ABSTRACTThe binding of complement factor H (fH) to meningococci was recently found to be specific for human fH. Therefore, passive protective antibody activity measured in animal models of meningococcal bacteremia may overestimate protection in humans, since in the absence of bound fH, complement activation is not downregulated. We developed an ex vivo model of meningococcal bacteremia using nonimmune human blood to measure the passive protective activity of stored sera from 36 adults who had been immunized with an investigational meningococcal multicomponent recombinant protein vaccine. Before immunization, human complement-mediated serum bactericidal activity (SBA) titers of ≥1:4 against group B strains H44/76, NZ98/254, and S3032 were present in 19, 11, and 8% of subjects, respectively; these proportions increased to 97, 22, and 36%, respectively, 1 month after dose 3 (P< 0.01 for H44/76 and S3032). Against the two SBA-resistant strains, NZ98/254 and S3032, passive protective titers of ≥1:4 were present in 11 and 42% of sera before immunization, respectively, and these proportions increased to 61 and 94% after immunization (P< 0.001 for each strain). Most of the sera with SBA titers of <1:4 and passive protective activity showed a level of killing in the whole-blood assay (>1 to 2 log10decreases in CFU/ml during a 90-min incubation) similar to that of sera with SBA titers of ≥1:4. In conclusion, passive protective activity was 2.6- to 2.8-fold more frequent than SBA after immunization. The ability of SBA-negative sera to killNeisseria meningitidisin human blood where fH is bound to the bacteria provides further evidence that SBA titers of ≥1:4 measured with human complement may underestimate meningococcal immunity.

Sera from healthy infants (under 1 year old), toddlers (3–4 years) and adults (18–65 years) were assayed for their ability to bind to inner core (ic) lipopolysaccharide (LPS) epitopes of Neisseria meningitidis. Antibodies (Abs) reacting to inner core structures, including different substitutions of the first heptose (HepI) and second heptose (HepII) residues of the LPS backbone, truncated and fully extended LPS glycoforms, were detected and for each structure, these inner core antibodies showed an age-related pattern of acquisition. A novel column-based methodology was used to affinity purify IgG antibodies in which purified inner core LPS (derived from a mutant MC58) was covalently linked to Sepharose 4B. Comparison of reactivity before and after affinity purification of the pooled sera showed that the purified Abs bound to the surface of N. meningitidis organisms displaying truncated and extended LPS with a homologous inner core region, promoted the deposition of C3b, were opsonophagocytic in vitro and decreased bacteraemia when used to passively protect infants rats. In addition, the purified Abs were bactericidal in vitro against the mutant strain displaying truncated LPS with a homologous inner core region. These results demonstrate that naturally occurring serum human antibodies to N. meningitidis LPS can access inner core epitopes of encapsulated organisms with a fully extended LPS.

ABSTRACT The role of opsonophagocytosis (OP) in protection against meningococcal disease is controversial because patients with deficiencies in terminal complement proteins whose sera support OP but not bactericidal activity (BA) are at greatly increased risk of disease. We assayed complement-mediated BA and OP bactericidal activity in sera from 32 adults immunized with an outer membrane vesicle vaccine given alone or combined with an investigational recombinant protein, genome-derived neisserial antigen (GNA2132). The sera were heat inactivated to remove internal complement activity, and BA was measured with exogenous nonimmune human serum as a complement source. OP was measured with human polymorphonuclear cells (PMNs) and C6-depleted complement, which without PMNs did not support BA. Before immunization, 9 to 19% of sera from subjects in both vaccine groups combined had BA titers of ≥1:4, which increased to 41 to 72% after immunization (P < 0.01 against each of three test strains). The percentages of sera with OP titers of ≥1:5 were 3 to 16%, which increased to 55 to 72% (P < 0.001 for each strain). Most postimmunization BA-positive sera were OP positive, but 10 to 37% of BA-negative sera also were OP positive. Comparing the two vaccine groups, there were no significant differences in the percentages of sera with BA or OP activity except for a higher percentage of OP against one strain in postimmunization sera from subjects in the combination vaccine group (P ≤ 0.02). The data support independent roles for serum BA and OP bactericidal activity in protection against group B disease.

