No abstract

SummaryWe have identified a gene, lpt-3, that is required for the addition of phosphoethanolamine to the 3-position (PEtn-3) on the b-chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn-3 substituent is characteristic of the LPS of a majority (ª 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface-accessible epitope. All strains of Nm that have PEtn-3 possess the lpt-3 gene. In some lpt-3-containing strains, the 3-position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt-3 resulted in loss of PEtn-3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt-3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a L9 (Jennings et al., 1983) have been elucidated. There has been significant success in identifying genes involved in LPS biosynthesis, including lgtA, lgtB, lgtE (Jennings et al., 1995a), lgtC (Gotschlich, 1994), rfaC (Stojiljkovic et al., 1997), lgtF, rfaK (Kahler et al., 1996a,b), rfaF (Jennings et al., 1995b) and lgtG (Banerjee et al., 1998). A characteristic of meningococcal LPS is reversible, highfrequency phase variation of its outer core structures mediated by slippage-like mechanisms in homopolymeric DNA tracts present in LPS biosynthetic genes . These homopolymeric tracts are absent in inner core LPS biosynthetic genes such as rfaC, rfaK, rfaF and lgtF, making this structure relatively stable and an attractive candidate for incorporation into a vaccine.In an attempt to investigate LPS epitopes for potential inclusion into a vaccine against serogroup B meningococcal disease, we have previously identified an inner core LPS epitope defined by a monoclonal antibody designated B5 (mAb B5). The cognate epitope of mAb B5 was found to be present on 70% of all Nm strains (Plested et al., 1999). mAb B5 recognises LPS inner core structures that contain phosphoethanolamine (PEtn) attached specifically at the 3-position (PEtn-3) on the b-chain heptose (HepII). L1, L3, L7, L8 and L9 LPS immunotypes of Nm containing PEtn-3 were mAb B5 reactive (B5+), whereas those containing PEtn in an exocyclic position (L2, L4 and L6 immunotypes) or glycoforms that completely lack PEtn (L5 immunotype) were mAb B5 nonreactive (B5-).Recent studies have revealed that antibodies specific for the mAb B5+ epitope are present in sera from infants recovering from invasive meningococcal disease (Plested et al., 2000). These antibodies exhibited functional activity towards meningococci in an in vitro opsonophagocytic (OP) assay . Taken together, these findings indicate that the mAb B5+ epitope is a target for protective antibodies, and that the inner core...

A T cell-dependent immune response to group C meningococcal capsular polysaccharide (CPS) can be elicited when CPS is conjugated to the class 3 neisserial porin (CPS-porin). Treatment of CPS-porin-immunized mice with B7-2 blocking monoclonal antibody (MAb) caused a dramatic reduction in the CPS-specific IgG response, treatment with anti-B7-1 MAb had no effect, and concurrent blockade of B7-1 and B7-2 resulted in a synergistic abrogation of the CPS-specific IgG response while the CPS IgM response was unaffected. Anti-CD40L MAb treatment caused a significant reduction of both CPS-specific IgG and IgM levels. In contrast, blockade of CTLA4 interactions resulted in increases in both CPS IgG and IgM responses in CPS-porin-immunized mice. These data support the hypothesis that the ability of neisserial porins to improve the immune response to poorly immunogenic antigens (e.g., polysaccharides) is related to porin-induced increases in B7-2 expression on antigen-presenting cells and enhanced B/T cell interactions.

A recently described flow cytometric opsonophagocytic assay (OPA) was adapted to quantify the functional activity of serum antibodies specifically directed against serogroup B inner core lipopolysaccharide (LPS) of Neisseria meningitidis. The percentage of human peripheral polymorphonuclear leukocytes and monocytes (PMNms) ingesting fluorescently labeled, ethanol-fixed N. meningitidis organisms (phagocytic activity) in the presence of human sera was measured to reflect the serum opsonic activity against the bacterium. The contribution to opsonophagocytic activity of antibodies to inner core LPS was estimated by comparing the opsonic activities of adult and infant sera before and after adsorbing anti-LPS antibodies from the sera using purified LPS extracted from an LPS mutant (galE) of N. meningitidis strain MC58 (B:15:P1.7,16:L3). The specificity of the assay was further investigated using monoclonal antibody (MAb) B5, which binds to an inner core LPS epitope of N. meningitidis. A dose-dependent decrease in phagocytic activity was observed when MAb B5 was incubated with LPS from an inner core LPS (galE) mutant. Similarly, the number of PMNms ingesting fluorescently labeled polystyrene beads coated with inner core (galE) LPS decreased in a dose-dependent fashion when MAb B5 was incubated with various concentrations of the homologous inner core LPS. Strong correlations were found between the concentration of serum antibodies to inner core LPS (galE) versus the phagocytic activity using healthy adult sera (r 2 ‫؍‬ 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r 2 ‫؍‬ 0.99) using polystyrene beads coated with inner core LPS and opsonized with the same sera using the oxidative burst indicator system dihydrorhodamine123/rhodamine 123. OPA results were also found to correlate closely with the results of the serum bactericidal assay using MAb B5 against the N. meningitidis MC58 galE mutant in the presence of human complement (r 2 ‫؍‬ 0.994, P ‫؍‬ 0.003, two-tailed test). These studies demonstrate that functional antibodies are produced in humans against meningococcal inner core LPS and that the OPA is a useful approach to study the opsonic activity of antibodies to inner core LPS in health and disease.