RESEARCH ARTICLE Outer membrane vesicles from group B Neisseria meningitidis ˜gna33 mutant: Proteomic and immunological comparison with detergent-derived outer membrane vesicles Germano Ferrari 1 , Ignazio Garaguso 1 , Jeannette Adu-Bobie 1 , Francesco Doro 1 , Anna Rita Taddei 2 , Alessia Biolchi 1 , Brunella Brunelli 1 , Marzia Monica Giuliani 1 , Mariagrazia Pizza 1 , Nathalie Norais 1 and Guido Grandi 1 1 Biochemistry and Molecular Biology Unit, Chiron Vaccines, Siena, Italy 2 C.I.M.E., Università degli Studi della Tuscia, Largo dell’Università, Viterbo, Italy We compared the proteome of detergent-derived group B Neisseria meningitidis (MenB) outer membrane vesicles (DOMVs) with the proteome of outer membrane vesicles (m-OMVs) sponta- neously released into culture supernatant by MenB Dgna33, a mutant in which the gene coding for a lytic transglycosylase homologous to the E. coli MltA was deleted. In total, 138 proteins were identified in DOMVs by 1- and 2-DE coupled with MS; 64% of these proteins belonged to the inner membrane and cytoplasmic compartments. By contrast, most of the 60 proteins of m- OMVs were classified by PSORT as outer membrane proteins. When tested for their capacity to elicit bactericidal antibodies, m-OMVs elicited a broad protective activity against a large panel of MenB strains. Therefore, the identification of mutations capable of conferring an OMV-releasing phenotype in bacteria may represent an attractive approach to study bacterial membrane com- position and organization, and to design new efficacious vaccine formulations. Received: March 17, 2005 Revised: September 27, 2005 Accepted: September 29, 2005 Keywords: Neisseria meningitidis / Outer membrane vesicles / Vaccines 1856 Proteomics 2006, 6, 1856–1866 1 Introduction Meningococcal meningitis and sepsis are devastating dis- eases that can kill children and young adults within hours of infection. These diseases are caused by Neisseria meningitidis, a capsulated, gram-negative bacterium that has been classi- fied into five major serogroups (A, B, C, W-135 and Y) on the basis of the composition of its capsular polysaccharide. While very effective conjugated capsular poly- saccharide-based vaccines against serogroups A, C, W-135 and Y have been developed [1–6], no vaccines are available for group B N. meningitidis (MenB). This is of particular concern for industrialized countries where MenB is responsible for a large proportion of all meningococcal meningitis [7, 8]. One proposed anti-MenB vaccine is constituted by outer membrane vesicles (OMVs), portions of bacterial outer membranes typically organized in spheroid structures 50– 200 nm in diameter, which are obtained by detergent extrac- tion and differential centrifugation of bacterial cells [9]. Detergent-extracted OMVs (DOMVs) have been recently tes- ted, with encouraging results [10–12], in phase III trials to Correspondence: Dr. Guido Grandi, Biochemistry and Molecular Biology Unit, Chiron Vaccines, Via Fiorentina 1, 53100 Siena, Italy E-mail: guido_grandi@chiron.com Fax: 139-0577-278514 Abbreviations: BCA, complement-mediated bactericidal activity; CFU, colony-forming units; DOMV, detergent-derived OMV; FACS, fluorescence-activated cell sorter; GNA, genome-derived Neisseria antigen; LPS, lipopolysaccharide; MenB, Neisseria meningitidis serogroup B; m-OMV, Dgna33 knock-out mutant- derived OMV; NOMV, natural OMV; OMV, outer membrane vesi- cle DOI 10.1002/pmic.200500164  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com