Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes

Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Tn916-generated, lipooligosaccharide mutants of Neisseria meningitidis and Neisseria gonorrhoeae.

A library of Tn916-generated, tetracycline-resistant (Tc) mutants of the group B Neisseri meningitidis strain NMB was screened by using monoclonal antibodies (MAbs) that recognize structural differences in neisserial lipooligosaccharide (LOS). The LOS of parental strain NMB had a relative molecular mass of 4.5 kDa, reacted with MAbs 3F11 and 6B4 but not with MAb 4C4 or 6E4, and contained a lacto-N-neotetrose unit. Two phenotypically stable mutants, SS3 and R6, altered in LOS, were identified by colony immunoblots, electrophoresis, and Western immunoblots. The LOS of mutant SS3 was 3.4 kDa and reacted with MAbs 4C4 and 6E4 but not MAb 3E11 or 6B4. The LOS of mutant R6 was 3.1 to 3.2 kDa and reacted with MAb 6E4 but not MAb 3F11, 6B4, or 4C4. Thus, the LOSs of the R6 and SS3 mutants were predicted to contain different truncations of the core oligosaccharide. The LOS phenotype of each mutant was linked to Tc(r), as determined by transformation of the parent strain with DNA from the mutant. Southern hybridizations and single-specific-primer PCR revealed in each mutant a single truncated tn916 insertion which had lost genes required for mobilization. Tn916 mutagenesis was used to identify two distinct genetic sites in the meningococcal chromosome involved in biosynthesis of the oligosaccharide chain of LOS and to create genetically defined LOS mutants of N. meningitidis and Neisseria gonorrhoeae.     

The lipooligosaccharide immunotype as a virulence determinant in Neisseria meningitidis.

We have studied the antigenic (immunotype) and physical characteristics of the lipooligosaccharide (LOS) of epidemiologically related Neisseria meningitidis case (36) and carrier (76) isolates associated with a virulent clone of meningococci (ET-5 complex). LOS immunotypes were determined by dot blotting using immunotype specific monoclonal antibodies and physical characteristics were determined from silver stained SDS-PAGE following proteinase K digestion. The genetic similarity of the different isolates was confirmed by analysis of the restriction fragment length polymorphisms. An association between LOS immunotype expression and invasive disease was found; 97% of case isolates expressed the L3,7,9 immunotype, of which 13% additionally expressed the L1,8,10 determinant. The LOS immunotypes of carrier strains were much more heterogeneous. The predominant immunotype was L1,8,10 (70%) and only 24% expressed L3,7,9 alone. Genotypically related case isolates from Norway (6) and Austria (18) expressed the L3,7,9 immunotype with similar frequency to the U.K. isolates. The combination of LOS immunotype and capsule expression appears to be related to the virulence of these meningococcal strains.     

Microheterogeneity of Neisseria lipooligosaccharide: analysis of a UDP-glucose 4-epimerase mutant of Neisseria meningitidis NMB.

Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11+, 6B4+, and 4C4-) that was designated NMB-SS3 (monoclonal antibodies 3F11-, 6B4-, and 4C4+). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-->(GlcNAc)-->Hep2PEA-->KDO2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex-->(HexNAc)-->Hep2PEA-->KDO2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hexn-->(HexNAc)-->Hep2PEA-->KDO2 (n = 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.     

Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway

Meningococcal lipopoly(oligo)saccharide (LOS) is a major inflammatory mediator of fulminant meningococcal sepsis and meningitis. Highly purified wild-type meningococcal LOS and LOS from genetically defined mutants of Neisseria meningitidis that contained specific mutations in LOS biosynthesis pathways were used to confirm that meningococcal LOS activation of macrophages was CD14/Toll-like receptor 4 (TLR4)-MD-2 dependent and to elucidate the LOS structural requirement for TLR4 activation. Expression of TLR4 but not TLR2 was required, and antibodies to both TLR4 and CD14 blocked meningococcal LOS activation of macrophages. Meningococcal LOS alpha or beta chain oligosaccharide structure did not influence CD14/TLR4-MD-2 activation. However, meningococcal lipid A, expressed by meningococci with defects in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis or transfer, resulted in an approximately 10-fold (P < 0.0001) reduction in biologic activity compared to KDO2-containing meningococcal LOS. Removal of KDO2 from LOS by acid hydrolysis also dramatically attenuated cellular responses. Competitive inhibition assays showed similar binding of glycosylated and unglycosylated lipid A to CD14/TLR4-MD-2. A decrease in the number of lipid A phosphate head groups or penta-acylated meningococcal LOS modestly attenuated biologic activity. Meningococcal endotoxin is a potent agonist of the macrophage CD14/TLR4-MD-2 receptor, helping explain the fulminant presentation of meningococcal sepsis and meningitis. KDO2 linked to meningococcal lipid A was structurally required for maximal activation of the human macrophage TLR4 pathway and indicates an important role for KDO-lipid A in endotoxin biologic activity.     

