Antibodies against the majority subunit of type IV pili disperse nontypeable Haemophilus influenzae biofilms in a LuxS‐dependent manner and confer therapeutic resolution of experimental otitis media

Abstract

Summary Despite resulting in a similar overall outcome, unlike antibodies directed against the DNABII protein, integration host factor (IHF), which induce catastrophic structural collapse of biofilms formed by nontypeable Haemophilus influenzae (NTHI), those directed against a recombinant soluble form of PilA [the majority subunit of Type IV pili (Tfp) produced by NTHI], mediated gradual ‘top-down’ dispersal of NTHI from biofilms. This dispersal occurred via a mechanism that was dependent upon expression of both PilA (and by inference, Tfp) and production of AI-2 quorum signaling molecules by LuxS. The addition of rsPilA to a biofilm-targeted therapeutic vaccine formulation comprised of IHF plus the powerful adjuvant dmLT, and delivered via a non-invasive transcutaneous immunization route, induced an immune response that targeted two important determinants essential for biofilm formation by NTHI. This resulted in significantly earlier eradication of NTHI from both planktonic and adherent populations in the middle ear, disruption of mucosal biofilms already resident within middle ears prior to immunization, and rapid resolution of signs of disease in an animal model of experimental otitis media. These data support continued development of this novel combinatorial immunization approach for resolution and/or prevention of multiple diseases of the respiratory tract caused by NTHI.

Transcutaneous Immunization with a Band-Aid Prevents Experimental Otitis Media in a Polymicrobial Model

“…The resulting equilibrium imbalance promotes dissociation of additional DNABII proteins from the eDNA matrix, destabilization of the eDNA lattice, and subsequent collapse of the biofilm structure (17). Earlier, we showed that therapeutic TCI with IHF promotes rapid clearance of preformed NTHI biofilms from the middle ears of chinchillas (39), as did infusion of the NTHI-challenged chinchilla middle ear with IgG-enriched polyclonal serum against IHF or monoclonal antibodies directed against the DNA-binding tips of IHF (16). It is expected that NTHI also establish biofilms rapidly in vivo, and as indicated by the data presented herein, the presence of IHF-specific antibody early in this process likely hindered the development of a robust biofilm structure, exposed NTHI to clearance by host immune mediators, and prevented the onset of OM.…”

“…Antibodies directed against an OMP P5-directed immunogen, called LB1, in addition to antibodies against PilA prevent adherence of NTHI to respiratory epithelial cells (37,38). Moreover, antibodies against NTHI PilA inhibit biofilm formation in vitro and induce dispersal of NTHI from established biofilms via a LuxS-mediated quorum signaling event (37,39). In vivo, parenteral immunization with LB1 or rsPilA, a soluble recombinant and N-terminally truncated PilA molecule, show significant protective efficacy in experimental models of NTHI-induced OM (14,30,39), and TCI with LB1 or rsPilA induces resolution of ongoing experimental OM (18)(19)(20).…”

Therapeutic Transcutaneous Immunization with a Band-Aid Vaccine Resolves Experimental Otitis Media

“…At that time, the fur directly caudal to each pinna (postauricular region) or at the nape of the neck was plucked, to permit resolution of any nonspecific inflammation induced by hair removal prior to immunization. Four days later, when middle ear biofilms are well established and clinically relevant signs of OM are observed (19,25,26), animals were immunized by placement of a circular adhesive bandage (CVS Pharmacy brand) in each postauricular region (two band-aid vaccines were applied, and the total formulation dosage was divided equally between the bandages) or at the nape of the chinchilla neck (one band-aid was applied). All band-aid vaccines were removed after 24 h. Animals received band-aid vaccine boosters 7 days later.…”

“…The adherent middle ear mucosal biofilms were also collected and individually homogenized in sterile saline solution. It is well established in chinchilla models of OM that each middle ear should be considered independently (18,19,25,26,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40); therefore, MEF and biofilm homogenates collected from the left and right middle ears were individually plated on chocolate agar to semiquantitatively assess the relative numbers of NTHI in the planktonic and adherent populations. MEF samples were then clarified by centrifugation, and supernatants were stored at Ϫ80°C until determination of immunoglobulin profiles as described below.…”

“…Overall signs of OM were blindly rated on a scale of 0 to 4, and middle ears with scores of Ն2.0 were considered positive for OM, as middle ear fluid (MEF) was visible behind the tympanic membrane (27). According to established protocol, each middle ear was considered independently (18,19,26,(28)(29)(30), and the percentage of middle ears with OM was calculated for each cohort.…”