Pneumococcal Haemophilus influenzae Protein D Conjugate Vaccine Induces Antibodies That Inhibit Glycerophosphodiester Phosphodiesterase Activity of Protein D

Unencapsulated, nontypeable Haemophilus influenzae (NTHI) is a frequent commensal of the human nasopharynx but is also the common cause of respiratory tract infections, such as otitis media (OM), sinusitis, bronchitis, and pneumonia (12, 22). Prevention of NTHI infections would provide considerable health and economic benefits. Thus, efforts have been directed toward identifying bacterial structures with potential as vaccine antigens. Of these, the outer membrane protein D (PD) is one of the most promising (25). PD (also known as LPD) is a conserved 42-kDa outer membrane-associated lipoprotein (8). It belongs to the glycerophosphodiester phosphodiesterase (GlpQ) protein family and shows 78% amino acid similarity to the periplasmic nonlipidated GlpQ protein in Escherichia coli (21) and 90% amino acid similarity to the lipoprotein homologue in Pasteurella multocida (17). Similar to other members of this protein family, PD displays GlpQ activity, catalyzing the hydrolysis of glycerophosphodiesters to sn-glycerol 3-phosphate and the corresponding alcohol (21). The hpd (or glpQ) gene, encoding lipo-PD, has been cloned and sequenced from several strains (4, 9, 29). The protein is genetically and antigenically conserved (4, 29) and is present in all typeable H. influenzae and NTHI strains tested thus far (3). Deviating from the nonlipidated GlpQ homologue in E. coli (15) and the lipidated GlpQ homologues in P. multocida (17) and Treponema pallidum (27), which are all located in the periplasm, in NTHI PD is proposed to be exposed to the cell surface (3). The specific function(s) of PD is not known; however, previous in vivo and in vitro studies suggest that it is involved in NTHI pathogenesis. In an experimental rat OM model, a 100-fold higher concentration of PD-deficient mutant than PD-expressing wild-type bacteria was required to induce OM after direct injection of bacteria into the middle ear (10). Likewise, in a human nasopharyngeal tissue culture model using the same wild-type and mutated bacteria, the PD-deficient mutant caused much less damage to ciliated epithelial cells and loss of cilia than the wild-type, PD-expressing bacteria did (7). The mechanism(s) behind PD's virulence properties is not clear but may involve its GlpQ activity, either directly or indirectly (6). Recently, a recombinant nonacylated form of PD (rPD) was used successfully as a novel carrier protein in a pneumococcal conjugate vaccine (Pnc-PD) (25). In a pediatric efficacy trial in the Czech Republic and in Slovakia, an efficacy of 35.3% (95% confidence interval [CI], 1.8% to 57.4%) against acute OM caused by NTHI was detected, associated with a 41.4% (95% CI, −4.9% to 67.3%) reduction in the nasopharyngeal NTHI carriage rate (25). The mechanism(s) for how PD induces protective immunity is currently unclear, but it seems to be antibody mediated, as passive immunization with a pediatric human serum pool generated against polysaccharide-PD conjugate vaccines conferred approximately 34% protection against the development of ascending NTHI-induced OM in a chinchilla viral-bacterial coinfection model (23). The development of PD-based vaccines against NTHI would be facilitated if there was a functional assay correlating with protective efficacy. To study if PD-induced protection could be due to antibodies that inhibit, i.e., neutralize, its enzymatic activity, a GlpQ enzyme inhibition assay was developed, and pre- and postvaccination serum samples collected from infants given three or four doses of Pnc-PD vaccine during a previous immunogenicity and safety study in Finland (24) were analyzed for enzyme inhibition and anti-PD immunoglobulin G (IgG) antibody concentrations.