An Outer Membrane Receptor of Neisseria meningitidis Involved in Zinc Acquisition with Vaccine Potential

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract

Evaluation of truncated NhhA protein as a candidate meningococcal vaccine antigen

NhhA (Neisseria hia homologue) is an outer membrane protein from Neisseria meningitidis, the causative agent of meningococcal disease. The protein is surface exposed and its expression in a wide range of meningococcal strains suggests it is a promising vaccine candidate. In addition, immunization of mice with outer membrane vesicles of strains that overexpress NhhA in conjunction with one of TbpA, Omp85 or NspA results in synergistic bactericidal responses. We previously showed that the NhhA sequence is highly conserved between strains, with the majority of the differences localized to four distinct variable regions located in the amino-terminal region of the mature protein. In this study, N. meningitidis strains were constructed that over-express wild-type NhhA. Strains expressing truncated versions of NhhA, with deletions from the amino-terminal region that removed the most variable regions, were also made. These expression strains were also modified so that immunodominant, phase- and antigenically-variable outer membrane proteins were not expressed, truncated lipooligosaccharide (LOS) expression was genetically fixed (no phase variability), and capsular polysaccharide expression abolished. Outer membrane vesicles derived from these strains were used to immunize mice. As previously observed, a synergistic effect involving another antigen, TbpA, was required to demonstrate bactericidal activity. The highest bactericidal response against a heterologous strain was obtained with a truncated variant of NhhA. These results indicate that removal of (a) variable region(s) does not reduce bactericidal responses against NhhA, and that bactericidal targets exist in regions other than the variable N-teminus. This provides the basis for future examination of responses against truncated NhhA in protecting against heterologous NhhA strains, and further evaluation of truncated NhhA as a candidate for inclusion in a vaccine against all serogroups of N. meningitidis

Workshop cluster 1+γδ T-cell receptor+ T cells from calves express high levels of interferon-γ in response to stimulation with interleukin-12 and -18

γδ T-cell receptor+ T lymphocytes are an important element of the innate immune system. Early production of interferon (IFN)-γ by γδ T cells may have a role in linking innate and adaptive immune responses and contribute to T helper-1 bias. We investigated the role of cytokines in the activation and induction of IFN-γ secretion by bovine workshop cluster 1+ (WC1+) γδ T cells. The effects of culture with interleukin (IL)-12, IL-18, IL-15 and IL-2 were investigated; these cytokines are known to influence murine and human γδ T cells. We report that bovine WC1+γδ T cells are synergistically stimulated by IL-12 and IL-18 to secrete large quantities of IFN-γ. Neonatal calves were shown to have significantly higher numbers of circulating WC1+γδ T cells than adult animals. In addition, the response of peripheral blood WC1+γδ T cells was significantly higher in neonatal calves compared with adult animals. However, in adult animals the response of lymph node WC1+γδ T cells to IL-12/IL-18 was more pronounced than that of peripheral blood WC1+γδ T cells. We hypothesize that the induction of IFN-γ secretion from WC1+γδ T cells by IL-12 and IL-18 is likely to be an important element of the innate response to pathogens such as Mycobacterium bovis. The high numbers of WC1+γδ T cells in neonatal calves, and their inherent ability to respond to inflammatory cytokines, could be a key factor in the enhanced responses seen in calves to BCG vaccination

Dual Pili Post-translational modifications synergize to mediate meningococcal adherence to platelet activating factor receptor on human airway cells

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, twitching motility, auto-aggregation, and DNA transformation. Pili of N. meningitidis are subject to several different post-translational modifications. Among these pilin modifications, the presence of phosphorylcholine (ChoP) and a glycan on the pilin protein are phase-variable (subject to high frequency, reversible on/off switching of expression). In this study we report the location of two ChoP modifications on the C-terminus of N. meningitidis pilin. We show that the surface accessibility of ChoP on pili is affected by phase variable changes to the structure of the pilin-linked glycan. We identify for the first time that the platelet activating factor receptor (PAFr) is a key, early event receptor for meningococcal adherence to human bronchial epithelial cells and tissue, and that synergy between the pilin-linked glycan and ChoP post-translational modifications is required for pili to optimally engage PAFr to mediate adherence to human airway cells