Carlos M. Ferreirós

In vitro induction of memory-driven responses against Neisseria meningitidis by priming with Neisseria lactamica.

Natural immunity against Neisseria meningitidis is acquired during childhood and youth through successive colonizations by commensal Neisseria, carrier N. meningitidis, and other bacterial genera sharing cross-reactive antigens with the meningococci. We have analyzed in mice the ability of Neisseria lactamica strains to induce immunological memory so that, upon a later contact with N. meningitidis, quickly raise protective responses against antigens that show cross-reactivity with meningococcal surface proteins. Sera obtained from mice immunized with N. lactamica and boosted with N. meningitidis were able to kill meningococci, with bactericidal activities variable depending on the immunizing strains used in the assays. Different mixtures of those sera resulted in higher killing activities, which agrees with the idea that successive colonizations by N. lactamica enhance the anti-meningococcal response. The existence of such outer membrane cross-reactive antigens has to be kept in mind when using outer membrane vesicle (OMV)-based anti-meningococcal vaccines because their use can affect colonization by N. lactamica and other species, hampering the natural mechanisms of acquisition of immunity to the meningococci, and leaving its ecological niche free for colonization by undesirable microorganisms.

Cooperation between the components of the meningococcal transferrin receptor, TbpA and TbpB, in the uptake of transferrin iron by the 37-kDa ferric-binding protein (FbpA).

Meningococcal TbpAB complexes TbpA, TbpB and FbpA were purified and used to study their role in the uptake of iron from transferrin to FbpA. Purification was achieved by affinity chromatography techniques, yielding homogeneous, non-denatured and functional material. TbpA could not be separated from TbpB and had to be purified from a TbpB-defective mutant strain. FbpA was able to bind iron from transferrin only when TbpAB complexes, TbpA and/or TbpB, were also present during the interaction. The highest uptake efficiences were obtained with TbpAB complexes or TbpA/TbpB mixtures. We conclude that the TbpA and TbpB molecules form true functional transferrin receptors, that FbpA is able to take iron directly from transferrin when in the presence of the components of the receptor, and that both Tbps are necessary for an optimal operation of the uptake system.

The neisserial 37 kDa ferric binding protein (FbpA)

The ferric binding protein (FbpA) is one of the major proteins regulated by the level of environmental iron in the genus Neisseria. Its conservation in all species of pathogenic Neisseria has been demonstrated, and the possible role that it plays in the iron uptake mechanisms in these bacteria has been postulated. Similar proteins in Haemophilus influenzae (HitA) and in Serratia marcescens (SfuA) have been described, but relationships with the meningococcal FbpA could not be proven. Although supposedly periplasmic, the exact location of FbpA remains controversial because some molecules, or parts of them, have been found exposed to the bacterial outer surface. The DNA sequence downstream of the fbpA gene has been recently analysed, finding an operon composed of three open reading frames: fbpA, encoding for FbpA; fbpB, that codifies a cytoplasmic permease, and fbpC, that contains the information for a nucleotide binding protein. These proteins would form an iron transport system through the periplasmic space. FbpA is highly antigenic in mice when injected in purified form, shows intraspecies and interspecies antigenic homogenicity, and specific anti-FbpA antibodies are fully cross-reactive; nevertheless, the in vivo induction of anti-FbpA antibodies in man is still polemical. Recent studies reveal that the purified FbpA induces a fair response of bactericidal antibodies in mice.

Analysis of TbpA and TbpB functionality in defective mutants of Neisseria meningitidis

Iron uptake analysis suggested that the Neisseria meningitidis transferrin (Tf) binding proteins, TbpA and TbpB, form only one type of receptor complex. Mutants defective in the synthesis of either TbpA or TbpB, but not defective in both proteins, can bind Tf, suggesting that both proteins are surface exposed and function in Tf binding. Also, iron uptake from Tf into the meningococci did not require the presence of both Tbps. The TbpB-defective mutant incorporated c. 37% of the iron taken up by the wild-type strain, but this was insufficient for bacterial growth. The TbpA-defective mutant incorporated c. 50% of the iron taken up by the wild-type strain and was able to grow with Tf as the only iron source. Mouse antibodies specific for TbpA were able to block c. 70% of the iron uptake from Tf in the wild-type strain, whereas they blocked only 22% of iron uptake in the TbpB-defective mutant and did not block uptake in the TbpA-defective strain. These results emphasise that TbpA should be considered in future vaccine trials in which iron-restricted proteins are to be included in the vaccine formulation.

EVALUATION OF CROSS-REACTIVE ANTIGENS AS DETERMINANTS OF CROSS-BACTERICIDAL ACTIVITY IN PATHOGENIC AND COMMENSAL NEISSERIA

Several antisera raised against outer membane vesicles obtained from invasive and carrier Neisseria meningitidis strains and commensal Neisseria and Moraxella catharralis species were assayed to test cross-bactericidal activity on Neisseria meningitidis strains. Results demonstrate that, despite the wide antigenic cross-reactivity previously shown by Western-blotting for the major outer membrane antigenic proteins of all Neisseria species, complement mediated killing shows very variable patterns that can not be predicted on the basis of antigenic cross-reactivity. Results of antibody tritations on homologous and heterologous strains, isotyping, and bactericidal activity of sera raised against denatured purified outer-membrane vesicle proteins, suggest that the responsibility for most of the bactericidal activity of the sera must be conformational and/or shared epitopes not detectable by Western-blotting.

