Fine mapping of outer membrane protein P2 antigenic sites which vary during persistent infection by Haemophilus influenzae

Abstract

Antigenic drift of the major outer membrane protein (MOMP) P2 of nonencapsulated Haemophilus influenzae as observed during persistent infections in patients with chronic bronchitis was mimicked in a rabbit model in which H. influenzae persisted in subcutaneous cages. The antigenic drift resulted from amino acid substitutions in potentially surface-exposed loops of MOMP P2. Since in a rabbit model the appearance of antigenic variants was associated with the presence of strain-specific bactericidal antibodies (L. Vogel, B. Duim, F. Geluk, P. Eijk, H. Jansen, J. Dankert, and L. van Alphen, Infect. Immun. 64:980-986, 1996), we determined the epitope specificities of these bactericidal antibodies. The eight loops of MOMP P2 of H. influenzae d1 were separately expressed as fusion proteins with glutathione S-transferase. Sera of rabbits persistently infected with H. influenzae reacted with the loop 5 and loop 6 fusion proteins in immunoblotting and enzyme-linked immunosorbent assay. For fine mapping of the epitopes with pepscan analysis, overlapping synthetic peptides consisting of 12 amino acids were made. Rabbit sera contained antibodies reacting with peptides derived from loop 5 and peptides containing amino acids of the side of loop 6. In addition, MOMP P2 variant-specific reactions with the amino acids located at the tip of loop 6 were detected. The rabbit sera showed variantspecific complement-dependent bactericidal activities, which were eliminated by affinity chromatography with fusion proteins of loop 6 but not of loop 5. We conclude that, during persistence of H. influenzae in rabbits, variant-specific bactericidal antibodies are elicited to the variable tip of MOMP P2 loop 6.

Molecular determinants of the pathogenesis of disease due to non-typableHaemophilus influenzae

“…Survival of the population is accomplished by frequent non‐synonymous nucleotide changes within the region encoding the surface‐exposed loop 6. Following the alteration in loop 6 structure, the original antibodies are no longer bactericidal [193]. Interestingly, mutations in this region occur at a significantly higher rate than would be expected by random occurrence.…”

“…Further analysis has revealed that variation in P2 sequence also occurs within a clonal population during the course of persistent infection. Both in individual children with chronic otitis media and adults with chronic bronchitis due to non‐typable H. influenzae , specific regions of the P2 gene vary at a relatively high frequency [193–195]. These changes correspond to a single surface‐exposed loop referred to as loop 6, which is also the major epitope against which antibodies are targeted.…”

Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae

“…Survival of the population is accomplished by frequent non-synonymous nucleotide changes within the region encoding the surface-exposed loop 6. Following the alteration in loop 6 structure, the original antibodies are no longer bactericidal [193]. Interestingly, mutations in this region occur at a signi¢cantly higher rate than would be expected by random occurrence.…”

The identification and characterization of P5 and P2 colonization proteins in Haemophilus parasuis and the targeted binding of carcinoembryonic antigen (CEA)

“…The P2 protein is a porin and is strongly immunogenic ). Surface exposed regions of P2 vary greatly in sequence and this antigenic drift allows the organism to evade immune clearance contributing to the development of chronic infection Duim et al, 1996;. Synthetic peptides of these surface exposed regions have also been shown to activate the mitogen-activated protein kinase (MAPK) cascade .…”

“…The changes are linked to the surfaced-exposed loops of the porin protein and are primarily found in loops 5 and 6. These loops appear at the carboxy terminal end of the protein and the changes have been shown to render the original host antibodies ineffective (Duim et al, 1996). Smith Vaughn et al…”