Sandra Sánchez

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.

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.

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 outer membrane porin complexes of Neisseria meningitidis in wild‐type and specific knock‐out mutant strains

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1‐D blue native (BN) electrophoresis, 2‐D BN/SDS‐PAGE and 2‐D diagonal electrophoresis, followed by LC/MS‐MS (for 1‐D gels) or MALDI‐TOF (for 2‐D gels) revealed at least six porin complexes in the wild‐type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild‐type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (≈︂800 kDa). Our results suggest that PorA and PorB could form a ‘basic’ template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.

Study of the stability of proteoliposomes as vehicles for vaccines against Neisseria meningitidis based on recombinant porin complexes

Although effective against epidemic serogroup B Neisseria meningitidis strains, vaccines based on outer membrane vesicles continue to present important limitations, and great efforts are currently being focused in the development of a variety of new vaccine candidates and in the reformulation of currently existing ones. In this work, three N. meningitidis proteins, the PorA and PorB porins and the RmpM protein, were cloned, purified and incorporated into liposomes to build defined systems. The ability of proteoliposomes to allow the refolding porin complexes, and their stability during storage at 4°C and after lyophilization in presence of two cryoprotection agents, glucose and trehalose, were evaluated. This approach allowed to mimic the porin complexes present in natural OMVs, reducing the content of hypervariable protein PorA. During storage at 4°C, our systems showed some changes in the morphology and aggregation after three months, while after lyophilization the systems maintained their properties during the whole nine months of storage checked, with glucose allowing the best preservation of the antigenic properties of the proteins in the proteoliposomes.

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.

Characterisation and immune responses to meningococcal recombinant porin complexes incorporated into liposomes

We have analysed the structure of meningococcal outer membrane complexes and found that the main complexes are formed by different combinations of PorA and/or PorB molecules, associated to other proteins such as RmpM. In view of the growing knowledge of the importance of conformational epitopes in the immune responses to many pathogens, our aim in this study was to analyse the interactions of PorA and PorB by reconstitution of both recombinant porins into liposomes and determine the relevance of these interactions for the immune response. Recombinant PorA and PorB incorporated into liposomes associate forming complexes that are homomeric when only one of the porins is present, but heteromeric when both neisserial porins are present, mimicking those found previously in native outer membrane vesicles (OMVs). Association of PorA and PorB to form heterocomplexes modifies the immunogenicity of at least PorB, allowing the production of antibodies that recognise conformational epitopes, and produces new epitopes that react with a 50 kDa outer membrane protein not yet identified.

High resolution clear native electrophoresis (hrCNE) allows a detailed analysis of the heterotrimeric structure of recombinant Neisseria meningitidis porins inserted into liposomes.

Three recombinant proteins of Neisseria meningitidis, rPorB, rPorA, and rRmpM, were purified and incorporated into liposomes prepared by dialysis-extrusion. The protein complexes formed using different combinations of recombinant proteins were studied by high resolution clear native electrophoresis (hrCNE) and 2-D hrCNE/SDS-PAGE, analyzing the influence of the stoichiometry of the two porins in the formation of complexes and comparing them with native porin complexes present in OMVs from five different N. meningitidis strains. Insertion of the recombinant proteins into liposomes allowed a complete refolding of porin complexes, and the electrophoretic analyses showed that, when the three recombinant proteins are present, the pattern of porin complexes obtained is similar to that observed in native OMVs. We could show homocomplexes of each individual porin and PorA/PorB, RmpM/PorB, and PorA/PorB/RmpM heterocomplexes. Our results suggest that RmpM binds only to PorB, confirm the trimeric structure of N. meningitidis pores, and demonstrate that insertion into liposomes restores the native structure of porin complexes.

Interspecific neisserial high molecular weight proteins able to induce natural immunity responses are strongly correlated with in vitro bactericidal activity

Human sera from healthy volunteers and patients convalescent from meningitis were used to search for widely cross-reactive antigens implicated in vivo protective responses. Using the type strain Neisseria meningitidis B16B6 and a wide variety of both N. meningitidis and N. lactamica strains, several cross-reactive antigens and bactericidal sera were found, although the cross-reactivity patterns did not correlate with bactericidal activity, a total correlation was found between bactericidal activity and reaction with one or two high molecular weight proteins (162 and 138 kDa), and a mouse serum against the purified proteins showed a high bactericidal activity. Our results suggest that the high molecular weight proteins found are immunogenic and cross-reactive, eliciting bactericidal responses during infection and as a result of natural immunity. These proteins should be taken into account in studies for future vaccine formulations.

High resolution clear native electrophoresis is a good alternative to blue native electrophoresis for the characterization of the Escherichia coli membrane complexes.

Blue native electrophoresis (BNE) has become the most popular method for the global analysis of membrane protein complexes. Although it has been shown to be very useful for that purpose, it can produce the dissociation of complexes with weak interactions and, due to the use of Coomassie Brilliant Blue, does not allow the subsequent application of fluorimetric and/or enzymatic techniques. Recently, we have successfully used the high resolution clear native electrophoresis (hrCNE) for the analysis of Neisseria meningitidis outer membrane porin complexes. The aim of this study was to determine the composition of the complexome of the Escherichia coli envelope by using hrCNE and to compare our results with those previously obtained using BNE. The bidimensional electrophoresis approaches used, hrCN/hrCNE and hrCN/SDS-PAGE, coupled to mass spectrometry allowed a detailed analysis of the complexome of E. coli membranes. For the first time, the three subunits of the formate dehydrogenase FDH-O were identified forming a single complex and hrCNE also allowed the identification of both the HflK and HflC proteins as components of the HflA complex. This technique also allowed us to suggest a relationship between OmpF and DLDH and, although OmpA is considered to be monomeric in vivo, we found this protein structured as homodimers. Thus hrCNE provides a good tool for future analyses of bacterial membrane proteins and complexes and is an important alternative to the commonly used BNE.