Glycoconjugates were prepared by covalently linking the immunogenic protein carrier CRM197 to O-deacylated lipopolysaccharide (LPS) derived from Neisseria meningitidis (strain H44/76), immunotype L3 galE LPS. This mutant strain elaborates a truncated LPS structure that displays immunological epitopes characteristic of 76% of Group B meningococcal (NmB) strains. CRM197 was covalently linked either to the reducing glucosamine residue of the lipid A region of the O-deacylated LPS or to a 2-keto-3-deoxy-octulosonic acid (Kdo) residue in the inner core region of the O-deacylated LPS. In both rabbits and mice a much stronger IgG response to the immunising antigen was generated in those animals that received conjugates linked via the lipid A region. Sera from mice that were immunized with these conjugates were assayed for their reactivity with LPS, both mutant and wild-type, of several homologous and heterologous NmB strains. Sera obtained from mice immunized with conjugates in which the carrier protein was linked via the Kdo moiety were only able to react with O-deacylated, but not fully acylated (native), LPS from the homologous strain. However, sera obtained from mice that were immunized with conjugates, in which the carrier protein was coupled to the lipid A region, reacted predominately with inner core epitopes that contained phosphoethanolamine at the same 3-position of the distal heptose residue (HepII) of the inner core LPS as was present on the immunising antigen. Additionally it was observed that sera from rabbits immunised with lipid A linked conjugates, unlike the mice responses, were generally not as specific for LPS antigens that contained phosphoethanolamine at the same 3-position as was present on the immunising antigen, but showed a broader inner core recognition, whereas those rabbits that received the Kdo-linked conjugates gave only a very weak non-specific response to all immunotypes. Finally, the sera from two out of six mice that had received lipid A linked conjugates had bactericidal activity against L3 wild-type NmB strain 8047 and one of these was able to passively protect against meningococcal infection in an infant rat model. This study demonstrates evidence towards the proof-in-principle that by using Nm inner core LPS conjugates coupled via the lipid A region with an intact phosphoethanolamine at the O-3 position of the HepII of the inner core LPS, it is possible to elicit functional and protective antibodies against meningococcal infection.

A panel of six murine monoclonal antibodies (MAbs) recognizing inner core lipopolysaccharide (LPS) epitopes of Neisseria meningitidis was prepared and characterized in order to determine the diversity of inner core LPS glycoforms among disease and carrier isolates. Two of these MAbs, L2-16 (immunoglobulin G2b [IgG2b]) and LPT3-1 (IgG2a), together with a third, previously described MAb, L3B5 (IgG3), showed reactivity, either individually or in combination, with all except 3 of 143 disease and carriage isolates (125 of 126 strains from blood, cerebrospinal fluid, or skin biopsy samples and 15 of 17 from nasopharyngeal cultures). MAbs L3B5, L2-16, and LPT3-1 were further characterized in an indirect immunofluorescence assay. All three MAbs bound to the bacterial cell surface, findings that correlated strongly with whole-cell enzyme-linked immunosorbent assay and immunodot blots. However, in contrast to our findings with L3B5, cell surface binding of L2-16 or LPT 3-1 did not correlate with functional activity as determined by bactericidal or infant rat passive protection assays against wild-type N. meningitidis strains. These findings are provocative with respect to the requirements for protective activity of antibodies and the development of inner core LPS vaccines against invasive meningococcal disease.

Inner-core lipopolysaccharide (LPS) from Neisseria meningitidis is under investigation as a vaccine for prevention of meningococcal disease caused by N. meningitidis serogroup B (NmB). We investigated the functional activity of murine monoclonal antibody (MAb) B5 that recognizes a highly conserved (galE) LPS epitope. Three patterns of MAb reactivity were observed in N. meningitidis by Western blot, depending on the relative prevalence of sialylated, nonsialylated, and/or truncated LPS glycoforms. Three representative N. meningitidis strains (8047, M986, and 2996) were investigated with MAb B5 in functional assays in vitro and in vivo. MAb B5 completely protected infant rats against bacteremia caused by 8047, partially protected against 2996, and had no protective activity against M986. Thus, an inner-core LPS epitope can be a target for protective immunity, but the affinity of MAb B5 may only be sufficient to mediate protection against NmB strains possessing at least some truncated glycoforms.

