|Home | About | Journals | Submit | Contact Us | Français|
Analysis of the specificity of bactericidal antibodies in normal, convalescent, and postvaccination human sera is important in understanding human immunity to meningococcal infections and can aid in the design of an effective group B vaccine. A collection of human sera, including group C and group B convalescent-phase sera, normal sera with naturally occurring cross-reactive bactericidal activity, and some postvaccination sera, was analyzed to determine the specificity of cross-reactive bactericidal antibodies. Analysis of human sera using a bactericidal antibody depletion assay demonstrated that a significant portion of the bactericidal activity could be removed by purified lipopolysaccharide (LPS). LPS homologous to that expressed on the bactericidal test strain was most effective, but partial depletion by heterologous LPS suggested the presence of antibodies with various degrees of cross-reactivity. Binding of anti-L3,7 LPS bactericidal antibodies was affected by modification of the core structure, suggesting that these functional antibodies recognized epitopes consisting of both core structures and lacto-N-neotetraose (LNnT). When the target strain was grown with 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA) to increase LPS sialylation, convalescent-phase serum bactericidal titers were decreased by only 2- to 4-fold, and most remaining bactericidal activity was still depleted by LPS. Highly sialylated LPS was ineffective in depleting bactericidal antibodies. We conclude that natural infections caused by strains expressing L3,7 LPS induce persistent, protective bactericidal antibodies and appear to be directed against nonsialylated bacterial epitopes. Additionally, subsets of these bactericidal antibodies are cross-reactive, binding to several different LPS immunotypes, which is a useful characteristic for an effective group B meningococcal vaccine antigen.
Neisseria meningitidis is a Gram-negative bacterium utterly adapted to the human host. The species is highly variable in antigenic types and expression of surface antigens, including outer membrane proteins (OMPs) and polysaccharide capsules. Yet only A, B, C, X, Y, and W135 capsular serogroups (of 13 total) are considered pathogenic for healthy humans. Most people experience N. meningitidis through benign colonization of the nasopharynx starting in childhood with a series of antigenically distinct strains. Thereafter, many adults develop protective immunity mainly due to increased bactericidal antibody titers to surface antigens, though opsonic antibodies can also contribute to protection (18, 43). For the licensed vaccines (against A, C, Y, and W135), a bactericidal titer of 1:4 measured with human complement has been established as the standard correlate of protective immunity (18). Those individuals, especially those <5 and 16 to 21 years of age, who encounter virulent N. meningitidis without protective immunity (acquired by nasopharyngeal colonization or immunization) can rapidly develop sepsis or meningitis, which is sometimes fatal, or may result in developing serious sequelae, such as tissue necrosis leading to amputations, permanent neurologic, or muscular damage.
The lipopolysaccharide (LPS) of Gram-negative bacteria, including N. meningitidis, is an abundant and consistently expressed outer membrane component. As such, it might be expected to play an important role in the immunology and the pathology of the organism. N. meningitidis LPS is known to have potent endotoxin activity and to be responsible for much of the pathology associated with systemic infections (7). When sialylated, it has been reported to be a virulence factor (51, 53). Structurally the LPS of Neisseria does not have the O side chain that is associated with the LPSs of many Gram-negative bacteria and is therefore often referred to as lipooligosaccharide (LOS) since the “polysaccharide” part consists only of short branched oligosaccharides consisting of 7 to 12 sugar residues. In this paper we use the traditional term lipopolysaccharide.
The role of the LPS in human immunity to meningococcal disease has received less attention than its role in pathogenesis. As an antigen, the LPS is known to exhibit antigenic variation from strain to strain within a serogroup. This occurs both as a result of differences in the repertoire of biosynthetic genes possessed by the strain (26, 60) and phase variation in expression of the genes that are present (4). Twelve different LPS immunotypes, L1 to L12, were initially identified using a set of polyvalent rabbit sera (36, 64). One, L12, was not identified on strains other than the prototype strain and may represent a spontaneous mutant. Three immunotypes (L9, L10, and L11) were associated mostly with serogroup A strains, and the other eight were associated with all other serogroups. These immunotypes were subsequently confirmed by structural analysis of the respective oligosaccharides (9, 16, 20, 24, 29, 37, 38, 42). Since that time, it has become evident that additional variations in structure, not identified by the initial immunotyping scheme, such as the substitution of glycine at the 7 position of Hep II, occur among N. meningitidis strains (25, 44).