Effect of adjuvants in the isotypes and bactericidal activity of antibodies against the transferrin-binding proteins of Neisseria meningitidis

Twenty-eight Neisseria meningitidis strains of different serogroups, serotypes, and TbpB isotypes were used to test the effect of five adjuvant formulations on the immune response to the meningococcal transferrin-binding proteins (Tbps) in mice. Levels of anti-Tbps antibodies were relatively low when purified TbpA-TbpB complexes were used for immunization, those obtained with the RAS adjuvant being the highest, and the isotype distribution reveals a prevalence of the non-bactericidal IgG1. Specific anti-Tbps antibody levels were five to 125 times higher immunizing with whole outer membrane vesicles, with bactericidal isotypes prevailing, which suggests that presentation of these antigens in their natural conformation is crucial to elicit a good response. Nevertheless, bactericidal activity did not correlate with these characteristics, confirming that it must be also influenced by other factors, and direct evaluation of the killing ability is necessary to draw conclusions about the efficacy of antigens or adjuvants in vaccine design.

Adherence of psychrotrophic bacteria to dairy equipment surfaces.

Psychrotrophic bacteria isolated from raw milk were tested for their ability to adhere to steel, two types of rubber, and glass, materials employed in the construction of milking equipment. The adherence assays were carried out by exposure of the materials to radioactively labelled bacteria in both a buffering solution (Ringers) and milk. The degree of adherence of Gram-positive bacteria was lower (P less than 0.001) than that of Gram-negative bacteria. Glass was the material least prone to bacterial adherence (P less than 0.001); there were no significant differences between the other three materials. Milk was found to inhibit adhesion significantly (P less than 0.05), this inhibition being more evident with the most adherent bacteria. There was no statistically significant correlation between bacterial surface hydrophobicity and adherence. Our results suggest that intrinsic bacterial adherence cannot be considered a relevant factor in the contamination of milking equipment.

Antigenicity, cross-reactivity and surface exposure of the Neisseria meningitidis 37 kDa protein (Fbp)

The 37 kDa iron-repressible protein, Fbp, was purified from two Neisseria meningitidis strains by metal-affinity chromatography and used to obtain mouse monospecific polyclonal immune sera. Dot-blot, immunoblotting and whole cell ELISA results demonstrate that the Fbp is present in all 16 N. meningitidis and four commensal Neisseria species tested, is highly antigenic in mouse when injected in pure form, and shows intra- and inter-species antigenic homogeneity, anti-Fbp antibodies being fully cross-reactive using the techniques mentioned. We also found that Fbp molecules (or parts of them) are surface exposed, in disagreement with the proposed exclusively periplasmic localization, although anti-Fbp antibodies seem unable to block iron uptake or to induce complement-mediated killing of the meningococci. Taken along with the high immunogenicity of the purified protein and the complete cross-reactivity of the antibodies elicited, this suggests that the protective effect of the purified Fbp must be further studied to evaluate its inclusion in future vaccine trials.

Analysis of the expression of outer-membrane proteins in Neisseria meningitidis in iron-replete and iron-deficient media

A total of 103 Neisseria strains, including 42 carrier and 40 invasive N. meningitidis and 21 commensal species were studied. Outer-membrane proteins from carrier and invasive N. meningitidis showed similar molecular mass distributions (except in the range from 78 to 82 kDa in which 90% of the invasive but only 47.6% of the carrier strains expressed proteins), many strains showing neither class II (41 kDa) nor class III (38 kDa) proteins. When grown in iron-restricted conditions proteins were induced mainly in the ranges from 62 to 92 kDa with no significant differences between groups. Commensal species, both in normal and in iron-restricted conditions, showed outer-membrane protein patterns different from those of N. meningitidis in several molecular mass ranges. Cluster analysis based on expression of principal outer-membrane proteins allowed the differentiation of commensal species and N. meningitidis, although not that of the invasive and carrier groups within the latter.

Factor Analysis in the Evaluation of the Relationship between Bacterial Adherence to Biomaterials and Changes in Free Energy

The relative surface charge and free energy of forty-one coagulase-negative staphylococci were found to be normally distributed; therefore, they can be considered a homogeneous group under strict statistical criteria. The adherence of these bacteria to eight different biomaterials (seven synthetic and one biologic) was found to be independent of charge and variations in free energy during adhesion. Adherence can be explained as a thermodynamic process (free energy decreased with adherence), except in the case of bovine pericardium in which free energy increases. With these biomaterials, a correlation was found between adherence and bacterial charge. Bacterial adherence and bacterial charge correlate with the surface parameters of the biomaterials. This correlation does not occur when the relationships between parameters are evaluated by means of factors analysis, thus indicating the importance of the statistical method selected for the evaluation of bacterial adherence.