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1.
Expert Rev Vaccines ; 20(10): 1235-1256, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34524062

ABSTRACT

INTRODUCTION: Species of the genus Neisseria are important global pathogens. Neisseria gonorrhoeae (gonococcus) causes the sexually transmitted disease gonorrhea and Neisseria meningitidis (meningococcus) causes meningitis and sepsis. Liposomes are self-assembled spheres of phospholipid bilayers enclosing a central aqueous space, and they have attracted much interest and use as a delivery vehicle for Neisseria vaccine antigens. AREAS COVERED: A brief background on Neisseria infections and the success of licensed meningococcal vaccines are provided. The absence of a gonococcal vaccine is highlighted. The use of liposomes for delivering Neisseria antigens and adjuvants, for the purposes of generating specific immune responses, is reviewed. The use of other lipid-based systems for antigen and adjuvant delivery is examined briefly. EXPERT OPINION: With renewed interest in developing a gonococcal vaccine, liposomes remain an attractive option for delivering antigens. The discipline of nanotechnology provides additional nanoparticle-based options for gonococcal vaccine development. Future work would be needed to tailor the composition of liposomes and other nanoparticles to the specific vaccine antigen(s), in order to generate optimal anti-gonococcal immune responses. The potential use of liposomes and other nanoparticles to deliver anti-gonococcal compounds to treat infections also should be explored further.


Subject(s)
Gonorrhea , Meningococcal Vaccines , Neisseria meningitidis , Gonorrhea/prevention & control , Humans , Liposomes , Neisseria , Neisseria gonorrhoeae
2.
Methods Mol Biol ; 1997: 77-85, 2019.
Article in English | MEDLINE | ID: mdl-31119618

ABSTRACT

Gonococcal colony typing is part a science and part an art that has been central to studies which have identified crucial virulence antigens and also demonstrated the ability of the bacteria to undergo rapid phase and antigenic variation. Without this fundamental work, modern molecular biological studies of gonococcal pathogenesis would not have been possible. Indeed colony typing is still essential when performing biological experiments with clinical and laboratory isolates and for monitoring their outcome. In this chapter, the methods for performing colony typing and techniques to optimize the process are described.


Subject(s)
Bacterial Typing Techniques/methods , Neisseria gonorrhoeae/classification , Antigenic Variation/immunology , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , DNA, Bacterial/isolation & purification , Neisseria gonorrhoeae/pathogenicity , Virulence Factors/immunology , Virulence Factors/isolation & purification
3.
Expert Rev Vaccines ; 14(12): 1633-49, 2015.
Article in English | MEDLINE | ID: mdl-26468663

ABSTRACT

Peptidyl prolyl cis/trans isomerases (PPIases) are a superfamily of proteins ubiquitously distributed among living organisms, which function primarily to assist the folding and structuring of unfolded and partially folded polypeptide chains and proteins. In this review, we focus specifically on the Macrophage Infectivity Potentiator (MIP)-like PPIases, which are members of the immunophilin family of FK506-binding proteins (FKBP). MIP-like PPIases have accessory roles in virulence and are candidates for inclusion in vaccines protective against both animal and human bacterial pathogens. A structural vaccinology approach obviates any issues over molecular mimicry and potential cross-reactivity with human FKBP proteins and studies with a representative antigen, the Neisseria meningitidis-MIP, support this strategy. Moreover, a dual approach of vaccination and drug targeting could be considered for controlling bacterial infectious diseases of humans and animals.


Subject(s)
Bacterial Proteins/immunology , Bacterial Vaccines/immunology , Macrophages/immunology , Peptidylprolyl Isomerase/immunology , Tacrolimus Binding Proteins/immunology , Vaccines, Subunit/immunology , Amino Acid Sequence , Animals , Bacterial Infections/immunology , Bacterial Infections/prevention & control , Drug Design , Humans , Legionella pneumophila/immunology , Meningitis, Meningococcal/immunology , Meningitis, Meningococcal/microbiology , Meningitis, Meningococcal/prevention & control , Molecular Sequence Data , Neisseria meningitidis/immunology , Neisseria meningitidis/pathogenicity
4.
Vaccine ; 33(36): 4486-94, 2015 Aug 26.
Article in English | MEDLINE | ID: mdl-26207592

ABSTRACT

The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.


Subject(s)
ATP-Binding Cassette Transporters/immunology , Antibodies, Bacterial/blood , Bacterial Proteins/immunology , Blood Bactericidal Activity , Immunity, Heterologous , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/immunology , ATP-Binding Cassette Transporters/genetics , Adjuvants, Immunologic/administration & dosage , Aluminum Hydroxide/administration & dosage , Animals , Bacterial Proteins/genetics , Cloning, Molecular , Cross Reactions , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Liposomes/administration & dosage , Meningococcal Vaccines/administration & dosage , Meningococcal Vaccines/genetics , Mice, Inbred BALB C , Neisseria meningitidis, Serogroup B/genetics , Rabbits , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
5.
Infect Immun ; 83(2): 730-42, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25452551

ABSTRACT

A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (-LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines.


Subject(s)
Antibodies, Bacterial/immunology , Bacterial Proteins/immunology , Meningitis, Meningococcal/immunology , Meningococcal Vaccines/immunology , Neisseria meningitidis/immunology , Tacrolimus Binding Proteins/immunology , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Cloning, Molecular , Cross Reactions/immunology , Humans , Meningitis, Meningococcal/prevention & control , Mice , Mice, Inbred BALB C , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Vaccination
6.
Vaccine ; 32(11): 1280-6, 2014 Mar 05.
Article in English | MEDLINE | ID: mdl-24486354

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/immunology , Cross Reactions/immunology , Meningococcal Vaccines/immunology , Proteome/immunology , Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Clinical Trials, Phase I as Topic , Humans , Neisseria meningitidis, Serogroup B , Randomized Controlled Trials as Topic , Serum Bactericidal Antibody Assay
8.
Vaccine ; 31(22): 2584-90, 2013 May 24.
Article in English | MEDLINE | ID: mdl-23566947

ABSTRACT

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.


Subject(s)
Chaperonin 60/immunology , Meningococcal Infections/immunology , Meningococcal Vaccines/pharmacology , Neisseria meningitidis/immunology , Amino Acid Sequence , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology , Antibody Formation , Antigens, Bacterial/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Proteins/pharmacology , Blotting, Western , Chaperonin 60/genetics , Chaperonin 60/pharmacology , Cross Reactions , Fluorescent Antibody Technique , Humans , Meningococcal Infections/microbiology , Meningococcal Infections/prevention & control , Meningococcal Vaccines/genetics , Meningococcal Vaccines/immunology , Mice , Mice, Inbred BALB C , Mitochondrial Proteins/immunology , Neisseria meningitidis/genetics , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/pharmacology
9.
mBio ; 4(2)2013 Feb 26.
Article in English | MEDLINE | ID: mdl-23443003

ABSTRACT

UNLABELLED: The acp gene encoding the 13-kDa adhesin complex protein (ACP) from Neisseria meningitidis serogroup B strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant ACP (rACP) was used for immunization studies. Analysis of the ACP amino acid sequences from 13 meningococcal strains, isolated from patients and colonized individuals, and 178 strains in the Bacterial Isolate Genome Sequence (BIGS) database showed the presence of only three distinct sequence types (I, II, and III) with high similarity (> 98%). Immunization of mice with type I rACP in detergent micelles and liposomes and in saline solution alone induced high levels of serum bactericidal activity (SBA; titers of 1/512) against the homologous strain MC58 and killed strains of heterologous sequence types II and III with similar SBA titers (1/128 to 1/512). Levels of expression of type I, II, or III ACP by different meningococcal strains were similar. ACP functioned as an adhesin, as demonstrated by reduced adherence of acp knockout (MC58 ΔACP) meningococci to human cells in vitro and the direct surface binding of rACP and by the ability of anti-rACP sera to inhibit adherence of wild-type bacteria. ACP also mediated the invasion of noncapsular meningococci into human epithelial cells, but it was not a particularly impressive invasin, as the internalized bacterial numbers were low. In summary, the newly identified ACP protein is an adhesin that induces cross-strain bactericidal activity and is therefore an attractive target antigen for incorporation into the next generation of serogroup B meningococcal vaccines. IMPORTANCE: Infections caused by Neisseria meningitidis serogroup B are still significant causes of mortality and morbidity worldwide, and broadly protective vaccines of defined antigen composition are not yet licensed. Here, we describe the properties of the adhesin complex protein (ACP), which we demonstrate is a newly recognized molecule that is highly conserved and expressed to similar levels in meningococci and facilitates meningococcal interactions with human cells. We also report that a recombinant ACP protein vaccine induces murine antibodies that significantly kill meningococci expressing different ACP. Taken together, these properties demonstrate that ACP merits serious consideration as a component of a broadly protective vaccine against serogroup B meningococci.


Subject(s)
Adhesins, Bacterial/immunology , Meningococcal Infections/microbiology , Meningococcal Infections/prevention & control , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/immunology , Adhesins, Bacterial/chemistry , Adhesins, Bacterial/genetics , Animals , Bacterial Adhesion , Blood Bactericidal Activity , Cell Line , Cloning, Molecular , Epithelial Cells/microbiology , Escherichia coli , Gene Expression , Genetic Variation , Humans , Meningococcal Infections/immunology , Meningococcal Vaccines/administration & dosage , Meningococcal Vaccines/genetics , Mice , Mice, Inbred BALB C , Microbial Viability , Molecular Weight , Neisseria meningitidis, Serogroup B/genetics , Rabbits , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
10.
PLoS One ; 7(8): e42660, 2012.
Article in English | MEDLINE | ID: mdl-22900037

ABSTRACT

BACKGROUND: Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of life-threatening neonatal meningitis and survivors often suffer permanent neurological damage. How this organism interacts with the meninges and subsequently with astrocytes that constitute the underlying cortical glia limitans superficialis is not known. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we demonstrate dose-dependent adherence of GBS over time to human meningioma cells and fetal astrocytes in vitro, which was not influenced by expression of either ß-haemolysin/cytolysin (ß-h/c) toxin, different capsule serotypes or by absence of capsule (p>0.05). Internalization of GBS by both cell types was, however, a slow and an infrequent event (only 0.02-0.4% of associated bacteria were internalised by 9 h). Expression of ß-h/c toxin did not play a role in invasion (p>0.05), whereas capsule expression lead to a reduction (p<0.05) in the numbers of intracellular bacteria recovered. GBS strains induced cytotoxicity as demonstrated by the measurement of lactate dehydrogenase (LDH) enzyme release by 9 h and by viable staining. Increasing levels of meningioma cell death correlated with bacterial growth and the phenotype of ß-h/c toxin production, i.e. from weakly, to normo- to hyper-haemolytic. However, cytotoxicity was significantly greater (p<0.05) towards astrocytes, and infection with initial MOI≥0.003 induced 70-100% LDH release. By comparing wild-type (ß-h/c(+)) and mutant (ΔcylE ß-h/c(-)) strains and ß-h/c toxin extracts and by using the surfactant dipalmitoylphosphatidylcholine in cytotoxicity inhibition experiments, ß-h/c toxin was demonstrated as principally responsible for cell death. CONCLUSIONS/SIGNIFICANCE: This study has described key events in the interactions of GBS with meningeal cells and astrocytes in vitro and a major virulence role for ß-h/c toxin. Understanding the mechanisms involved will help to identify potential therapies for improving patient survival and for reducing the incidence and severity of neurological sequelae.


Subject(s)
Astrocytes/metabolism , Astrocytes/microbiology , Meninges/metabolism , Meninges/microbiology , Streptococcus agalactiae/metabolism , Bacterial Adhesion , Bacterial Toxins/metabolism , Cell Death , Cell Line, Tumor , Hemolysin Proteins/metabolism , Humans , Streptococcus agalactiae/pathogenicity , Tumor Cells, Cultured
11.
J Biomed Biotechnol ; 2012: 491298, 2012.
Article in English | MEDLINE | ID: mdl-22318778

ABSTRACT

BACKGROUND: A role for pilus during attachment of Neisseria gonorrhoeae to epithelia of the female reproductive tract is currently assumed. However, Pil⁻ gonococci have been observed during infection of the reproductive tract, which prompted us to examine the effect of pili on the dynamics of infection and the inflammatory responses of mucosal explants of the human fallopian tube. METHODS: Mucosal explants were infected in vitro with Opa negative Pil⁻ and Pil⁺N. gonorrhoeae strains. RESULTS: Piliation enhanced gonococcal adherence to the epithelium within 3 h of infection (P < 0.05) but thereafter did not offer advantage to gonococci to colonize the epithelial cell surface (P > 0.05). No differences were found between the strains in numbers of gonococci inside epithelial cells. Pil⁻ bacteria induced higher levels (P < 0.05) of IL-1ß, TNF-α, GM-CSF, MCP-1, and MIP-1ß than Pil⁺ bacteria. There were no differences between both strains in LOS pattern, and Pil expression did not change after coincubation with mucosal strips. CONCLUSIONS: Results show that gonococcal invasion of the human fallopian tube can occur independently of pilus or Opa expression, and suggest that pilus, by inhibition of several key elements of the initial inflammatory response, facilitates sustained infection of this organ.


Subject(s)
Epithelium/microbiology , Fallopian Tubes/microbiology , Fimbriae, Bacterial/genetics , Neisseria gonorrhoeae/genetics , Bacterial Outer Membrane Proteins/genetics , Cytokines/genetics , Cytokines/metabolism , Female , Gene Expression Regulation, Bacterial , Humans , Inflammation/metabolism , Inflammation/microbiology , Neisseria gonorrhoeae/growth & development , Neisseria gonorrhoeae/pathogenicity , Organ Culture Techniques
12.
Methods Mol Biol ; 799: 343-60, 2012.
Article in English | MEDLINE | ID: mdl-21993655

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/immunology , Immune Sera/immunology , Neisseria meningitidis/immunology , Proteomics/methods , Bacterial Vaccines/immunology , Blotting, Western , Electrophoresis, Gel, Two-Dimensional/methods , Electrophoresis, Polyacrylamide Gel/methods , Humans , Isoelectric Focusing/methods
13.
Infect Immun ; 79(9): 3784-91, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21708989

ABSTRACT

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.


Subject(s)
Antibodies, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Meningitis, Meningococcal , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/immunology , Amino Acid Sequence , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Blood Bactericidal Activity , Blotting, Western , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Macrophages , Meningitis, Meningococcal/immunology , Meningitis, Meningococcal/prevention & control , Meningitis, Meningococcal/therapy , Mice , Mice, Inbred BALB C , Neisseria meningitidis, Serogroup B/genetics , Rabbits , Recombinant Proteins/immunology , Serum Bactericidal Antibody Assay , Vaccines, Synthetic/immunology
14.
Biol Res ; 43(1): 39-50, 2010.
Article in English | MEDLINE | ID: mdl-21157631

ABSTRACT

In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40% reduction in LDH release, which correlated with a ~50% reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.


Subject(s)
Fallopian Tubes/microbiology , L-Lactate Dehydrogenase/metabolism , Neisseria gonorrhoeae/enzymology , Nitric Oxide Synthase/metabolism , Nitric Oxide/metabolism , RNA, Messenger/metabolism , Cells, Cultured , Fallopian Tubes/pathology , Female , Humans , Time Factors
15.
Genome Med ; 2(7): 43, 2010 Jul 22.
Article in English | MEDLINE | ID: mdl-20670394

ABSTRACT

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.

16.
Inflamm Res ; 59(2): 105-13, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19685205

ABSTRACT

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.


Subject(s)
Inflammation/physiopathology , Meninges/microbiology , Meninges/physiopathology , Meningitis, Meningococcal/physiopathology , Neisseria meningitidis/drug effects , alpha-MSH/pharmacology , Bacterial Adhesion/drug effects , Cell Line , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Humans , Inflammation/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Meninges/drug effects , Meningitis, Meningococcal/metabolism , Up-Regulation/drug effects
17.
Biol. Res ; 43(1): 39-50, 2010. graf
Article in English | LILACS | ID: lil-548028

ABSTRACT

In the present study, we investigated whether cellular damage, as demonstrated by lactate dehydrogenase (LDH) release in the human fallopian tube (FT) infected by Neisseria gonorrhoeae (Ngo), correlated with high levels of nitric oxide synthase (NOS) mRNA and enzyme activity. Infection with Ngo induced a significant increase (~35-fold) in mRNA transcripts of the inducible isoform of NOS. Paradoxically, a reduction in NOS enzyme activity was observed in infected cultures, suggesting that gonococcal infection possibly influences translation of iNOS mRNA to the enzyme. In addition, treatment with the NOS inhibitor TRIM did not prevent gonococcal-induced cellular damage. In contrast, the addition of the inhibitor L-NAME induced a 40 percent reduction in LDH release, which correlated with a ~50 percent reduction in gonococcal numbers. Moreover, treatment of normal FT explants with an exogenous NO donor, SNAP, did not induce significant cellular damage. Taken together, our data suggest that NO does not contribute to cellular damage during infection of the human FT with Neisseria gonorrhoeae.


Subject(s)
Female , Humans , Fallopian Tubes/microbiology , L-Lactate Dehydrogenase/metabolism , Neisseria gonorrhoeae/enzymology , Nitric Oxide Synthase/metabolism , Nitric Oxide/metabolism , RNA, Messenger/metabolism , Cells, Cultured , Fallopian Tubes/pathology , Time Factors
18.
Infect Immun ; 77(11): 5080-9, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19737898

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Carrier State/immunology , Meningococcal Infections/immunology , Meningococcal Vaccines/immunology , Neisseria meningitidis, Serogroup B/immunology , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Blotting, Western , Carrier State/blood , Electrophoresis, Polyacrylamide Gel , Humans , Meningococcal Infections/blood , Proteomics
19.
Infect Immun ; 76(7): 3156-63, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18443097

ABSTRACT

Septicemia caused by Neisseria meningitidis is characterized by increasing levels of meningococcal lipopolysaccharide (Nm-LPS) and cytokine production in the blood. We have used an in vitro human whole-blood model of meningococcal septicemia to investigate the potential of CyP, a selective Toll-like receptor 4 (TLR4)-MD-2 antagonist derived from the cyanobacterium Oscillatoria planktothrix FP1, for reducing LPS-mediated cytokine production. CyP (> or = 1 microg/ml) inhibited the secretion of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1beta (IL-1beta), and IL-6 (by >90%) and chemokines IL-8 and monocyte chemoattractant protein 1 (by approximately 50%) induced by the treatment of blood with pure Nm-LPS, by isolated outer membranes, and after infection with live meningococci of different serogroups. In vitro studies with human dendritic cells and TLR4-transfected Jurkat cells demonstrated that CyP competitively inhibited Nm-LPS interactions with TLR4 and subsequent NF-kappaB activation. These data demonstrate that CyP is a potent antagonist of meningococcal LPS and could be considered a new adjunctive therapy for treating septicemia.


Subject(s)
Bacteremia/immunology , Cyanobacteria/immunology , Cytokines/antagonists & inhibitors , Lipopolysaccharides/antagonists & inhibitors , Neisseria meningitidis/pathogenicity , Toll-Like Receptor 4/antagonists & inhibitors , Bacteremia/microbiology , Cytokines/biosynthesis , Dendritic Cells , Humans , Jurkat Cells , Lipopolysaccharides/pharmacology , Toll-Like Receptor 4/genetics , Transfection
20.
Infect Immun ; 76(1): 334-8, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17967859

ABSTRACT

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.


Subject(s)
Antibodies, Bacterial , Bacterial Vaccines/immunology , Meningococcal Infections/prevention & control , Neisseria meningitidis/immunology , Porins , Vaccines, DNA/immunology , Animals , Epitopes , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Plasmids
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