Interest in LPS as a potential vaccine antigen has been somewhat limited due to its toxicity and the observation that most LPS immunotypes expressed by meningococcal case isolates contain the tetrasaccharide lacto-N-neotetraose (LNnT), which is also found on human blood cells (34), where it is usually capped with sialic acid or other sugars. The presence of this tetrasaccharide both on human cells and in meningococcal LPS raises the possibility of inducing autoimmunity with a LPS-based vaccine containing the LNnT group. Nevertheless, autoimmune disorders based on this shared antigen have not been reported in association with natural meningococcal infections or following vaccination with meningococcal vaccines containing LPS with LNnT. It is likely that the immunogenicity of meningococcal LPS that contains LNnT is reduced for epitopes that cross-react with human blood cells, but there are enough unique epitopes on the LPS for induction of a protective immune response without inducing autoimmunity. Since most LNnT on human blood cells is sialylated, the LPS structures containing sialylated LNnT may be expected to be the least immunogenic.
The presence of bactericidal antibodies to meningococcal LPS in human sera has been demonstrated by several investigators (13, 19, 23, 54). These antibodies are typically specific for the wild-type LPS that contains the LNnT. However, the fine specificity of these antibodies and the role they play in cross-reactive protection have not been fully explored. The fact that the LPS structures are subject to phase variation by multiple biosynthetic genes turning on and off suggests that LPS variation may well play a role in immune evasion (1).
In this study, we have used a bactericidal antibody depletion assay to demonstrate the presence and often the dominance of anti-LPS bactericidal antibodies in the cross-reactive bactericidal activity of sera from naturally immune and vaccinated individuals. Our data suggest that these antibodies persist over relatively long periods of time and play an important role in natural immunity.
Convalescent-phase sera were obtained by attending physicians in the course of treatment and follow-up of cases of group B and group C meningococcal disease that occurred in U.S. military personnel and dependents over the past 15 to 20 years. The sera were rendered anonymous by assigning code numbers not linkable to the donors.
Postvaccination sera from several different vaccine studies were analyzed. A small number of pre- and postvaccination sera obtained as part of a large efficacy trial that was conducted in Iquique, Chile, between 1987 and 1989 were analyzed (6). The trial was a double-blind controlled study involving subjects 1 to 21 years of age. The experimental vaccine consisted of purified outer membrane proteins (less than 1% LPS) from a strain typical of the epidemic clone, 8529(B:15:P1.7-2,3:L3,7), noncovalently complexed to group C capsular polysaccharide. The proteins were extracted from the outer membrane, purified with buffer containing Empigen BB (Albright and Wilson, Ltd., Whitehaven, United Kingdom), and purified by gel filtration to remove low-molecular-weight proteins, such as the class 5 proteins, leaving multimeric aggregates (61). The control vaccine was the commercially available tetravalent meningococcal A, C, Y, and W135 polysaccharide vaccine (Menomune; Sanofi Pasteur, then Connaught Laboratories, Inc., Swiftwater, PA), which was essentially free of LPS and protein. Postvaccination sera from a clinical trial of an intranasal vaccine consisting of native outer membrane vesicles (NOMV) from a synX (disruption, capsule-negative) mutant of strain 9162(B:15:P1.7-2,3:L3,7) were included in the analysis (12). Sera from a clinical study of a vaccine consisting of approximately equal amounts of purified, detoxified (de-O-acylated) L8-5 LPS and purified outer membrane proteins from strain 9162 incorporated into liposomes (2) were also analyzed. A pooled sample of postvaccination serum was also analyzed; the 8-week postvaccination sera were pooled from five subjects immunized with an experimental vaccine consisting of about equal amounts of purified, detoxified (de-O-acetylated) L3,7 LPS noncovalently complexed to purified outer membrane proteins from two group B strains, H44/76(B:15:P1.7,16:L3,7) and 8047(B:2b:P1.5,2:L3,4,7) (65). Also, adult normal human sera were obtained from individuals who were excluded from participation in clinical studies of experimental group B vaccines due to preexisting high bactericidal titers against the vaccine strain.
The use of human sera was done under an Institutional Review Board-approved human use protocol. Informed consent was obtained from all individuals, and prior to use in this study, the sera were codified to render them anonymous.
