Development of a modular and multiplexed assay to predict the coverage of BexseroTM against Neisseria meningitidis type B

Development of a modular and multiplex assay on Luminex platform to predict the coverage of BexseroTM against Neisseria meningitidis type B a,b Sara Favale, b Brunella Brunelli, b Bruno Galletti, b Francois Legay, b Marzia Giuliani, a Developmental and Cellular Biology Department, La Sapienza, University of Rome, 00185 Roma, Piazzale Aldo Moro 5, Italy bGlaxoSmithKline, 53100 Siena, Via Fiorentina 1, Italy e-mail: sara.favale@uniroma1.it Keywords: N. meningitidis, meningitis, BexseroTM, MATS, Luminex. BexseroTM vaccine against Neisseria meningitidis type B has been approved in several countries. It is composed by three sub-capsular antigens, selected by Reverse Vaccinology approach: factor H binding protein (fHbp) variant 1.1, Neisserial Heparin Binding Antigen (NHBA) peptide 2 and Neisserial Adesin A (NadA) variant 3. Bexsero vaccine includes also Outer Membrane Vescicles (OMVs) derived from the New Zealand strain NZ 98/254, expressing PorA serosubtype P1.4. Evaluation of antigen expression by circulating N. meningitidis strains is a very critical step in order to predict the vaccine coverage and, a specific test has been set up for this purpose (Ref. 1). The Meningococcal Antigen Typing System (MATS) was designed to measure immunologic cross-reactivity and quantify antigen expression by target strains of N. meningitidis B. MATS results from a combination of three sandwich Enzyme-Linked Immunosorbent Assay (ELISA) assays, one for each vaccine antigen, plus sequencing of PorA P1.4 (Ref.2). Although working, conventional ELISA makes immunogenicity evaluation of a multi-component vaccine difficult, laborious, time-consuming and expensive, since only one immunogen per assay run can be tested. xMap Luminex Technology allows the development of multiplex immunoassays where, multiple antibody types can be determined simultaneously in one assay run. Taking the case of MATS-ELISA assay and BexseroTM vaccine as reference, this PhD project aim to: (i) develop a flexible multiplexed and quantitative sandwich assay (on Luminex platform) allowing the simultaneous measurement of all vaccine antigens expressed by bacterial strains, in order to predict the coverage of BexseroTM; (ii) qualify the multiplex assay performance (reproducibility, sensitivity, accuracy, precision, intra/inter assay variability); (iii) evaluate comparability of the new assay with the currently accepted ones, taking also into account results of the Serum Bactericidal Assay (SBA), the only accepted reference test for functional antibodies directed to meningococcus. We developed and qualified a multiplex 4-plex assay based on Luminex technology able to simultaneously quantify BexseroTM vaccine antigens in Men B isolates. The assay was also designed to measure the OMVs content, avoiding PorA, sequencing in order to speculate on a possible role of the entire OMVs in the coverage of BexseroTM, avoiding MATS underestimation (Ref.3). The new multiplex assay RPs shows a good correlation with MATS RPs for each BexseroTM antigen. It represents a promising assay to obtain information about BexseroTM coverage in easy, fast, cheap and more reproducible way. Due to the high flexibility of this technology in the future it will be possible to increase the antigens panel (from 4 up to 100 microspheres in the single well) and detect other vaccine antigens expressed on bacterial strain to predict the coverage of a new generation multi-component vaccine. Ref.1 G. Boccadifuoco, B. Brunelli, M. G. Pizza, and M. M. Giuliani, “A combined approach to assess the potential coverage of a multicomponent protein-based vaccine,” Journal of Preventive Medicine and Hygiene, vol. 53, no. 2, pp. 56–60, 2012 Ref.2 Serruto, D., M. J. Bottomley, et al. (2012). "The new multicomponent vaccine against meningococcal serogroup B, 4CMenB: immunological, functional and structural characterization of the antigens." Vaccine 30 Suppl 2: B87-97. Ref.3 Frosi, G. et al. Bactericidal antibody against a representative epidemiological meningococcal serogroup B panel confirms that MATS underestimates 4CMenB vaccine strain coverage. (2013). Vaccine 31:4968– 4974