Immunization with HMW1 and HMW2 adhesins protects against colonization by heterologous strains of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and results in significant morbidity. The pathogenesis of NTHi disease begins with nasopharyngeal colonization, and therefore, the prevention of colonization represents a strategy to prevent disease. The NTHi HMW1 and HMW2 proteins are a family of conserved adhesins that are present in 75 to 80% of strains and have been demonstrated to play a critical role in colonization of the upper respiratory tract in rhesus macaques. In this study, we examined the vaccine potential of HMW1 and HMW2 using a mouse model of nasopharyngeal colonization. Immunization with HMW1 and HMW2 by either the subcutaneous or the intranasal route resulted in a strain-specific antibody response associated with agglutination of bacteria and restriction of bacterial adherence. Despite the specificity of the antibody response, immunization resulted in protection against colonization by both the parent NTHi strain and heterologous strains expressing distinct HMW1 and HMW2 proteins. Pretreatment with antibody against IL-17A eliminated protection against heterologous strains, indicating that heterologous protection is IL-17A dependent. This work demonstrates the vaccine potential of the HMW1 and HMW2 proteins and highlights the importance of IL-17A in protection against diverse NTHi strains.

Nontypeable (nonencapsulated) Haemophilus influenzae (NTHi) is a common cause of acute otitis media (AOM), otitis media with effusion, sinusitis, and exacerbations of underlying lung disease in children (1). NTHi is also frequently associated with community-acquired pneumonia and exacerbations of chronic obstructive pulmonary disease in adults (2).The pathogenesis of NTHi disease begins with colonization of the nasopharynx. Colonization occurs early in life and is common throughout childhood and into adulthood (3). NTHi spreads contiguously within the respiratory tract to cause localized disease, typically in the setting of a viral respiratory infection or allergic disease (4–6). Recent evidence indicates that colonization in early infancy may predispose to the development of neutrophilic asthma and allergic airway disease (7). Given the burden of NTHi disease and the association between early life NTHi colonization and asthma, there is interest in developing a vaccine against NTHi.Currently, vaccines against H. influenzae are limited to polysaccharide conjugate vaccines targeting the capsule of type b strains (Hib). While these vaccines have been largely successful in eliminating Hib invasive disease, there has been no effect on strains of NTHi, which lack a capsule. Efforts to identify highly conserved antigens in NTHi and develop a vaccine against NTHi have been significantly more difficult. While Hib and other encapsulated strains of H. influenzae are clonal, NTHi strains exhibit much greater genetic diversity and heterogeneity (8). Across NTHi strains, common surface antigens exhibit high degrees of antigenic variation, resulting in variable and strain-specific antibody responses. In studies of mice, antibodies acquired during NTHi infection, including IgG in nasal washes directed against outer membrane proteins, are associated with reduced nasopharyngeal colonization density following subsequent challenge by NTHi (9). This suggests a protective role for antibodies at the mucosal surface. However, evidence in children indicates that despite the presence of antibody against an infecting strain, children remain susceptible to subsequent infections by new strains, suggesting that the antibody response may only protect against infection by the same strain.In combination with antibody responses, cell-mediated immunity has been increasingly recognized as a key driver of protection against airway infection by respiratory pathogens. The production of IL-17A by host cells mediates many effector functions at mucosal surfaces, including the production of antimicrobial peptides, proinflammatory cytokines, chemotactic factors, and granulopoietic factors. These effector functions result in increased recruitment of macrophages and neutrophils and enhanced cytotoxicity and phagocytosis (10–12). There is also evidence that IL-17–producing cells drive humoral immunity, including the generation of antibody in mucosal secretions (13, 14). IL-17 production is known to be essential for defense against various mucosal pathogens, including Mycoplasma pneumoniae, Streptococcus pneumoniae, Klebsiella pneumoniae, and Bordetella pertussis (15–17). In studies of Staphylococcus aureus, clearance of bacteria colonizing the nasopharynx is driven by IL-17–mediated neutrophil influx and antimicrobial peptide production (16, 18). Thus far, it is unclear whether IL-17 production directly contributes to host defense against nasopharyngeal colonization by NTHi.NTHi colonization of the nasopharynx begins with bacterial adherence to respiratory epithelial cells, which is dependent on NTHi adhesive proteins. The HMW1 and HMW2 high-molecular weight proteins are surface-exposed glycoproteins and are the predominant adhesins in ∼75 to 80% of NTHi strains (19–23). HMW1 and HMW2 also facilitate upper respiratory tract colonization in rhesus macaques as highlighted in studies comparing a wild-type strain and an isogenic mutant lacking both of these proteins (24). HMW1 and HMW2 are highly homologous to each other, sharing ∼70% identity and 80% similarity. HMW1 and HMW2 are also highly homologous among diverse NTHi strains (25, 26).In children recovering from AOM, HMW1 and HMW2 are the major targets of the serum antibody response to infection. Similarly, rhesus macaques colonized with HMW1/HMW2-expressing strains develop serum antibody responses against HMW1 and HMW2, and serum antibodies against HMW1 and HMW2 are present in adults, suggesting that these adhesins are highly immunogenic. The development of HMW1/HMW2-specific antibodies in humans also coincides with increased serum bactericidal activity (27). However, antibody-mediated killing appears to be directed primarily against the homologous infecting strain rather than broadly acting against heterologous strains (28). The conservation of HMW1 and HMW2 among diverse strains, the immunogenicity of HMW1 and HMW2, the surface localization of HMW1 and HMW2, and the role of HMW1 and HMW2 in adherence to respiratory epithelial cells and in colonization make these adhesins promising antigens for a vaccine.In this study, we evaluated whether immunization with HMW1 and HMW2 protects against nasopharyngeal colonization. Using a mouse model of immunization and nasopharyngeal challenge, we found that immunization with the HMW1 and HMW2 proteins results in protection against colonization by homologous and heterologous strains, despite a highly strain-specific antibody response. Protection against nasopharyngeal colonization is mediated by both antibody and T cell responses.