We identified Neisseria meningitidis lipooligosaccharide (LOS) as an acceptor for complement component C4b (C4b). Phosphoethanolamine (PEA) residues on the second heptose (HepII) residue in the LOS core structure formed amide linkages with C4b. PEA at the 6-position of HepII (6-PEA) was more efficient than 3-PEA in binding C4b. Strains bearing 6-PEA bound more C4b than strains with 3-PEA and were more susceptible to complement-mediated killing in serum bactericidal assays. Deleting 3-PEA from a strain that expressed both 3- and 6-PEA simultaneously on HepII did not decrease C4b binding. Glycose chain extension of the first heptose residue (HepI) influenced the nature of the C4b-LOS linkage. Predominantly ester C4b-LOS bonds were seen when lacto-N-neotetraose formed the terminus of the glycose chain extension of HepI with 3-PEA on HepII in the LOS core. Related LOS species with more truncated chain extensions from HepI bound C4b via amide linkages to 3-PEA on HepII. However, 6-PEA in the LOS core bound C4b even when the glycose chain from HepI bore lacto-N-neotetraose at the terminus. The C4A isoform exclusively formed amide linkages, whereas C4B bound meningococci preferentially via ester linkages. These data may serve to explain the preponderance of 3-PEA-bearing meningococci among clinical isolates, because 6-PEA enhances C4b binding that may facilitate clearance of 6-PEA-bearing strains resulting from enhanced serum killing by the classical pathway of complement.

We describe the development of a flow-cytometric-based opsonophagocytosis (OP) assay that is capable of looking at antigen-specific antibodies, that is, complement-dependent, not complement-mediated. Using the OP assay we are able to look at uptake of bacteria by neutrophils simultaneously with oxidative burst within the cell. This assay using human donor peripheral blood polymorphonuclear cells (PMNs) is rapid, reproducible, and specific. Other OP assays described use HL-60 cell lines that are technically more difficult to use and require tissue culture facilities ().

The structure of the lipopolysaccharide (LPS) from three Neisseria meningitidis strains was elucidated. These strains were nonreactive with mAbs that recognize common inner-core epitopes from meningococcal LPS. It is well established that the inner core of meningococcal LPS consists of a diheptosyl-N -acetylglucosamine unit, in which the distal heptose unit (Hep II) can carry P Etn at the 3 or 6 position or not at all, and the proximal heptose residue (Hep I) is substituted at the 4 position by a glucose residue. Additional substitution at the 3 position of Hep II with a glucose residue is also a common structural feature in some strains. The structures of the O-deacylated LPSs and core oligosaccharides of the three chosen strains were deduced by a combination of monosaccharide analysis, NMR spectroscopy and MS. These analyses revealed the presence of a structure not previously identified in meningococcal LPS, in which an additional beta-configured glucose residue was found to substitute Hep I at the 2 position. This provided the structural basis for the nonreactivity of LPS with these mAbs. The determination of this novel structural feature identified a further degree of variability within the inner-core oligosaccharide of meningococcal LPS which may contribute to the interaction of meningococcal strains with their host.

Lipopolysaccharide (LPS) is important for the virulence of Neisseria meningitidis, and is the target of immune responses. We took advantage of a monoclonal antibody (Mab B5) that recognises phosphoethanolamine (PEtn) attached to the inner core of meningococcal LPS to identify genes required for the addition of PEtn to LPS. Insertional mutants that lost Mab B5 reactivity were isolated and characterised, but failed to yield genes directly responsible for PEtn substitution. Subsequent genetic linkage analysis was used to define a region of DNA containing a single intact open reading frame which is sufficient to confer B5 reactivity to a B5 negative meningococcal isolate. The results provide an initial characterisation of the genetic basis of a key, immunodominant epitope of meningococcal LPS.

