systematic analysis of fhbp sequence coverage by antibodies against meningococcal gmma from ...

126 Pages·2016·2.54 MB·English

S A FH
YSTEMATIC NALYSIS OF BP
S C
EQUENCE OVERAGE BY
A
NTIBODIES AGAINST
M GMMA
ENINGOCOCCAL FROM
A G W S
FRICAN ROUP TRAINS

Maria Grazia Aruta

Dottorato in Scienze Biotecnologiche – XXVIII ciclo
Indirizzo Biotecnologie Industriali e Molecolari
Università di Napoli Federico II

Dottorato in Scienze Biotecnologiche – XXVIII ciclo
Indirizzo Biotecnologie Industriali e Molecolari
Università di Napoli Federico II

S A FH
YSTEMATIC NALYSIS OF BP
S C
EQUENCE OVERAGE BY
A
NTIBODIES AGAINST
M GMMA
ENINGOCOCCAL FROM
A G W S
FRICAN ROUP TRAINS

Maria Grazia Aruta

Dottorando: Maria Grazia Aruta

Relatore: Prof. Giovanni Sannia,
Dr. Oliver Koeberling

Coordinatore: Prof. Giovanni Sannia

Il miglior riconoscimento
per la fatica fatta
non è ciò che se ne ricava,
ma ciò che si diventa
grazie ad essa.

John Ruskin

Index

Summary ..................................................................................................................................... 1
Riassunto .................................................................................................................................... 3
II Introduction .............................................................................................................................. 7
2.1 Neglected diseases ............................................................................................................ 7
2.2 Meningitis .......................................................................................................................... 7
2.2 Neisseria meningitidis ........................................................................................................ 9
2.2.1 Biology and microbiology ............................................................................................. 9
2.2.2 Epidemiology ............................................................................................................. 13
2.2.3 Pathogenesis ............................................................................................................. 16
2.2.4 Neisseria interaction with complement components .................................................. 18
2.3 Meningococcal vaccines .................................................................................................. 20
2.3.1 State of the art in developed countries....................................................................... 20
2.3.2 State of the art in Africa ............................................................................................. 21
2.3.3 Native outer membrane vesicles (NOMV) and Generalized Modules for Membrane
Antigens (GMMA) ............................................................................................................... 22
2.3.3 Factor H binding protein ............................................................................................ 24
III Aim of thesis .......................................................................................................................... 29
IV Material and methods ............................................................................................................ 31
4.1 Bacterial growth conditions .............................................................................................. 31
4.1.1 Escherichia Coli ......................................................................................................... 31
4.1.2 Neisseria Meningitidis ................................................................................................ 31
4.2 Molecular biological DNA techniques ............................................................................... 31
4.2.1 Polymerase chain reaction (PCR) .............................................................................. 31
4.2.2 Colony PCR............................................................................................................... 34
4.2.3 Agarose gel electrophoresis ...................................................................................... 34
4.2.4 DNA purification and quantitation .............................................................................. 35
4.2.5 Enzymatic restriction of DNA ..................................................................................... 36
4.2.6 Gel extraction of DNA ................................................................................................ 36
4.2.7 Ligation of DNA fragments ......................................................................................... 37
For ligation reactions T4 DNA Ligase (Invitrogen) was used to ligate DNA fragments. ....... 37
4.2.8 Neisseria genomic DNA prep .................................................................................... 38
4.2.9 Plasmidic Miniprep .................................................................................................... 39
4.2.10 Escherichia Coli transformation ............................................................................... 39
4.2.11 Neisseria Meningitidis transformation ...................................................................... 40
4.2.12 Preparation of bacterial glycerol stocks for long term storage .................................. 40
4.3 Generation of Neisseria Meningitidis mutants .................................................................. 40
4.3.1 Overview of definitions and objectives of mutations in Neisseria meningitidis genome
........................................................................................................................................... 40
4.3.2 Plasmids and primers ................................................................................................ 41
4.4 Determination of protein concentration by Lowry assay ................................................... 47
4.5 SDS-Polyacrylamid gel electrophoresis of proteins .......................................................... 47
4.6 Western Blotting............................................................................................................... 47
4.7 Dot blotting....................................................................................................................... 48
4.8 Coomassie blue stain of proteins ..................................................................................... 48
4.9 FACS analysis of fHbp surface expression on live bacteria .............................................. 49
4.10 Preparation and purification of GMMA ........................................................................... 49
4.11 Characterization and quality control of GMMA ............................................................... 50
4.11.1 Negative Staining Trasmission Electron Microscopy ................................................ 50
4.11.2 Monocyte Activation Test (MAT) .............................................................................. 50
4.11.3 MALDI-TOF and MS/MS analysis on lipid A ............................................................ 51
4.11.4 HPLC-SEC MALLS analysis of GMMA samples ...................................................... 52
4.12 Mice immunization ......................................................................................................... 55
4.13 Characterization of immune responses by Serum Bactericidal Assay (SBA) .................. 55
4.14 FHbp sequence diversity analysis by pairwise amino acid comparison .......................... 56
V – Results ................................................................................................................................ 59
5.1 Generation and characterization of GMMA from a serogroup W strain with over-expressed
fHbp v.2 ................................................................................................................................. 59
5.1.1 Over-expression of native fHbp variant 2 in MenW .................................................... 60
5.1.2 Deletion of capsule in MenW strains over-expressing fHBp v.2, ................................ 62
5.1.3 Deletion of lpxL1 ........................................................................................................ 63
5.1.4 Deletion of gna33 ...................................................................................................... 64
5.2 Comparative characterization of Group W GMMA over-expressing two different variants of
fHbp ....................................................................................................................................... 65
5.2.1 Detection of total protein and fHbp in GMMA by SDS PAGE, Comassie blue and
western blotting .................................................................................................................. 65
5.2.2 MALDI-TOF analysis of lipid A from N. meningitidis GMMA lpxL1 knock out ............. 67
5.2.3 Evaluation of endotoxin activity of Neisseria GMMA after genetic manipulation ......... 68
5.2.4 Analysis of GMMA samples from group W mutants with over-expressed fHbp fHbp v.2
by HPLC-SEC MALLS ........................................................................................................ 69
5.3 Analysis of the breadth of fHbp sequence coverage by antibodies against fHbp in GMMA
.............................................................................................................................................. 71
5.3.1 Structure and sequence analysis of fHbp v.1, v.2 and v.3 .......................................... 72
5.3.2 Investigation of functional activity of antibodies against fHbp on a strain panel
expressing highly diverse fHbp sequences ......................................................................... 80
VI Discussion ............................................................................................................................ 95
VII Reference List .................................................................................................................... 101
Ringraziamenti ........................................................................................................................ 115