Immunoresponses to Neisseria meningitidis epitopes: primary versus secondary antiphosphorylcholine responses

Specific antiphosphorylcholine immune responses were found to be elicited by different Neisseria meningitidis group B M986 preparations. Our results suggest the functional presence of phosphorylcholine in the bacteria. The immune responses, mostly immunoglobulin M, were measured with a plaque-forming cell assay. The secondary phosphorylcholine-specific immune response induced by intact meningococci was significantly lower than the primary phosphorylcholine-specific immune response induced by the same antigens. This suppression is priming time dependent and does not represent an early switching to the expression of other classes of immunoglobulins.     

Relationship of Serogroups of Neisseria meningitidis II. R Variants of Neisseria meningitidis

Meningococci isolated in primary cultures from nasopharyngeal carriers occasionally consisted of mixtures of smooth (S) and rough (R) strains. The R strains were separated from the S strains and their morphological and serological characteristics were studied. Some of these R strains reverted spontaneously to S strains which subsequently produced group-specific polysaccharide. Several R strains, grown in the presence of deoxyribonucleic acid from either an R strain of known parentage or an S strain, formed recombinants with serological group specificity.     

Organism resembling Neisseria gonorrhoeae and Neisseria meningitidis.

A problem isolate resembling Neisseria gonorrhoeae and Neisseria meningitidis is reported. Growth and biochemical characteristics indicated the organism to be N. meningitidis, whereas serological characteristics indicated it to be N. gonorrhoeae. This vaginal isolate may be a genetically transformed gonococcus with the ability to utilize maltose. Conversely, it may be a meningococcus which has acquired antigenic determinants of N. gonorrhoeae.     

Differential role of lipooligosaccharide of Neisseria meningitidis in virulence and inflammatory response during respiratory infection in mice

Meningococcal lipooligosaccharide (LOS) induces a strong proinflammatory response in humans during meningococcal infection. We analyzed the role of LOS in the inflammatory response and virulence during the early infectious process in a mouse model of meningococcal respiratory challenge. An lpxA mutant strain (serogroup B) devoid of LOS (strain Z0204) could not persist in the lungs and did not invade the blood. The persistence in the lungs and invasion of the bloodstream by a rfaD mutant expressing truncated LOS with only lipid A and 3-deoxy-d-manno-2-octulosonic acid molecules (strain Z0401) was intermediate between those of the wild-type and Z0204 strains. Both LOS mutants induced acute pneumonia with the presence of infiltrating polymorphonuclear leukocytes in lungs. Although tumor necrosis factor alpha production was reduced in mice infected with the mutant of devoid LOS, both LOS mutants induced production of other proinflammatory cytokines, such as interleukin-1beta (IL-1beta), IL-6, and the murine IL-8 homolog KC. Together, these results suggest that meningococcal LOS plays a role during the early infectious and invasive process, and they further confirm that other, nonlipopolysaccharide components of Neisseria meningitidis may significantly contribute to the inflammatory reaction of the host.     

Molecular analysis of lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A HindIII gene bank of Neisseria gonorrhoeae MUG116 was constructed in the cosmid vector pHC79. A cosmid (pSY81) was isolated that was able to convert N. gonorrhoeae FA5100 to reactivity with monoclonal antibody (MAb) 2-1-L8. Several MAb-reactive transformants were isolated and characterized with respect to lipooligosaccharide (LOS) production as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their ability to react with two other LOS-specific MAbs (3F11 and O6B4), and Southern blot analysis. Escherichia coli containing the clone had altered lipopolysaccharide expression as determined by electrophoretic analysis; however, no reactivity was seen with gonococcus-specific MAbs. The introduction of pSY81 into FA5100 had a pleiomorphic effect, giving rise to transformants having the full parental phenotype or transformants lacking reactivity to a combination of LOS-specific MAbs. Southern blot analysis indicated that the LOS biosynthetic mutation in FA5100 was not due to chromosomal rearrangement or large deletions.     

