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1.
Int J Pharm ; 443(1-2): 1-8, 2013 Feb 25.
Article in English | MEDLINE | ID: mdl-23305865

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/chemistry , Drug Carriers/chemistry , Meningitis, Meningococcal/prevention & control , Meningococcal Vaccines/administration & dosage , Neisseria meningitidis, Serogroup B/immunology , Porins/chemistry , Proteolipids/chemistry , Antigens, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , Drug Stability , Drug Storage , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Meningitis, Meningococcal/immunology , Meningitis, Meningococcal/microbiology , Meningococcal Vaccines/chemistry , Microscopy, Electron, Transmission , Particle Size , Plasmids , Porins/genetics , Surface Properties
2.
J Proteome Res ; 12(2): 777-84, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23259616

ABSTRACT

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.


Subject(s)
Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/chemistry , Liposomes/chemistry , Neisseria meningitidis/chemistry , Porins/chemistry , Electrophoresis, Polyacrylamide Gel , Protein Binding , Protein Multimerization , Protein Refolding , Recombinant Proteins/chemistry
3.
J Proteome Res ; 9(1): 611-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19888731

ABSTRACT

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. In this work, using a recently described native separation technique -high resolution Clear Native Electrophoresis-and different bidimensional approaches, we were able to demonstrate the presence of relevant outer membrane complexes which could be resolved with a higher resolution than in previous analysis. The most relevant were nine porin complexes formed by different combinations of the meningococcal PorA, PorB and RmpM proteins, and comparison with the complexes formed in specific knockout mutants allowed us to infer the relevance of each porin in the formation of each complex.


Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Electrophoresis, Gel, Two-Dimensional/methods , Neisseria meningitidis/chemistry , Databases, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
4.
Vaccine ; 27(39): 5338-43, 2009 Aug 27.
Article in English | MEDLINE | ID: mdl-19607954

ABSTRACT

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.


Subject(s)
Bacterial Outer Membrane Proteins/immunology , Liposomes/immunology , Meningococcal Vaccines/immunology , Porins/immunology , Animals , Mice , Neisseria meningitidis/immunology , Recombinant Proteins/immunology
5.
Proteomics ; 9(3): 648-56, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19137557

ABSTRACT

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 (approximately 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.


Subject(s)
Bacterial Outer Membrane Proteins/analysis , Neisseria meningitidis/metabolism , Porins/analysis , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Tandem Mass Spectrometry
6.
Enferm Infecc Microbiol Clin ; 26(9): 564-72, 2008 Nov.
Article in Spanish | MEDLINE | ID: mdl-19100177

ABSTRACT

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. Technical skills will become highly refined, so that any antigen or epitope can be presented in a highly immunogenic form within a vaccine. Modern technology has led to the formulation of a new paradigm in vaccine development, in which the genomic and/or proteomic aspects of diseases are analyzed a priori to identify factors implicated in the immune response that may serve as promising vaccine candidates.


Subject(s)
Vaccination/trends , Vaccines , Animals , Antibodies, Catalytic/immunology , Antibodies, Monoclonal/immunology , Bacterial Vaccines , Drug Design , Female , Forecasting , Guinea Pigs , Humans , Immunization, Passive , Infection Control/methods , Infection Control/trends , Male , Vaccines, Attenuated , Vaccines, Conjugate , Vaccines, Contraceptive , Vaccines, DNA , Vaccines, Subunit , Viral Vaccines
7.
Enferm. infecc. microbiol. clín. (Ed. impr.) ; 26(9): 564-572, nov. 2008. tab
Article in Es | IBECS | ID: ibc-70174

ABSTRACT

La prevención de muchas enfermedades infecciosas y otras patologías, como las alergias, las enfermedades autoinmunes y el cáncer, suponen todavía un gran reto en el siglo XXI. Se han conseguido ya grandes logros, como la erradicación de algunas enfermedades y el control de otras muchas, y los avances en nuevas tecnologías en las áreas de la biología molecular, el ADN recombinante, la bioquímica de proteínas, la bacteriología y la inmunología nos permiten predecir que en los próximos años se seguirá progresando. Los aspectos técnicos serán refinados de tal forma que cualquier antígeno o epitopo podrá ser presentado de manera altamente inmunogénica. Estas tecnologías modernas han llevado a la formulación de un paradigma en la investigación de vacunas en el que la genómica y/o proteómica de las enfermedades se analizan a priori con el fin de identificar los factores que estarían implicados en la respuesta inmune, y que podrían ser adecuados como candidatos vacunales (AU)


