Antibodies Specific for the Hia Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity

The Hia autotransporter proteins are highly immunogenic surface adhesins expressed by nontypeable Haemophilus influenzae (NTHI). The objective of our study was to assess the opsonophagocytic activity of anti-Hia antibodies against homologous and heterologous NTHI. A segment of the hia gene that encodes a surface-exposed portion of the H. influenzae strain 11 Hia protein was cloned into a pGEMEX-2 expression vector. Escherichia coli JM101 was transformed with the resulting pGEMEX-Hia BstEII del recombinant plasmid, and recombinant fusion protein was recovered. An immune serum against recombinant GEMEX-Hia (rGEMEX-Hia)-mediated killing of the homologous NTHI strain 11 at a 1:160 titer and five heterologous Hia-expressing strains at titers of ≥1:40. Immune serum did not mediate killing of two Hia-knockout strains whose hia genes were inactivated but did mediate killing of one knockout strain at a high titer after the strain was transformed with a plasmid containing the hia gene. Immune serum did not mediate killing of HMW1/HMW2-expressing NTHI strains, which do not express the Hia adhesin. However, when two representative HMW1/HMW2-expressing strains were transformed with the plasmid containing the hia gene, they expressed abundant Hia and were susceptible to killing by the immune serum. Immune serum did not mediate killing of HMW1/HMW2-expressing strains transformed with the plasmid without the hia gene. Our results demonstrate that the Hia proteins of NTHI are targets of opsonophagocytic antibodies and that shared epitopes recognized by such antibodies are present on the Hia proteins of unrelated NTHI strains. These data argue for the continued investigation of the Hia proteins as vaccine candidates for the prevention of NTHI disease.Otitis media remains a significant health problem for children in this country and elsewhere in the world (10, 11). Most children in the United States have had at least one episode of otitis by their third birthdays, and one-third have had three or more episodes (34). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (33, 41). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for the prevention of otitis media in young children (20). Although the total prevention of disease will be a difficult goal to achieve, the prevention of even a portion of cases would be beneficial, given the magnitude and costs of the problem.Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of the cases of acute otitis media (16, 35). Streptococcus pneumoniae is the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (16, 35). Nontypeable Haemophilus influenzae (NTHI) is the second-most-common bacterium recovered and accounts for 20% to 30% of the cases of acute otitis media and a larger percentage of the cases of chronic and recurrent disease (26). Interestingly, since the introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccine schedule, NTHI has become an even more common cause of acute and recurrent middle ear disease, often surpassing S. pneumoniae in the frequency of recovery from middle ear specimens (12, 26). Many different antigens have been suggested as possible NTHI vaccine candidates (1, 3, 18, 29, 30, 42). Outer membrane proteins appear to be the principal targets of bactericidal and protective antibodies (22), and as a group, they have been the major focus of vaccine development efforts. Table ​Table11 summarizes the relevant characteristics of some of the leading vaccine candidates currently under active investigation.

TABLE 1.

Potential vaccine antigens of NTHI

Antigen	Molecular mass (kDa)	Osonophagocytic or bactericidal antibody	Protects animals	Adherence factor	Reference(s) or source
LOS	3-5	Yes	Yes	Yes	23, 46
PilA	16	Not known	Yes	Yes	2
OMP P6	16	Yes	Yes	No	17, 30
OMP 26	26	Not known	Yes	No	18
OMP P5 fimbrin	36	Not known	Yes	Yes	4
OMP P2	36-42	Yes	Yes	Yes	29
Protein D	42	Not known	Yes	No	36
HMW1/HMW2	100-150	Yes	Yes	Yes	5, 44
Hia	100->250	Yes	Not known	Yes	This study

Open in a separate windowIn our early work, we demonstrated that the development of bactericidal antibodies in the sera of children recovered from acute NTHI otitis media was associated with the appearance of serum antibodies directed against highly immunogenic high-molecular-weight proteins (6). This work subsequently led to the identification and characterization of the HMW1/HMW2 family of proteins (7). The HMW1/HMW2 proteins have subsequently been shown to be major adhesins of NTHI (37), as well as targets of opsonophagocytic (43, 44) and protective antibodies (5). The HMW1/HMW2-like proteins are expressed by approximately 75% of NTHI strains (7, 38). The 25% of NTHI strains that do not express HMW1/HMW2-like proteins express immunogenic high-molecular-weight proteins that are recognized by human convalescent-phase serum antibodies (6). Almost all such HMW1/HMW2-negative strains have subsequently been shown to express a second distinct class of adhesin known as Hia (9). Nearly all NTHI strains that lack HMW1/HMW2 proteins contain an hia gene and express an Hia protein, and conversely, strains that express HMW1/HMW2 proteins lack an hia gene (9, 38).The Hia proteins are members of a large family of bacterial proteins known as autotransporters that are found in many gram-negative bacteria (24, 48). Autotransporters are typically expressed as precursor proteins with three functional domains, an N-terminal signal peptide, an internal “passenger domain,” and a C-terminal translocator or beta domain (24, 27). The signal peptide directs the protein across the inner bacterial membrane, and the translocator or beta domain forms a β-barrel structure in the outer membrane through which the passenger domain is extruded to the bacterial surface (24, 27). On the bacterial surface, the passenger domain is usually cleaved, but in the case of the Hia protein, the protein remains uncleaved and cell associated, and it functions on the cell surface as an important adhesin for Hia-expressing NTHI strains (25, 48). At present, members of the autotransporter family expressed by other gram-negative bacteria are under active investigation as possible vaccine candidates (13, 28, 40).No information is currently available concerning the functional activity of antibodies directed against the Hia proteins of NTHI. The objective of the present study was to assess the ability of antibodies directed against the Hia proteins to mediate opsonophagocytic activity. In the work described here, we demonstrated that the Hia proteins are indeed targets of opsonophagocytic antibodies, and furthermore, we demonstrated that epitopes recognized by such antibodies are also present on the Hia proteins of heterologous NTHI strains.