Urethritis caused by Neisseria meningitidis.

A glucose-negative group B strain of Neisseria meningitidis isolated from a meningitis case is described. A brief review of Neisseria identification procedures is also presented.     

Purification and characterization of H.8 antigen from group B Neisseria meningitidis.

The surface antigen (H.8) common to the pathogenic Neisseria species was purified by a simple procedure by use of high-performance liquid chromatography. The purified H.8 antigen was characterized as to its amino acid composition, susceptibilities to several proteolytic enzymes, isoelectric point, and susceptibilities to an acid and a base. The amino acid composition of purified H.8 antigen from two strains of Neisseria meningitidis group B, namely, 44/76 and 8047, were compared. It was found that glutamic acid, alanine, and proline accounted for about 80% of the total amino acids in each case. A preliminary analysis of the lipid content of this protein was made. It showed the presence of a lipid component that moves between C9 and C11 straight-chain fatty acids in the gas chromatograph. Limited amino acid sequence data were obtained by sequencing a fragment of the H.8 antigen that was isolated after partial acid hydrolysis. The H.8 antigen epitope was found to be labile to treatment with both a mild acid and a mild base.     

Lipopolysaccharide banding patterns of Neisseria meningitidis and Neisseria gonorrhoeae.

The lipopolysaccharides of Neisseria meningitidis and Neisseria gonorrhoeae were examined by electrophoresis after whole-cell lysis and proteinase K digestion. The banding patterns observed from clinical isolates and laboratory strains demonstrated lipopolysaccharide which included a small number of smooth high-molecular-weight molecules as well as the previously reported lower-molecular-weight rough lipopolysaccharide.     

Affinity-purified human immunoglobulin G that binds a lacto-N-neotetraose-dependent lipooligosaccharide structure is bactericidal for serogroup B Neisseria meningitidis

Despite technological advances, no vaccine to prevent serogroup B meningococcal disease is available. The failure to develop a vaccine has shifted the focus to an alternative outer membrane structure, lipooligosaccharide (LOS), because disseminated disease induces bactericidal immunoglobulin G (IgG) that binds LOS. The purpose of this study was to identify the LOS structure(s) that induces human bactericidal IgG by purification and characterization of these antibodies. Human LOS IgG antibodies were affinity purified by passage of intravenous immunoglobulin through purified, type-specific LOS having a known structure coupled to epoxy-activated Sepharose 6B. Pathogenic group B strains representing the major LOS serotypes were used to examine the binding and bactericidal activities of four LOS-specific IgG preparations. All four LOS-specific IgG preparations bound to strains expressing homologous, as well as heterologous, LOS serotypes as determined by flow cytometry and an enzyme-linked immunosorbent assay. With human complement, IgG that was purified with L7 LOS was bactericidal for strains expressing L3,7 and L2,4 LOS, serotypes expressed by the majority of disease-associated group B and C meningococci. In conclusion, we purified human LOS-specific IgG that binds meningococci across LOS glycose-specific serotypes. An antigen that is dependent on the glycose lacto-N-neotetraose induces IgG in humans that is bactericidal for L2, L3, L4, and L7 strains. A vaccine containing this antigen would have the potential to protect against the vast majority of group B meningococcal strains.     

Genomics of Neisseria meningitidis

An important feature of disease caused by Neisseria meningitidis is the propensity to invade the meninges. Much progress has been made in our understanding of how this pathogen circumvents the physical properties of this cellular barrier. This review will address the new possibilities offered by the recent availability of meningococcal genome sequences.     

