Redirecting the immune response towards immunoprotective domains of a DNABII protein resolves experimental otitis media.

The chronicity and recurrence of many bacterial diseases is largely attributable to the presence of a biofilm, and eradication of these structures is confounded by an extracellular DNA-rich matrix. DNABII proteins, including integration host factor (IHF), are critical components of the matrix formed by all human pathogens tested to date. Whereas the natural adaptive immune response to IHF is against non-protective epitopes within the carboxyl-terminal region, antibodies against the DNA-binding "tips" induce biofilm collapse. We designed a "tip-chimer" immunogen to mimic the DNA-binding regions within the α-subunit and ß-subunit of IHF from nontypeable Haemophilus influenzae (IHFNTHi). Re-direction of the natural adaptive immune response toward immunoprotective domains disrupted NTHi biofilms in vitro and in an experimental model of otitis media. Our data support the rational design of a powerful therapeutic approach, and also that of a DNABII-directed vaccine antigen that would avoid augmentation of any pre-existing natural, but nonprotective, immune response.

Biofilms can be dispersed by focusing the immune system on a common family of bacterial nucleoid-associated proteins.

Bacteria that cause chronic and/or recurrent diseases often rely on a biofilm lifestyle. The foundation of the biofilm structure is the extracellular polymeric substance (EPS) that acts as a barrier to both effectors of the immune system and antimicrobial agents. Recent work has highlighted extracellular DNA (eDNA) as a key component common to many pathogenic biofilms. Here, we show that the DNABII family of proteins, well known for their strong structural influences on intracellular DNA, was also critical for the integrity of the EPS matrix of biofilms that contain eDNA. In fact, antisera derived against a purified Escherichia coli DNABII family member rapidly disrupts the biofilm EPS formed by multiple human pathogens in vitro. In addition, when a member of this family of proteins was used as an immunogen in an animal model in which the bacteria had already formed a robust biofilm at the site of infection, the resultant targeted immune response strongly ameliorated this biofilm disease in vivo. Finally, this methodology to debulk the biofilm of EPS was shown to work synergistically with otherwise ineffective traditional anti-microbial approaches in vitro. We discuss the prospects for targeting DNABII family members as a potential universal strategy for treating biofilm diseases.

Transcutaneous immunization as preventative and therapeutic regimens to protect against experimental otitis media due to nontypeable Haemophilus influenzae.

We have developed three nontypeable Haemophilus influenzae (NTHI) adhesin-derived immunogens that are significantly efficacious against experimental otitis media (OM) due to NTHI when delivered parenterally. We now expanded our preventative immunization strategies to include transcutaneous immunization (TCI) as a less invasive, but potentially equally efficacious, regimen to prevent OM due to NTHI. Additionally, we examined the potential of TCI as a therapeutic immunization regimen to resolve ongoing experimental OM. Preventative immunization with NTHI outer membrane protein (OMP) P5- and type IV pilus-targeted immunogens, delivered with the adjuvant LT(R192G-L211A), induced significantly earlier clearance of NTHI from the nasopharynges and middle ears of challenged chinchillas compared with receipt of immunogen or adjuvant alone. Moreover, therapeutic immunization resulted in significant resolution of established NTHI biofilms from the middle ear space of animals compared with controls. These data advocate TCI with the adhesin-directed immunogens as an efficacious regimen for prevention and resolution of experimental NTHI-induced OM.

Passive transfer of antiserum specific for immunogens derived from a nontypeable Haemophilus influenzae adhesin and lipoprotein D prevents otitis media after heterologous challenge.

We recently determined that passive transfer of serum directed against a synthetic peptide called LB1 or a recombinant fusion protein immunogen [LPD-LB1(f)(2,1,3)] could prevent otitis media after challenge with a homologous nontypeable Haemophilus influenzae (NTHI) isolate. NTHI residing in the nasopharynx was rapidly cleared from this site, thus preventing it from ascending the eustachian tube and inducing otitis media in chinchillas compromised by an ongoing viral upper respiratory tract infection. While LB1 is based solely on one NTHI adhesin, the latter immunogen, LPD-LB1(f)(2,1,3), was designed to incorporate two NTHI antigens shown to play a role in the pathogenesis of otitis media; lipoprotein D (LPD) and the P5-homologous fimbrin adhesin. The design of LPD-LB1(f)(2,1,3) also accommodated for the recently demonstrated existence of three major groupings, based on amino acid sequence diversity, in the third surface-exposed region of P5-fimbrin. LPD-LB1(f)(2,1,3) was thus designed to potentially confer broader protection against challenge by diverse strains of NTHI. Chinchillas were passively immunized here with serum specific for either LB1 or for LPD-LB1(f)(2,1,3) prior to challenge with a member of all three groups of NTHI relative to diversity in region 3. The transferred serum pools were also analyzed for titer, specificity, and several functional activities. We found that both serum pools had equivalent ability to mediate C'-dependent killing and to inhibit adherence of NTHI strains to human oropharyngeal cells. When passively transferred, both serum pools significantly inhibited the signs and incidence of otitis media (P

Epitope mapping of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae.

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.

Protection against development of otitis media induced by nontypeable Haemophilus influenzae by both active and passive immunization in a chinchilla model of virus-bacterium superinfection.

Three separate studies, two involving active-immunization regimens and one involving a passive-transfer protocol, were conducted to initially screen and ultimately more fully assess several nontypeable Haemophilus influenzae outer membrane proteins or their derivatives for their relative protective efficacy in chinchilla models of otitis media. Initial screening of these antigens (P5-fimbrin, lipoprotein D, and P6), delivered singly or in combination with either Freund's adjuvant or alum, indicated that augmented bacterial clearance from the nasopharynx, the middle ears, or both anatomical sites could be induced by parenteral immunization with P5-fimbrin combined with lipoprotein D, lipoprotein D alone, or the synthetic chimeric peptide LB1 (derived from P5-fimbrin), respectively. Data from a second study, wherein chinchillas were immunized with LB1 or lipoprotein D, each delivered with alum, again indicated that clearance of nontypeable H. influenzae could be augmented by immunization with either of these immunogens; however, when this adjuvant was used, both antibody titers in serum and efficacy were reduced. A third study was performed to investigate passive delivery of antisera directed against either LB1, lipoprotein D, nonacylated lipoprotein D, or a unique recombinant peptide designated LPD-LB1(f)2,1,3. The last three antiserum pools were generated by using the combined adjuvant of alum plus monophosphoryl lipid A. Passive transfer of sera specific for LB1 or LPD-LB1(f)2,1,3 to adenovirus-compromised chinchillas, prior to intranasal challenge with nontypeable H. influenzae, significantly reduced the severity of signs and incidence of otitis media which developed (P