Towards a Neisseria meningitidis B vaccine Introducing systems biology in process development Gino J.E. Baart Stellingen 1. Realistische modellen van celmetabolisme bevatten altijd onderbepaaldheden (dit proefschrift). 2. Het berekenen van numerieke waarden voor onderbepaalde fluxen is een wiskundige oefening (dit proefschrift). 3. In vitro celkweek is een typisch voorbeeld van de tragedie van de meent [1,2]. [1] MacLean RC, Gudelj I. 2006. Resource competition and social conflict in experimental populations of yeast. Nature 441:498–501 [2] Hardin G. 1968. The Tragedy of the Commons. Science 162:1243–1248. 4. Biotechnologie is een trukendoos. Variant op de stellingname: De truc van ruimtevaart is vallen (André Kuipers, 2005). 5. Het zou de groenste universiteit van Nederland sieren als eerste het drukken van proefschriften af te schaffen. 6. De slogan ”leuker kunnen we het niet maken, wel makkelijker” kan de belastingdienst beter vervangen door ”leuker kunnen we het nooit maken, ook niet makkelijker”. Stellingen behorende bij het proefschrift: Towards a Neisseria meningitidis B vaccine Introducing systems biology in process development Gino J.E. Baart 17 oktober 2008 Towards a Neisseria meningitidis B vaccine Introducing systems biology in process development Gino J.E. Baart Promotor Prof. dr. ir. J. Tramper Hoogleraar in de bioprocestechnologie, Wageningen Universiteit, Nederland Co-promotoren Dr. ir. D.E. Martens Universitair docent sectie proceskunde, Wageningen Universiteit, Nederland Dr. E.C. Beuvery Bionnovations, Nederland Promotiecommissie Prof. dr. ir. J.J. Heijnen Technische Universiteit Delft, Nederland Prof. dr. ir. J. van der Oost Wageningen Universiteit, Nederland Prof. dr. ir. E. Vandamme Universiteit Gent, België Prof. dr. B.A.M. van der Zeijst Nederlands Vaccin Instituut; Universiteit Leiden, Nederland Dit onderzoek werd uitgevoerd op het Nederlands Vaccin Instituut te Bilthoven, Nederland, binnen de onderzoekschool VLAG. Gino Johannes Elisabeth Baart Towards a Neisseria meningitidis B vaccine Introducing systems biology in process development Proefschrift Ter verkrijging van de graad van doctor op gezag van de Rector Magnificus van Wageningen Universiteit, Prof. dr. M.J. Kropff in het openbaar te verdedigen op vrijdag 17 oktober 2008 des namiddags te half twee in de Aula Baart GJE. 2008. Towards a Neisseria meningitidis B vaccine - Introducing systems biology in process development Ph.D. thesis, Wageningen University, Wageningen, The Netherlands – With summary in Dutch ISBN 978-90-8504-997-5 Aan mijn geliefden voor mezelf Life comes down to a few moments. This is one of them. Oliver Stone CONTENTS Chapter 1 General introduction 1 Chapter 2 Modeling Neisseria meningitidis metabolism: 35 from genome to metabolic fluxes Chapter 3 Modeling Neisseria meningitidis B metabolism 107 at different specific growth rates Chapter 4 Heterologous expression of phosphofructokinase 153 in Neisseria meningitidis Chapter 5 Scale-up for bulk production of vaccine 199 against meningococcal disease Chapter 6 General discussion 227 Summary 241 Samenvatting 249 Glossary 257 Dankwoord 265 About the author 273 Chapter 1 General introduction 2 General introduction NEISSERIA MENINGITIDIS Meningococcal disease The genus Neisseria belongs to the family Neisseriaceae of the β-proteobacteria [1] and is named after Albert Neisser who discovered the gonococcus in 1879 [2]. The genus includes a group of closely related cocci that are primarily commensal organisms of mucosal surfaces of mammals [3]. Two species: Neisseria gonorrhoeae (the gonococcus) and Neisseria meningitidis (the meningococcus), are important human pathogens [4]. Anton Weichselbaum first identified N. meningitidis as the causative agent of bacterial meningitis in 1887 [5]. N. gonorrhoeae is considered to be always pathogenic and causes the sexually transmitted disease gonorrhea, while N. meningitidis colonizes oro- and nasopharyngeal mucosa, its sole ecological niche, asymptomatically, which means that not all people who harbor the meningococcus develop disease [6, 7]. Transfer of meningococci from one person to another can occur via direct contact with nasal or oral secretions or through inhalation of droplets [8]. Acquisition of meningococci may result in invasive disease, which normaly occurs 1-14 days after acquisition or lead to colonization (carriage) [8]. Carriage leads to the induction of ‘natural acquired’ immunity against the organisms [9]. In non-epidemic situations approximately 10% of the adult population is colonized by meningococci [10-13], but increased carriage rates have been observed during epidemics and close contacts [12, 14]. The highest incidence of meningococcal disease occurs in young children under the age of four. In addition, an incidence peak is observed among teenagers, most likely as a result of increased exposure to environmental risk factors, such as the number and closeness of contacts (e.g. intimate kissing) [15] and (passive) smoking [16]. By a mechanism that remains unknown, some N. meningitidis strains are able to cross the mucosa into the bloodstream from where they can cause invasive meningococcal disease with severe clinical syndromes [17]. Understanding how the meningococcus can both be a harmless commensal and a devastating human pathogen has been a major quest in the biology and in the design of prevention strategies for N. meningitidis. 