Electromorphic characterization and description of conserved epitopes of the lipooligosaccharides of group A Neisseria meningitidis

We studied the lipooligosaccharides (LOS) of 28 group A Neisseria meningitidis of epidemiologically diverse origins to investigate whether each of the LOS serotypes found in serogroup A could be identified physically as well as antigenically. Using a dot blot assay with LOS-specific monoclonal antibodies (MAbs), we identified four epitopes that were serotype specific. The LOS from strains of each serotype were electromorphically and antigenically distinct when analyzed by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The LOS of L8 strains contained a 3,600-Mr component that bound the L8 MAb. The LOS of L9 strains contained two major components of 4,500 and 4,200 Mr. They bound the L9 MAb to the larger component. The LOS of L10 strains had a single major component of 4,000 Mr that bound the L10 MAb. The LOS of L11 strains contained a major 3,600-Mr component that could not be distinguished from the 3,600-Mr LOS of L8 strains by SDS-PAGE but that bound the L11 MAb. LOS of group A strains contained a highly conserved epitope in addition to a serotype-specific epitope. This was identified by a MAb that bound to all the strains on dot-blots and to multiple LOS components of various Mrs on immunoblots. We conclude that the LOS which bear the L9, L10, and L11 determinants are physically distinct and can be identified by SDS-PAGE or MAb binding or both. L8 and L11 are both borne on a 3.6-kilodalton LOS and can only be distinguished serologically.     

Immunoresponses to Neisseria meningitidis epitopes: suppression of secondary response to phosphorylcholine is carrier specific

Results of our previous work have shown that Neisseria meningitidis serogroup B M986 can induce a phosphorylcholine (PC)-specific plaque-forming cell immunoresponse in mice. Also, a single injection of a relatively low dose of meningococci in NBF1 female mice induced a priming time-dependent suppression on subsequent meningococcus challenge. This suppression was not due to switching to another class of immunoglobulin nor to the presence of a capsule on N. meningitidis. In this study we show that suppression induced by meningococcus is carrier specific. Furthermore, we offer evidence suggesting that the structure(s) on meningococcus that trigger this suppression is heat labile and different from the antigenic structure(s) recognized by the suppressed B cells. In addition, we found that there is a gradual increase in antibody secretion rates of N. meningitidis-induced anti-PC plaque-forming cells that correlates with N. meningitidis priming time. Rather unexpected was the fact that pretreatment of mice with PC-keyhole limpet hemocyanin (thymus-dependent antigen) had a great influence on the subsequent PC-specific immunoresponses induced by N. meningitidis and PC-coupled heat-inactivated meningococcus [PC-(NMB)HI], as shown by (i) a striking decrease in T15 idiotype expression, (ii) concomitant direct anti-PC plaque-forming cells reduction, (iii) switching to immunoglobulin G (N. meningitidis-induced immunoresponse) or immunoglobulin G plus immunoglobulin A [PC-(NMB)HI-induced immunoresponse], and (iv) a significant increase in heterogeneity of plaque-forming cell secretion rates. The possibility that N. meningitidis, PC-(NMB)HI, and PC-KLH stimulate B lymphocytes pertaining to three different subpopulations embedded in distinct regulatory circuits is discussed, with emphasis on the interrelationships between T-dependent and T-independent lymphocyte compartments. We focus on the possibility of the existence of high-level regulatory circuits in which lymphocyte subpopulations or sets of lymphocyte subpopulations with different requirements of activation are connected.     

Morphological differences in Neisseria meningitidis pili.

Disease and carrier isolates of Neisseria meningitidis were examined for their ability to adhere to human buccal epithelial cells and human cell lines and to hemagglutinate human erythrocytes, properties thought to be associated with the presence of pili. Seventy percent (7 of 10) of carrier isolates were found to be highly adherent to human buccal epithelial cells and to agglutinate human A, B, O, Rh-, and Rh+ erythrocytes. In contrast, 60% of the disease isolates adhered poorly to human buccal epithelial cells and 80% failed to agglutinate human erythrocytes. No adherence of either disease or carrier isolates was observed when several human cell lines were tested. When the meningococcal strains were examined by electron microscopy, 7 of 10 disease isolates were found to possess large bundles of aggregated pili (alpha-type pili), while 7 of 10 carrier isolates were found to have numerous unaggregated pili (beta-type pili). A monoclonal antibody against meningococcal pili and one against gonococcal pili reacted with 6 of 10 piliated carrier isolates and 4 of 10 piliated disease isolates. These results suggest that meningococci, like gonococci, possess different types of pili which differ in morphological, antigenic, and binding properties. In addition, antigenic and morphological differences between pili from carrier and disease isolates were observed as well as differences in adherence and hemagglutinating properties.