Liposomes or traditional adjuvants: induction of bactericidal activity by the macrophage infectivity potentiator protein (Mip) of Neisseria meningitidis.

Currently, one of the main approaches to achieve a vaccine for serogroup B Neisseria meningitidis is based on outer membrane proteins with low antigenic variability among strains. Since these proteins tend to be minor components of the outer membrane, recombinant production is required to obtain them in sufficient amounts for evaluation and development of vaccines. In this study, we analysed the ability of recombinant macrophage infectivity potentiator (rMip) protein to induce protective bactericidal activity in mice. The rMip protein was cloned from N. meningitidis strain H44/76 and was used to immunise mice, and the sera obtained were tested against the homologous and several heterologous N. meningitidis strains. The sera were obtained using the rMip alone, with adjuvant Al(OH)3 , or after inclusion into liposomes. Bactericidal activity was variable depending on the strain, although high titres were seen against strains H44/76 and NmP27. Liposomes enhanced fourfold the reactivity against the homologous strain. The results presented suggest that the rMip protein should be considered a promising candidate for the improvement of future protein-based vaccines.

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.

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.

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.

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. 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.

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.

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 (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.

[Classic vaccinology and advances in vaccine design]. / Vacunología clásica y nuevas tecnologías en el diseño de vacunas.

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.

Vacunología clásica y nuevas tecnologías en el diseño de vacunas / Classic vaccinology and advances in vaccine design

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)

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. 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.

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. 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.

Locus NMB0035 codes for a 47-kDa surface-accessible conserved antigen in Neisseria / El locus NMB0035 codifica un antígeno de superficie conservado de 47 kDa en 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. 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)

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. 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.

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. 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.

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. 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.

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.

[Influence of adjuvants on the ability of anti-Tbp antibodies to block transferrin binding, iron uptake and growth of Neisseria meningitis]. / Influencia de adyuvantes en la capacidad de los anticuerpos anti-Tbps de bloquear la unión de transferrina, la asimilación de hierro y el crecimiento en Neisseria meningitidis.

OBJECTIVE: To evaluate the effect of five adjuvants on the ability of specific anti-TbpA/B to block iron uptake in Neisseria meningitidis. MATERIALS AND METHODS: Transferrin binding complexes (TbpA/B) purified from a TbpB isotype II Neisseria meningitidis strain were used to obtain sera with five different adjuvant formulations in mice in order to test the effect of the adjuvant on the ability of specific anti-TbpA/B antibodies to block transferrin binding, iron uptake and growth by meningococci. RESULTS: Levels of anti-TbpA/B antibodies were relatively low (1:125 in most cases), the highest being obtained with the RAS adjuvant (1:3125). Despite the relatively low responses, all sera were able to significantly inhibit transferrin binding, iron uptake and growth in the homologous strain. Nevertheless, the effect on a strain with a TbpB isotype different from that of the immunizing strain was almost nil, a result in keeping with the described division of the meningococci into at least two TbpB groups (isotypes I and II). CONCLUSIONS: In contrast to previous results for another important meningococcal protein, FbpA, the use of various adjuvants in the immunization of mice with TbpA/B complexes did not produce differences in the immune responses elicited, except in relation to antibody titers.

Influencia de adyuvantes en la capacidad de los anticuerpos anti-Tbps de bloquear la unión de transferrina, la asimilación de hierro y el crecimiento en Neisseria meningitidis / Influence of adjuvants on the ability of anti-Tbp antibodies to block transferrin binding, iron uptake and growth of Neisseria meningitidis

OBJETIVO. Analizar el efecto de cinco adyuvantes en la capacidad de los sueros anti-TbpA/B para bloquear la asimilación de hierro en Neisseria meningitidis. MATERIAL Y MÉTODOS. Se han ensayado 5 formulaciones de adyuvantes para obtener, en ratones, sueros específicos contra los complejos proteicos de unión de transferrina (TbpA/B) purificados de una cepa de N. meningitidis y analizar la influencia del adyuvante en la capacidad de los anticuerpos generados de bloquear la unión de transferrina y, en consecuencia, la asimilación de hierro y el crecimiento de los meningococos. RESULTADOS. Todos los sueros fueron capaces de inhibir de manera significativa la unión de transferrina, la consiguiente asimilación de hierro y el crecimiento en la cepa homóloga, aunque se observó un claro incremento en los títulos de anticuerpos cuando se emplea RAS como adyuvante (1/3.125 frente a 1/125 con los otros adyuvantes). El efecto de estos sueros en una cepa heteróloga, con una TbpB de isotipo diferente al de la cepa inmunizante, fue casi nulo, lo que concuerda con la división descrita para el meningococo en 2 grupos en función de la TbpB que posean (isotipos I y II).CONCLUSIONES. Contrariamente a lo ya demostrado para otra importante proteína meningocócica (FbpA), el uso de diferentes adyuvantes en la inmunización de ratones con los complejos TbpA/B no ofrece diferencias en la respuesta inmunitaria producida, excepto en lo relativo al título de anticuerpos (AU)

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.