In vitro evaluation of the antibacterial and osteogenic activity promoted by selenium-doped calcium phosphate coatings.

Selenium is an essential trace element present in 25 selenoenzymes, playing critical roles in a variety of physiological processes, such as anti-oxidative defense and the modulation of cell proliferation and differentiation. This paper characterizes selenium-doped calcium phosphate coatings and evaluates their effects on the osteogenic activity, the proliferation of osteosarcoma cells and biofilm formation. To do so, the structure and elemental composition of the obtained coatings were analyzed, in addition to their thicknesses, and they were compared to pure calcium phosphate coatings. Moreover, the dose-effect ratio of two coatings with the lower (0.6 at%) and the higher (2.7 at%) selenium content was studied in terms of osteogenic, anti-biofilm and cancerous anti-proliferative properties. The results showed the incorporation of selenium in the form of selenite groups into the hydroxyapatite structure, with a similar crystalline pattern to the latter and increased roughness of the coatings. The calcium phosphate coatings with 2.7 at% of selenium resulted in significant osteogenic activity (p < 0.01) of healthy pre-osteoblasts (MC3T3-E1) over long periods of incubation, a significant anti-proliferative effect (p < 0.01) on cancerous osteoblasts (MG63) in a preliminary study, and anti-biofilm properties (p < 0.01) against Staphylococcus epidermidis and Staphylococcus aureus bacterial strains, which are responsible for most infections after orthopedic surgeries.

Induction of immune responses by purified outer membrane protein complexes from Neisseria meningitidis.

A broad-spectrum vaccine against disease caused by serogroup B of Neisseria meningitidis is still a challenge due to antigenic variability. In the present study outer membrane protein complexes and their components were analysed using non-denaturing 2D electrophoresis and identified using LC/MS-MS and MALDI-TOF. Outer membrane protein complexes were purified from both the wild-type strain H44/76 and their knock-out mutants lacking PorA, PorB, RmpM or FetA. The immune responses elicited by the whole outer membrane vesicles (OMV) and the purified complexes were analysed for bactericidal activity, antibody surface binding, antibody-mediated C3b/iC3b deposition, membrane attack complex (MAC) deposition and induction of opsonophagocytosis, both on the homologous and several heterologous strains. The main antigenic complexes found were homomeric, formed by the 60 kDa chaperonin (MSP63) or PorB, or heteromeric, formed by different combinations of PorA, PorB and/or RmpM. The lack of some of these proteins in the OMVs from the knock-out mutants did not affect significantly the immune responses analysed except MAC, which was significantly reduced in the anti-PorA- and anti-PorB- sera, and bactericidal activity, which was absent in the anti-PorA- serum. The sera against purified native complexes showed variable activities against the homologous strain, with greatest responses observed for anti-chaperonin and anti-PorA/PorB/RmpM sera. When tested against heterologous strains, the only anti-complex serum showing consistent responses was that against the 60 kDa chaperonin. The comparison of the responses elicited by the different sera suggests an important role of conformational epitopes, present only in native complexes, in the induction of more effective responses against N. meningitidis.

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis.

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.

Analysis of the expression of the putatively virulence-associated neisserial protein RmpM (class 4) in commensal Neisseria and Moraxella catarrhalis strains.

The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185,H-8), having a molecular mass ( approximately 31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.

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.

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species.

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.

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.

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.

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.

Bactericidal activity of antibodies elicited against the Neisseria meningitidis 37-kDa ferric binding protein (FbpA) with different adjuvants.

