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.

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.

Identification of Neisseria meningitidis outer membrane vesicle complexes using 2-D high resolution clear native/SDS-PAGE.

The identification and characterization of meningococcal outer membrane vesicle complexes can be important for gaining an in-depth understaining of their structure and functionality. Analysis of the vesicle complexome by 'traditional' 2-D analysis, in which isoelectrofocusing is used for separation in the first dimension, is hampered by the high hydrophobicity and extreme isoelectric points of many relevant proteins. Analysis of the meningococcal outer membrane vesicle complexome using Blue Native (nondenaturing) electrophoresis instead of isoelectrofocusing in the first dimension showed several porin complexes, but their composition could not be clearly resolved after separation by SDS-PAGE in the second dimension.

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).

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.

[Classic vaccinology and advances in vaccine design].

The prevention of many infectious diseases, allergies, autoimmune diseases, and cancer continues to be a challenge in the twenty-first century. Nonetheless, considerable advances have already been made, such as the eradication of certain infectious diseases and effective control of many others, and new technology is being developed in areas related to molecular biology, recombinant DNA, protein biochemistry, microbiology, and immunology. The current trends point to continued progress in coming years.

A cross-reactive neisserial antigen encoded by the NMB0035 locus shows high sequence conservation but variable surface accessibility.

The meningococcal NMB0035 locus encodes a 47 kDa outer-membrane protein that is highly conserved antigenically, and is able to induce antibodies during infection and bactericidal responses in vitro. This study analysed the surface exposure of this protein using specific antibodies in flow cytometry assays and determined its nucleotide sequence in 33 Neisseria strains. Genomic analyses revealed no significant differences in the nucleotide or amino acid sequences, but flow cytometry showed that surface accessibility was highly variable among the strains.

Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N.

Locus NMB0035 codes for a 47-kDa surface-accessible conserved antigen in Neisseria.

A47 kDa neisserial outer-membrane antigenic protein (P47) was purified to homogeneity and used to prepare polyclonal anti-P47 antisera. Protein P47 was identified by MALDI-TOF fingerprinting analysis as the hypothetical lipoprotein NMB0035. Two-dimensional diagonal SDS-PAGE results suggested that, contrary to previous findings, P47 is not strongly associated with other proteins in membrane complexes.

Cross-linking analysis of antigenic outer membrane protein complexes of Neisseria meningitidis.

Polysaccharide-based approaches have not enabled the development of effective vaccines against meningococci of serogroup B, and the most promising current research is focused on the use of outer membrane vesicles. Due to the toxicity of the outer membrane oligosaccharides, new vaccines based on purified proteins are being sought, but despite the application of advanced techniques, they remain elusive, perhaps due to the fact that standard techniques for analysis of antigens overlook conformational epitopes located in membrane complexes. Membrane complex antigens have been analyzed in Neisseria gonorrhoeae, and a study published on Neisseria meningitidis has reported the in vitro formation of 800-kD complexes by deposition of a purified protein (MSP63) onto synthetic lipid layers; however, no studies to date have attempted to identify membrane complexes present in vivo in N.

Analysis of outer membrane protein complexes and heat-modifiable proteins in Neisseria strains using two-dimensional diagonal electrophoresis.

Two-dimensional diagonal SDS-PAGE was used to resolve membrane complexes and identify proteins with temperature-dependent mobility in Neisseria meningitidis and N. lactamica. The main membrane complexes were composed of porins and were formed by heteromers of PorA, PorB and RmpM in N.