The production and characterization of monoclonal antibodies against the protein III of Neisseria gonorrhoeae

Monoclonal antibodies were produced against gonococcal protein III. Antibodies of two different specificities were obtained. One reacted with all Neisseria species tested (N. gonorrhoeae, N. meningitidis and five non-pathogenic species), whereas the other was specific for Neisseria gonorrhoeae and may provide the basis for improved diagnostic reagents.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Conserved lipoprotein H.8 of pathogenic Neisseria consists entirely of pentapeptide repeats

The pathogenic Neisseria, N. gonorrhoeae and N. meningitidis, possess an outer membrane protein (OMP), designated H.8, with a conserved monoclonal antibody (MAb)-binding epitope. We determined the DNA sequence of a gonococcal H.8 gene, and confirmed the relationship between the cloned gene and the H.8 OMP by constructing a gonococcal mutant lacking H.8. The predicted H.8 OMP is a lipoprotein 71 amino acids in length, composed of 13 repeats of a consensus sequence AAEAP with perfect 5-residue periodicity. The AAEAP units form a repeating epitope that comprises the entire predicted sequence of the protein.     

Antigenic variation of outer membrane protein II in colonial variants of Neisseria gonorrhoeae P9

Antibodies were detected by an enzyme-linked immunosorbent assay (ELISA) in sera from rabbits immunized wtih outer membranes from colonial opacity variants in Neisseria gonorrhoeae P9. ELISA-inhibition experiments with purified antigens revealed approximately equal proportions of antibodies directed against each of the three major surface antigens, lipopolysaccharide, the major outer membrane protein (protein I) and protein II, the variable protein associated with colonial opacity. Inhibition experiments with intact gonococci showed considerable antigenic diversity which could be correlated with differences between the protein II species present. Despite their considerable structural homology, different protein II species from colonial variants of the same strain showed little cross-reactivity with specific anti-protein II sera, thus demonstrating the considerable variation in that part of the antigen which is exposed on the surface of the gonococcus and is closely involved in pathogenic mechanisms.     

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis

Abstract When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M _r of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N . meningitidis strains induce immune responses in vivo.     

Immune-enhanced phagocytosis of Neisseria gonorrhoeae by macrophages: characterization of the major antigens to which opsonins are directed

antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab')2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.     

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.     

The lipopolysaccharide (R type) as a common antigen of Neisseria gonorrhoeae. II. Use of hen antiserum to gonococcal lipopolysaccharide in a rapid slide test for the identification of N. gonorrhoeae from primary isolates and secondary cultures.

An antiserum has been prepared in hens to R-type gonococcal lipopolysaccharide (LPS) and used in a simple slide-agglutination test for the identification of Neisseria gonorrhoeae. Anti-LPS serum agglutinated gonococcal cells representative of the four colony types of N. gonorrhoeae. Absorption of the antiserum with LPS removed the agglutinating activity. Secondary cultures (1120) were tested without observation of the colony type and all were agglutinated. No agglutination occurred with strains of Neisseria meningitidis, Neisseria lactamica, non-pathogenic Neisseria. Pseudomonas aeruginosa, Branhamella catarrhalis, or with species of lactobacilli and Acinetobacter. Cross-reactivity of the antiserum occurred with some streptococci. The anti-LPS serum was used to identify N. gonorrhoeae in primary isolates from the cervix, urethra, and pharynx. Of 251 gonococcal isolates tested, 249 were agglutinated by the antiserum, while all of the corresponding second cultures were agglutinated. The antiserum did not agglutinate N. meningitidis found in primary isolates from pharyngeal specimens. Anti-LPS hen serum should be useful for the rapid identification of N. gonorrhoeae in primary isolates or secondary cultures.     

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.     

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

Characterization of a stress protein from group B Neisseria meningitidis.

Increased levels of a 65-kDa stress protein (Msp65) were observed in group B Neisseria meningitidis grown under stationary-growth conditions. Electron microscopy showed two apposing rings of seven subunits, a structure typical of Escherichia coli GroEL. Msp65 was not found in either the periplasmic space or the outer membrane. Several important differences between the GroEL analogs of N. meningitidis and Neisseria gonorrhoeae are discussed.     

