Neisseria gonorrhoeae Pilus Attenuates Cytokine Response of Human Fallopian Tube Explants

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-1β, TNF-α, GM-CSF, MCP-1, and MIP-1β 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

Immunization with a multiple antigen peptide containing defined B- and T-cell epitopes: production of bactericidal antibodies against group B Neisseria meningitidis

Previous analysis of the class 1 outer-membrane (OM) protein of Neisseria meningitidis has identified discrete epitopes to be potential targets for immune attack. The conformation of these epitopes is important for inducing antibodies which can react with the native protein and promote complement-mediated lysis of the meningococcus. The multiple antigen peptide (MAP) system, which consists of an oligomeric branching lysine core to which are attached dendritic arms of defined peptide antigens, confers some conformational stability and also allows for the preparation of immunogens containing both B-cell and T helper (Th)-cell epitopes. In this study, MAPs were synthesized to contain (i) the subtype P1.16b meningococcal class 1 protein B-cell epitope (B-MAP), and (ii) the P1.16b epitope in tandem with a defined Th-cell epitope, chosen from tetanus toxin (BT-MAP). The B-MAP was nonimmunogenic in animals. In contrast, incorporation of the Th-cell epitope into BT-MAP induced a strong humoral response towards the class 1 protein B-cell epitope. Antisera from immunized mice and rabbits reacted in ELISA with synthetic peptides containing the B-cell epitope, and also cross-reacted with meningococcal OMs from strains of subtype P1.16b and P1.16a. Murine and rabbit antisera showed similar reactivity and epitope specificity, but did not react with denatured class 1 protein in Western blotting, indicating the predominance of antibodies directed towards conformational epitopes. The antisera from rabbits immunized with BT-MAP promoted complement-mediated bactericidal killing not only of the homologous meningococcal subtype P1.16b strain but also of subtype P1.16a

Expression of the Class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag

The class 1 protein (PorA) is a major component of the outer membrane of Neisseria meningitidis and functions as a cationic porin. The protein is particularly effective in generating a bactericidal immune response following infection and is therefore under investigation as a potential antigen for inclusion in new meningococcal vaccines. Studies on the vaccine potential of PorA would be facilitated by the production of pure protein, free from other components of the meningococcal outer membrane. In the current study, PorA was expressed from the heterologous host Escherichia coli as a C-terminal fusion to an inducible protein-splicing element (intein) with an N-terminal chitin-binding domain (CBD) (IMPACT-TWIN system). The CBD acted as an affinity tag and allowed binding of the fusion protein to a chitin bead column, after which self-cleavage of the intein at its C-terminus was induced, resulting in the release of mature PorA. Cleavage of the fusion protein was temperature- and time-dependent, and was optimal at pH 7.0 after 5 days of storage at 4 °C. Efficient cleavage was also dependent on the addition of a minimal amino acid sequence (Gly–Arg–Ala) to the N-terminus of the mature PorA protein. This represented a significant improvement on the large N-terminal sequences introduced by other expression systems previously used to prepare recombinant PorA, and the yields of PorA purified with the IMPACT-TWIN system were similar. Thus, the IMPACT-TWIN system provides a facile method for producing recombinant PorA and may also be useful for the production of other bacterial outer-membrane proteins for vaccine studies

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

Effect of adjuvant composition on immune response to a multiple antigen peptide (MAP) containing a protective epitope from Neisseria meningitidis class 1 porin

A variety of adjuvants with the potential for use with experimental human vaccines were used for immunisation of mice, in an attempt to augment the humoral immune response to a multiple antigen peptide (MAP) containing a protective epitope from the sero-subtype specific class 1 porin protein of Neisseria meningitidis, in tandem with a Th-cell epitope. Surface plasmon resonance showed that combinations of the immunomodulators pluronic block co-polymer, muramyl dipeptide and monophosphoryl lipid A (MPL), increased the magnitude and avidity of the immune response in comparison with both Al(OH)3 and Freund-type adjuvants. In addition, the incorporation of MPL was essential for the induction of a broad distribution of antibody isotypes. The antibodies induced recognised the native protein in meningococcal outer membranes in a subtype-specific manner. The formulations containing these multiple immunomodulators which have already been used in human phase I/II trials with experimental vaccines, are candidates for inclusion in future human vaccines based on synthetic peptides containing defined, protective epitopes

The potential utility of liposomes for Neisseria vaccines

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.</p

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