Genomic fingerprinting of Neisseria meningitidis associated with group C meningococcal disease in Canada.

A single electrophoretic type (ET15) of Neisseria meningitidis has been associated with an increased incidence of group C meningococcal disease in Canada. Genomic fingerprinting through pulsed-field gel electrophoresis of chromosomal DNA was used to characterize the clonal relationship among meningococcal isolates of different electrophoretic types and among isolates within ET15. The genomic fingerprints of the ET15 isolates, while similar as a group, were sufficiently distinct to confirm linkage for four pairs of strains from focal outbreaks and differed markedly from those of the other common electrophoretic types, ET5, ET9, and ET21.     

An outbreak of severe invasive meningococcal disease due to a capsular switched Neisseria meningitidis hypervirulent strain B:cc11

Objectives

The aim was to investigate an outbreak of invasive meningococcal disease (IMD) in Southern Sardinia.

Aberrant strain of group G 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.     

Applications of restriction endonuclease fingerprinting of chromosomal DNA of Neisseria meningitidis.

Restriction endonucleases are bacterial enzymes that cleave DNA at specific sites. The resulting DNA fragments may be separated electrophoretically in gel to form specific restriction patterns. In the present study, the restriction endonuclease method was successfully adapted to the analysis of the chromosomal DNA of Neisseria meningitidis. The endonucleases HindIII and EcoRI provided optimal restriction patterns of ca. 50 well-separated lines. The pattern of each bacterial isolate was characteristic, stable, and reproducible. Despite some general similarity, the restriction patterns of the closely related B15 meningococci were surprisingly heterogeneous.     

Activation of murine B lymphocytes by Neisseria meningitidis and isolated meningococcal surface antigens.

Heat-killed Neisseria meningitidis was found to be a potent mitogen for mouse splenic lymphocytes. Results obtained with different cell separation techniques indicated that the bacteria acted to selectively induce proliferation of B lymphocytes. First, partial or total depletion of T lymphocytes by treatment with various anti-T-cell antisera plus complement did not affect the ability of the remaining spleen cells to proliferate in response to N. meningitidis. Second, T lymphocytes purified by affinity chromatography through an immunoglobulin-antiimmunoglobulin-coated glass bead column were unresponsive to meningococcal stimulation, even when provided with a source of macrophages (irradiated or mitomycin C-treated spleen cells). Finally, treatment of spleen cells with soy bean agglutinin showed that, whereas the soy bean agglutinin-positive population (B-enriched lymphocytes) was highly responsive to stimulation by N. meningitidis, the soy bean agglutinin-negative population (T-enriched lymphocytes) displayed only a background level of proliferation when exposed to the bacteria. Isolated meningococcal surface antigens such as lipopolysaccharide (LPS) and outer membranes also possessed mitogenic activity and induced proliferation of B lymphocytes in a dose-dependent manner. Both LPS and non-LPS components contributed to the mitogenicity of outer membranes since the addition of outer membrane preparations to spleen cells from the low LPS responder C3H/HeJ mouse strain gave rise to a high level of proliferative activity.     

Antibody responses to the capsular polysaccharide of Neisseria meningitidis serogroup B in patients with meningococcal disease.

