Immuno-proteomic analysis of human immune responses to experimental Neisseria meningitidis outer membrane vesicle vaccines identifies potential cross-reactive antigens.

Human volunteers were vaccinated with experimental Neisseria meningitidis serogroup B vaccines based on strain H44/76 detoxified L3 lipooligosaccharide (LOS)-derived outer membrane vesicles (OMV) or the licensed Cuban vaccine, VA-MENGOC-BC. Some volunteers were able to elicit cross-bactericidal antibodies against heterologous L2-LOS strain (760676). An immuno-proteomic approach was used to identify potential targets of these cross-bactericidal antibodies using an L2-LOS derived OMV preparation. A total of nine immuno-reactive spots were detected in this proteome: individuals vaccinated with the detoxified OMVs showed an increase in post-vaccination serum reactivity with Spots 2-8, but not with Spots 1 and 9. Vaccination with VA-MENGOC-BC induced sera that showed increased reactivity with all of the protein spots. Vaccinees showed increases in serum bactericidal activity (SBA) against the heterologous L2-LOS expressing strain 760676, which correlated, in general, with immunoblot reactivity. The identities of proteins within the immuno-reactive spots were determined. These included not only well-studied antigens such as Rmp, Opa, PorB and FbpA (NMB0634), but also identified novel antigens such as exopolyphosphatase (NMB1467) and γ-glutamyltranspeptidase (NMB1057) enzymes and a putative cell binding factor (NMB0345) protein. Investigating the biological properties of such novel antigens may provide candidates for the development of second generation meningococcal vaccines.

Immunization with recombinant Chaperonin60 (Chp60) outer membrane protein induces a bactericidal antibody response against Neisseria meningitidis.

Sera from individuals colonized with Neisseria meningitidis and from patients with meningococcal disease contain antibodies specific for the neisserial heat-shock/chaperonin (Chp)60 protein. In this study, immunization of mice with recombinant (r)Chp60 in saline; adsorbed to aluminium hydroxide; in liposomes and detergent micelles, with and without the adjuvant MonoPhosphoryl Lipid A (MPLA), induced high and similar (p>0.05) levels of antibodies that recognized Chp60 in outer membranes (OM). FACS analysis and immuno-fluorescence experiments demonstrated that Chp60 was surface-expressed on meningococci. By western blotting, murine anti-rChp60 sera recognized a protein of Mr 60kDa in meningococcal cell lysates. However, cross-reactivity with human HSP60 protein was also observed. By comparing translated protein sequences of strains, 40 different alleles were found in meningococci in the Bacterial Isolate Genome Sequence database with an additional 5 new alleles found in our selection of 13 other strains from colonized individuals and patients. Comparison of the non-redundant translated amino acid sequences from all the strains revealed ≥97% identity between meningococcal Chp60 proteins, and in our 13 strains the protein was expressed to high and similar levels. Bactericidal antibodies (median reciprocal titres of 32-64) against the homologous strain MC58 were induced by immunization with rChp60 in liposomes, detergent micelles and on Al(OH)3. Bactericidal activity was influenced by the addition of MPLA and the delivery formulation used. Moreover, the biological activity of anti-Chp60 antisera did not extend significantly to heterologous meningococcal strains. Thus, in order to provide broad coverage, vaccines based on Chp60 would require multiple proteins and specific bactericidal epitope identification.

Analysis of the immune response to Neisseria meningitidis using a proteomics approach.

The availability of Neisseria genome sequences together with improvements in proteomic technologies provide the opportunity to study at high resolution the immune response to Neisseria meningitidis. In this chapter, we describe a protocol that combines two-dimensional (2D) SDS-PAGE of meningococcal outer membranes with western blotting of human antisera to identify proteins associated with the development of protective antibody responses. This methodology can identify putative vaccine candidates for incorporation in a multi-component serogroup B meningococcal vaccine.

The Neisseria meningitidis macrophage infectivity potentiator protein induces cross-strain serum bactericidal activity and is a potential serogroup B vaccine candidate.

A gene encoding a 29-kDa protein from Neisseria meningitidis serogroup B strain MC58 with homology to the macrophage infectivity potentiator (MIP) protein of Legionella pneumophila was cloned and expressed in Escherichia coli, and the purified soluble recombinant protein (rMIP) was used for immunization studies. Analysis of the predicted amino acid sequences of MIP from 13 well-characterized meningococcal strains, isolated from carriers or patients and differing in serogroup, serotype, and subtype, showed that the protein was highly conserved (98 to 100%), with only three distinct sequence types (designated I, II, and III) found. Western blotting showed that the MIP protein was expressed at similar levels by all of these strains. Immunization of mice with type I MC58 rMIP in detergent micelles and liposomes containing monophosphoryl lipid A (MPLA) induced high levels of surface-reactive antibodies with serum bactericidal activity (SBA) titers of 1/1,024 against the homologous strain. Bactericidal antibodies were also induced with the protein in saline alone and liposomes alone (titers, 1/128) but not following adsorption to Al(OH)(3). Significantly, antisera raised against type I rMIP administered in saline or liposomes killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/256). Taken together, these findings suggest that rMIP can provide cross-strain protection against meningococci and should be considered a potential antigen for inclusion in new vaccines against meningococcal infection.

The influence of genomics and proteomics on the development of potential vaccines against meningococcal infection.

There is a particular need for an effective vaccine against life-threatening meningitis and septicemia caused by Neisseria meningitidis (meningococcus) serogroup B strains. Vaccine strategies incorporating capsular polysaccharide have proved effective against other meningococcal serogroups, but are not applicable to serogroup B. Attention has therefore focused on the subcapsular outer membrane protein antigens as potential vaccine components. The sequencing of genomes from three serogroups and the availability of the corresponding translated protein databases, combined with the development of sensitive proteomic techniques, have opened up new avenues of meningococcal vaccine research. This has resulted in the identification of potential candidate antigens for incorporation into multicomponent meningococcal vaccines.

Neuropeptide alpha-MSH exerts pro-inflammatory effects on Neisseria meningitidis infection in vitro.

OBJECTIVE AND DESIGN: alpha-Melanoycte stimulating hormone (alpha-MSH), a neuropeptide hormone with reported anti-microbial and immuno-modulatory properties in vitro, has previously been detected in the cerebrospinal fluid of children with bacterial meningitis. We investigated the therapeutic effects of alpha-MSH administration on Neisseria meningitidis infection of human meningeal cell cultures in vitro. MATERIALS AND METHODS: Meningeal cell lines (n = 2) were infected with meningococci (10(2)-10(8) cfu/monolayer), isolated bacterial outer membranes (OM; 1 microg/ml) or lipo-oligosaccharide (LOS; 1 microg/ml) with and without alpha-MSH (10(-5)-10 microM). Bacterial adherence was quantified at 6 h, and cytokine production and microbicidal activity of alpha-MSH for meningococci were assessed at 24 h. RESULTS: Compared with infection by meningococci alone, alpha-MSH (10 microM) up-regulated secretion of IL-6 and IL-8 (mean values increased from approximately 33 to 60 ng/ml), RANTES (mean values increased from approximately 26 to 105 ng/ml) and GM-CSF (mean values increased from approximately 0.3 to 1 ng/ml; P < 0.05). Upregulated secretion correlated with a neuropeptide-mediated rapid and >5-fold increase (P < 0.05) in bacterial adherence to cells and was dependent on OM components including LOS acting synergistically with alpha-MSH. Meningococci were resistant to the anti-microbial activity of alpha-MSH at all concentrations tested. CONCLUSIONS: Our study demonstrates that a potentially therapeutic neuropeptide exerts pro-inflammatory effects during meningococcal infection in vitro and its use in the treatment of meningitis is contra-indicated.

Immunoproteomic analysis of the development of natural immunity in subjects colonized by Neisseria meningitidis reveals potential vaccine candidates.

The potential protective effect of existing vaccines against serogroup B meningococci, based on outer membrane proteins, is limited by strain restriction and apparent short duration of immune responses. In contrast, meningococcal colonization is known to stimulate the production of cross-protective antibodies as defined by the development of serum bactericidal activity (SBA) against heterologous serogroup B strains. In the current study, a resource of human serum samples and meningococcal carriage strains from studies of longitudinal carriage has been subjected to immunoproteomic analysis to investigate the outer membrane protein antigens associated with the development of SBA to both homologous and heterologous meningococcal serogroup B strains. Proteins from outer membranes of homologous and heterologous strains were separated by two-dimensional electrophoresis and reacted with paired sera which showed an increase in SBA following colonization. Individuals showed differing patterns of reactivity upon colonization, with an increase in SBA being associated with increases in the number of spots detected before and after colonization and/or with increases in the intensity of individual spots. Analysis of immunoreactive spots by mass spectrometry resulted in the identification of 43 proteins potentially associated with the development of SBA against both homologous and heterologous strains. The list of protein immunogens generated included not only well-established antigens but also novel proteins that represent potentially new candidates for inclusion in defined, multicomponent serogroup B vaccines.

A DNA fusion vaccine induces bactericidal antibodies to a peptide epitope from the PorA porin of Neisseria meningitidis.

An experimental DNA plasmid vaccine was developed based on a well-characterized and protective peptide epitope derived from a bacterial porin protein. For this study, we used the P1.16b serosubtype epitope, located in variable region (VR)2 in loop 4 of the PorA outer membrane (OM) porin from Neisseria meningitidis serogroup B strain MC58. A plasmid that encoded the entire loop (pPorAloop4) was prepared, as well as a fusion plasmid that encoded the loop in tandem with the fragment C (FrC) immunostimulatory sequence from tetanus toxin (pPorAloop4-FrC). The constructs were used for intramuscular immunization without exogenous adjuvant. Murine antisera raised to the pPorAloop4-FrC DNA fusion plasmid reacted significantly with OMs in enzyme-linked immunosorbent assay and with whole bacteria by immunofluorescence, whereas antisera raised to the pPorAloop4 DNA plasmid and to control plasmid showed little or no reactivity. Significantly, only the pPorALoop4-FrC plasmid induced bactericidal antibodies, demonstrating that the intrinsic immunostimulatory sequence was essential for inducing a protective immune response. The antibodies raised to the P1.16b pPorALoop4-FrC plasmid were serosubtype specific, showing no significant immunofluorescence reactivity or bactericidal activity against other PorA variants. These data provide proof of principle for a DNA fusion plasmid strategy as a novel approach to preparing vaccines based on defined, protective epitopes.

Proteomic analysis of outer membranes and vesicles from wild-type serogroup B Neisseria meningitidis and a lipopolysaccharide-deficient mutant.

Current experimental vaccines against serogroup B Neisseria meningitidis are based on meningococcal outer membrane (OM) proteins present in outer membrane vesicles (OMV) in which toxic lipopolysaccharide is depleted by detergent extraction. Knowledge of the composition of OM and OMV is essential for developing new meningococcal vaccines based on defined antigens. In the current study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nanocapillary liquid chromatography-tandem mass spectrometry were used to investigate the proteomes of OM and OMV from meningococcal strain MC58 and OM from a lipopolysaccharide-deficient mutant. The analysis of OM revealed a composition that was much more complex than the composition that has been reported previously; a total of 236 proteins were identified, only 6.4% of which were predicted to be located in the outer membrane. The most abundant proteins included not only the well-established major OM proteins (PorA, PorB, Opc, Rmp, and Opa) but also other proteins, such as pilus-associated protein Q (PilQ) and a putative macrophage infectivity protein. All of these proteins were also present in OMV obtained by extraction of the OM with deoxycholate. There were markedly increased levels of some additional proteins in OM from the lipopolysaccharide-deficient mutant, including enzymes that contribute to the tricarboxylic acid cycle. In all the preparations, the proteins not predicted to have an OM location were predominantly periplasmic or cytoplasmic or had an unknown location, and relatively few cytoplasmic membrane proteins were detected. However, several proteins that have previously been identified as potential vaccine candidates were not detected in either OM preparations or in OMV. These results have important implications for the development and use of vaccines based on outer membrane proteins.

Multivalent liposome-based vaccines containing different serosubtypes of PorA protein induce cross-protective bactericidal immune responses against Neisseria meningitidis.

Four serosubtypes (P1.7, 16, P1.7-2, 4, P1.19, 15 and P1.5-1, 10-4) of the PorA outer-membrane protein of Neisseria meningitidis were purified as recombinant proteins and incorporated into liposomes to investigate their immunogenicity. Each serosubtype induced high levels of bactericidal activity against the homologous strain. In addition, liposome preparations containing multiple serosubtypes induced high levels of bactericidal activity against each of the four strains. Significantly, antisera raised against monovalent and multivalent liposomes also showed cross-reactive bactericidal activity against heterologous strains. These data demonstrate that multivalent liposome vaccines, containing multiple PorA serosubtypes, have the potential to provide protection against a broad range of meningococcal strains.

Development of immunity to serogroup B meningococci during carriage of Neisseria meningitidis in a cohort of university students.

Understanding the basis of protective immunity is a key requirement for the development of an effective vaccine against infection with Neisseria meningitidis of serogroup B. We have conducted a longitudinal study into the dynamics of meningococcal acquisition and carriage in first-year university students. The detection of carriage of serogroup B meningococci correlated with an increase in detection of serum bactericidal activity (SBA) against both colonizing and heterologous serogroup B strains. Once induced, SBA remained high throughout the study. Although students showed increases in antibodies reactive with capsular polysaccharide and lipopolysaccharide (LPS), these antibody responses were transitory, and their decline was not accompanied by a corresponding decline in SBA. In contrast, there was a significant correlation between the presence of antibodies to the PorA outer membrane protein and SBA against both homologous and heterologous strains. SBA induced by a PorA-negative mutant confirmed the contribution of PorA to heterologous activity. Increases in SBA against a range of serogroup B strains were also observed in students in whom no meningococcal carriage was detected. This heterologous protection could not be associated with the presence of antibodies reacting with capsule, LPS, PorA, PorB, Rmp, Opa, Opc, or pilin, demonstrating that other, as yet unidentified, antigens contribute to the development of immunity to serogroup B meningococci. Identification of such antigens with the ability to induce an effective cross-reactive bactericidal response to a range of strains would be a major step in the production of a universally effective vaccine against infections caused by serogroup B meningococci.

Recombinant meningococcal PorA protein, expressed using a vector system with potential for human vaccination, induces a bactericidal immune response.

The PorA protein of Neisseria meningitidis (subtype P1.7,16) was expressed as a recombinant protein using three vectors; pTWIN, pQE30 and pRSETA, which introduce different sized N-terminal leader sequences to the mature protein. The immunogenicity of these proteins was compared following incorporation into liposomes and ZW-micelles. All of the recombinant PorA (rPorA) preparations induced high titres of antibody that recognised the homologous PorA within the outer membrane (OM) and on the surface of meningococci. Antisera raised against liposomes and micelles containing the different rPorA proteins induced high and comparable levels of complement-mediated killing of the homologous, but not heterologous, strain. Furthermore, the bactericidal effect was greater when rPorA were incorporated into liposomes rather than detergent micelles. The minimal addition of three N-terminal amino acids in rPorA purified from the pTWIN vector represents a significant improvement over rPorA purified from vectors pQE30 and pRSETA, plus other previously purified rPorA, when considering use of these proteins in vaccines for human use.

Serological correlates of protection against meningococci in a cohort of university students, before and during an outbreak of serogroup C infection.

The association between individual meningococcal antigens and the development of protective immunity to both serogroup C and B meningococci was studied before and during an outbreak of serogroup C infection among university students. Persons who became infected showed, in serum taken either before infection or on admission to the hospital, low levels of bactericidal activity against the outbreak strain; patients who survived infection developed bactericidal activity that correlated with production of antibodies to serogroup C capsular polysaccharide but not to either lipopolysaccharide or major outer-membrane proteins. Uninfected classmates also showed a strong correlation between bactericidal activity and the presence of anti-capsular antibodies. In contrast, bactericidal activity against serogroup B did not correlate with the presence of antibodies to capsular polysaccharide but did correlate with antibodies reacting with the porin proteins PorA and PorB. These studies support the introduction of conjugate MenC vaccines, validate strategies for prevention of serogroup B infection that are based on vaccines containing PorA, and suggest that PorB may also be an important component of such vaccines.

Immunization with the recombinant PorB outer membrane protein induces a bactericidal immune response against Neisseria meningitidis.

Infections with Neisseria meningitidis are characterized by life-threatening meningitis and septicemia. The meningococcal porin proteins from serogroup B meningococci have been identified as candidates for inclusion in vaccines to prevent such infections. In this study, we investigated the vaccine potential of the PorB porin protein free of other meningococcal components. The porB gene from a strain of Neisseria meningitidis expressing the class 3 outer membrane porin protein (PorB3) was cloned into the pRSETB vector, and the protein was expressed at high levels in a heterologous host Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and used for immunization after incorporation into liposomes and into micelles composed either of zwitterionic detergent or nondetergent sulfobetaine. The immunogenicity of these preparations was compared to recombinant PorB protein adsorbed to Al(OH)(3) adjuvant as a control. Although sera raised against the protein adsorbed to Al(OH)(3) reacted with the purified recombinant protein, sera raised against liposomes and micelles showed greater activity with native protein, as measured by enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Reactivity with native protein was considerably enhanced by incorporation of the adjuvant monophosphoryl lipid A into the liposome or micelle preparations. Recognition of the native protein was in a serotype-specific manner and was associated with the ability of the antisera to promote high levels of serotype-specific complement-mediated killing of meningococci. These results demonstrate that the PorB protein should be considered as a component of a vaccine designed to prevent serogroup B meningococcal infection.

Detection of meningococcal carriage by culture and PCR of throat swabs and mouth gargles.

The standard method for detecting meningococcal carriage is culture of throat swabs on selective media, but the levels of carriage determined depend heavily on the skills of the individuals taking the swab and interpreting the cultures. This study aimed to determine the most sensitive detection method for meningococcal carriage. Throat swabs and saline mouth gargles, obtained from 89 university students, were processed in parallel by conventional culture and TaqMan ctrA PCR. Carriage of meningococci, as detected by the combined methods, was 20%. The sensitivities of throat swab culture, throat swab PCR, gargle culture, and gargle PCR were 72, 56, 56, and 50%, respectively, and the probabilities that these techniques would correctly identify the absence of carriage (negative predictive value [NPV]) were 93.4, 89.9, 89.9, and 88.8%. Culturing both throat swabs and gargles increased the NPV to 98.6%. The further addition of throat swab PCR increased this to 100%. Testing gargles by both culture and PCR was as sensitive as testing throat swabs by both methods, suggesting that gargles may be a suitable alternative for large-scale screening studies when throat swabs are difficult to obtain, although they required more lengthy laboratory processing. PCR was a useful adjunct to culture for detecting nasopharyngeal carriage, but it failed to detect some nongroupable strains. For maximum sensitivity, a combination of techniques was required. This study indicates the confidence with which health care professionals involved in meningococcal screening can regard laboratory results.