Renaturalización de complexos proteicos de membrana externa de Neisseria meningitidis en liposomasestrutura e potencial vacinal

  1. Freixeiro Díaz, Paula
unter der Leitung von:
  1. Sandra Sánchez Poza Doktormutter
  2. Carlos M. Ferreirós Domínguez Doktorvater

Universität der Verteidigung: Universidade de Santiago de Compostela

Fecha de defensa: 24 von April von 2015

Gericht:
  1. Miguel Blanco Álvarez Präsident
  2. Alejandro Sánchez Barreiro Sekretär
  3. Julio A. Vázquez Moreno Vocal
  4. Concepción Herrero Vocal
  5. Andrew Richard Gorringe Vocal
Fachbereiche:
  1. Departamento de Microbioloxía e Parasitoloxía

Art: Dissertation

Zusammenfassung

Although vaccines based on outer membrane vesicles are effective against epidemic serogroup B N. meningitidis strains, they 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 improvement of currently existing ones, which immune response is mainly directed against porin complexes. This work aims to deeper understanding of N. meningitidis porin complexes and the role of the RmpM by refolding of protein complexes into liposomes and the evaluation of the vaccine potential of proteoliposome formulations containing porins and RmpM. Dialysis-extrusion proteoliposomes containing different combinations of the recombinant proteins PorB, PorA, and RmpM allowed the refolding of porin complexes with a profile very similar to that of the complexes present in OMVs produced naturally by the bacteria. Analysis showed that RmpM binds to porin complexes through interaction between PorB and its N-terminal domain, where a six amino acid peptide (VSGQSN) is crucial for this interaction. Due to its interaction with other protein complexes, an important role of the RmpM in ions transport across cell membrane, in protein translocation through the periplasm and in adhesion processes to host cells is suggested for this protein. All proteoliposome formulations induced a good immune response against the homologous strain, even when PorA was absent, but the cross-reactivity against heterologous strains tested was low. The formulations showed good long-term stability at room temperature after lyophilization, with no differences between the two cryoprotectants tested, glucose and trehalose. Therefore, proteoliposomes are stable systems with a well defined composition and so of great interest as an alternative to current OMVs-based vaccines and as vehicles for new protein subunits vaccines.