The 37-kDa ferric binding protein, FbpA, from three Neisseria meningitidis strains was purified to homogeneity with iron-affinity chromatography and used for immunisation of mice employing four different adjuvants: aluminium hydroxide, Freund's, the saponin Quil-A, and a Ribi adjuvant system (RAS). Controls immunised without adjuvant were also included. All sera obtained were monospecific for the meningococcal FbpA, with antibody titres higher when RAS and Quil-A were used (256), PBS resulting in titres similar to those of Freund's (64), and, surprisingly, with no antibodies elicited when aluminium hydroxide, the only approved adjuvant for use in humans, was used. All anti-FbpA sera bound to intact meningococcal cells, showing a complete cross-reactivity, but the bactericidal activity of anti-FbpA antibodies, demonstrated for the first time in this work, was low (32% of killing with the homologous strain), and the analysis of immunoglobulin isotypes showed that the non-bactericidal IgG1 was predominant. The results confirm that the FbpA is surface-exposed, antigenic, and able to elicit bactericidal antibodies, although, in the conditions and with the adjuvants tested, killing efficacy was low and cross-killing was very variable, not supporting the inclusion of this protein in vaccine formulations. Nevertheless, given the high conservation of the FbpA in the genus Neisseria, its surface exposure and its antigenicity, studies on immunisation with peptides corresponding to the exposed epitopes and/or new adjuvant systems could improve the bactericidal response to this protein, making it suitable for vaccine development.

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.

Blocking of iron uptake by monoclonal antibodies specific for the Neisseria meningitidis transferrin-binding protein 2.

The existence of epitopes common to different strains in the Neisseria meningitidis transferrin (Tf)-binding protein 2 (TBP2), combined with the ability of polyclonal anti-TBP2 antibodies to inhibit Tf binding and block iron uptake in this species, led to this study on the effect of anti-TBP1+2 monoclonal antibodies (MAbs) to determine the presence of epitopes inside the Tf-binding region. All MAbs used reacted exclusively with the homologous strain when tested by dot-blots of outer membrane vesicles, with the reaction being specific for TBP2 after SDS-PAGE and electroblotting. In contrast, ELISA and iron-uptake blocking assays were also positive with heterologous strains belonging to Rokbi's group II (high mol.wt TBP2). The results confirmed the two group classification proposed by Rokbi and, in contrast to other studies, indicated the existence of epitopes in the Tf-binding region that are common only to strains of Rokbi's group II. These epitopes may become denatured after drying for dot-blot assays or after SDS-PAGE and electroblotting.

Blocking of iron uptake from transferrin by antibodies against the transferrin binding proteins in Neisseria meningitidis.

Neisseria meningitidis, when cultured in iron-restricted environments, synthesises new outer-membrane proteins, many of which are necessary for their survival and growth. Some of these proteins e.g. transferrin-binding proteins 1 and 2 (Tbp1 and Tbp2), are required for the acquisition of iron from transferrin and are examples of important iron-regulated meningococcal surface antigens which are not expressed after growth in common laboratory media. The antigenicity and antigenic heterogeneity of these proteins have been extensively studied, and the bactericidal activity of antibodies directed to them have been studied. In this work we analysed the ability of such antibodies to inhibit transferrin binding and to block iron uptake from human transferrin. Antisera from mice immunized with either meningococcal outer membrane vesicles, purified Tbp1/2 complexes, or purified Tbp2, were incorporated in radiolabeled-iron uptake assays. Uptake was blocked by more than 80% in the homologous strains, but blocked much less efficiently in some heterologous strains, correlating well with inhibition of transferrin binding and with an inhibitory effect on bacterial growth. Inhibition of iron uptake from citrate was unaffected which suggests that this effect is due to antibodies against the components of the transferrin binding system, specially Tbp2. Our results support the importance of these proteins and their suitability to be considered in the development of effective vaccines against serogroup B meningococci.

Reliability of laboratory models in the analysis of TBP2 and other meningococcal antigens.

