Synthesis and Characterization of Lipooligosaccharide-Based Conjugate Vaccines for Serotype B Moraxella catarrhalis

Abstract

Moraxella catarrhalis is an important cause of otitis media in children and respiratory tract infections in the elderly. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium that elicits bactericidal antibodies. Serological studies show that three major LOS types (A, B, and C) have been identified among clinical isolates. Our previous studies demonstrated that the type A LOS-based conjugates were immunogenic in animals. In this study, LOS from type B strain 26397 was detoxified and conjugated to tetanus toxoid (TT) or a cross-reactive mutant (CRM) of diphtheria toxin to form detoxified LOS (dLOS)-TT and dLOS-CRM, respectively, as vaccine candidates. The molar ratios of dLOS to TT and CRM in the conjugates were 43:1 and 19:1, respectively, while both weight ratios were around 0.9. The antigenicity of the conjugates was similar to that of the LOS, as determined by enzyme-linked immunosorbent assay using a rabbit antiserum to strain 26397. Subcutaneous immunization with each conjugate elicited a 180-to 230-fold rise of serum anti-LOS immunoglobulin G in mice and >2,000-fold rise in rabbits. In addition, both mouse and rabbit antisera showed elevated complement-mediated bactericidal activity against the homologous strain, and a representative rabbit antiserum showed bactericidal activity against nine of twelve clinical isolates studied. The bactericidal activity of the rabbit antiserum can be fully inhibited by the type B LOS but not the A or C LOS. These results indicate that the type B LOS-based conjugates can be used as vaccine components for further investigation.Moraxella catarrhalis is a gram-negative diplococcus, currently the third leading cause of otitis media and sinusitis in children along with Streptococcus pneumoniae and nontypeable Haemophilus influenzae (4,5,15,17). Up to 80% of children below 3 years of age will be diagnosed with at least one episode of acute otitis media, and 15 to 20% of these middle-ear infections are caused by M. catarrhalis (18,43). Sinusitis, however, accounts for 5 to 10% of upper respiratory tract infections in early childhood (53). In addition, M. catarrhalis is a frequent cause of lower respiratory tract infections in the elderly, particularly in those with a compromised immune system or chronic obstructive pulmonary disease, where M. catarrhalis can cause severe infections such as pneumonia that can be life-threatening (44). Presently, treatment of the diseases has largely relied on antimicrobial agents. However, with growing antibiotic resistance observed in clinical isolates (34), attention has been focused on the possibility of vaccination against M. catarrhalis.

Synthesis, Characterization, and Immunological Properties of LPS-Based Vaccines Composed of O-Polysaccharides Conjugated with Recombinant Exoprotein A from <i>Pseudomonas aeruginosa</i>

“…The highest IgM and IgG titers against O-PS moieties were obtained when the conjugates were emulsified with MPL adjuvant, in agreement with many previous studies (Figure 2) [12] [34] [35]. A mixture of unconjugated rEPA and O-PS emulsified with MPL adjuvant elicited the highest level of anti-rEPA IgG, followed by conjugates emulsified with MPL (Figure 3).…”

Biological and Immunological Characteristics of Lipooligosaccharide-Based Conjugate Vaccines for Serotype C Moraxella catarrhalis

“…The immunogenicity of the conjugates was better in rabbits than in mice, especially with an addition of Ribi adjuvant. These data are for the most part consistent with our previous studies with M. catarrhalis serotype A and B, meningococcal, and nontypeable Haemophilus influenzae LOS conjugate vaccines (11,12,13,43). We believe that rabbits may respond better to the LOS-based conjugates, as well as Ribi adjuvant, than mice.…”

“…Both mice and rabbits immunized with the conjugates developed anti-LOS IgG antibodies with bactericidal activity (11,43). Active or passive immunization with the serotype A conjugates or their antiserum generated protection against homologous and heterologous strains in a mouse model of pulmonary clearance (17).…”

“…Binding activities of the dLOS and conjugates with rabbit antiserum against strain 26404 were tested by using an enzyme-linked immunosorbent assay (ELISA) (43). The reactions were read by using a microplate autoreader at A 405 after 1 h of incubation with a substrate.…”

“…Mouse and rabbit antisera were inactivated at 56°C for 30 min and tested for bactericidal activity against M. catarrhalis by a microbactericidal assay as described previously (43). The antiserum was diluted at 1:5, and then twofold continuous dilution with Dulbecco's phosphate-buffered saline containing calcium, magnesium, and 0.1% gelatin was performed.…”