We measured antibody responses to meningococcal serogroup B (MenB) polysaccharide (PS) by enzyme-linked immunosorbent assay (ELISA) in sera from 94 patients from The Netherlands with disease caused by Neisseria meningitidis group B. The patients ranged in age from 3 to 73 years (mean age, 18.8 years). In initial studies we showed that the binding of a panel of MenB PS-reactive human immunoglobulin M (IgM) paraproteins to biotinylated MenB PS bound to avidin-coated microtiter wells was inhibited > 90% by the addition of soluble MenB PS or encapsulated group B meningococci. In contrast, inhibition of IgM anti-MenB PS antibody-binding activity in many of the patient sera was less than 50% (range, 20 to 94%). These data suggested a high frequency of nonspecific binding in the patient sera. Therefore, all serum samples were assayed in replicate in the presence or absence of soluble MenB PS, and only the inhibitable fraction of the binding signal was used to calculate the anti-MenB PS antibody concentrations. In 17 control patients with meningococcal disease caused by serogroup A or C strains, there was no significant difference in the respective IgM or IgG anti-MenB PS antibody concentrations in paired acute- and convalescent-phase sera. In contrast, in patients with MenB disease, the geometric mean IgM anti-MenB PS antibody concentration increased from 3.9 units/ml in acute-phase serum to 10.5 units/ml in convalescent-phase serum (P < 0.001). The corresponding geometric mean IgG anti-MenB PS antibody titers were 1:27 and 1:36 (P < 0.05). There was only a weak relationship between age and the magnitude of the logarithm of the antibody concentrations in convalescent-phase sera (for IgM, r2 = 0.06 and P < 0.05; for IgG, r2 = 0.08 and P < 0.01). Our data indicate that precautions are needed to avoid nonspecificity in measuring serum antibody responses to MenB PS by ELISA. Furthermore, although this PS is thought to be a poor immunogen, patients as young as 3 years of age recovering from MenB disease demonstrate both ImG and IgG antibody responses in serum.     

Protection against group B Neisseria meningitidis disease: preparation of soluble protein and protein-polysaccharide immunogens.

Although effective polysaccharide vaccines have been developed for meningococcal groups A, C, Y, and W135, the purified group B polysaccharide has proven to be nonimmunogenic. Earlier studies indicated that serotype 2 outer membrane protein vaccines induced bactericidal antibodies in animals and protected them from meningococcal challenge. However, a similar vaccine induced only low levels of antiprotein antibodies in both adults and children (C.E. Frasch et al., in J.B. Robbins et al., ed., Seminars in Infectious Disease vol. 4, p. 263-267, 1982). Methods were therefore developed to produce more immunogenic serotype 2 protein vaccines. We found that, by growing the organism for 65 to 72 h at 32 degrees C, three to four times more outer membrane protein was released into the culture medium than could be extracted from overnight-grown cells. The outer membranes were therefore purified directly from the broth by ultrafiltration followed by ammonium sulfate precipitation. Most of the lipopolysaccharide was selectively removed from the membranes by treatment with the nonionic detergent Brij-96. The Brij-96 was then removed and the resulting vaccine was filter sterilized. Some vaccines were prepared by combining equal parts of detergent-treated membrane protein and high-molecular-weight group B polysaccharide producing highly soluble vaccines. These new vaccines were compared by using an enzyme-linked immunosorbent inhibition assay to an insoluble vaccine (E-06) found to be poorly immunogenic in humans. A human serum with serotype 2 specificity was used in the inhibition assay, and 5 microgram of E-06 was required for 50% inhibition, whereas less than 1 microgram of the soluble vaccines was required. Addition of group B polysaccharide slightly increased the inhibitory capacity of the protein component.     

Peptide mimotopes of Neisseria meningitidis group B capsular polysaccharide

The antigenic similarity between Neisseria meningitidis group B (NMGB) capsular polysaccharide (PS) and human polysialic acid (PSA) has hampered the development of a NMGB PS-based vaccine. But the possibility of a safe vaccine based on NMGB PS has been demonstrated by the existence of the NMGB PS-associated nonautoreactive epitope, which is distinct from those present on human PSA. To obtain peptide mimotopes of NMGB PS, we used HmenB3, a protective and nonautoreactive monoclonal antibody, to screen a phage library with 12 amino acids. We obtained 23 phage clones that bound to HmenB3 but not in the presence of E. coli K1 PS [which is alpha(2-8)-linked PSA like NMGB PS]. The clones contained 3 mimotopes and differed from previously described NMGB PS mimotopes. Immunization with a synthetic peptide of one mimotope elicited anti-NMGB antibodies in BALB/c mice. These mimotopes may be useful in the development of group B meningococcal vaccines.     

