Antisera Against Certain Conserved Surface-Exposed Peptides of Nontypeable Haemophilus influenzae Are Protective

Abstract

Nontypeable Haemophilus influenzae (NTHi) cause significant disease, including otitis media in children, exacerbations of chronic obstructive pulmonary disease, and invasive disease in susceptible populations. No vaccine is currently available to prevent NTHi disease. The interactions of NTHi and the human host are primarily mediated by lipooligosaccharide and a complex array of surface-exposed proteins (SEPs) that act as receptors, sensors and secretion systems. We hypothesized that certain SEPs are present in all NTHi strains and that a subset of these may be antibody accessible and represent protective epitopes. Initially we used 15 genomic sequences available in the GenBank database along with an additional 11 genomic sequences generated by ourselves to identify the core set of putative SEPs present in all strains. Using bioinformatics, 56 core SEPs were identified. Molecular modeling generated putative structures of the SEPs from which potential surface exposed regions were defined. Synthetic peptides corresponding to ten of these highly conserved surface-exposed regions were used to raise antisera in rats. These antisera were used to assess passive protection in the infant rat model of invasive NTHi infection. Five of the antisera were protective, thus demonstrating their in vivo antibody accessibility. These five peptide regions represent potential targets for peptide vaccine candidates to protect against NTHi infection.

Fig 1. Protection afforded by anti-HxuC antisera in the infant rat model of NTHi bacteremia.

Panel A) Percentage of infected infant rats pretreated with pentavalent anti-HxuC antisera with detectable bacteremia 48 hours after infection. Twenty-four hours prior to infection cohorts of 10 infant rats were pretreated with phosphate-buffered saline (PBS), pre-immune serum (PIS) or peptide-specific antiserum (PSAS). Using Fisher’s exact test to compare percentages of bacteremic pups P = 0.0011 for PBS vs PSAS and P = 0.0198 for PIS vs PSAS. Panel B) Percentage of infected infant rats pretreated with antisera against specific HxuC peptides with detectable bacteremia 48 hours after infection. Twenty-four hours prior to infection cohorts of infant rats were pretreated with phosphate- buffered saline (PBS; cohort of 8 rats), HxuC1 specific antisera (HxuC1; cohort of 10 rats), or HxuC2 specific antisera (HxuC2; cohort of 10 rats). Using Fisher’s exact test to compare percentages of bacteremic pups P = 0.0011 for PBS vs HxuC1 and P = 0.0001 for PBS vs HxuC2. Panel C) Bacteremic titers in infected infant rats pretreated with antisera against specific HxuC peptides 48 hours after infection. Filled dots represent the bacteremic titer in each individual animal in a cohort. The unfilled dot represents the average bacteremic titers in all members of the cohort. Values of 1 or below represent animals with no detectable bacteremia. Using the Kruskal-Wallis test to compare bacteremic titers(means ±SD) P = 0.002 for PBS vs HxuC1 and P = 0.0004 for PBS vs HxuC2.

Fig 2. Distribution of sequenced NTHi isolates.

Neighbor joining dendogram of NTHi strains used in this study. The tree is rooted with Eschericia coli MG1655 and is based on sequence comparisons of the concatenated adk, pgi, recA, infB and 16s rRNA gene sequences, with bootstrap values of greater than 50% of 1,000 bootstraps indicated. Also included are several non NTHi sequences; Hp (H. parainfluenzae T3T1), HH (H. haemolyticus ATCC 33390) and the H. influenzae strains Rd KW20 and 10810.

Fig 3. Outer membrane loops of an NTHi SEP.

3D computer-predicted spatial topography of the gated TonB-dependent porin HxuC. The ribbon area corresponds to the external membrane embedded barrel. Also shownare surface exposed external loops. Arrows point to two specific loops designated OM loops 6 and 8. Alignments of both loop regions from five NTHi strains are shown. OM loop 8 is highly heterologous while OM loop 6 is conserved.

Fig 4. Protection afforded by antisera raised against ComE and Hel derived peptides in the infant rat model of NTHi bacteremia.

Panel A) Percentage of infected infant rats pretreated with anti-ComE1 antiserum with detectable bacteremia 24 hours after infection. Twenty-four hours prior to infection cohorts of infant rats were pretreated with phosphate-buffered saline (PBS; cohort of 9 rats), pre-immune serum (PIS; cohort of 9 rats) or anti-ComE1 antiserum (ComE1; cohort of 10 rats). Using Fisher’s exact test to compare percentages of bacteremic pups P = 0.0031 for PBS vs ComE1 and P = 0.0698 for PIS vs ComE1. Panel B) Bacteremic titers in infant rats pretreated with anti-ComE1 antisera 24 hours after infection. Filled dots represent the bacteremic titer in each individual animal in a cohort. The unfilled dot represents the average bacteremic titers in all members of the cohort. Values of 1 or below represent animals with no detectable bacteremia. Using the Kruskal-Wallis test to compare bacteremic titers (mean ±SD) P = 0.07 for PBS vs PIS, P = 0.0002 for PBS vs ComE1 and P = 0.01 for PIS vs ComE1. Panel C) Percentage of infected rats pre-treated with anti-Hel1 antisera with detectable bacteremia 24 hours after infection. Twenty-four hours prior to infection cohorts of infant rats were pretreated with phosphate-buffered saline (PBS; cohort of 10 rats), pre-immune serum (PIS; cohort of 9 rats) or anti-Hel1 antiserum (Hel1; cohort of 10 rats). Using Fisher’s exact test to compare percentages of bacteremic pups P = 0.0325 for both PBS vs Hel1 and PIS vs Hel1. Panel D) Bacteremic titers in infant rats pretreated with anti-Hel1 antiserum with detectable bacteremia 24 hours after infection. Filled dots represent the bacteremic titer in each individual animal in a cohort. The unfilled dot represents the average bacteremic titers in all members of the cohort. Values of 1 or below represent animals with no detectable bacteremia. Using the Kruskal-Wallis test to compare bacteremic titers (mean ±SD) P = 0.15 for PBS vs PIS, P = 0.0003 for PBS vs Hel1 and P = 0.0005 for PIS vs Hel1.