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. Technical skills will become highly refined, so that any antigen or epitope can be presented in a highly immunogenic form within a vaccine. Modern technology has led to the formulation of a new paradigm in vaccine development, in which the genomic and/or proteomic aspects of diseases are analyzed a priori to identify factors implicated in the immune response that may serve as promising vaccine candidates (AU)


Subject(s)
Humans , Vaccination/trends , Molecular Biology/trends , Vaccines/administration & dosage , Genomics/trends , Proteomics/trends , Biomedical Research/trends , Vaccines, Inactivated , Immunization, Passive/trends
8.
J Med Microbiol ; 57(Pt 1): 80-87, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18065671

ABSTRACT

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. These results suggest that masking by and/or association with lipo-oligosaccharides or other membrane molecules can be crucial for antigen accessibility, which must be thoroughly analysed in new vaccine candidates.


Subject(s)
Antigens, Bacterial/isolation & purification , Antigens, Surface/analysis , Bacterial Outer Membrane Proteins/isolation & purification , Conserved Sequence , Neisseria meningitidis/immunology , Antigens, Bacterial/immunology , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/immunology , Flow Cytometry , Meningococcal Infections/prevention & control , Neisseria meningitidis/chemistry , Neisseria meningitidis/metabolism
9.
Int Microbiol ; 10(1): 5-11, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17407055

ABSTRACT

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. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.


Subject(s)
Bacterial Outer Membrane Proteins/analysis , Neisseria lactamica/metabolism , Neisseria meningitidis/metabolism , Proteomics/methods , Antigens, Bacterial/analysis , Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Computational Biology/methods , Electrophoresis, Gel, Two-Dimensional , Humans , Neisseria lactamica/chemistry , Neisseria meningitidis/chemistry , Porins/analysis , Porins/chemistry , Porins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
10.
Int. microbiol ; 9(4): 273-280, dic. 2006. ilus, tab
Article in En | IBECS | ID: ibc-055836

ABSTRACT

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. Western blotting with the polyclonal monospecific serum showed that linear P47 epitopes were expressed in similar amounts in the 27 Neisseria meningitidis strains tested and, to a lesser extent, in commensal Neisseria, particularly N. lactamica. However, dot-blotting assays with the same serum demonstrated binding variability between meningococcal strains, indicating differences in surface accessibility or steric hindrance by other surface structures. Specific anti-P47 antibodies were bactericidal against the homologous strain but had variable activity against heterologous strains, consistent with the results from dot-blotting experiments. An in-depth study of P47 is necessary to evaluate its potential as a candidate for new vaccine designs (AU)


El tratamiento con Triton X-114, seguido de la separación por SDS-PAGE permitió la purificación de una proteína antigénica de 47 kDa (P47) de la membrana externa de Neisseria de forma homogénea, lo que permitió la preparación de sueros policlonales anti-P47. La P47 se identificó como la lipoproteína (hipotética) NMB0035 mediante el análisis de la huella molecular realizado por MALDI-TOF. Los resultados obtenidos por SDS-PAGE diagonal bidimensional sugieren que, a diferencia de otros resultados previos, la P47 no parece estar fuertemente asociada a otras proteínas en complejos de membrana. Análisis de inmunoelectrotransferencia (Western blot) empleando el suero policlonal monoespecífico demostró que los epítopos lineales de la P47 se expresaron de forma similar en las 27 cepas de Neisseria meningitidis ensayadas y, aunque con menor intensidad, también lo hicieron en algunas Neisseria comensales, especialmente en N. lactamica. Sin embargo, resultados obtenidos por transferencia de mancha con el mismo suero demostraron que entre las cepas meningocócicas existe variabilidad en la capacidad de unión, lo que sugiere que o bien hay diferencias en la accesibilidad en superficie o bien una ocultación estérica producida por otras estructuras de superficie. Los anticuerpos específicos anti-P47 tenían efecto bactericida contra la cepa homóloga pero mostraron una actividad variable contra cepas heterólogas, lo cual concuerda con los resultados de la transferencia de mancha, y hace pensar que es necesario estudiar esta proteína más profundamente para evaluar su potencial como candidata para nuevos diseños de vacunas (AU)