The following strains of N. meningitidis were used in the study as bactericidal test strains, as vaccine strains, or as sources of antigens used in the depletion studies: 8532(B:15:P1.7-2,3:L3,7,8) or phase variants expressing L8-5 or L8; 9162(B:P15:P1.7-2,3:L3,7); 8047(B:2b:P1.5,2:L3,4,7); H44/76(B:15:P1.7,16:L3,7); 6275(B:2a:P1.5,2:L3,7); 8529(B:15:P1.7-2,3:L3,7); 8570(B:4:P1.19,15:L3-5,7-5); 8900(B:15:P1.7-2,3:L3,7); B16B6(B:2a:P1.5,2:L2); 126E(C:8,19:P1.5,2:L1); and 99M(B:2a:P1.5,2:L3,7). Typing was done by dot blotting using monoclonal antibodies; therefore, the older strain designations are used. The test strain typing was recently reconfirmed to ensure that surface antigens, including PorA and PorB, were present at normal levels. The typing scheme is defined as follows, (serogroup: serotype: subtype: immunotype), corresponding to the following surface antigens, (capsule: PorB: PorA: LPS). We did not perform multilocus sequence typing (MLST) or other genetic analyses on these strains. The traditional typing designation (15) includes the LPS immunotype, which is critical for this study. The strains were obtained mostly from the strain collection of the Walter Reed Army Institute of Research. Strain H44/76 was obtained from the Norwegian Institute of Public Health, and strain B16B6 was obtained from the Center for Biologics Evaluation and Research, FDA. The PorA-deficient mutant of strain 8529 was isolated as a spontaneous mutant, identified by colony blotting with a PorA-specific monoclonal antibody.
Monoclonal antibodies 9-1-L379, 9-2-L379, 12-1-L379, 2-1-L8, and 25-1-LC1 to meningococcal LPS were prepared in our laboratory. The monoclonal antibody 1B2-1B7 was obtained from the American Type Culture Collection (Bethesda, MD).
NOMV were isolated by nondetergent extraction from pelleted cells that were briefly warmed to 56°C, exposed to hypotonic buffer, and sheared to increase the yield of vesicles as described previously (46, 67). LPS was purified by the phenol-water method of Westphal et al. (55). When used as an antigen in the bactericidal depletion assay, it was noncovalently complexed to an equal weight of fatty acid free bovine serum albumin to aid in binding to the test plate and in optimal presentation for binding of antibodies (12). Conjugated LNnT and sialylated LNnT were obtained from IsoSep AB (Tullinge, Sweden).
The bactericidal assay and bactericidal depletion assay were performed as previously described (66) using normal human sera lacking bactericidal activity toward the test strain as a source of complement. The bactericidal titer was reported as the highest tested dilution of serum (in a 2-fold dilution series) that resulted in 50% or greater reduction in viable meningococci (compared to the heat-destroyed complement control). For the bactericidal depletion assay, dilution series of one or more antigens to be tested were bound to wells of a microtiter plate, blocked, and used to specifically remove bactericidal antibodies from a serum diluted to the 50 to 60% kill point. The previously determined bactericidal titer was used to estimate the dilution resulting in 50 to 60% killing. After incubation of the diluted sera in wells of the antigen-coated plate, the sera were transferred to a clean plate, and residual bactericidal activity in the diluted sera was determined by addition of complement and bacterial cells. The percentage of the original bactericidal activity removed from each diluted serum was taken as the percent depletion. Protein concentration was determined by the method of Lowry et al. (33), and LPS concentration was determined based on dry weight or by the 2-keto-3-deoxyoctulosonic acid (KDO) method of Karkhanis et al. (27) using KDO and intact meningococcal LPS (dry weight basis) as standards.
The mean percentage of bactericidal activity removed by a particular antigen was averaged for groups of similar sera and standard deviation, standard error, and/or the range of the values in the group determined. Depletion curves could not routinely be done in duplicate or triplicate because of the small amount of human serum available in most cases. An example of an experiment done in duplicate is given (see Fig. 4) and serves to provide an estimate of the reproducibility of the depletion curves.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining was performed by the method of Laemmli (32), and silver staining was performed by the method of Tsai and Frasch (50). Western blotting was performed by the method of Burnette (8) using sequential staining of blots with alkaline phosphatase-labeled second antibody with Fast Red TR/Naphthol AS MX and BCIP/NBT substrate systems and peroxidase-labeled second antibody with the 4CN membrane system (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
