the pathogenesis of various allergic diseases. The successful development of the anti-IgE antibody, omalizumab, has validated IgE as an effective therapeutic target for the treatment of various IgE-mediated allergic diseases. Two research groups have reported that mAbs specific for certain parts of CɛmX, a domain of 52 aa residues in human membrane-bound IgE (mIgE), can cause the lysis of mIgE-B cells by apoptosis and antibody-dependent cellular cytotoxicity (ADCC). Herein, we explore virus-like particles formed by hepatitis B virus core antigen (HBcAg) that harbors the entire CɛmX peptide or its fragments as immunogens for inducing anti-CɛmX antibodies. The results showed that mice immunized subcutaneously with these immunogens produced antibodies that bind to recombinant CɛmX-containing human IgE.Fc in ELISA and Western blot analyses, and to genetically engineered human mIgE-expressing Ramos B cell line in fluorescence flow cytometric assays. The IgG antibodies purified from the sera of immunized mice were able to cause the apoptosis of mIgE-expressing Ramos cells through a BCR-dependent caspase pathway. Furthermore, the IgG could mediate ADCC in human mIgE-expressing A20 murine B-cell lymphoma. These studies suggest that HBcAg-CɛmX peptide immunogens warrant further investigation as a therapeutic modality for modulating IgE in patients with IgE-mediated allergic diseases.

Lipid rafts hinder binding of antibodies to the extracellular segment of the membrane-anchor peptide of mIgA.

Membrane-bound IgA (mIgA) is associated with Igα/Igß as the B cell receptor (BCR) complex on mIgA-expressing B cells. The α chain of mIgA (mα) contains a C-terminal membrane-anchor peptide, which encompasses extracellular, transmembrane and intracellular segments. The extracellular segment, referred to as the mIg isotype-specific (migis-α) segment or the extracellular membrane proximal domain of mα, has been proposed to be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. In this study, we developed several anti-migis-α monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-α. The mAbs bound strongly to synthetic peptides of migis-α and to various recombinant proteins containing migis-α as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-α region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-α-specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells.

Exploring the P2 and P3 ligand binding features for hepatitis C virus NS3 protease using some 3D QSAR techniques.

Several three-dimensional quantitative structure-activity relationship (3D-QSAR) models have been constructed using the comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and catalyst pharmacophore feature building programs for a series of 26 truncated ketoacid inhibitors designed particularly for exploring the P2 and P3 binding pockets of HCV NS3 protease. The structures of these inhibitors were built from a structure template extracted from the crystal structure of HCV NS3 protease. The structures were aligned through docking each inhibitor into the NS3 active site using program GOLD. The best CoMSIA model was identified from the stepwise analysis results and the corresponding pharmacophore features derived were used for constructing a pharmacophore hypothesis by the catalyst program. Pharmacophore features obtained by CoMFA and CoMSIA are found to be in accord with each other and are both mapped onto the molecular 5K surface of NS3 active site. These pharmacophore features were also compared with those obtained by the catalyst program and mapped onto the same NS3 molecular surface. The pharmacophore building process was also performed for 20 boronic acid based NS3 inhibitors characterized by a long hydrophobic side chain attached at position P2. This latter pharmacophore hypothesis built by the catalyst program was also mapped onto the molecular surface of NS3 active site to define a second hydrophobic feature at position P2. The possibility of using the pharmacophore features mapped P2 and P3 binding pocket to design more potent depeptidized NS3 inhibitors was discussed.