Subject(s)
Neisseria/pathogenicity , Anti-Bacterial Agents , Counterimmunoelectrophoresis , Electrophoresis, Polyacrylamide Gel , Lipoproteins , Antigens, Surface , Membrane Proteins , Antibody Formation
11.
Res Microbiol ; 157(2): 136-42, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16135405

ABSTRACT

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. meningitidis. In the present study, cross-linking with formaldehyde was used to identify outer membrane protein associations in various N. meningitidis and Neisseria lactamica strains. In N. meningitides, complexes of about 450 kD (also present in N. lactamica), 165 and 95 kD were detected and shown to be made up of the proteins MSP63, PorA/PorB/RmpM/FetA, and PorA/PorB/RmpM, respectively. In western blots, the 450-kD complex was identified by mouse antibodies raised against outer membrane vesicles, but not by antibodies raised against the purified complex, demonstrating the importance of conformational epitopes, and thus suggesting that the analysis of antigens in their native conformation may be useful or even essential for the design of effective vaccines against meningococci.


Subject(s)
Antigens, Bacterial/analysis , Bacterial Outer Membrane Proteins/analysis , Macromolecular Substances/analysis , Neisseria meningitidis/chemistry , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Bacterial Outer Membrane Proteins/immunology , Bacterial Outer Membrane Proteins/isolation & purification , Blotting, Western , Cross-Linking Reagents/pharmacology , Electrophoresis, Polyacrylamide Gel , Formaldehyde/pharmacology , Macromolecular Substances/immunology , Molecular Weight , Neisseria lactamica/chemistry , Porins/isolation & purification , Protein Binding
12.
Int Microbiol ; 9(4): 273-80, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17236161

ABSTRACT

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. Western blotting with the polyclonal monospecific serum showed that linear P47 epitopes were expressed in similar amounts in the 27 Neisseria meningitidis strains tested and, to a lesser extent, in commensal Neisseria, particularly N. lactamica. However, dot-blotting assays with the same serum demonstrated binding variability between meningococcal strains, indicating differences in surface accessibility or steric hindrance by other surface structures. Specific anti-P47 antibodies were bactericidal against the homologous strain but had variable activity against heterologous strains, consistent with the results from dot-blotting experiments. An in-depth study of P47 is necessary to evaluate its potential as a candidate for new vaccine designs.


Subject(s)
Antigens, Bacterial/isolation & purification , Antigens, Surface/analysis , Bacterial Outer Membrane Proteins/isolation & purification , Neisseria lactamica/genetics , Neisseria meningitidis/genetics , Neisseriaceae Infections/microbiology , Animals , Antigens, Bacterial/immunology , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/immunology , Neisseria lactamica/immunology , Neisseria lactamica/metabolism , Neisseria meningitidis/immunology , Neisseria meningitidis/metabolism , Rabbits , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
13.
J Proteome Res ; 4(1): 91-5, 2005.
Article in English | MEDLINE | ID: mdl-15707362

ABSTRACT

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. meningitidis, and by PorB and RmpM in N. lactamica. Also, other proteins, including Opa, with temperature-dependent mobility were clearly demonstrated. The method allows improved detection of the components of membrane complexes and proteins with temperature-dependent mobility which is difficult to resolve with other analytical approaches.


Subject(s)
Bacterial Outer Membrane Proteins/analysis , Electrophoresis, Gel, Two-Dimensional , Hot Temperature , Neisseria/chemistry , Bacterial Proteins/analysis , Multiprotein Complexes/analysis , Porins/analysis , Proteomics
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