Studying interactions involving the T-cell antigen receptor by surface plasmon resonance.

T-lymphocyte activation is initiated by the interaction of the alpha beta TCR with a complex consisting of a class I or class II MHC-encoded molecule and an antigenic peptide, displayed on the surface of an antigen-presenting cell. Real-time binding measurements using surface plasmon resonance have revealed kinetic and equilibrium parameters for the interactions between purified MHC molecules and peptides, between TCR and MHC-peptide complexes, and between TRC and superantigens. The MHC-peptide interaction is characterized by its high affinity and long half-life, the TCR-MHC/peptide interaction by its low affinity and short half-life, and the TCR-superantigen interaction by its low-to-moderate affinity, which is dependent on the particular superantigen involved. The consistent finding is that both MHC-peptide complexes and superantigens interact with TCR with a low affinity attributable to rapid dissociation. That an MHC-peptide complex that encounters a single TCR only briefly can still deliver the necessary activation signals offers a mechanistic conundrum for which several solutions have been proposed.

Measuring interactions of MHC class I molecules using surface plasmon resonance.

To examine the molecular interactions between major histocompatibility complex (MHC)-encoded molecules and peptides, monoclonal antibodies (mAbs), or T cell receptors, we have developed model systems employing genetically engineered soluble MHC class I molecules (MHC-I), synthetic peptides, purified mAbs, and engineered solubilizable T cell receptors. Direct binding assays based on immobilization of one of the interacting components to the dextran modified gold biosensor surface of a surface plasmon resonance (SPR) detector have been developed for each of these systems. The peptide binding site of the MHC-I molecule can be sterically mapped by evaluation of a set of peptides immobilized through the thiol group of cysteine substitutions at each peptide position. Kinetic binding studies indicate that the MHC-I/peptide interaction is characterized by a low to moderate apparent kass (approximately 5000-60000 M-1 s-1) and very small kdis (approximately 10(-4)-10(-6) s-1) consistent with the biological requirement for a long cell surface residence time to permit engagement with T cell receptors. Several mAb directed against different MHC-I epitopes were examined, and kinetic parameters of their interaction with MHC molecules were determined. These showed characteristic moderate association rate constants and moderate dissociation rate constants (kass approximately 10(4)-10(6) M-1 s-1 and kdis approximately 10(-2)-10(-4) s-1), characteristic of many antibody/protein antigen interactions. The interaction of an anti-idiotypic anti-TCR mAb with its purified cognate TCR was of moderate affinity and revealed kinetic binding similar to that of the anti-MHC mAbs. The previously determined interaction of a purified T cell receptor with its MHC-I/peptide ligand is characterized by kinetic constants more similar to those of the antibody/antigen interaction than of the MHC-I/peptide interaction, but is remarkable for rapid dissociation rates (apparent kdis approximately 10(-2) s-1). Such binding studies of reactions involving the MHC-I molecules offer insight into the mechanisms responsible for the initial specific events required for the stimulation of T cells.

Direct detection of major histocompatibility complex class I binding to antigenic peptides using surface plasmon resonance. Peptide immobilization and characterization of binding specificity.

We have developed model systems in which the binding of purified, genetically engineered, soluble analogues of major histocompatibility complex (MHC) class I molecules to immobilized antigenic peptides can be monitored in real time using surface plasmon resonance (SPR). Synthetic analogues of several peptides known to bind different mouse and human MHC class I molecules were prepared with cysteine residues substituted at appropriate positions. The analogue peptides were immobilized via the bifunctional reagent N-gamma-maleimidobutyryloxy-succinimide to amino groups generated on the dextran-modified gold surface of a biosensor flow cell. Using this approach, each position in the sequence of an H-2Ld-specific viral peptide, pMCMV (YPHFMPTNL), was used for coupling, and the resulting surfaces were tested for binding of the soluble analogue of H-2Ld, H-2Lds. In accord with our previously described H-2Ld/pMCMV three-dimensional structural model, only those residues of the peptide that remain exposed following binding (positions 4-8) can be replaced by cysteine and used for coupling. Stable binding of soluble MHC class I molecules, H-2Lds, H-2Dds, H-2Kbs, and HLA-A2s to their respective immobilized cognate peptides was detected by SPR. Specificity of the peptide/MHC interaction was characterized both by direct binding using immobilized peptides and by competition with peptides in solution, and in general was consistent with known immunological reactivity. Some peptides bound not only their cognate MHC molecule, but others at lower apparent affinity. Measurement of real time binding of MHC class I molecules to peptides immobilized through specific side chains suggests the application of a similar approach to the study of the interaction of peptides with a wide variety of peptide-binding macromolecules.