During the analysis of the bactericidal antibody responses to an experimental group B vaccine tested in an efficacy trial in Iquique, Chile (6), we observed that some children in the control group had preexisting bactericidal antibodies that appeared to persist over relatively long periods of time. Some other children that were vaccinated with the experimental vaccine appeared to acquire bactericidal antibodies 6 months or more after initial vaccination without receiving additional vaccine (Table 1). This antibody response was presumably induced as a consequence of exposure to N. meningitidis or other bacteria with cross-reacting antigens. The specificity of the bactericidal antibodies in these sera was investigated using the bactericidal depletion assay. A strain, 8532(B:15:P1.7-2,3:L3,7,8), isolated from a case of group B meningococcal disease that occurred in Iquique during the epidemic outbreak was used as the target strain. Native outer membrane vesicles (NOMV) homologous to the test strain (expressing either L3,7 or L8 LPS) as well as purified L3,7 and L1 LPS were used as solid-phase antigens to deplete bactericidal antibodies from the sera. An example of the depletion curves obtained with one of the analyzed sera (volunteer 145, 18-month serum sample, 145-18) is shown in Fig. 1. Two of the antigens were tested only at concentrations of 100 μg/ml and 50 μg/ml to give the maximum depletion values. A summary of the results for the nine sera analyzed is shown in Fig. 2. The homologous NOMV expressing L3,7 LPS were used as a positive control and removed 80 to 100% (mean, 91.9%) of the bactericidal activity. NOMV from a L8-expressing phase variant of the same strain were much less effective, removing only 10 to 50% of the bactericidal antibody from all sera except a control serum, 17-8, that had B vaccine-induced anti-protein bactericidal antibodies. Serum 17-8 had 96% of the bactericidal activity removed by the 8532 L8 NOMV, and purified LPS removed very little bactericidal antibody. These results would be expected for vaccine-induced bactericidal antibodies, as the experimental vaccine contained little LPS, if any. In the other eight sera containing naturally induced antibodies, purified L3,7 LPS was able to remove between 45% and 79% (mean, 65.5%) of the bactericidal antibodies (Fig. 2). L1 LPS was used as a negative control and removed very little of the bactericidal activity from any of the sera. Thus, on average, about two-thirds of the bactericidal activity in these sera was due to antibodies to the L3,7 LPS. Similar results were obtained using an alternative test strain, 9162(B:15:P1.7-2,3:L3,7), also from the Chile epidemic (data not shown).
Sera from healthy adults in the United States who had serum bactericidal activity at various titers toward different group B test strains were examined using the bactericidal depletion assay to determine how much of the bactericidal activity could be removed with purified LPS homologous to the test strain. Purified L1 LPS served as a negative control, and NOMV homologous to the test strain were the positive control. Twenty-two sera from different individuals were tested using the bactericidal test strain 9162 L3,7, and three sera were tested against one of two other strains. Since the carriage history of these individuals was unknown, the serum antibodies bactericidal for the test strain were most likely cross-reactive antibodies. The geometric mean titers and mean depletion of bactericidal activity by homologous NOMV and purified homologous LPS are shown in Table 2. As expected, the positive control NOMV homologous to the test strain reduced bactericidal killing by ≥89%. Purified homologous LPS reduced bactericidal killing by 40 to 100%, with a mean depletion of ≥70%, suggesting that the majority of the cross-reactive, bactericidal antibodies in the normal subjects were directed against LPS.
In the example experiment shown in Fig. 3, strain 9162(B:15:P1.7-2,3:L3,7) was killed by a normal human serum (HN026) which was first exposed to one of a series of different solid-phase antigens, including whole cells from strain 99M L3,7 grown on normal medium or in the same medium supplemented with 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA). NOMV from strain 8532 L3,7 were used as a positive control. This strain is very similar to the test strain (same epidemic clone) and was able to remove all of the bactericidal antibodies. Purified homologous LPS was able to remove about 60% of the bactericidal activity and a purified L4 LPS (LNnT alpha chain but different core) was able to remove about 30%. Highly sialylated 99M(B:2a:P1.5,2:L3) cells were not able to remove any activity, while 99M L3,7 cells grown on normal medium were able to remove about 65% of the bactericidal activity.
Convalescent-phase sera from patients with systemic disease caused by serogroup B and serogroup C meningococci were obtained from military installations over a period of several years. These sera were examined to determine the specificity of the cross-reactive bactericidal antibodies present. One group of sera was associated with a small focal outbreak of serogroup C disease at Fort Leonard Wood, MO, in 2002. Case isolates were obtained from most of these patients and serotyped using monoclonal antibodies. All the strains from this outbreak were found to be C:2a:P1.5,2:L3,7 (or L2). The sera were tested for bactericidal activity against a panel of serogroup B bactericidal test strains for the presence of cross-reactive bactericidal antibodies. The results (Table 3) showed a considerable amount of bactericidal activity against the group B strains of different serotype (PorB variant) and serosubtype (PorA variant). Remarkably, none of the sera except serum HCC007 showed bactericidal activity against B16B6, which shares the same serotype and serosubtype as the case isolate. The case isolate associated with serum HCC007 expressed an L2 LPS, which is also expressed by strain B16B6.
