Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides

The major pore-forming outer membrane proteins (Omps) of gram-negative bact eria demonstrate numerous immunomodulating properties and are involved in t he virulence of pathogenic strains. Because Escherichia coli OmpF is the be st-characterized porin in terms of structural and functional characteristic s, in vitro B-cell and T-cell responses to this porin in six different stra ins of mice were analyzed. Mice were immunized with purified OmpF trimers o r overlapping synthetic polypeptides (20-mers) spanning the entire 340-amin o-acid sequence of the OmpF monomer. T-cell proliferative responses and imm unoglobulin G antibody responses to native OmpF and the peptide analogues w ere determined. For each strain, patterns of T-cell proliferation were simi lar regardless of whether native OmpF or synthetic peptides were inoculated , although all strains recognized one or more cryptic determinants. Mice ex hibited several haplotype-specific responses, but genetically permissive ep itopes were also identified. Four peptides (75-94, 265-284, 295-314, and 30 5-324) elicited strong T-cell proliferative responses from all strains of m ice when mice were presensitized with native OmpF or a homologous peptide. In general, 10 or fewer peptides were recognized by sera from mice immunize d with native OmpF or synthetic peptides, and most sera from peptide-immuni zed mice reacted poorly with the native protein. Four peptides spanning ami no acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from pept ide-immunized mice, Peptides 245-264 and 305-324 were universally recognize d by sera from peptide-immunized mice, but these sera reacted weakly or wer e negative when tested against the native protein. Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response. T-cell and B -cell responses have been investigated based on the assumption that differe nces in epitope specificity could influence protective or pathologic host r eactions. Because of the high level of structural homology of OmpF to porin s isolated from other enteric pathogens, the identification of T- and B-cel l-stimulatory determinants of E. coli OmpF may have broader application.