In this study, we immunized mice subcutaneously with recombinant proteins corresponding to the C-terminal region of Hap S from H. influenzae strains N187, P860295, and TN106 and examined the resulting immune response. Antisera against the recombinant proteins from all three strains not only recognized native Hap S purified from strain P860295 but also inhibited H. influenzae Hap-mediated adherence to Chang epithelial cells. Furthermore, when mice immunized intranasally with recombinant protein plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization. These observations demonstrate that the C-terminal region of Hap S is capable of eliciting cross-reacting antibodies that reduce nasopharyngeal colonization, suggesting utility as a vaccine antigen for the prevention of nontypeable H. influenzae diseases.Nontypeable Haemophilus influenzae (NTHi), a nonencapsulated gram-negative bacterium, is the cause of a number of human respiratory tract diseases, such as otitis media, sinusitis, bronchitis, and pneumonia (15, 16). Otitis media is among the most common infections in young children. By 3 years of age, approximately 80% of children have had at least one episode of acute otitis media (25). Recurring bouts of otitis media may lead to significant hearing loss, which in turn may result in developmental delay. A vaccine that prevents nontypeable H. influenzae disease would provide major benefits to the health of children and the general population.The pathogenesis of H. influenzae disease begins with colonization of the nasopharynx. Subsequently, organisms spread to other sites in the respiratory tract, including the middle ear, sinuses, and lower airways (21). Based on in vitro and animal studies, a number of factors appear to influence the process of colonization. One such factor is the Hap adhesin, which promotes bacterial interaction with human respiratory epithelial cells and extracellular matrix proteins as well as mediates bacterial aggregation and microcolony formation (10,23).Hap belongs to the autotransporter family of proteins common among gram-negative pathogens (9). It is synthesized as a 155-kDa precursor protein, which consists of an N-terminal 25-amino-acid signal peptide, an internal 110-kDa passenger domain called Hap S , and a C-terminal 45-kDa outer membrane domain called Hap␤ (9). Hap S has serine protease activity and is released from the precursor protein via autoproteolysis. Of note, autoproteolysis is inhibited by secretory leukocyte protease inhibitor, which is a natural component of respiratory secretions. The Hap S domain is responsible for all the adhesive properties of Hap (10, 23). Furthermore, purified Hap S is immunogenic in mice, eliciting significant anti-Hap S antibody titers. In a mouse intranasal challenge model, animals immunized with purified Hap S from NTHi strain P860295 or N187 in the presence of mutant cholera toxin CT-E29H as an adjuvant are protected against nasopharyngeal colonization (5). These findings sug...

The outer membrane protein CD of Moraxella catarrhalis is considered to be a potential vaccine antigen against Moraxella infection. We purified the native CD from isolate O35E, administered it to mice, and detected considerable titers of anti-CD antibodies. Anti-CD sera were cross-reactive towards six different M. catarrhalis isolates and promoted bacterial clearance of O35E in a pulmonary challenge model. To circumvent the difficulty of generating large quantities of CD from M. catarrhalis for vaccine use, the CD gene from O35E was cloned into Escherichia coli, and the recombinant CD, expressed without a signal sequence or fusion tags, represented ϳ70% of the total E. coli proteins. The recombinant CD formed inclusion bodies that were solubilized with 6 M urea and then purified by ion-exchange chromatography, a procedure that produced soluble CD of high purity and yield. Mice immunized with the purified recombinant CD had significant titers of anti-CD antibodies that were cross-reactive towards 24 different M. catarrhalis isolates. Upon challenge, these mice showed enhanced bacterial clearance of both O35E and a heterologous M. catarrhalis isolate, TTA24. In an in vitro assay, antisera to either the native or the recombinant CD inhibited the binding activity of CD to human tracheobronchial mucin in a serum concentration-dependent manner, and the extent of inhibition appeared to correlate with the corresponding anti-CD antibody titer and whole-cell enzyme-linked immunosorbent assay titer. Our results demonstrate that the recombinant CD is a promising vaccine candidate for preventing Moraxella infection.Moraxella catarrhalis is an important human mucosal pathogen of the respiratory tract (20,29,44). It is the third most common cause of bacterial otitis media in infants and young children (3, 40), following Streptococcus pneumoniae and nontypeable Haemophilus influenzae. Among adults, M. catarrhalis is often associated with bronchitis, laryngitis, and other respiratory diseases (1, 5). Patients with chronic obstructive pulmonary disease (COPD) are particularly vulnerable to exacerbations caused by M. catarrhalis (1,6,35). Interest in the development of a Moraxella vaccine is further stimulated by the increasing prevalence of antibiotic resistance among M. catarrhalis strains (2,8,19).The CD outer membrane protein of M. catarrhalis has been identified as a potential vaccine against Moraxella infection (9, 26) and is a safe and effective carrier for M. catarrhalis detoxified lipooligosaccharide (LOS)-based conjugates (18). Serum immunoglobulin G (IgG) antibodies specific to CD are present in infants with otitis media (25) and in children with otitis media with effusion (11). Analysis of salivary immunoglobulin A (IgA) in children with acute respiratory tract infection indicates that CD may be one of the outer membrane antigens eliciting a mucosal immune response (27). IgA antibodies against CD as well as several other surface components of M. catarrhalis are also detected in the saliva of healthy adults (28). Furthe...

We have defined the nature of the covalent linkages in a Haemophilus influenzae type b oligosaccharide-CRM197 conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative, N epsilon-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed between H. influenzae type b oligosaccharides (HbO) and the lysyl epsilon-amino groups of CRM197 (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM197 molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM197 was also demonstrated by the coincidence of immunoreactivity of gel-electrophoresed HbOC on a Western blot probed with anti-CRM197 and anti-saccharide antisera.

The gene products from an 8-kb region adjacent to the 3 end of the ptx operon are required by Bordetella pertussis for the export of pertussis holotoxin. At least one of these gene products (PtlC) is specifically required for the export of assembled holotoxin from the periplasmic space. ptlC mutants exhibit a 20-fold reduction in the amount of holotoxin present in the culture supernatant but have no effect upon the assembly or steady-state level of holotoxin present in the periplasmic space. Impaired export of holotoxin from the ptlC strain blocks expression of toxin at a posttranscriptional level, and wild-type levels of ptx mRNA are detected in the mutant strain. The transcription of ptl is subject to modulation by MgSO 4 in the same manner as ptx is; however, in B. pertussis strains containing an E. coli tac promoter in place of the native ptx promoter, wild-type levels of ptx mRNA are present and holotoxin is synthesized and exported even in the presence of MgSO 4. Promoter mapping of the region extending from the ptxS3 coding region to the ptlC coding region failed to detect the ptl transcription initiation site. Additional RNase protection experiments with ptx promoter deletion and substitution strains indicate that the ptl operon is transcribed from the ptx promoter as part of a >11-kb mRNA.