A bacterial vaccine polypeptide protective against nontypable Haemophilus influenzae.

Nontypeable strains of Haemophilus influenzae (NTHi) are one of the most common cause of otitis media and the most frequent infection associated with exacerbations of chronic obstructive pulmonary disease; there is currently no vaccine in the U.S. to prevent NTHi. Using bioinformatics and structural vaccinology, we previously identified several NTHi species-conserved and sequence-conserved peptides that mediate passive protection in the rat model of infection. Using these, and similar peptides, we designed Hi Poly 1, a Bacterial Vaccine Polypeptide, comprising 9 unique peptides from 6 different surface proteins. Recombinant Hi Poly 1 was purified by affinity chromatography. Forty chinchillas were immunized three times with 200 µg of Hi Poly 1 with alum adjuvant; similarly, 41 controls were immunized with adjuvant alone. The average Log2 IgG titer among immunized animals was 17.04, and IgG antibodies against each component peptide were detected. In the infant rat model, antisera from immunized chinchillas provided significant passive protection compared to PBS (p = 0.01) and pre-immune sera (p = 0.03). In the established chinchilla model of NTHi otitis media, the vaccinated group cleared infection faster than the control group as indicated by significantly decreased positive findings on video-otoscopy (p < 0.0001) and tympanometry (p = 0.0002) on day 7, and for middle ear fluid obtained by aspiration (p = 0.0001) on day 10 post-infection. Using 12 representative NTHi strains in a Live-Cell ELISA, greater antibody binding to each strain was detected with post Hi Poly 1 than the pre-immune chinchilla antisera. The data from this proof-of-principle study demonstrate the effectiveness of Hi Poly 1 against the NTHi in two relevant preclinical models of bacteremia and otitis media as well as surface antibody binding across the species. The Bacterial Vaccine Polypeptide approach to a vaccine against NTHi also serves as a paradigm for development of similar vaccines to protect against other bacteria.

Draft Genome Sequences of Eight Nontypeable Haemophilus influenzae Strains Previously Characterized Using an Electrophoretic Typing Scheme.

Nontypeable Haemophilus influenzae is an important cause of human disease. Strains were selected for genome sequencing to represent the breadth of nontypeable strains within the species, as previously defined by the electrophoretic mobility of 16 metabolic enzymes.

Draft Genome Sequences of Six Nontypeable Haemophilus influenzae Strains That Establish Bacteremia in the Infant Rat Model of Invasive Disease.

Haemophilus influenzae is an important cause of invasive disease. The infant rat is the accepted model of invasive H. influenzae disease. Here, we report the genome sequences of six nontypeable H. influenzae strains that establish bacteremia in the infant rat.

Haemophilus influenzae OxyR: characterization of its regulation, regulon and role in fitness.

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness.