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.     

Antigenic and molecular heterogeneity of the transferrin-binding protein of Neisseria meningitidis

The transferrin-binding protein in 35 Neisseria meningitidis isolates was examined using a binding assay involving 125I-transferrin. The results show that most strains have a binding protein with a Mr between 78 kDa and 83 kDa; only 4 strains had a binding protein with a Mr of about 68 kDa. The side of the protein appears unrelated to the serogroup or serotype of the organism. Using antibodies raised to whole cells of N. meningitidis grown under iron restriction we show also that considerable antigenic heterogeneity exists amongst the transferrin-binding proteins. This makes it a less than promising vaccine candidate antigen, although conserved antigenic domains are now being sought.     

Purification of the Neisseria meningitidis transferrin binding protein-2 (TBP2) to homogeneity using column chromatography

Abstract A method for purifying TBP2 from N. meningitidis has been developed using affirnity chromatography on Tf-agarose followed by ion exchange chromatography; the Tf-binding activity of fractions was evaluated by a dot-blot technique. This method allowed the purification of the TBP2 in milder conditions than those used previously and to a high degree of homogeneity as was demonstrated by Coomassie brilliant blue or Silver training after SDS-PAGE electrophoresis. The SDS-PAGE profile of the material obtained in the Tf-agarose affinity chromatography step shows only two detectable proteins, identified as the TBP1 and the TBP2, with a small amount of contamination. Passage through a MonoQ HR anion exchange column, allowed the isolation of TBP2 in the absence of TBP1. Our results demonstrate the conservation of the antigenic reactivity of this protein, which produces monospecific serum with the antibodies elicited reacting exclusively with the TBP2 in whole outer membrane vesicles.     

Neisseria meningitidis transferrin-binding protein 1 expressed in Escherichia coli is surface exposed and binds human transferrin

Abstract A gene library of Neisseria meningitidis B15 P1.16 DNA was established in λ Zap II and clones containing DNA encoding transferrin binding protein 1 (TBP-1) identified following hybridisation with a 63-bp DNA probe based on the codon assignment for the first 21 N-terminal amino acids of TBP-1. Sequencing of the cloned DNA demonstrated that all of the intergenic DNA (i.e. upstream of bp-1 running through to the 3' end of the transferrin-binding protein 2 gene) and approx. 15% of tbp-1 had been cloned. The complete gene was generated using a polymerase chain reaction, with the primer for the 3' end being based on tbp-A of N. gonorrhoeae , and the approx. 2.9-kb DNA product cloned into pGem-3Z. The expressed protein (approx. 100 kDa) reacted with antiserum to an N-terminal peptide of TBP-1. In addition, the native product was surface-expressed by Escherichia coli and bound human transferrin.     

Identification of a 13-kDa protein associated with the polyhydroxyalkanoic acid granules from Acinetobacter spp.

Abstract Transferrin-binding proteins from Neisseria meningitidis vary among different isolates. We have identified and studied a hypervariable region adjacent to the carboxyl-end of the transferrin-binding domain of the Tbp2 molecule. The tbp2 genes from six strains of N. meningitidis were cloned and sequenced in this particular region. Sequence analysis of these regions along with five other sequences available from pathogenic Neisseria showed a common organisation of seven highly variable nucleotide stretches interspersed with six conserved nucleotide stretches. The variable regions correlated with the location of immunoreactive epitopes in polyclonal antisera raised to transferrin-binding proteins identified by peptide pin technology. Sequence analysis suggested a mosaic-like organisation of the tbp2 genes. Taken together, these data suggest that the antigenic variation in this part of the protein may result from a strong host immune pressure.     

Cloning and partial sequence of transferrin-binding protein 2 of Neisseria meningitidis using a novel method: Twin N-terminal PCR

Abstract The genes encoding transferrin-binding proteins (TBPs) 1 and 2 of Neisseria meningitidis and N. gonorrhoeae were used as model loci in a novel method of cloning (twin N-terminal polymerase chain reaction; TNT-PCR) involving amplification between the 5' ends of two genes. Primers were based on N-terminal amino-acid sequences. A 2.1-kb product amplified from N. meningitidis strain SD (B15 P1.16) was cloned into a plasmid vector and partially sequenced. Translated sequence immediately downstream of the primer at both ends of this product correlated to the additional known N-terminal amino acids of TBP-1 and 2. The protein encoded by the cloned sequence reacted with TBP-2-specific antiserum. The size of products generated in TNT-PCR correlated exactly with the different sized TBP-2 produced by 10 strains of the Neisseria spp. examined, indicating successful cloning of the gene for TBP-2 and showing it to be adjacent to and preceding TBP-1 on the chromosome for both N. meningitidis and N. gonorrhoeae .     

Human immune response to epitopes on the meningococcal outer membrane class 5 protein following natural infection

Abstract Two monoclonal antibodies (mAbs) were produced against a serogroup B Neisseria meningitidis strain. These mAbs recognized two epitopes in the class 5 outer membrane proteins (OMP), designated P5.7 and P5.Bm, and were able to kill the homologous strain through complement activation. Both epitopes were surface exposed and 68% of group B meningococcal clinical isolates had one or both epitopes present in their class 5 OMP. Antibodies to one or both epitopes were demonstrated in 17 patients with meningococcal meningitis using an ELISA inhibition assay. Of the 17 paired sera, 41% and 29% of the acute-phase sera had antibodies to the P5.7 and P5.Bm epitopes, respectively. Immunoglobulin G to P5.Bm were found in all 17 convalescent-phase sera while specific antibodies against P5.7 were only found in 6 of these sera. These results demonstrate the potential importance of the P5.Bm and P5.7 epitopes on the class 5 OMP as candidates for vaccine composition.     

A human transferrin-binding protein of Staphylococcus aureus is immunogenic in vivo and has an epitope in common with human transferrin receptor

To understand human immune responses against the human transferrin-binding protein of Staphylococcus aureus (SA-tbp), we examined cell wall proteins from S. aureus ATCC 6538 using human convalescent sera, and a monoclonal antibody specific for human transferrin receptor (McAb-HTR). The SA-tbp, detected by immunoblot assay, was iron-repressible, reacted with the convalescent sera, and cross-reacted with McAb-HTR. Immunoelectron microscopy probed with McAb-HTR showed a reaction zone around the test strain from the deferrated BHI. After being preincubated with an S. aureus -bacteremic serum, the electroblot of the SA-tbp still reacted with McAb-HTR, but not with human transferrin-horseradish peroxidase conjugate. We conclude, there are at least two kinds of epitopes in the SA-tbp; one able to bind to human transferrin is immunogenic in humans, but the other sharing epitopes common with human transferrin receptor is not immunogenic in humans.     

Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide-P64k protein conjugates

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.     

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.     

Analysis of the human Ig isotype response to lactoferrin binding protein A from Neisseria meningitidis

An effective vaccine for serogroup B meningococci has yet to be developed and attention has turned to subcapsular antigens of the meningococcus as possible vaccine candidates. Iron binding proteins are being studied, with most interest focused on the transferrin binding proteins (TbpA and TbpB) and the ferric binding protein (FbpA). This study describes the purification of lactoferrin binding protein A (LbpA) from two meningococcal strains and assesses the human isotype-specific serum antibody response to these proteins in patients with proven meningococcal disease due to a range of phenotypes. Overall, fewer than 50% of sera contained IgG that recognised LbpA isolated from either strain and this antibody response was not uniform between the two proteins. There was some evidence that the antibody response varied between meningococcal phenotypes. This study demonstrates that LbpA does not induce a highly cross-reactive antibody response, indicating that it is unlikely to be an effective vaccine antigen.     

重组B群脑膜炎球菌fHBP融合蛋白的表达和免疫原性研究

采用PCR构建编码B群脑膜炎球菌fHBP蛋白V<,1>变异型全长序列和V<,2>变异型C结构域抗原表位融合蛋白的基因片段.并克隆入pET30a<'(+)>载体,转化大肠杆菌BL21(DE3),在其实现表达,表达量约占菌体蛋白总量的30%.表达产物经离子交换层析纯化后,获得了纯度达到90%以上的融合蛋白.将融合蛋白免疫小...     

Calorimetric studies of the interaction between the insulin-enhancing drug candidate bis(maltolato)oxovanadium(IV) (BMOV) and human serum apo-transferrin

Bis(maltolato)oxovanadium(IV) (BMOV), and its ethylmaltol analog, bis(ethylmaltolato)oxovanadium(IV) (BEOV), are candidate insulin-enhancing agents for the treatment of type 2 diabetes mellitus; in mid-2008, BEOV advanced to phase II clinical testing. The interactions of BMOV and its inorganic congener, vanadyl sulfate (VOSO₄), with human serum apo-transferrin (hTf) were investigated using differential scanning calorimetry (DSC). Addition of BMOV or VOSO₄ to apo-hTf resulted in an increase in thermal stability of both the C- and N-lobes of transferrin as a result of binding to either vanadyl compound. A series of DSC thermograms of hTf solutions containing different molar ratios of BMOV and VOSO₄ were used to determine binding constants; at 25 °C the binding constants of BMOV to the C- and N-lobes of apo-hTf were found to be 3 (±1) × 10⁵ and 1.8 (±0.7) × 10⁵ M⁻¹, respectively. The corresponding values for VOSO₄ were 1.7 (±0.3) × 10⁵ and 7 (±2) × 10⁴ M⁻¹. The results show that the vanadium species initially presented as either BMOV or VOSO₄ had similar affinities for human serum transferrin due to oxidation of solvated vanadyl(IV) prior to complexation to transferrin. Binding of metavanadate () was confirmed by DSC and isothermal titration calorimetry (ITC) experiments of the interaction between sodium metavanadate (NaVO₃) and hTf.     

Epitopes of serogroup B Neisseria meningitidis analysed in vitro and directly from cerebrospinal fluid

Two type B15 P1.16 strains of Neisseria meningitidis were examined by immunogold electron microscopy for accessibility of two outer membrane protein (OMPs) to monoclonal antibodies. Both strains exhibited cell-to-cell variation of one epitope of the Class 3 OMP (P3.15) and one of the Class 1 OMPs (P1.16) when grown in vitro. One strain, a nasopharyngeal isolate revealed this variation to be growth-phase independent and double labelling of both epitopes showed independent variation. CSF containing N. meningitidis was stored in liquid nitrogen without laboratory processing at the time of isolation of the second strain. Direct analysis of the organisms showed no cell-to-cell variation and immunoglobulin G on the surface. However, while there were similar labelling densities of the Class 1 epitope in vivo compared with either strain grown in vitro, there was a lower labelling density of the Class 3 epitope in vivo that was not caused by freeze-thawing. This reduction may be due to decreased expression of this epitope in vivo which casts doubts on the use of the Class 2/3 OMP as a vaccine candidate.     

Immunologic memory response induced by a meningococcal serogroup C conjugate vaccine using the P64k recombinant protein as carrier

In this study, we used an adoptive lymphocyte transfer experiment to evaluate the ability of the P64k recombinant protein to recruit T-helper activity and induce immunologic memory response to the polysaccharide moiety in a meningococcal serogroup C conjugate vaccine. Adoptive transfer of splenocytes from mice immunized with the glycoconjugate conferred antipolysaccharide immunologic memory to naive recipient mice. The observed anamnestic immune response was characterized by more rapid kinetics, isotype switching from IgM to IgG and higher antipolysaccharide antibody titers compared with those reached in groups transferred with splenocytes from plain polysaccharide or phosphate-immunized mice. The memory response generated was also long lasting. Sera from mice transferred with cells from conjugate-immunized mice were the only protective in the infant rat passive protection assay, and also showed higher bactericidal titers. We demonstrated that priming the mice immune system with the glycoconjugate using the P64k protein as carrier induced a memory response to the polysaccharide, promoting a switch of the T-cell-independent response to a T-cell dependent one.     

The class 3 outer membrane protein (PorB) of Neisseria meningitidis: gene sequence and homology to the gonococcal porin PIA

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promoter.