Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa.

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

Immune adjuvant effect of V. cholerae O1 derived Proteoliposome coadministered by intranasal route with Vi polysaccharide from Salmonella Typhi.

The proteoliposome derived from Vibrio cholerae O1 (PLc) is a nanoscaled structure obtained by a detergent extraction process. Intranasal (i.n) administration of PLc was immunogenic at mucosal and systemic level vs. V. cholerae; however the adjuvant potential of this structure for non-cholera antigens has not been proven yet. The aim of this work was to evaluate the effect of coadministering PLc with the Vi polysaccharide antigen (Poli Vi) of S. Typhi by the i.n route. The results showed that Poli Vi coadministered with PLc (PLc+Poli Vi) induce a higher IgA response in saliva (p<0.01) and faeces (p<0.01) than Poli Vi administered alone. Likewise, the IgG response in sera was higher in animals immunised with PLc+Poli Vi (p<0.01). Furthermore, IgG induced in sera of mice immunised with PLc+Poli Vi was similar (p>0.05) to that induced in a group of mice immunised by the parenteral route with the Cuban anti-typhoid vaccine vax-TyVi, although this vaccine did not induce a mucosal response. In conclusion, this work demonstrates that PLc can be used as a mucosal adjuvant to potentiate the immune response against a polysaccharide antigen like Poli Vi.

Una vacuna de vesículas de membrana externa de Vibrio cholerae es aún una promesa: Caracterización proteómica / Outer membrane vesicle vaccine from Vibrio cholerae is still a promise: Proteomic characterization

Las epidemias de cólera afectan a un gran número de países africanos, asiáticos y del Caribe. Los cambios climatológicos y las constantes migraciones hacen que esta enfermedad se extienda, por lo que resulta necesario disponer de vacunas protectoras. En el presente trabajo se caracterizó una nueva vacuna de vesículas de membrana externa (VMEs) obtenidas de Vibrio cholerae O1 biotipo El Tor serotipo Ogawa cepa C7258, en el Instituto Finlay de vacunas (Cuba), a través de métodos proteómicos. Se identificaron 53 proteínas presentes en las VME (4 proteínas por banda electroforética) separadas por electroforesis unidimensional (1D) y digeridas con tripsina. Los fragmentos obtenidos fueron separados por cromatografía líquida de alta resolución (HPLC) acoplada a espectrometría de masa, secuenciados e identificados mediante bases de datos de proteínas Swiss-Prot y TrEMBL. El patrón proteico obtenido presentó algunas de las proteínas (12 proteínas citoplasmáticas y 5 proteínas de membrana externa) sugeridas dentro del proteoma de buena calidad para candidatos vacunales. Se estudiaron las mejores condiciones para la separación de las proteínas a través de electroforesis bidimensional. Las VME evaluadas cuentan con una composición fundamentada en proteínas necesarias para garantizar una respuesta inmune que proteja contra Vibrio cholerae O1 biotipo El Tor serotipo Ogawa.

Cholera epidemics affect a large number of African, Asian and Caribbean countries. The climate changes and the constant migrations cause this disease to spread, making it is necessary to obtain protective vaccines. In the present work, a new vaccine of outer membrane vesicles (OMV) from V. cholerae O1 El Tor biotype Ogawa serotype strain C7258 at Finlay Institute of vaccines (Cuba) was characterized by proteomic methods. A total of 53 proteins present in the OMV (approximate ratio of 4 proteins by electrophoresis band) were identified, separated by one dimension electrophoresis and digested by tripsin method. The fragments were separated by high performance liquid chromatography (HPLC) coupled to mass spectrometry, sequenced and identified, using Swiss-Prot and TrEMBL protein databases. The pattern showed some proteins (12 cytoplasmic proteins and 5 outer membrane proteins) suggested within the highest quality proteome for vaccine candidate. The best conditions for proteins separation by two dimension electrophoresis were studied. The OMV composition was based on proteins described to the immunity response and protection against V. cholerae O1 El Tor biotype Ogawa serotype.