Specificity of the immune response to the group B polysaccharide of Neisseria meningitidis.

A panel of monoclonal antibodies (mAb) and polyclonal sera of murine, human and equine origin, of IgM isotype and with specificity for Neisseria meningitidis group B polysaccharide, an alpha(2----8)-linked homopolymer of sialic acid, were examined for their antigenic and biological specificities. The nature of the antigenic determinants on B polysaccharide was investigated using a series of N-acyl derivatives of B polysaccharide, two sialic acid polymers containing alpha(2----9)-linkages and a series of polynucleotides. The panel of antibodies recognized an array of unrelated antigenic determinants on the B polysaccharide, despite its structural simplicity, and all but one were highly effective in an in vitro bactericidal assay and/or in an in vivo murine passive protection model. There was no evidence that B polysaccharide induced antibody capable of blocking biological activity (blocking antibody).     

Conjugation of meningococcal lipopolysaccharide R-type oligosaccharides to tetanus toxoid as route to a potential vaccine against group B Neisseria meningitidis.

Oligosaccharides were obtained by the mild acid hydrolysis of the lipopolysaccharides from a number of different strains of Neisseria meningitidis, serotypes L2, L3, L4, L5, and L10. The dephosphorylated oligosaccharides were conjugated to tetanus toxoid as their 2-(4-isothiocyanatophenyl)-ethylamine derivatives, which resulted in the incorporation of from 18 to 38 oligosaccharides per molecule of tetanus toxoid. When injected in rabbits, the conjugates produced oligosaccharide-specific antibodies which were predominantly serologically specific but which also exhibited some cross-reactivity. These serological results can be attributed to regions of structural dissimilarity and similarity within the oligosaccharides. The oligosaccharide-specific antibodies were also lipopolysaccharide serotype specific, thus indicating that the oligosaccharides are the determinants associated with this serotype specificity. Consistent with the serological results, the conjugate antisera were bactericidal for the homologous serotype meningococcal organisms and in some cases for heterologous serotype organisms.     

Outer membrane antigens of Neisseria meningitidis group B serotype 2 studied by crossed immunoelectrophoresis.

This study shows that the capsular polysaccharide, protein, and lipopolysaccharide antigens from the outer membrane of Neisseria meningitidis group B serotype 2 may be identified by crossed immunoelectrophoresis. By using this technique, seven precipitates were resolved when outer membrane preparations were reacted against goat anti-whole cell group B type 2 antiserum. Most of these precipitates were identified by comparison with purified reference preparations. Different outer membrane preparations, reflecting different growth conditions, varied in their compositions of lipopolysaccharide, protein, and polysaccharide. Detergent treatment altered the protein and lipopolysaccharide precipitation patterns. In the presence of detergent, the lipopolysaccharide did not precipitate, and the electrophoretic migration of the protein antigens decreased. Crossed immunoelectrophoresis is a useful qualitative method for analysis of the antigenic components of the meningococcal outer membrane. The crossed immunoelectrophoresis with intermediate gel technique is presently being used to measure the human immune response to the different cell surface components.     

Protection against group B Neisseria meningitidis disease: effect of serogroup B polysaccharide and polymyxin B on immunogenicity of serotype protein preparations.

The inability to prepare an effective polysaccharide vaccine against group B Neisseria meningitidis was the impetus for these studies. Outer membrane protein vaccines used in our initial studies failed to induce bactericidal antibodies in humans. The particulate nature of these vaccines may have led to their clearance before effective immune stimulation. Less denaturing procedures, therefore, were developed for preparation of serotype 2 protein-containing vaccines. These procedures included isolation of naturally released outer membrane vesicles and selective removal of lipopolysaccharide from the vesicles by the nonionic detergent Brij-96. The resultant protein vaccines were evaluated with and without noncovalently complexed group B meningococcal polysaccharide or polymyxin B sulfate or both. The new vaccines were at least 10-fold more immunogenic in mice and guinea pigs than the previous vaccines when assayed for bactericidal and enzyme-linked immunosorbent assay antibodies. The protein vaccines alone protected guinea pigs from intrachamber infection, and a single 0.1-microgram injection prevented meningococcal bacteremia in mice. Addition of group B polysaccharide to the protein significantly improved the immunogenicity of the protein, and this combined vaccine showed a greater protective effect. Polymyxin B generally reduced the immunogenicity of the vaccines in both mice and guinea pigs.     

