ImmunoMethods最新文献

Several bacterial proteins have been recently identified as superantigens which stimulate large numbers of T cells expressing particular T-cell receptor (TCR) Vβ elements. This suggests important roles for these proteins in the pathogenesis of bacterial infections. However, before the superantigenic property can be assigned to a particular protein, the purity needs to be carefully evaluated and several measures need to be taken to rule out the presence of minute amounts of contamination by other superantigens such as bacterial toxins. Here we describe a technique that can be used effectively to address this problem. This technique exploits the fact that superantigens have a signature Vβ specificity and, therefore, analysis by PCR of TCR Vβ gene expression which shows the Vβ expansion profile can be used to determine the superantigenicity of a protein and to detect cross-contamination by other superantigens. 1993 Academic

Due to the biologic implications for clinical infection, defining the Fab-binding specificities of certain bacterial Ig-binding proteins has been an area of considerable interest. To elucidate the structural and genetic correlates of the Fab-binding specificity of a prototypic Fab-binding bacterial protein, staphylococcal protein A (SpA), we have developed several new approaches. In earlier studies, we used a panel of peptide-induced serologic reagents to identify the V-region family usage in monoclonal Ig and then assessed their binding interaction with the Fab-specific binding site of SpA. To identify the conserved V-region sequences that correlate with SpA binding, the SpA-binding abilities of a group of purified, B-cell-line derived, monoclonal Ig of known sequence were also assessed. More recently, we have studied the binding of SpA with a combinatorial Ig expression library made with a phage surface-display vector. These studies have rigorously demonstrated that SpA binding is restricted to V_H3 Fab: The vast majority of V_H3 Fab bind SpA, and diverse V_H3 genes can encode for SpA binding. We then used the labeled Fab-specific SpA as a V_H3-specific phenotypic marker in multiparameter flow cytometric analyses to study human B-cell repertoire expression. These studies indicate that SpA possesses the Fab-binding specificity predicted for a B-cell superantigen, and we speculate that this type of unconventional antigen may have potent capabilities of influencing the formation of human immune repertoires.

Methods for analyzing the functional heterogeneity of type II IgG-binding proteins expressed by group A streptococci are described. Evidence for two major antigenic classes of type II IgG-binding proteins is presented. Heterogeneity in functional binding profiles was found to be associated with proteins belonging to either antigenic class. The antigenic class of IgG-binding protein was found to correlate with the antigenic class of M protein expressed by the same group A isolate. For certain group A isolates, the IgG-binding protein and M protein were shown to be a single molecule.

Bacterial Fc-binding proteins (FcBPs) such as staphylococcal protein A and streptococcal protein G possess IgG fine specificity strikingly similar to that of rheumatoid factors (RFs) derived from patients with rheumatoid arthritis (RA). They were shown to bind to the C_H2-C_H3 interface region of IgG. It has also been shown that peripheral blood lymphocytes can be selectively induced to produce RF by protein A. Several hypotheses, including idiotypic mimicry, have been proposed to explain the relationship of RF and bacterial FcBPs. Although convincing evidence for the involvement of bacterial infection in the etiology of RA has not been available, viral infection has frequently been strongly suspected as the agent possibly triggering RA. Herpes family viruses possess FcBPs reacting with the same C_H2-C_H3 interface region of IgG. Fab fragments of monoclonal antibodies (II-481, 88-S) to the IgG-bindlng site of glycoprotein E (gE), the FcBP glycoprotein of herpes simplex viruses, showed strong binding to RF. The epitope on gE reacting with mAb II-481 showed significant overlap with the IgG Fc-binding site. Antibodies to cytomegalovirus FcBP have been detected in a substantial proportion of sera from patients with RA. These observations may imply that some RFs may be produced as anti-idiotype antibodies to anti-viral FcBP antibodies. Thus, bacterial or viral FcBP could provide a link between RF production and a possible infectious etiology of RA.

Protein crystallography offers a powerful means of analyzing the molecular mechanisms that underlie the action of bacterial immunoglobulin-binding proteins. Successful approaches used to date involve the isolation of individual IgG-binding domains from the immunoglobulin-binding protein under study and the crystallization of these on their own or in complex with Fc or Fab fragments. Two structures of complexes that have been determined to high resolution by protein crystallography are compared. A single IgG-binding domain from protein A (from Staphylococcus) binds to a human Fc fragment through formation of two α-helices, which bind in the cleft between the CH2 and the CH3 domains. Recognition is mediated by side chains on protein A which interact with conserved side chains on the surface of the antibody, ensuring binding to IgG molecules from different subclasses and species. A similar analysis of the complex of a single IgG-binding domain from protein G (from Streptococcus) with an Fab fragment from mouse IgG1 reveals that the same problem in molecular recognition is tackled in a different way. Protein G binds via an antiparallel alignment of β-strands from the IgG-binding domain and the CH1 domain in Fab: this main chain-main chain interaction is supported by a number of specific hydrogen bonds between the side chains in both proteins. By recognition of a high proportion of main-chain atoms, protein G minimizes the effects of IgG sequence variability in a way that is distinct from that adopted by protein A.

