title: Design and assessment of pre-clinical vaccine candidates against otitis media due to nontypeable Haemophilus influenzae and the development of a non-invasive vaccine delivery strategy
Creators: name: Novotny, Laura A
Publisher: name: Griffith University
Year: 2019
Subjects: subject: Otitis media
subject: chronic suppurative otitis media
subject: Nontypeable Haemophilus influenzae
subject: vaccines
subject: immunisation

description: Otitis media (OM) is one of the most common bacterial diseases of childhood and include both acute and chronic forms of disease (1). Whereas acute OM (AOM) is a disease with rapid onset and is short in duration, chronic forms of OM can persist for weeks to months. The most severe form, chronic suppurative OM (CSOM) occurs upon OM with tympanic membrane rupture and is characterized by purulent drainage from the middle ear space that persists for at least 2 weeks or longer. There are 709 million cases of acute OM and 65 million to 330 million episodes of chronic suppurative OM (CSOM) occurring each year, worldwide (2, 3). Complications of CSOM result in the deaths of at least 50,000 children under 5 years of age in resource-poor regions across the world (4, 5) and morbidity associated with OM is significant for all children worldwide. Hearing loss due to OM is associated with developmental delays in behavior, language acquisition and education, all factors with profound influence that last well into adulthood (6-8). Significantly, prevention of the first incidence of OM curb extensive antibiotic prescription and surgical intervention in young children and is projected to reduce subsequent episodes, limiting the disease-related sequellae (9). Nontypeable Haemophilus influenzae (NTHI) is a primary causative agent of OM, in addition to multiple upper and lower respiratory tract diseases (10). In children, NTHI is the predominant bacterium implicated in chronic OM, recurrent OM and OM for which treatment has failed (11-14). Moreover, whereas Streptococcus pneumoniae was the primary pathogen in OM prior to the use of pneumococcal conjugate vaccines, NTHI now also predominates during acute OM (15-17). NTHI, a typically benign commensal inhabitant of the human nasopharynx, possesses numerous determinants that facilitate its persistence, including outer membrane adhesive proteins and lipooligosaccharide (18). Perturbation of innate and/or physical immune defenses can result in disease distally, for example within the middle ear during OM, sinuses in rhinosinusitis, lungs in chronic cough and during episodes of exacerbations of chronic obstructive pulmonary disease (COPD) and cystic fibrosis (19). Moreover, the ability of this bacterium to rapidly form a biofilm, a community of bacteria that is frequently polymicrobial and is highly recalcitrant to the action of antibiotics and resistant to clearance by host immune effectors (20) promotes disease chronicity. This compilation of my research, presented for consideration herein, describes the pre-clinical development of vaccines against NTHI-induced OM with a focus on two critical adhesive proteins expressed by NTHI, outer membrane protein P5 (OMP P5) and the Type IV pilus (Tfp), selected from my research performed over the past 22 years. Adhesin OMP P5 binds to mucin, intercellular adhesin molecule- 1 (ICAM1) and carcinoembryonic antigen-related adhesion molecule-1 (CEACAM1) (21-25), and I identified that NTHI Tfp engages ICAM1 (26). Furthermore, NTHI Tfp performs multiple biological functions which include competence, twitching motility and biofilm formation (27-31). I show that antibodies directed against the majority protein subunit of NTHI Tfp, PilA, induces active dispersal of NTHI from established biofilms, an outcome that is dependent on and coordinated with expression of the quorum signaling molecule, LuxS (29). Toward development of NTHI OMP P5 and Tfp-directed vaccine immunogens, I performed epitope mapping studies that revealed minimal immunodominant and immunoprotective domains within each native protein (32-34). These data guided the subsequent design of a novel chimeric immunogen, as a multi-component vaccine may be necessary to provide maximal protection against this heterogeneous bacterial species (35). I validated pre-clinical efficacy of each immunogen in chinchilla models of NTHI-induced experimental OM (29, 34-36). Although immunisation via intramuscular or subcutaneous injection is wellestablished and proven to be one of the most effective strategies for disease prevention, non-invasive delivery methods have the potential to promote greater compliance, reduce costs associated with production and administration and extend the reach of vaccines to underserved regions (37). To explore this premise, I considered transcutaneous immunisation (TCI). My first efforts involved rubbing vaccine formulations on to the pinnae (or outer ears) of chinchillas (38, 39), a strategy that was later refined to utilize a small circular bandaid applied to the postauricular region behind the ear as a delivery device and administration route (40, 41). I examined the influence of adjuvantation, skin hydration and homing of antigenpresenting cell to local lymphoid tissues as essential factors toward induction of protective immunity. I characterised the durability of immunity induced by skin vaccination and validated TCI as a highly effective strategy to induce protective immune responses in experimental models of NTHI-induced OM (38-41). Improving vaccination methodologies for young children, particularly those in resource-poor regions of the world, was a key consideration in TCI conceptualization and refinement. Collectively, these data demonstrate the simplicity of bandaid immunisation which, combined with highly effective antigens, that target two critical NTHI adhesive proteins admixed with a potent adjuvant, has tremendous potential to enable greater reach of vaccines against NTHI and diseases of the respiratory tract, including OM.