ABSTRACT Macrophages (Mφ) play a key role in the pathogenesis of invasive meningococcal infections. The roles of two pattern recognition molecules, the Mφ scavenger receptor (SR-A) and Toll-like receptor 4 (TLR-4), have been investigated using bone marrow culture-derived Mφ (BMMφ). Surprisingly, a comparison of BMMφ from wild-type and SR-A knockout (SR-A −/− ) mice showed that nonopsonic phagocytosis of meningococci was mediated almost exclusively via SR-A. Previous studies have demonstrated only a partial involvement of the receptor in the uptake of other bacteria, such as Escherichia coli . Interestingly, we also show that lipopolysaccharide (LPS) was not the ligand for the receptor on these organisms. Further study of the downstream events of SR-A-mediated ingestion of Neisseria meningitidis demonstrated that SR-A was not required for cytokine production. To determine the bacterial and host factors required to stimulate Mφ activation, we examined TLR-4-deficient Mφ from C3H/HeJ mice and LPS-deficient meningococci. TLR-4-deficient cells elaborated reduced amounts of tumor necrosis factor alpha, interleukin-12 (IL-12), and IL-10, even though ingestion via SR-A was unaffected in these cells. Similarly, although there was no change in SR-A-mediated ingestion of LPS-deficient meningococci, the mutant failed to stimulate a Mφ-dependent cytokine response. Thus, we show that Mφ SR-A mediates opsonin-independent uptake of N. meningitidis independently of lipid A and that this activity is uncoupled from the Mφ secretion of proinflammatory cytokines, which provides a basis for further investigation of the role of this receptor in meningococcal disease in humans.

SummaryWe have identified a gene, lpt‐3, that is required for the addition of phosphoethanolamine to the 3‐position (PEtn‐3) on the β‐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 (≈ 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 candidate immunogen for inclusion in an LPS‐based vaccine to protect against invasive meningococcal disease.

ABSTRACTA 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) ofNeisseria meningitidis. The percentage of human peripheral polymorphonuclear leukocytes and monocytes (PMNms) ingesting fluorescently labeled, ethanol-fixedN. meningitidisorganisms (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) ofN. meningitidisstrain 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 ofN. 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 (r2= 0.89). There was a correlation between phagocytic ingestion and initiation of intracellular oxidative burst (r2= 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 theN. meningitidisMC58galEmutant in the presence of human complement (r2= 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.

We have developed a solid-phase ELISA to study the human immune response to inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm) using structurally defined glycolipids from a genetically defined mutant (galE) of a serogroup B Nm strain. Previous studies had demonstrated that a galE (inner core) LPS epitope is conserved in ∼70% Nm strains and was accessible to antibody in fully encapsulated wild-type Nm strains. A murine monoclonal antibody, MAb B5, raised to a galE mutant of serogroup B Nm strain, immunotype L3 (B.15.P1.7,16) was used to determine the specificity of the inner core LPS ELISA by inhibition studies using purified galE LPS and human sera. The intra-assay coefficient of variation (CV) was 5–6% and inter-assay CV was 19–22%. Using this ELISA, significant differences in the geometric mean titres (GMTs) of naturally occuring serum antibodies (specific to inner core LPS) between healthy adults (18–65 years, N=54) and healthy infants (3–4 months, N=144) of both IgG and IgM classes were found (P<0.0001). GMTs were expressed in galE arbitrary units (AU/ml) (95% confidence intervals): IgG antibodies in adults 5.7 (5.0,6.9) and in infants 1.1 (1.0,1.3); IgM antibodies in adults 7.7 (5.7,10.4), and in infants 0.85 (0.7,1.1). In age-matched children aged 26–113 months a difference (P=0.04) in specific IgG was found in healthy infants and infants in the acute phase of invasive Nm disease (GMT (95%CI) in AU/ml: in healthy infants 7.7 (5.3,11.0), in acute phase infants 4.2 (2.5,7.2). However, there was no difference in specific IgM (P=0.98) between these groups healthy infants 4.7 (3.1,7.0), acute phase 4.6 (2.9, 7.4). In eleven children (5–181 months) there were differences in the GMTs of specific IgG and IgM (P=0.02, P=0.008 respectively) between paired acute and convalescent sera (GMT) (95%CI) in AU/ml: IgG acute 1.95 (0.98, 3.8), convalescent 5.2 (2.2,12.4); IgM acute 1.78 (1.05,3.0), convalescent 4.38 (2.6,7.3). We conclude that ELISA is a specific, sensitive and reproducible method for the detection of antibodies to inner core LPS of Nm and that an epitope defined by MAb B5 can be immunogenic in infants and adults. These findings are relevant to the potential candidacy of inner core LPS as a vaccine.