As epidemias de cólera afetam um grande número de países africanos, asiáticos e caribenhos. As mudanças climáticas e as constantes migrações fazem com que esta doença se espalhe, portanto é necessário ter vacinas protectoras. No presente trabalho, uma nova vacina de vesículas de membrana externa (VMEs) obtidas de Vibrio cholerae 01 biotipo El Tor sorotipo Ogawa cepa C7258, no Instituto de Vacinas Finlay (Cuba), através de métodos proteômicos. Foram identificadas 53 proteínas presentes nas VME (4 proteínas por banda eletroforética) separadas por eletroforese unidimensional (1D) e digeridas com tripsina. Os fragmentos obtidos foram separados por cromatografia de alta resolução (HPLC) acoplada a espectrometria de massa, sequenciados e identificados usando bancos de dados de proteínas Swiss-Prot e TrEMBL. O padrão proteico obtido apresentou algumas das proteínas (12 proteínas citoplasmáticas e 5 proteínas de membrana externa) sugeridas dentro do proteoma de boa qualidade para candidatos vacinais. As melhores condições para a separação de proteínas através de eletroforese bidimensional foram estudadas. As VME avaliados possuem uma composição baseada em proteínas necessárias para garantir uma resposta imune que proteja contra Vibrio cholerae O1 biotipo El Tor sorotipo Ogawa.

A new oral vaccine candidate based on the microencapsulation by spray-drying of inactivated Vibrio cholerae.

The aim of this work was to evaluate the microencapsulation by spray-drying of inactivated Vibrio cholerae, using methacrylic copolymers Eudragit® L30D-55 and FS30D. The microparticles obtained presented a particle size around 3.0 µm. The preparation temperature affected the morphology and the antigenicity of microparticles, but it did not affect the V. cholerae content. In vitro release studies showed that in acid medium less than 5% of bacteria was released, and in neutral medium, Eudragit® L30D-55 microparticles released 86% after 24 h, whereas FS30D released less than 30%. Rats inoculated with microparticles exhibited vibriocidal antibody titres. Microencapsulation by spray-drying of inactivated V. cholerae could be proposed as a method to obtain an oral vaccine which provides controlled release of the bacteria.

Estandarización de ensayos inmunoenzimáticos (ELISA) para la cuantificación de anticuerpos IgG inducidos por una vacuna de vesículas de membrana externa de los serogrupos A y W135 de Neisseria meningitidis / Standardization of immunoenzimatic assays (ELISA) for the quantitative determination of IgG antibodies elicited by an-outer-membrane-vesicle vaccine from Neisseria meningitidis serogroups A and W13

La enfermedad meningocócica es una afección invasiva, de amplia incidencia mundial, cuyo agente causal es la bacteria gramnegativa Neisseria meningitidis . Existen vacunas polisacarídicas sin conjugar o conjugadas, contra cuatro de los cinco serogrupos responsables del 95 por ciento de los casos en el mundo. Para el serogrupo B, cuyo polisacárido es pobremente inmunogénico, se han evaluado varios candidatos vacunales producidos a base de vesículas de membrana externa. La determinación de la actividad bactericida y la cuantificación de IgG por ELISA han sido los métodos más utilizados en la medición de la respuesta inmune generada por vacunas contra la meningitis meningocócica. El segundo de estos métodos es utilizado en el Instituto Finlay como ensayo de inmunogenicidad para la liberación de lotes de VA-MENGOC-BC®. Como parte de una colaboración con investigadores noruegos, se trabaja en la obtención de un candidato vacunal contra los serogrupos A y W135 , basado en vesículas de membrana externa. En el presente trabajo se describe la estandarización de un ELISA para ser utilizado en la evaluación de la respuesta inmune del candidato vacunal bivalente

Invasive meningococcal disease constitutes a worldwide health problem. Gram negative bacteria, Neisseria meningitidis is the causal agent of this disease. Plain or conjugated polysaccharide vaccines are available against four of five serogroups responsible of more than 95 percent of cases in the world. Because serogroup B polysaccharide is non immunogenic, some vaccine candidates, based on outer membrane proteins have been obtained and evaluated in clinical trials. Bactericidal activity determination and ELISA IgG have been used to evaluate immune response generated by antimeningococcal vaccines. The latter has been used, as potency test, to release VA-MENGOC-BC® vaccine lots, produced at Finlay Institute. As a result of the collaboration with Norwegian researchers, a vaccine candidate based on outer membrane proteins against serogroups A and W135, has been obtained and evaluated in animal models. The current work describes the standardization of an ELISA method to be used in the immune response evaluation of the bivalent vaccinal candidate(AU)