The sera were further analyzed using the bactericidal depletion assays. The bactericidal depletion assays were generally designed to facilitate analysis of cross-reactive bactericidal antibodies by use of group B test strains heterologous in serotype and serosubtype to the case isolate. In each of four convalescent-phase sera tested with several different test strains, 80 to 100% (mean, 95.4%) of the bactericidal activity was removed by purified LPS homologous to that expressed by the test strain (Table 4). Depletion curves were obtained in each case, and both positive and negative controls were included. An example of the depletion curves obtained with these sera is shown in Fig. 4. In this experiment, which was done in duplicate, strain 9162 L3,7 was killed with the convalescent-phase serum HCC010 used at a dilution of 1:200. The diluted serum was incubated with homologous NOMV or L3,7 LPS from the test strain or with NOMV from a PorA-negative L3,7 strain closely related to the test strain. About 95 to 100% of the bactericidal antibody was removed from the serum by the higher concentrations of purified 9162 L3,7 LPS or by NOMV from the test strain. NOMV from the PorA deletion mutant were also able to efficiently remove bactericidal antibodies, indicating that PorA-specific antibody did not contribute to the bactericidal activity.
In addition to these serogroup C case sera, four serogroup B case sera that were obtained from isolated cases of meningococcal disease were analyzed, with similar results. These four sera, for which corresponding case isolates were not available, were tested against strain 9162 L3,7. A mean of 88.5% of the bactericidal activity was depleted by purified L3,7 LPS compared to 97.3% depleted by the homologous NOMV (data not shown).
In past years, several different experimental vaccines were evaluated in phase 1 clinical studies. Three of these vaccines contained LPS as a vaccine component (2, 12, 65). Several postvaccination sera from each study were evaluated for the presence of LPS-specific bactericidal antibodies. Two of the vaccines contained detoxified (de-O-acetylated) LPS noncovalently complexed to purified outer membrane proteins and, in one case, incorporated into liposomes (2, 65). The third vaccine consisted of NOMV containing unmodified L3,7 LOS (about 25% relative to protein) and was given intranasally (12). The results of these analyses are shown in Table 5. No matter what formulation, on the average, about 60% of the bactericidal activity was removed by purified LPS homologous to the test strain, indicating that vaccine formulations containing LPS were capable of inducing a bactericidal antibody response to LPS.
The effectiveness of bactericidal antibodies to LPS in protection against disease may be affected by the degree of sialylation of the test strain. LPS sialylation of strains grown in the laboratory can vary widely from strain to strain. Since LPS sialylation in vivo may be relatively high as a consequence of endogenous sialyl transferase, the effect of high sialylation on bactericidal activity was investigated for monoclonal antibodies to LPS and for a number of human sera. To compare the ability of anti-LPS antibodies to kill test strains with high and moderate to low levels of LPS sialylation, a test strain was grown with or without addition of 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA) to the growth medium. First, the effect of added CMP-NANA at different concentrations on LPS sialylation was determined by examining the LPS expressed by the organisms by SDS-PAGE with silver staining (see Fig. S1 in the supplemental material). The presence of CMP-NANA at 1 μg/ml or greater in the growth medium resulted in most of the LPS becoming sialylated, as evidenced by the shift of the L7 band to a slightly slower-migrating position characteristic of L3 LPS.
Four human convalescent-phase sera that had been shown by bactericidal depletion tests to contain primarily bactericidal antibodies specific for LPS and four LPS-specific murine monoclonal antibodies were tested for bactericidal activity against meningococci grown with or without 20 μg/ml of CMP-NANA in the medium (Table 6). The results of these tests showed that growth in the presence of CMP-NANA (greater sialylation) generally made the strain more resistant to killing. However, the decrease in bactericidal activity for the highly sialylated strains varied considerably among the monoclonal antibodies. Those that were most specific for the LNnT group (1B2-1B7 and 12-1-L379) were most affected by sialylation, becoming essentially nonbactericidal toward the highly sialylated strains. Two other monoclonal antibodies showed about an 8-fold reduction in bactericidal titers against the highly sialylated strains. However, the effect of high sialylation on the bactericidal titers of the human convalescent-phase sera was less pronounced, showing a 0- to 4-fold reduction in bactericidal titers, suggesting that naturally acquired polyclonal immunity is less sensitive to sialylation of target meningococcal strains than the mouse monoclonal antibodies. The ability of anti-LPS antibodies in convalescent-phase sera to kill highly sialylated strains suggests that these antibodies would be effective in providing protection in vivo.