Sialic acid of group B Neisseria meningitidis regulates alternative complement pathway activation.

The effect of meningococcal cell-associated sialic acid on activation of the human alternative complement pathway was examined by using a quantitative fluorescence immunoassay to assess alternative pathway-mediated C3 binding to a group B strain of Neisseria meningitidis from which graded amounts of sialic acid had been removed with neuraminidase. Using human serum absorbed with strain B16B6 (B:2a:L2,3) and chelated with 10 mM MgCl2 and 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we found an increase in the amount of C3 bound by enzymatically desialylated B16B6 organisms over the amount bound by fully sialylated organisms. This increase was proportional to the amount of sialic acid cleaved from the bacteria. Enhanced C3 binding was accompanied by an increase in factor B deposition. A sialic acid-deficient mutant of strain B16B6, designated 2T4-1, bound C3 via the alternative pathway at a level equivalent to that bound by wild-type meningococci from which 88% of the sialic acid had been removed. Strain B16B6 was resistant to the alternative pathway-mediated bactericidal activity of both absorbed and hypogammaglobulinemic human sera, whereas noncapsular variant 2T4-1 was sensitive to these sera. The addition of purified immune immunoglobulin M (IgM) and IgG significantly increased the alternative pathway-mediated killing of strain B16B6 organisms. IgM mediated increased bactericidal activity without an increase in C3 or factor B deposition. In contrast, the IgG-mediated killing was associated with increased binding of C3 and factor B to the organisms. Absorption studies showed that the IgM bound to the sialic acid capsule, whereas the IgG bound to noncapsular surface antigens. We conclude from these results that the group B meningococcal sialic acid capsule inhibits activation of the alternative pathway in the nonimmune host and that both IgM and IgG, although specific for different surface antigens, are capable of augmenting the alternative pathway-mediated killing of group B meningococci.     

Acquisition of heme iron by Neisseria meningitidis does not involve meningococcal transferrin-binding proteins.

Similarities in size between hemin-binding protein 1 (HmBP1) and transferrin-binding protein 1 (TBP1) of Neisseria meningitidis suggest that these proteins are functionally homologous. However, a meningococcal mutant lacking the transferrin-binding proteins retained the capacity to acquire iron from heme and hemoglobin. In immunoblots, hyperimmune polyclonal antiserum against TBP1 did not react with HmBP1.     

Immunoresponses to Neisseria meningitidis epitopes: Immunodulation by meningococcus B acts on more than one meningococcal surface epitope

Neisseria meningitidis group B strain M986 (serotypes 2a, 7) (NMB) elicits a specific primary antiphosphorylcholine immune response in mice but not a secondary response. The ability of other serotype and serogroup meningococci to induce similar primary responses in mice was studied, as was the immunogenicity of trinitrophenyl coupled NMB (TNP-NMB) in primary and secondary antitrinitrophenyl responses. Except for NMB, all other strains tested (three serogroup B and one serogroup A meningococcal strains) were found to be very poor phosphorylcholine immunogens. TNP-NMB, however, though proving to be a very good TNP antigen, was only a weak phosphorylcholine antigen. Priming NBF₁ female mice with TNP-NMB one month or more before challenging them with the same antigen induced a strong depression of anti-TNP response in the subsequent challenge. However, this effect was not observed with Xid NBF₁ male mice. Furthermore, priming mice with NMB weakly affected the anti-TNP response, but greatly depressed the antiphosphorylcholine response, after TNP-NMB challenge. In addition, whereas apparently only one TNP-specific B cell subpopulation was responding in unprimed mice challenged simultaneously with TNP-NMB and TNP-Ficoll (non-additive response), priming mice with NMB appeared to facilitate the independent activation of two different TNP-specific B cell subpopulations (additive response).     

