Galactose Residues on the Lipooligosaccharide ofMoraxella catarrhalis26404 Form the Epitope Recognized by the Bactericidal Antiserum from Conjugate Vaccination

Abstract

Lipooligosaccharide (LOS) fromMoraxella catarrhalis has the potential to elicit bactericidal antibodies against the pathogen. We generated LOS-based conjugate vaccines that elicited bactericidal antibodies in animal models. However, epitopes on the LOS recognized by the functional anti-LOS antibodies remain unidentified. In this study, a mutant strain, D4, which lost the recognition by a bactericidal anti-LOS rabbit serum in Western blotting was generated from a serotype C strain 26404 by random transposon mutagenesis. Sequence analysis revealed there was an insertion of a kanamycin resistance gene in the lgt2 gene of D4, which encodes ␤(1-4)-galactosyltransferase. An isogenic lgt2 mutant, 26404lgt2, was constructed. Structural analysis indicated that the mutant strain produced a truncated LOS lacking terminal galactoses from 4-and 6-linked oligosaccharide chains of strain 26404. Further studies showed that the antiserum lost the recognition of both mutant cells and LOSs in Western blotting, an enzyme-linked immunosorbent assay (ELISA), or a flow cytometry assay. The antiserum also lost the ability to kill the mutant strain in a bactericidal assay, whereas it showed a bactericidal titer of 1:80 to strain 26404. In an inhibition ELISA, D-(؉)-galactose or 26404lgt2 LOS showed no inhibition. However, the 26404 LOS and a serotype A O35E LOS with terminal galactoses on its 6-linked oligosaccharide chain showed >90% inhibition, while a serotype B 26397 LOS showed >60% inhibition. These studies suggest that the terminal ␣-Gal-(134)-␤-Gal on the 6-linked oligosaccharide chain of 26404 LOS plays a critical role in forming the epitope recognized by the bactericidal antiserum induced by immunization with our conjugate vaccine.Moraxella catarrhalis is a gram-negative human respiratory tract pathogen that causes 15 to 20% of acute otitis media in children, or a total of 3 to 4 million cases annually in the United States (37). The organism's only known host is the human, and its importance as a host-adapted pathogen is now widely recognized. In addition to causing childhood ear infections, this bacterium is also responsible for 10 to 35% of lower respiratory tract infections in adults with chronic obstructive pulmonary disease, the fourth leading cause of death in the United States (25). The present treatment of these diseases has relied largely on antimicrobial agents. However, with growing antibiotic resistance observed in clinical isolates all over the world (10) and the emergence of fatal neonatal meningitis (4), attention has been focused on the possibility of vaccination to protect humans from M. catarrhalis infections (3, 24).There have been a number of putative vaccine candidates described for M. catarrhalis, and most of them are outer membrane proteins (12,16,23). Lipooligosaccharide (LOS) is another prominent surface component of M. catarrhalis. It has been implicated as a virulence factor important in the pathogenesis of this organism (5,11,28). Clinical investigations demonstrated that serum antibodies to LOS deve...

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera

“…CE-ES-MS analysis of LPS-OH from the wild-type and mutant strains (Table 1) revealed that LPS-OH from the lgt2 mutant was 324.6 Da smaller than LPS-OH from the wild-type serotype A strain, corresponding to a loss of two hexoses, which is consistent with mutation of the lgt2 gene as previously shown [19]. LPS-OH from the lgt2/lgt4 mutant was 203.2 Da smaller than LPS-OH from the lgt2 mutant corresponding to a loss of an additional N-acetylhexosamine residue which is consistent with mutation of the lgt4 gene.…”

“…Previous studies on LPS based vaccines to combat M. catarrhalis have been carried out in the laboratories of Gu [13][14][15][16][17][18][19]. However the work of Gu has focused upon immunisation with conjugates derived from wild-type strains, thus raising the possibility of auto-immunity, and in the most recent paper indeed identified that the terminal galactose disaccharide, part of the pK antigen as the immunodominant epitope [19].…”

“…We showed that antibodies derived in this way were bactericidal and protective against wild-type N. meningitidis strains [10]. Previous studies on LPS based vaccines to combat M. catarrhalis have been carried out in the laboratories of Gu [13][14][15][16][17][18][19]. This elegant research has documented that it is indeed possible to raise bactericidal antibodies to M. catarrhalis following immunisation with LPS-based glycoconjugates.…”

Use of Moraxella catarrhalis Lipooligosaccharide Mutants To Identify Specific Oligosaccharide Epitopes Recognized by Human Serum Antibodies

“…It is also important that this common LOS contains the ␣-D-Galp-(1-4)-␤-D-Galp moiety, which is involved in resistance to the bactericidal activity of human serum in one strain of M. catarrhalis (54). A recent investigation found that the loss of this same epitope decreased the recognition of bactericidal antibodies produced in mice against an M. catarrhalis conjugate vaccine (53). Our data suggest that some patients develop more antibodies to the terminal regions of M. catarrhalis LOS following infection; thus, the ␣-D-Galp- …”

“…Serotypes A and B are the predominant glycoforms expressed by most clinical isolates analyzed to date (12,46). In recently reported animal studies, other researchers suggested that detoxified M. catarrhalis LOS has potential as a vaccine antigen in a mouse pulmonary clearance model (16,19,52,53). While these data are both valuable and interesting, it is sometimes difficult to link observations of animals to those of humans (34).…”