3 Chapter 1 Meningococcal disease is a life-threatening illness with annual incidence rates varying from 1 to 1000 per 100 000 persons in different parts of the world [8, 12]. The disease is charachterized by a variety of clinical symptoms [17] and in the early phase mild symptoms like chills, fever and musle aches can change rapidly (within hours) into bacterial meningitis or septicaemia, potentially leading to shock and death [17]. Despite antibiotic treatment, the mortality rate is about 10 to 30% in the case of meningitis and sepsis, respectively [18]. Early antibiotic treatment should be applied as soon as possible when clinical diagnosis confirms meningococcal meningitis since effective antibiotics immediately stop proliferation of N. meningitidis [19]. Without antibiotic treatment, death rates can increase up to 80% [20]. About 5-20% of the surviving patients suffer from permanent damage, such as amputation of limbs, mental retardation, and deafness [20]. The rapid onset of invasive meningococcal disease [17], the high incidence in young children [19], the high mortality rate [18] and the severe permanent damage in patients recovering from disease [20] indicate the impor- tance of vaccine development against this devastating pathogen. Microbiology N. meningitidis is a heterotrophic, aerobic Gram-negative coccus, occurring as single coccus but often as diplococcus with adjacent sides flattened and ranging from 0.6–1.9 µm in diameter [1]. Meningococci are obligate human pathogens that have an optimal growth temperature of 35-37 °C. The cell envelop of N. meningitidis is typi- cal for a Gram-negative bacterium and composed of an outer membrane and an inner membrane, separated by the periplasmatic space that contains a peptidoglycan layer and proteins (Figure 1.1). The inner membrane is a phospholipid bilayer containing mainly proteins involved in transport of proteins and nutrients across the inner mem- brane, while the outer membrane is assymetrical containing mainly lipopolysaccharide (LPS) on the outer leaflet and phospholipids on the inner leaflet [21]. The outer membrane contains various outer membrane proteins (OMPs) of which the major ones are: Porin A (PorA), Porin B (PorB), reduction-modifiable protein M (RmpM), 4 General introduction opacity proteins (Opa, Opc) and the lactoferrin receptor proteins. Virulent bacteria are covered by a capsule [11] composed of polysaccharides. Through this capsule fimbriae (type IV pili) protrude. These pili are filamentous proteins and are found to play an important role in a number of processes such as: the initial attachement of meningococci to host cells [22], natural transformation [23], twitching motility (movement) [24] and biofilm formation [25]. Although type IV pili are immunogenic, Figure 1.1 Cross-sectional view of the N. meningitidis cell surface. The cell envelop is composed of an outer membrane (OM) and an inner membrane (IM), separated by the periplasmatic space (PS) that contains a peptidoglycan layer (PG). The cell surface is surrounded by capsular polysaccharide (CPS) that is anchored in the OM via a phosphatidyl glycerol moiety. Type IV pili (pilus) protrude through the entire envelope and extend several thousand nm from the surface [19]. The IM is a phospholipid (PL) bilayer containing mainly proteins involved in transport of proteins and nutrients across the inner membrane. The OM is assymetrical containing mainly phospholipids on the inner leaflet and lipopolysaccharide (LPS) on the outer leaflet. Furthermore, it harbors integral and surface exposed outer membrane proteins (OMPs). LbpA and LbpB are integral and cell-surface exposed OMPs respectively, which together constitute the lactoferrin receptor that is involved in the acquirement of iron from the environment. Opa: Opacity protein (class 5 OMP), PorA: protein Porin A (class 1 OMP), RmpM: reduction-modifiable protein M (class 4 OMP). Courtesy Wilma Witkamp, Netherlands Vaccine Institute. 5 Chapter 1 they are not suitable as vaccine candidates, because of the high degree of antigenic variability [20] and the phase variation of expression [23]. Flagella are absent in Neisseriaceae