Genetic diversity of the iron-binding protein (Fbp) gene of the pathogenic and commensal Neisseria

Abstract The pathogenic Neisseria and most commensal Neisseria species produce an iron-binding protein (Fbp) when grown under iron-limited conditions. In the current study, we confirmed the presence of Fbp, as well as DNA sequences homologous to the gonococcal fbp , in strains of N. gonorrhoeae, N. meningitidis, N. cinerea, N. lactamica, N. subflava, N. kochii and N. polysaccharea . The fbp genes from these strains were amplified by the polymerase chain reaction, digested with Stu I or Rsa I, and the restriction patterns examined. The patterns for the gonococcal and meningococcal fbp were virtually identical; however, variations were observed in the fbp sequences of the commensal Neisseria species. N. flavescens, N. mucosa, N. sicca, N. ovis and Branhamella catarrhalis , did not produce Fbp as detected by sodium dodecyl sulfate-polyacrylamide gel electropheris and reactivity with an Fbp specific monoclonal antibody, nor did they hybridize to an fbp -specific DNA probe.     

Involvement of phospholipid end groups of group C Neisseria meningitidis and Haemophilus influenzae type b polysaccharides in association with isolated outer membranes and in immunoassays.

There are several bacterial polysaccharides (PSs) which contain a terminal lipid moiety. It has been postulated that these terminal lipid moieties anchor the PSs to the outer membrane of the bacteria. Our studies have shown that incubation of native PS from group C Neisseria meningitidis or Haemophilus influenzae type b with isolated outer membrane vesicles results in association of a portion of the PS with the vesicles. Removal of the terminal lipid from the PS by treatment with phospholipase A2 or phospholipase D eliminates this association. In other studies, it was shown that delipidated PSs are not suitable as solid-phase antigens in a currently used enzyme-linked immunosorbent assay (ELISA). Measurement of antibody units in the reference sera by using delipidated PSs as antigens in an ELISA yielded negligible absorbance compared with native PSs when methylated human serum albumin was used to coat the PSs to the plate. Nevertheless, phospholipase A2 and phospholipase D treatment did not noticeably affect antigenic epitopes, since soluble group C PS without the terminal lipid bound antibody as effectively as the native PS did, as measured by a competitive inhibition assay. Both hydrophobic and electrostatic interactions are important for the binding of group C N. meningitidis PS to the ELISA plate, while charge interactions seem to be sufficient for binding the more negatively charged H. influenzae type b PS.     

Cloning, sequencing, and characterization of the gene encoding FrpB, a major iron-regulated, outer membrane protein of Neisseria gonorrhoeae.

FrpB (for Fe-regulated protein B) is a 76-kDa outer membrane protein that is part of the iron regulon of Neisseria gonorrhoeae and Neisseria meningitidis. The frpB gene from gonococcal strain FA19 was cloned and sequenced. FrpB was homologous to several TonB-dependent outer membrane receptors of Escherichia coli as well as HemR of Yersinia enterocolitica and CopB of Moraxella catarrhalis. An omga insertion into the frpB coding sequence caused a 60% reduction in 55Fe uptake from heme, but careful analysis suggested that this effect was nonspecific. While FrpB was related to the family of TonB-dependent proteins, a function in iron uptake could not be documented.     

Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species

The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes (porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.     

Detection of IgG and IgM to meningococcal outer membrane proteins in relation to carriage of Neisseria meningitidis or Neisseria lactamica

Carriage of non-serogroupable Neisseria meningitidis or Neisseria lactamica induces antibodies protective against meningococcal disease. Antibodies directed against outer membrane proteins are bactericidal and the serotype and subtype outer membrane protein antigens are being examined for their value as vaccine candidates for serogroup B disease. The aim of this study was to examine the effect of carriage of these two Neisseria species among children and young adults on induction of antibodies to outer membrane components from strains causing disease in Greece. Among 53 patients with meningococcal disease, IgG or IgM antibodies were detected by ELISA in 9 of 13 (69%) from whom the bacteria were isolated and 27 of 40 (67%) who were culture-negative. For military recruits (n = 604), the proportion of carriers of meningococci with IgM or IgG to outer membrane proteins was higher than non-carriers, P < 0.05 and P = 0.000000, respectively. Among school children (n = 319), the proportion with IgM or IgG to outer membrane proteins for carriers of meningococci was higher compared with non-carriers, P = 0.000000 and P = 0000043, respectively. Carriage of N. lactamica was not associated with the presence of either IgM or IgG to the outer membrane proteins in the children. The higher proportion of children (50%) with IgM to outer membrane proteins compared with recruits (10%) might reflect more recent exposure and primary immune responses to the bacteria. The lack of association between antibodies to outer membrane proteins and carriage of N. lactamica could reflect observations that the majority of N. lactamica isolates from Greece and other countries do not react with monoclonal typing reagents. Bactericidal antibodies to meningococci associated with high levels of IgG to N. lactamica were found in a previous study; these are thought to be directed to antigens other than outer membrane proteins or capsules and imply antigens such as lipo-oligosaccharide are involved in induction of antibodies cross-reactive with meningococci.