Epitope Mapping of the Outer Membrane Protein P5-Homologous Fimbrin Adhesin of Nontypeable Haemophilus influenzae

Abstract

To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae (NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8-to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8-to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.

Segregation of B and T Cell Epitopes of Treponema pallidum Repeat Protein K to Variable and Conserved Regions During Experimental Syphilis Infection

“…In this study the T cell epitopes of TprK recognized during infection were shown to be limited to regions that are highly conserved among T. pallidum subspecies pallidum isolates, while the B cell epitopes were focused primarily in the variable regions. Our results are consistent with a role for the variable regions in pathogenesis and a functional role for Abs to the variable regions, as seen in other bacterial infections (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

“…Analogies to other bacteria with molecules that have similar discrete variable regions suggest a possible surface exposure for the variable regions while the conserved regions are intramembrane domains. The discrete variable regions in neisserial Por and Opa, H. influenzae P2 and P5, C. trachomatis MOMP, and B. burgdorferi VlsE are all predicted to be surfaceexposed loops (15,21,22,(25)(26)(27). Under immunological pressure it is advantageous for antigenic targets not essential for structural integrity to be hypermutable.…”

“…Variable proteins from a number of bacteria, such as Chlamydia trachomatis major outer membrane protein (MOMP), nontypeable Hemophilus influenzae major outer membrane proteins P2 and P5, and Neisseria gonorrhoeae opacity proteins (Opa) and protein I (Por), have been shown to have critical functions in pathogenesis (such as pore forming or adhesion) and to be targets of variantspecific bactericidal, blocking, or opsonic Abs (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). In this study the T cell epitopes of TprK recognized during infection were shown to be limited to regions that are highly conserved among T. pallidum subspecies pallidum isolates, while the B cell epitopes were focused primarily in the variable regions.…”

Interaction between the P1 protein of Mycoplasma pneumoniae and receptors on HEp-2 cells

“…inhibited the adhesion of M. pneumoniae to host cells to varying degrees. A similar graduation of adhesion inhibition has been observed elsewhere in the investigation of the outer-membrane protein P5-homologous fimbrin adhesin protein from Haemophilus influenzae, in which both short and long peptides covering the protein show differing degrees of inhibition of bacterial adhesion (Novotny et al, 2000). The inhibition region, peptides 4-9, is flanked by the two membrane-associated regions, M4 and M5, in the extracellular D2 region (Fig.…”

Epitope-specific immune recognition of the nontypeableHaemophilus influenzaeouter membrane protein 26

“…Serum and OMP26 protein and peptide interactions were assessed by SPR using the Biacore 3000 instrument (GE Healthcare Life Sciences, USA) as previously described. 37 The Biacore system measures label-free binding of an analyte from a continuous flow to an immobilized ligand in real time. 38 Changes in refractive index due to variation of the mass on the sensor chip, which results from the interaction of the analyte and ligand, is expressed in relative Resonance Units (RU).…”