Obtención de extractos de membrana externa de Vibrio cholerae O1, mediante el uso de diferentes detergentes.

Obtaining Vibrio cholerae 01 outer membrane extracts with different detergents.

José Luis Pérez, Yamisley González, Gemma Año, Bárbara Cedré, Tania Valmaseda, Maydelis Alvarez, Daily Serrano, Ernesto Millián, Mildrey Fariñas, Arturo Talavera y Luis García.

Instituto Finlay. Centro de Investigación-Producción de Vacunas. Ave.27 No. 19805. La Lisa. A.P. 16017, C.P. 11600, Ciudad de La Habana, Cuba. E-mail: jlperez@finlay.edu.cu

RESUMEN

En la actualidad existen dos variantes principales de vacunas orales contra el cólera: una basada en células inactivadas de diferentes biotipos y serotipos y otra basada en la administración de cepas vivas genéticamente atenuadas. Una vacuna por subunidades pudiera ser una variante muy atractiva. Este trabajo describe la purificación parcial y caracterización preliminar de extractos de proteínas de membrana externa-lipopolisacárido (PME-LPS), obtenidos a partir de Vibrio cholerae O1, con el interés de seleccionar un proteoliposoma que posteriormente será estructurado en forma de cocleatos para su uso por vía oral en humanos. Las preparaciones fueron obtenidas a través del uso de diferentes detergentes. La cantidad de LPS en cada preparación fue estimada mediante la determinación de las unidades endotóxicas en el ensayo del Limulus (LAL). La composición de cada muestra fue evaluada mediante SDS-PAGE y Dot Blot. La inoculación intranasal (IN) en ratones Balb/c se utilizó para la evaluación de la inmunogenicidad de las preparaciones, y la respuesta inmune fue determinada por ELISA y el título de anticuerpos vibriocidas. El tamaño molecular de la preparación con mejores resultados en inmunogenicidad se estimó mediante la cromatografía en Sephacryl S-1000. Se obtuvieron diferentes perfiles electroforéticos de acuerdo con el tipo de detergente utilizado. El LPS fue identificado en todas las preparaciones y aquella obtenida con el SDS al 15% mostró la más baja relación proteínas/LPS y los mejores resultados en los ensayos de inmunogenicidad. Adicionalmente se comprobó que su tamaño molecular es similar al observado en el proteoliposoma de VAMENGOC- BC. La preparación obtenida con el SDS al 15% constituye un proteoliposoma, con capacidad para estimular altos niveles de anticuerpos IgG anti-LPS y altos títulos de anticuerpos vibriocidas, luego de su administración por vía intranasal en ratones. Estos resultados constituyen un importante paso en las investigaciones dirigidas a la obtención de una vacuna por subunidades, como alternativa preventiva contra el cólera.

Palabras claves: Cólera, vacunas, respuesta vibriocida, complejos PME-LPS.

Two oral vaccine variants have been developed to prevent cholera infection: one from genetically attenuated strains, and the other one from inactivated whole cells. A subunit vaccine could be another very attractive alternative. In this report, we describe the partial purification and preliminary characterization of V. cholerae O1 Outer Membrane Protein-Lipopolysaccharide (OMP-LPS) complexes, to select a proteoliposome that could be administered orally in humans as a cochleate. Preparations were obtained using different detergents. The quantity of LPS was estimated by determination of endotoxic units using Limulus test (LAL). The composition of the samples was visualized by SDS-PAGE and dot blot assay. Balb/c mice were inoculated intranasally (IN) and the immune response was evaluated using ELISA and vibriocidal antibody titer assay. The molecular size of the preparation giving the best results for immunogenicity assays was estimated by chromatography on Sephacryl S- 1000. Different electrophoretic profiles were obtained according to the type of detergent used. LPS was identified in all the preparations evaluated. The preparation obtained with 15% SDS showed the lowest OMP/LPS relation, the best results for the immunogenicity assay and a similar molecular size when compared to VA-MENGOC-BC proteoliposome. These results are very stimulating and constitute a very important step for research to obtain a subunit vaccine as a preventive alternative against cholera.

Keywords: Cholera, vaccines, vibriocidal response, OMP-LPS complexs.

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