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.     

The contrasting mechanisms of serum resistance of Neisseria gonorrhoeae and group B Neisseria meningitidis

Neisseria gonorrhoeae and Neisseria meningitidis have evolved intricate mechanisms to evade complement-mediated killing. Sialylation of gonococcal lipooligosaccharide (LOS) results in conversion of previously serum sensitive strains to unstable serum resistance, which is mediated by factor H binding. Porin (Por) is also instrumental in mediating stable serum resistance in gonococci. The 5th loop of certain gonococcal PorlAs binds factor H, which efficiently inactivates C3b to iC3b. Factor H glycan residues may be essential for factor H binding to certain Por1A strains. Por1A strains can also regulate the classical pathway by binding to C4b-binding protein (C4bp) probably via the 1st loop of the Por molecule. Certain serum resistant Por1 B strains can also regulate complement by binding C4bp through a loop other than loop 1. Purified C4b can inhibit binding of C4bp to Por 1B, but not Por1A, suggesting different binding sites on C4bp for the two Por types. Unlike serum resistant gonococci, resistant meningococci have abundant C3b on their surface, which is only partially processed to iC3b. The main mechanism of complement evasion by group B meningococci is inhibition of membrane attack complex (MAC) insertion by their polysaccharide capsule. LOS structure may act in concert with capsule to prevent MAC insertion. Meningococcal strains with Class 3 Por preferentially bind factor H, suggesting Class 3 Por acts as a receptor for factor H.     

Emergence of a virulent clone of Neisseria meningitidis serotype 2a that is associated with meningococcal group C disease in Canada.

Multilocus enzyme electrophoresis was used to characterize 378 isolates of Neisseria meningitidis serogroup C recovered during a period of an increase in group C meningococcal disease in Canada. Thirty-four enzyme electrophoretic types were found among the isolates, which were predominantly (96.0%) serotype 2a. One clone (ET 15), characterized by a rarely occurring allele for the enzyme fumarase, was responsible for a focal outbreak in Ontario followed by the spread of group C disease across the province. This clone, which occurred infrequently among strains isolated in 1986, accounted for over 65% of group C strains associated with meningococcal disease in Canada in 1990.     

Molecular analysis of anti-N-propionyl Neisseria meningitidis group B polysaccharide monoclonal antibodies

The capsular polysaccharide of Neisseria meningitidis group B (MBPS) is a polymer of alpha (2-->8) N-acetyl neuraminic acid, which is chemically identical to polysialic acid (PSA) expressed in human tissues. Antibodies from mice immunized with a MBPS-protein conjugate vaccine in which N-acetyl groups have been replaced by propionyl groups (N-Pr MBPS) can be bactericidal and show minimal or no cross-reactivity with human PSA. To investigate the molecular basis for antigen recognition, we cloned and sequenced the variable region (V) genes of five bactericidal anti-N-Pr MBPS murine mAbs and produced computer models of the combining sites. The results were compared to those reported in the literature for two autoreactive anti-MBPS. The V region genes of the anti-N-Pr MBPS mAbs and the anti-MBPS autoreactive mAbs are derived from a limited set of germline V, J, and D genes. However, the anti-N-Pr MBPS mAbs are more mutated than the anti-MBPS mAbs and the former use V-D-J editing that introduces arginine in H-CDR3. Models of the respective combining sites indicate that the anti-MBPS or anti-N-Pr MBPS mAbs that react with host PSA have relatively wide and shallow grooves with a high overall positive charge, consistent with recognition of extended helical polysaccharide structures recognized by the autoreactive mAbs. In contrast, anti-N-Pr MBPS mAbs that do not react with host PSA contain pockets and deep clefts that are consistent with recognition of discrete structural features of individual residues.