Purificación de lipopolisacárido de Neisseria meningitidis a partir de unafracción colateral del proceso de producción de VA-MENGOC-BC® / Purification of Neisseria meningitidis lipopolysaccharide starting from a collateral fraction of the process of the antimeningococcal vaccine, VA-MENGOC-BC production process

El trabajo tuvo como objetivo purificar lipopolisacáridos (LPS) de Neisseria meningitidis a partir de una fraccióncolateral del proceso de producción de la vacuna antimeningocócica VA-MENGOC-BC®, el sobrenadante que seobtiene del paso de ultracentrifugación durante el proceso de extracción de las proteínas de la membrana externa delmeningococo. La purificación se realizó mediante precipitación con etanol al 80 por ciento, extracción de las proteínas con fenol al 90 porciento entre 65-70 ºC y ultracentrifugación fraccionada a 105,000 g. Se obtuvieron tres lotes de LPS, en total 1,069 g, con un contenido de proteínas, ácidos nucleicos y ácido sálico respecto al LPS de 0,5 por ciento, 0,3 por ciento y 2,2 por ciento (m/m),respectivamente. La evaluación por cromatografía mostró una alta integridad molecular, con valores de constante de distribución reproducibles (0,36-0,38) y una posible asociación del ácido siálico al LPS. Se apreció homogeneidad en el perfil electroforético de los tres lotes y alta actividad endotóxica. El LPS purificado fue identificado fundamentalmente como del inmunotipo L3,7,9. El procedimiento de purificación empleado permite aprovechar una fracción colateral del proceso de producción de la vacuna, es escalable, no incluye métodos cromatográficos, y posibilita la obtención de gran cantidad de LPS de Neisseria meningitidis, no disponible en el mercado, con elevada pureza y alta actividad endotóxica(AU)

The work aimed at purifying lipopolysaccharides (LPS) of Neisseria meningitidis from a collateral fraction of the antimeningococcal BC vaccine, VAMENGOC-BC®. production process, the supernatant obtained from the ultra centrifugation stage during the proteins extraction process of the meningococcus outer membrane. The purification was carried out by precipitation 80 percent ethanol, protein extraction with 90 percent phenol from 65-70 ºC and fractional ultra centrifugation at 105.000 g. Three lots of LPS were obtained, in total 1.069 g, with a content of proteins, nucleic acids and sialic acid in respect to the LPS of 0.5 percent, 0.3 percent and 2.2percent (m/m) respectively. The assessment by chromatography showed a high molecular integrity. with constant valves of reproducible distribution (Kd 0.36 0.38) and a possible sialic acid association to the LPS. Homogeneitywas observed in the electrophoretic profile of the three lots and a high endotoxic activity. The purified LPS was mainly identified as the inmunotype L3,7,9. The purification procedure used allows making use of a collateral fraction of the vaccine production process, it is scalable, it does not include chromatographic methods and makes easy the obtainment of large quantityof LPS of Neisseria meningitidis, wihich it is non available on the market, with high purity and high endotoxic activity(AU)

Cuantificación de lipopolisacárido en un proteoliposoma obtenido a partir de la superficie externa de Vibrio cholerae O1 / Lipopolysaccharide quantification in a proteoliposome obtained from external surface of Vibrio

El lipopolisacárido (LPS) de Vibrio cholerae O1, a diferencia de la mayoría de las bacterias gramnegativas, no es reactivo en el ensayo del KDO, un método ampliamente utilizado para la cuantificación de LPS. Este LPS puede ser cuantificado por peso seco cuando se trata del antígeno en estado puro, pero cuando se necesita cuantificarlo, por ejemplo en un proteoliposoma, se requieren métodos de hidrólisis fuerte para que la molécula de KDO se exponga y sea reactiva en el ensayo antes mencionado. En este trabajo describimos un método rápido, sencillo y reproducible que nos permite cuantificar el LPS en un proteoliposoma obtenido a partir de la superficie externa de V. cholerae O1, El Tor Ogawa, mediante el uso de un anticuerpo monoclonal anti-LPS Ogawa en Western blot y la posterior evaluación del perfil reactivo en un densitómetro, utilizando LPS Ogawa purificado y cuantificado por peso seco como patrón de referencia. Este método puede ser aplicado a cualquier otra preparación que contenga LPS y que no pueda ser evaluada por KDO(AU)