To distinguish between the effect of CMP-NANA to increase production of the polysialic acid capsule (known to increase the overall serum resistance) versus the effect of CMP-NANA to increase sialylation of the LPS, a murine monoclonal antibody with specificity for the PorA outer membrane protein was tested to determine its bactericidal titer against the test strain grown with and without added CMP-NANA. A 4-fold reduction in titer was observed (Table 7) when the strain was grown in the presence of CMP-NANA, indicating a nonspecific increase in the serum resistance of the strain rather than a direct effect due to increased sialylation of the target antigen. Thus, the reduction in titers shown in Table 6 likely resulted from both a nonspecific increase in serum resistance and, in some cases, reduced affinity for the sialylated LPS. The antibodies in the human convalescent-phase serum that were still bactericidal toward the strain grown on CMP-NANA were tested by bactericidal depletion assay to determine the percentage that could be removed by purified L3,7 LPS. A total of 75% of the bactericidal activity was removed, compared to 93% removed by homologous NOMV. Thus, anti-LPS antibodies were still responsible for most of the bactericidal activity of the human serum toward the highly sialylated strain, suggesting human anti-LPS antibodies could be protective in vivo.
To further explore the specificity of human anti-LPS antibodies, a human convalescent-phase serum (HCC010) was depleted with synthetic conjugates of LNnT and sialylated LNnT to determine the effect of sialylation on an antigen's ability to bind bactericidal antibodies (Fig. 5). Purified homologous LPS was able to remove the bactericidal activity as effectively as the homologous NOMV. The negative control L1 LPS was unable to remove any of the antibody, and NOMV from strain 8532 (same epidemic clone as 9162), which expressed L8 LPS, were somewhat less effective. Interestingly, the unsialylated conjugate of LNnT was able to remove much of the bactericidal activity, but the potency cannot be directly compared with that of LPS because the concentrations plotted are not based on molar equivalents of the respective epitopes but rather on are based on weight. The sialylated conjugate of LNnT, however, was completely ineffective at removing the bactericidal antibodies. This result suggests that the human anti-LPS antibodies in this serum, which are specific for the alpha chain, do not bind to the sialylated form of the LPS.
Antibodies directed against the core of the LPS rather than the terminal sugars of the alpha chain could theoretically be protective, safer than epitopes containing LNnT, and less affected by sialylation. A bactericidal murine monoclonal antibody, 25-1-LC1 (35), with specificity for the L5 core structure was found to bind equally well on Western blots to LPS having the L5 core structure regardless of the length or composition of the alpha chain (see Fig. S2 in the supplemental material). Although monoclonal antibodies specific for L8 and L3,7 bound to only a single band, the monoclonal antibody 25-2-LC1 bound well to purified L8-5 as well as to several higher-molecular-weight bands of purified LPS from strains 6505 and 7835, which share the same core structure and exhibit bands on polyacrylamide gels that are consistent in size with structures containing sialylated and unsialylated LNnT (strain 6505) or unknown structures of comparable or larger size (strain 7835). Thus, core-specific antibodies may be equally effective in binding to short or full-length sialylated LPS.
The importance of antibodies to LPS is suggested by the fact that innate “natural” antibodies to LPS develop in the absence of antigenic exposure (3, 59) and their passive transfer to antibody-deficient mice protects from lethal endotoxin challenge (45). However, most anti-LPS antibodies in normal human sera have likely developed after exposure to antigen, either from pathogenic or commensal organisms (“naturally induced” antibodies) (14, 23, 40) or as a result of immunization (2, 11, 12, 39, 54, 65).
The abundance and consistent expression of LPS in the outer membrane suggests that LPS should be a good target for bactericidal antibodies and a potential vaccine component for N. meningitidis. But its toxicity and potential cross-reactivity with oligosaccharides on human cells have limited interest in it as a vaccine. Since the polysaccharide or oligosaccharide epitopes on LPS behave as T-cell-independent antigens, LPS would be expected to require association with protein(s) to be effective as a vaccine for infants (17). Consequently, candidate vaccines for group B meningococcal disease and other diseases that are based on LPS or contain LPS as an important antigen have been designed to circumvent this problem by presenting the LPS as a covalent conjugate, as noncovalent complexes with proteins, or as part of an intact outer membrane (2, 10, 11, 12, 14, 21, 22, 39, 41, 52, 56, 58, 62, 65).
The effectiveness of conjugating polysaccharides to protein to convert them to T-cell-dependent antigens is well established. However, T-cell-independent antigens, including LPS, when presented in the context of whole bacteria or isolated noncovalent complexes with protein also appear to exhibit T-cell-dependent properties (5, 39, 47). In any case, vaccines that induce anti-LPS antibodies have shown promise for inducing protection against diverse Gram-negative bacteria (5, 10, 11, 12, 21, 28, 39, 41, 58, 62). While efforts to develop an LPS-based vaccine for group B N. meningitidis have been reported (10, 22, 41, 52, 56), no vaccine candidate based solely on LPS has entered clinical trials. Some LPS-based vaccine efforts have focused on the inner core structures (10, 41) or a partially truncated LPS (56) to ensure safety or improve immunogenicity (31). Deoxycholate-extracted vesicles, which have been used for vaccination to respond to specific group B epidemics, have normally contained 5 to 7% residual LPS (14), usually of the L3,7 immunotype.
