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

Abstract

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.

Investigating the candidacy of lipopolysaccharide-based glycoconjugates as vaccines to combat Mannheimia haemolytica

“…The identification of two colony morphologies produced by only the ovine strain on exposure to serum or complement was perplexing and possibly affected the ability to obtain antibody-mediated bactericidal killing of this strain. We also observed a similar immune response to the linker molecules in our previous studies with LPS based vaccines for both N. meningitidis [31] and M. catarrhalis [22], and an ability to facilitate bactericidal killing of wild-type Moraxella strains, similar to that achieved in this study, was observed even in the presence of a response to the linkers, however, that was not the case with sera raised to meningococcal conjugates as only homologous strains were killed. Clearly the presence of an immune response to the linker molecules is not beneficial, but in the case of M. haemolytica and M. catarrhalis did not preclude facilitation of bactericidal killing.…”

“…We originally developed this methodology to prepare conjugates with N. meningitidis inner core LPS [29] and subsequently with M. catarrhalis [22], and achieved a similar high loading illustrating the reproducibility of this strategy. The loading achieved can be qualitatively and quantitatively characterised by SDS-PAGE/Western and MALDI MS techniques, respectively.…”

“…Similarly, we and others have derived polyclonal sera following immunisations in rabbits with glycoconjugates based on a conserved inner core LPS structure from Moraxella catarrhalis that were broadly cross-reactive and able to facilitate bactericidal killing [22,23]. In this study we confirm and extend the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. haemolytica wild-type strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen found in several veterinary species.…”

An Unusual Carbohydrate Conformation is Evident in Moraxella catarrhalis Oligosaccharides

“…The (1)(2)(3)(4)(5) glycosidic linkage between residue A and Kdo is the only linkage that is consistent between the two structures (lgt1/4Δ and lgt2Δ OS). This is interesting as each linkage that underwent a conformational change caused a corresponding variation in the The occurrence of a significant conformational change between a truncated and extended OS structure may have important implications on the strategy utilized to design carbohydrate vaccines composed of truncated OS that, although they may contain the core residues common to many strains, may not necessary possess the same three-dimensional structure as a wild type OS.…”

“…The lipooligosaccharide (LOS) component of the outer membrane of M. catarrhalis is a unique and complex class of glycolipid that is important in bacterial infection and represents an attractive vaccine target [3][4][5][6]. These amphiphilic molecules consist of a hydrophobic component, termed lipid A, that anchors the molecule into the outer membrane, and a hydrophilic oligosaccharide (OS) component.…”