This article describes expression systems based on staphylococcal protein A (SpA) and streptococcal protein G (SpG) which constitute attractive alternatives for the design and production of fusion proteins containing immunogenic structures. A dual expression system that allows the choice between two fusion partners, two synthetic IgG-binding domains (ZZ) of SpA and the serum albumin-binding region BB of SpG, was developed. Genes encoding antigens are expressed in Escherichia coli in parallel as fusions to ZZ and BB and the produced fusion proteins are affinity-purified on human IgG (ZZ fusions) or human serum albumin (BB fusions). The possibility of using ZZ fusions for immunization and the corresponding BB fusions for analysis of the induced immune responses provides a convenient strategy for the generation and analysis of immune responses to selected immunogenic structures. In addition, the cell surface-attaching regions of SpA have been utilized for cell surface display of heterologous antigens on the surface of the Gram-positive bacterium Staphylococcus xylosus. The dual expression system was used to express synthetic gene constructs and genomic gene fragments encoding immunogenic structures from blood-stage antigens of the malaria parasite Plasmodium falciparum. The fusion proteins produced were highly immunogenic in rabbits, mice, and monkeys and induced antibody and T-cell responses to the expressed antigens. Different applications of the SpA- and SpG-based expression systems are described and the immunological properties of the bacterial fusion partners SpA, ZZ, and BB are discussed.

Binding to β₁ chain integrin receptors can result in bacterial extracellular location or bacterial internalization In cultured cells. Using monoclonal antibodies (mAbs) to coat bacteria in vitro, we have shown that receptor-ligand affinity as well as receptor density determines the fate of the bacterium. In this report, we describe techniques used to coat the Gram-positive bacteria Staphylococcus aureus with anti-integrin mAbs and their application to the study of integrin-mediated bacterial internalization. The affinity of the purified anti-integrin mAb is estimated by determination of the ED₅₀ of the mAb by using cell monolayers, and the efficiency of mAb-promoted bacterial internalization is determined by a gentamicin survival assay. The efficiency of bacterial internalization promoted by various anti-β₁ chain integrin mAbs reflects differential expression of β₁ chain integrin receptors in various cell lines studied. Analysis of β₁ chain integrin receptor expression in HEp-2 cells transfectants shows that the level of bacterial internalization is directly related to the integrin receptor density.

A majority of group A streptococci (GAS) express immunoglobulin (Ig)-binding proteins. The genes encoding these proteins belong to either the emm or the emm-related (fcrA and enn) gene family. The present study presents oligonucleotide primers and protocols for the specific PCR-mediated amplification of fcrA and enn genes with and without their own promoters and transcription termination sites. The PCR system appears to be applicable to virtually every GAS serotype. Using these assays, some sequence variability in the 5′ untranslated region of fcrA and at the 5′ end of enn was demonstrated for a minority of GAS serotypes. The fcrA and enn genes of GAS serotype M49 were amplified by PCR, cloned into pJLA602, and expressed in Escherichia coli. The fcrA gene of the GAS serotype M49 strain CS101, including its adjacent regulatory sequences, was sequenced using a universal primer set by means of which emm-related genes can be completely sequenced with only five sequencing reactions. The fcrA49 sequence provides further evidence that fcrA genes are a separate entity from emm-related genes and exhibit a variability at their 5′ ends similar to that of proteins encoded by emm genes.

Antisense oligodeoxynucleotides (oligos or oligonucleotides) can be useful agents for studying the role of immunologically relevant genes. Dozens of laboratories have reported antisense inhibition of a wide variety of cytokines, growth factors, proto-oncogenes, and other targets of interest to immunologists. In addition to the potential applications for antisense oligos as research tools in immunologic studies, there is increasing interest in their possible therapeutic applications. Advances in the molecular biology of immunity and autoimmunity are leading to the identification of genes whose abnormal expression could contribute to disease pathogenesis. Antisense technology may provide therapies precisely targeted to the molecular genetic inducers of human disease. Despite this bright promise, some genes or experimental systems may not be amenable to the antisense approach. Potential explanations for failed antisense experiments include such factors as nonsequence-specific effects, oligonucleotide degradation, insufficient oligonucleotide uptake, inaccessibility of the RNA target region to the oligonucleotide, and a long half-life of the target protein. This article focuses on the practical factors that may be important in optimizing antisense efficacy.