A P5 peptide that is homologous to peptide 10 of OprF from Pseudomonas aeruginosa enhances clearance of nontypeable Haemophilus influenzae from acutely infected rat lung in the absence of detectable peptide-specific antibody

Abstract

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with otitis media and the exacerbation of chronic bronchitis. This study reports the vaccine potential of three peptides representing conserved regions of the NTHi P5 outer membrane protein which have been fused to a promiscuous measles virus F protein T-cell eptitope (MVF). The peptides correspond to a region in surface loop one (MVF/L1A), the central region of loop four (MVF/L4), and a C-terminal region homologous to peptide 10 of OprF from Pseudomonas aeruginosa (MVF/H3). Immunization of rats with MVF/H3 was the most efficacious in significantly reducing the number of viable NTHi in both the broncho-alveolar lavage fluid (74%) and lung homogenates (70%), compared to control rats. Importantly, despite significantly increased rates of clearance, immunization with MVF/H3 elicited poor antibody responses, suggesting that cell-mediated rather than humoral responses play an important role in the enhanced clearance of NTHi in this model.

FIG. 1

Sequences of the chimeric P5 peptides. Peptides were composed of MVF, a linker composed of a four-amino-acid β-turn and amino acids corresponding to regions in P5. Numbering above the P5 sequences refers to the amino acid number in P5, and the line below MVF/H3 indicates the intrachain disulfide bridge. The N-terminal cysteine of the peptides was used for coupling to a polylysine core to form multiple antigenic peptides.

FIG. 2

Alignment of the P5 H3 peptide from NTHi isolate UC19 with OprF peptide 10 from P. aeruginosa and the corresponding region in OmpA from E. coli. In order to identify the peptide 10-homologous region in P5, the sequences of P5 and OprF, together with OmpA, were aligned with the PileUp program (Genetics Computer Group, University of Wisconsin, accessed through the Australian National Genomic Information Service). Gaps in the sequence, to create the alignment, are indicated with dots, and the numbers refer to the amino acid number in the preprotein.

FIG. 3

Clearance of NTHi strain UC19 from BAL fluid (A) and homogenized lung (B) following challenge of animals immunized with the P5 chimeric peptides. Significant clearance was seen in the BAL fluid from MVF/H3-immunized rats and in the lung homogenates from MVF/L1A- and MVF/H3-immunized rats. ∗, P < 0.05; ▾, P < 0.005. The mean CFU recovered from the nonimmune group was given the value of 0% clearance. The percent clearance in the peptide-immunized groups was calculated as 100 minus the percent ratio of the mean CFU recovered from the immunized groups divided by the mean of the nonimmune group. Each group consisted of four to five animals, except for the control group, which consisted of eight animals. The error bars represent the standard errors of the mean expressed as percentages.

FIG. 4

Comparison of the clearance in P5 peptide-specific antibody-responding and -nonresponding rats. The percent clearance was calculated for individual animals as described in the legend for Fig. 3. The mean of the nonimmune group was given the value of 0% clearance. The animals with peptide-specific serum IgG are indicated with stars, and the means of each group are indicated by bars. Notably, the animal producing MVF/L1A-specific antibody had more viable NTHi in the BAL fluid than the nonimmune animals.