Aruta, Maria Grazia (2016) Systematic analysis of fHbp sequence coverage by antibodies against meningococcal GMMA from african group W strains. [Tesi di dottorato]

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FHbp sequence diversity expressed by African isolates is limited among currently circulating strains in sub-Saharan Africa but could change in the future. The primary aim of the project was to systematically investigate and dissect the breadth and nature of fHbp sequence coverage irrespective of the fHbp sequence prevalence. This was obtained by using a combination of bioinformatic tools and analysis of functional antibody raised against fHbp over-expressed in meningococcal GMMA from an African group W strain. The work is divided into three parts: 1. Comparative structure/sequence analysis of the amino acids involved in factor H binding including more than 700 individual existing and published fHbp sequences. The results obtained show a differential degree of variability of the factor H binding site between fHbp belonging to variant 1 versus those belonging to variant 2 or 3 with greater sequence diversification of the factor H binding site in the v.1 group. 2. Systematic selection of fHbp sequences based on a gradually different number of differences in the factor H (fH) binding site compared to fHbp variant 1 ID9 expressed in the group W GMMA. Applying these criteria a panel of 11 African and non-African wild type strains (belonging to serogroup A, C, W, Y or X) was selected that expressed a subset of the identified phylogenetically diverse fHbp sequences. Use of fHbp selection criteria to generate a panel of eight genetically defined isogenic strains that were engineered to express seven existing diverse fHbp v.1 sequences and one naturally existing and published fHbp v.1/2 hybrid protein. The wild type and isogenic strains as test strains in serum bactericidal assay to measure functional activity of antibodies raised against GMMA from a group W strain with over-expressed fHbp v.1 ID 9. The sera had bactericidal activity against 10 out of the 11 diverse wild type strains. However, against 8 of the susceptible strains also sera raised against GMMA that did not contain fHbp were bactericidal suggesting that other yet unknown target(s) contribute to the generation of bactericidal antibodies.The sera raised in mice against the group W GMMA with over-expressed fHbp v.1 also had bactericidal activity against the panel of eight isogenic strains engineered to express diverse fHbp sequences including the strain engineered to express the natural fHbp v.1/2 hybrid sequence. The results with the isogenic strains expressing diverse fHbp sequences suggest that differences in the fH binding site contribute to susceptibility of the isolate to killing by anti fHbp antibodies but also overall sequence diversity plays a role. 3. As proof of principle to broaden coverage of GMMA from a group W strain with over-expressed fHbp v.1 a mutant group W strain engineered to over-express the native fHbp v.2 sequence, which is predominant among African group W isolates was generated. GMMA from the mutant had approximately 10-fold higher amount of fHbp than in GMMA from wild type meningococcus; penta-acylation of lipidA after inactivation of lpxL1 was confirmed by MALDI-TOF analysis and Monocyte-Activation Test (MAT) assay showed an approximately 100-fold decreases ability of GMMA from the mutant to activate cytokine release from human blood cells. By HPLC-SEC MALLS integrity, size and impurities of the vesicles were characterized.

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