Vaccine 27 (2009) 5338–5343 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Characterisation and immune responses to meningococcal recombinant porin complexes incorporated into liposomes Sandra Sánchez a , Ana Abel a , Juan Marzoa a , Andrew Gorringe b , Teresa Criado a , Carlos M. Ferreirós a,∗ a Departamento de Microbiología y Parasitología, Facultade de Farmacia, Campus Sur, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain b Health Protection Agency Centre for Emergency Preparedness and Response, Porton Down, Salisbury SP4 0JG, United Kingdom article info Article history: Received 5 February 2009 Received in revised form 4 June 2009 Accepted 25 June 2009 Available online 14 July 2009 Keywords: Neisseria meningitidis Porin complexes Native electrophoresis 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 50kDa outer membrane protein not yet identified. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Neisseria meningitidis is the main cause of bacterial meningitis, a life threatening disease with a global distribution. The failure to obtain effective vaccines against serogroup B N. meningitidis using capsular antigens has focused investigations on the use of outer membrane antigens. Vaccine assays in which outer membrane vesicles (OMVs) were used showed that most of the immune response is directed to the PorA protein [1], a 44–47 kDa cationic porin that forms trimeric complexes. Studies carried out in our laboratory showed that in native conditions, N. meningitidis porin complexes are formed not only by PorA, but also by heteromers of PorA, PorB and some other proteins such as RmpM [2,3]. The structural rearrangement of these proteins in the outer mem- brane and their association to form pore complexes could create non-linear, shared or conformational epitopes, which could play a crucial role in the development of immune responses, as shown in studies carried out in other pathogens [4]. Studies performed using recombinant neisserial porins rPorA [5,6] or rPorB [7] showed that the production of protective antibodies can be obtained following refolding of the porins in their native configurations. The incor- ∗ Corresponding author. Tel.: +34 981 563 100x14946; fax: +34 981 594 631. E-mail address: carlos.ferreiros@usc.es (C.M. Ferreirós). poration of PorA, PorB or both porins into liposomes allows the recovery of their structure and function, mimicking their behaviour in the native outer membrane [8]. In addition, liposomes have been used extensively as vehicles in the formulation of vaccines utilising their capacity to be directed to specific targets, their ease of modification and their capacity as adjuvants of the humoral immune response [9]. Due to the significance of conformational epitopes, the char- acterisation of intact complexes is a crucial step in the search for potential vaccine candidates. Electrophoretic analysis of complexes is possible only in non-denaturing conditions. Blue- native electrophoresis in polyacrylamide gels (BN-PAGE) is a non-denaturing electrophoretic technique in which n-dodecyl-- d-maltoside (DDM), a mild non-ionic detergent, is used to solubilise and provide charge to hydrophobic proteins and complexes, pre- venting their dissociation [10,11]. Using SDS-PAGE in a second dimension after separation by BNE-PAGE (2D BN/SDS-PAGE) allows the dissociation of the complexes and the analysis of their compo- nents with a resolution greater than that obtained by other methods such as gel filtration or sucrose gradient separation [12]. Our aim in this study was to analyse PorA/PorB interactions by incorporation of both recombinant porins into liposomes followed by characterisation using native electrophoresis, and to determine the relevance of these interactions in the induction of the immune response. 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.06.086