Vaccines based on deoxycholate-extracted outer membrane vesicles typically induce bactericidal antibodies that are predominantly specific for PorA and are thus mostly subtype specific. A contribution of anti-LPS antibodies to the serum bactericidal activity of individuals vaccinated with deoxycholate-extracted outer membrane vesicle vaccines has been reported (54), but the extent of the contribution is unclear, as is its contribution to the induction of cross-reactive bactericidal antibodies by those vaccines (48). Part of the reason for the lack of understanding is the difficulty in determining the specificity of the bactericidal antibodies induced by complex vaccines, such as those based on outer membrane vesicles and by natural infections. By means of a bactericidal depletion assay (66) developed in our laboratory, we have been able to assess the contribution of anti-LPS antibodies to the bactericidal activity of a variety of human sera. This approach requires only a small amount of serum and does not require the affinity purification of the antibodies in order to directly test them for bactericidal activity.
Koeberling et al. (30) have shown the induction of anti-factor binding protein (fHBP) bactericidal antibodies after immunization of mice with NOMV, especially when expressed at levels 10-fold greater than the parent strain and compared to ΔfHBP NOMV. Higher bactericidal activity was detected against 5 strains expressing antigenically different porA and porB compared to the adjuvant-only control. Their method of adsorbing “bulk” quantities of sera immunized with 10× fHBP NOMV over a recombinant fHBP column and reporting a significant decrease in the bactericidal titer against several test strains is evidence for a strong anti-fHBP response. They did not detect a significant anti-LOS bactericidal response under those conditions, which were designed specifically to induce a strong anti-fHBP response. The reason Koeberling et al. did not see a significant bactericidal response to LPS is unclear. Such antibodies may have been difficult to detect in the presence of high titers of anti-fHBP bactericidal antibodies. They used a different bactericidal assay method which includes growth of the test strain in the presence of CMP-NANA to strongly sialylate the LPS. They also used different analytical methods to determine the specificity of the bactericidal antibodies, and in addition, 3 of 6 of their test strains expressed L1 LPS, which we have found to be poorly cross-reactive with L3,7 antibodies. It was not clear to what extent the LPS in their NOMV vaccines was sialylated. We have observed that sialylated LPS is less immunogenic than unsialylated LPS. Further work will be required to better understand the reasons for the different results.
In order to specifically identify broadly cross-reactive protective antigens, the bactericidal depletion assays summarized in this paper tested mostly human sera against strains heterologous to the strain that induced the bactericidal antibodies (e.g., convalescent-phase serum with the available case isolate). In other cases, such as normal human sera, the strain or strains that induced the bactericidal antibodies were unknown. Thus, we were less likely to detect strain- or type-specific bactericidal antibodies. For example, convalescent-phase sera from patients infected with group C organisms were, with a few exceptions, tested against group B test strains with serotype and serosubtype different from the causative strain. Nevertheless, titers against the two strains (6275 and B16B6) that shared the same PorA protein as the outbreak strain were not higher than those against PorA heterologous strains. Strain B16B6 was killed only by serum from the patient whose infecting strain expressed L2. The reason bactericidal antibodies to PorA were not present at titers comparable to the anti-LPS antibody titers in the convalescent-phase sera from the group C cases is not clear. PorA present on whole cells or in NOMV vaccines is likely less exposed than that present in deoxycholate-extracted vesicles from which much of the LPS, lipoprotein, and phospholipid have been removed. In NOMV vaccines which retain all the membrane-associated proteins, PorA is a significantly smaller percentage of the total protein. Thus, a dose of NOMV vaccine would contain a smaller amount of PorA than the deoxycholate-extracted vesicles. While some of the sera may have contained antibodies to the group B capsule, these antibodies are generally not bactericidal in conjunction with the human complement (63). In the human sera tested, antibodies to LPS appeared to play a major role in the cross-reactive bactericidal activity observed. As might be expected, the LPS immunotype homologous to that expressed by the test strain was always the most efficient in depleting the bactericidal activity against that strain but was not the only immunotype that depleted bactericidal activity. There are likely different populations of anti-LPS antibodies in the sera that have different specificities and thus different avidities for the LPS expressed by a given test strain. The presence of bactericidal antibodies with L3,7 specificity in pooled normal human sera was also demonstrated by Estabrook et al. by the affinity purification method (13).
