The Variable-Region Specificity of Bacterial Fab-Binding Proteins: The Search for B-Cell Superantigens

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.