Quantification by LC-MS(E) of outer membrane vesicle proteins of the Bexsero® vaccine.

Meningococcal disease is a major cause of morbidity and mortality worldwide. Its epidemiology is currently dominated by five capsular serogroups (A, B, C, W, and Y). While effective vaccines already exist for serogroups A, C, W and Y, except for under clonal outbreaks, no vaccine was available against serogroup B. Recently, a four component vaccine, Bexsero(®), designed to prevent infection caused by this serogroup, has been approved in Europe and other Countries for use in individuals from two months of age and older. The active components of this vaccine are three recombinant proteins identified by reverse vaccinology combined with detergent extracted outer membrane vesicles (DOMV) prepared from a New Zealand epidemic strain. Considering their intrinsic complexity, we performed additional characterization of DOMVs on top of the standard quality control testing carried out for batch release. We applied the Hi3 label-free LC-MS(E) methodology to qualitatively and quantitatively characterize the DOMV protein content. We first, successfully investigated the robustness and the accuracy of the methodology for the DOMV characterization and we then applied it to compare six DOMV production lots. Around 100 proteins were quantified from each preparation. When classified according to their predicted cellular localization, about 90% of the total protein amount belongs consistently to the outer membrane compartment. Using nonparametric hypothesis testing and complementary log-log linear regression, the quantifications of a subset of 21 proteins common to all lots and including approximately 90% (85-92%) of the total protein amount quantified in any DOMV lot were found consistent across lots. The relevance of these results is two-fold, showing that the Hi3 quantification methodology is robust for a broad range of proteins and indicating that the manufacturing process currently used for the production of the Bexsero(®) DOMV components is highly reproducible and consistent.

Introduction of zwitterionic motifs into bacterial polysaccharides generates TLR2 agonists able to activate APCs.

It was shown previously that bacterial polysaccharides (PS), which naturally contain both positive and negative charges, are able to activate T cells and APCs. However, the vast majority of bacterial PS are anionic and do not have these properties. In this study, we show that chemical introduction of positive charges into naturally anionic bacterial PS confers to the resulting zwitterionic PS (ZPS) the ability to activate pure human monocytes, monocyte-derived dendritic cells, and mouse bone marrow-derived dendritic cells, as do natural bacterial ZPS. Cells are induced to up-regulate MHC class II and costimulatory molecules and to produce cytokines. In mixed monocyte-T cell cocultures, ZPS induce MHC II-dependent T cell proliferation and up-regulation of activation markers. These stimulatory qualities of ZPS disappear when the positive charge is chemically removed from the molecules and thus the zwitterionic motif is destroyed. The ability of natural and chemically derived ZPS to activate APCs can be blocked by anti-TLR2 mAbs, and TLR2 transfectants show reporter gene transcription upon incubation with ZPS. In conclusion, the generation of a zwitterionic motif in bacterial PS confers the ability to activate both APCs and T cells. This finding has important implications for the design of novel polysaccharide vaccines.