It is not known to what extent the LPS on meningococci growing in vivo in a host is sialylated, but the presence of endogenous sialyltransferases suggests there may be a high degree of sialylation (35). If LPS is highly sialylated in vivo, then protective LPS antibodies must be able to bind to sialylated LPS. Yet the failure of sialylated LNnT versus unsialylated LNnT to remove bactericidal antibodies suggests that the L3,7-specific bactericidal antibodies to the LNnT epitope recognize mainly unsialylated (L7) LPS. This specificity is similar to monoclonal antibody (1B2-1B7) (Table 6), known to be specific for LNnT, which was not able to kill a highly sialylated strain. Nonetheless, anti-LPS antibodies in most of the human sera were bactericidal for highly sialylated meningococci, suggesting that the human antibodies are directed mainly at epitopes not including sialic acid, likely involving some core structure as well as LNnT sugars. Studies with monoclonal antibody 9-2-L379 (Table 6) have demonstrated a requirement for both the LNnT group and the L3 core structure for optimal binding (W. D. Zollinger, E. E. Moran, and B. L. Brandt, unpublished data). The bactericidal titer of this antibody was reduced but not eliminated by high sialylation of the test strain. In addition, if a subset of LPS-specific antibodies in human sera is fully directed to core structures, then it has the potential to be specific for unique LPS epitopes and yet bind to full-length, sialylated LPS on the meningococcus (see Fig. S2 in the supplemental material). Although most of the N. meningitidis strains used as bactericidal test strains expressed full-length LPS containing LNnT, often L3,7 LPS, we observed that sera with bactericidal activity toward such strains were also able to kill strains expressing shorter L8 or L8-like LPS. The observation that human sera are able to kill strains expressing short and full-length LPS suggests the presence of antibodies that recognize primarily the core structure of the LPS and are not dependent on the presence of the LNnT group. The LPS-specific bactericidal activity of most of the human sera we tested were reduced by 2- to 4-fold by high sialylation of the test strain resulting from growth on CMP-NANA, but we could not determine the relative contributions of LPS sialylation and a more general increase in serum resistance to the decreased titer.
Induction of antibodies able to bind LPS with similar cores yet different alpha chain lengths after vaccination with an LPS-based vaccine would seem to be the most desirable from a safety point of view. The monoclonal antibody 21-1-LC1 (49) appears to have these characteristics (see Fig. S2 in the supplemental material) in that it was made against an L5 strain expressing mostly the truncated L8-like form of the LPS (L8-5), as judged by silver-stained SDS-PAGE. The antibody appears to bind equally well on Western blots to full-length (including the sialylated form) and truncated L8-5 LPS. Vaccine-induced antibodies with these characteristics may be both safe and effective for protection against meningococcal disease. In the design of a multivalent group B vaccine based on NOMV, we have included the truncated L8 or L8-like LPS structures in the vaccine and have induced antibodies bactericidal for strains expressing full-length LPS (28, 62).
Some of the bactericidal antibody depletion data suggest that anti-LPS antibodies and anti-OMP antibodies may be able to cooperate in producing a bactericidal event (62). This type of cooperative event would not likely be detected when looking at the bactericidal activity of affinity-purified anti-LPS antibodies (13) or by simply trying to correlate binding on Western blots with bactericidal activity (54). Frequently, two different purified antigens, such as LPS and recombinant protein, were used in bactericidal depletion assays, and the total amount of bactericidal activity removed by the two antigens added up to greater than 100%. These results suggest cooperation between antibodies specific for two different antigens in the killing event. Complement deposition induced by bactericidal antibodies to the two different antigens acting together would require that the antigens are located close together on the bacterial surface. If antibodies of two different specificities are involved, the removal of either of these antibody specificities could potentially result in loss of bactericidal activity. The cooperative (even synergistic) action of antibodies to two different OMPs has been reported (57). Certainly, LPS is often closely associated with outer membrane proteins, so it would not be hard to imagine cooperation in killing between bactericidal antibodies specific for LPS and OMPs. Further work needs to be done to verify this hypothesis.
Our demonstration of the high prevalence of persistent anti-LPS bactericidal antibodies in normal, convalescent, and postvaccination human sera supports the use of appropriate forms of LPS as a vaccine or vaccine component.
This work was supported by the U.S. Army Medical Research and Materiel Command through the Military Infectious Disease Research Program office.
The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the U.S. Department of the Army or Department of Defense.
†Supplemental material for this article may be found at http://iai.asm.org/.
Published ahead of print on 18 July 2011.