MHC class I/peptide interactions: binding specificity and kinetics.

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class I molecules as well as in the application of real time biosensor technology have permitted the direct analysis of the binding of MHC class I molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as 'pepsicles', as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class I peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Detailed analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides reveals that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kd values for antigenic peptide/class I interactions at 25 degrees C are in the range of ca 10(-4)-10(-6)/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.

Comparison of adenoviral hexon polypeptides (monomers) and of native hexons (trimers) by SDS-polyacrylamide gel electrophoresis.

Purified hexons of 27 serotypes of human, simian, bovine and avian adenoviruses were analysed by SDS-PAGE. The apparent molecular weights of hexon polypeptides calculated by comparison with 5 non-hexon and 3 sequenced hexon polypeptide markers ranged from 98 kDa (for bovine adenovirus Ad bos7) to 118 kDa (for simian adenovirus Ad sim13; SV36). A stability of native hexon capsomers (trimers) in SDS at room temperature permitted us to resolve native (trimeric) hexon by SDS-PAGE and to distinguish them from denatured (monomeric) hexon polypeptides by electrophoretic mobilities. Hexon trimer bands with slow mobility in SDS-PAGE (unlike hexon monomer polypeptide bands) retained native hexon antigenicity as revealed by immunoblot analyses. Possible applications of simultaneous analyses of hexon trimers and monomers by SDS-PAGE are discussed.

Characterization of a series of monoclonal antibodies specific for bovine adenovirus serotype BAV3 hexon antigen and their use in an ELISA for detection of adenoviral hexons.

After immunization of mice with purified hexon (the main capsid antigen) of bovine adenovirus serotype BAV3 we have obtained a set of 16 individual hybridoma clones producing MAb's against BAV3 hexon. All MAb's were shown to belong to immunoglobulin G class. Specificity of the most avid MAb marked B3Hx-1 was tested on a panel of representative hexon antigens from 16 adenovirus serotypes of human and animal origin using several immunoassays. In Western blot analysis the MAb B3Hx-1 reacted only with native (trimeric) form of hexon protein and not with denaturated hexon polypeptide chains. The epitope defined by B3Hx-1 appeared stable against SDS at ambient temperature and against chloramine-promoted iodination. The specificity of the epitope was characterized as almost genus-crossreactive: it was absent from hexons of avian and of bovine subgroup 2 adenovirus serotypes and present in most hexons of bovine, canine, simian and human adenoviruses tested. Within the latter group its expression was weak or absent only for human subgenus C serotypes. Several variants of sandwich-type ELISA were developed using MAb B3Hx-1 and different polyclonal antibodies against hexons of mammalian adenoviruses. The level of hexon detection for different adenovirus serotypes varied in range 10(-9) to 10(-8) g per ml.

Study of avian adenovirus DNA infectivity in chick embryos.

Inoculation of CELO adenovirus deproteinized DNA into the allantoic cavity of 9-day-old chick embryos (CE) induced the synthesis of infectious viral particles. The produced virions appeared to be identical with CELO adenovirions in terms of morphology, electrophoretic and immunochemical properties of hexon major capsid protein and also of DNA dot-hybridization. High infectivity of CELO DNA (minimal infective dose equaled 40 ng) may be also related, at least in part, to the absence of deoxyribonuclease activity in the allantoic fluid (AF).