The lack of experimental models suitable for the study of meningococcal pathogenicity led us to investigate if those actually in use (culture in iron-restricted media and animal models) provide results comparable with the responses observed in vivo during infection. In this work we studied three invasive strains cultured both in laboratory media and in human plasma, analysing the immune responses elicited in mice against membrane antigens and comparing them with those seen using homologous human convalescent sera. Outer membrane protein profiles observed after culture in plasma were different and more complex than those obtained after growth in laboratory media. Analogous differences were observed in the antigenic profiles, detecting some antigens recognized by human, but not mouse sera, and vice versa. However, the response to one of the major iron-regulated outer membrane antigens, the transferrin binding protein 2 (TBP2), was unaffected by the culture medium or the model, human or mouse, used for the analysis. In conclusion, we have found that results of antigenic analysis change depending on the culture conditions and animal models used. For the meningococcal antigen TBP2, growth in iron-restricted laboratory media and a mouse model provide results which correlate well with those observed using convalescent human serum from individuals recovered from infections. We suggest that careful analysis and evaluation of experimental results and their comparison with in vivo elicited immune responses are essential in order to get accurate extrapolations for experimental vaccine designs.

In vivo human immune response to transferrin-binding protein 2 and other iron-regulated proteins of Neisseria meningitidis.

When grown under iron restriction, Neisseria meningitidis expresses new outer-membrane proteins, some of which are antigenic and potentially useful as vaccine components. This is particularly relevant to N. meningitidis serogroup B, against which neither polysaccharide nor conjugate vaccines are effective. We investigated recognition of N. meningitidis serogroup B outer-membrane antigens by three sera from patients recovered from meningitis. Recognition of antigens from the homologous strain provided information on in vivo expression during infection and immunogenicity, while cross-reactivity with outer membrane proteins from the other two strains and from another five strains in our collection allowed evaluation of antigenic heterogeneity. Our results demonstrate that transferrin-binding protein 2 (TBP2) is immunogenic in humans, to varying degrees depending on the strain, and that TBP2s (like the equivalent proteins of Haemophilus influenzae type b) are among the most important iron-regulated outer membrane antigens expressed during infection. Other immunogenic outer membrane proteins (some iron-regulated) are also expressed during infection; in a previous study in mouse, three of these proteins (with M(r) of 50, 70 and 77 kDa) did not induce an immune response. Our cross-reactivity data provide some support for Robki et al.'s two-group classification of N. meningitidis strains, and provide evidence against the possibility that the antigenic domains shared by the TBP2s of all N. meningitidis strains induce immune responses in vivo.

Characterization of the transferrin-iron uptake system in Neisseria meningitidis.

The transferrin-iron uptake system of six Neisseria meningitidis strains was characterized using 125I-transferrin in receptor assays and 55Fe-loaded transferrin in uptake assays. Receptors for transferrin varied among the strains both in number (from 700 to 4700 receptors per cell) and in their affinity constants for the protein (Ka ranged from 0.7 x 10(7) to 4.0 x 10(7) l mol-1). Neither receptor numbers nor affinity constants were significantly different in carrier and invasive strains, although the Ka seem to be somewhat higher in the latter. Iron uptake from transferrin was also variable among the strains, but showed the same lack of correlation with their origin.

Purification of the Neisseria meningitidis transferrin binding protein-2 (TBP2) to homogeneity using column chromatography.

A method for purifying TBP2 from N. meningitidis has been developed using affinity chromatography on Tf-agarose followed by ion exchange chromatography; the Tf-binding activity of fractions was evaluated by a dot-blot technique. This method allowed the purification of the TBP2 in milder conditions than those used previously and to a high degree of homogeneity as was demonstrated by Coomassie brilliant blue or Silver staining after SDS-PAGE electrophoresis. The SDS-PAGE profile of the material obtained in the Tf-agarose affinity chromatography step shows only two detectable proteins, identified as the TBP1 and the TBP2, with a small amount of contamination. Passage through a MonoQ HR anion exchange column, allowed the isolation of TBP2 in the absence of TBP1. Our results demonstrate the conservation of the antigenic reactivity of this protein, which produces monospecific serum with the antibodies elicited reacting exclusively with the TBP2 in whole outer membrane vesicles.

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.