Guasch Rodríguez, Marc

Universitat Autònoma de Barcelona. Facultat de Biociències

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      Guasch Rodríguez, Marc – Degree in Biotechnology Novel NOMV-based vaccine against Neisseria meningitidis serogroup B Factor H and the complement system Antigens that interact with factor H Serological assays    Relevant bibliography The role of the complement system against N. meningitidis is well established. Individuals deficient in components of the alternative (AP) and terminal complement pathways are highly predisposed to meningococcal infections5.  Factor H (fH) is a regulatory protein of the alternative complement pathway that can inhibit the complement system action. This protein binds to host cells due to its glycosaminoglycans (GAGs) affinity. Because of GAGs are not found on the bacterial cell surface, fH can inhibit the complement system attack in host cells preferably5. N. meningitidis use several mechanisms to evade killing by human complement. Capturing host fH permits meningococci disarm the AP and downregulate its action. Expression of surface proteins that bind to fH increases bacterium resistance to complement system attack5.     Figure 2. Molecular structure of the AP of complement components6. Three of the most important meningococcal proteins that bind to fH are:  • fHbp: N. meningitidis strains with a fHbp gene deletion lose binding capacity to fH and are more susceptible to complement attack. This antigen is a clear candidate for a meningococcal serogroup B vaccine; it is cell surface exposed, it is present in more than 99% of all invasive strains, it is expressed during infection and it generates protective bactericidal antibodies7.   • NspA: Recombinant NspA expressed in E. coli and purified does not have the same conformation as the protein present in the meningococcal outer membrane. For this reason, the first vaccine approximation does not elicit a bactericidal antibody response. Protective antibodies may be directed against conformational epitopes of the native protein8.   • PorB2: It is one of the most abundant outer membrane proteins in the bacterium. Because of its low affinity interaction with fH, its expression downregulates the AP of complement9.   Objectives Vaccine design Possible benefits Four strains are appropriately chosen to be genetically modified. Each strain naturally expresses the antigen which will be over-expressed (Table 1). Three genes are inactivated to improve vaccine safety and to reduce the possibility of inducing autoimmunity: ΔsynX (blocks sialic acid synthesis), ΔlgtA (blocks lacto-N-neotetraose expression) and ΔlpxL1 (reduces LOS endotoxicity)10. Enhanced expression of the desired antigen is achieved by inserting a second copy of the corresponding gene in the nadA locus (Fig. 3). Vaccine strain name Derived from ΔlpxL1 ΔsynX ΔlgtA Over-expression of S1 PMB1745 Yes Yes Yes fHbp A05 S2 M1573 Yes Yes Yes fHbp B01 S3 H44/76 Yes Yes Yes NspA S4 2996 Yes Yes Yes PorB2 Table 1. Vaccine strains with their genetic modifications. Figure 3. Plasmid used for the transformation of the meningococcal strains. Each strain is transformed with its corresponding genic construction, in which the gene of interest varies.  Antigens included in this vaccine fulfil the same function: Binding to fH. Moreover, these molecules   present low variability between meningococcal strains.   Achieving bactericidal antibodies against these proteins would produce loss of bacterial virulence and an increased susceptibility   of these bacteria towards elimination by the complement system.   The technique used in this work could be applied to a universal vaccine design against the main meningococcal serogroups, trough the expression of the appropriate antigens. 10 mice       Aluminum hidroxyde      (0.17 mg/dose) 10 mice         Aluminum hidroxyde (0.17 mg/dose) S1, S2, S3 and S4 NOMVs mixture (3 μg) 3 doses  (weeks 0, 4 and 8) Serum is taken at week 10 SBA assay 10 mice Individual NOMV components (3 μg) 3 doses  (weeks 0, 4 and 8) Serum is taken at week 10 ELISA assay         Aluminum hidroxyde (0.17 mg/dose) 10 mice 10 mice 10 mice 10 mice (x4) S1 NOMVs S2 NOMVs S3 NOMVs S4 NOMVs Control (NOMVs without enhanced protein expression)  (against 18 different meningococcal serogroup B strains) The main objective of this work is to develop a novel meningococcal serogroup B vaccine. This vaccine consists in NOMVs (Native Outer Membrane Vesicles) over-expressing these proteins: • fHbp (A05 and B01 variants) • NspA • PorB2     Figure 1. Worldwide distribution of the most prevalent Neisseria meningitidis serogroups4. Neisseria meningitidis is a Gram-negative diplococcus bacterium that causes a great number of deaths worldwide1. The most common form of meningococcal disease is meningitis, which often occurs as epidemics. Despite appropriate treatment, the case-fatality rate is around 10%2.  The highest incidence rates are found in the “meningitis belt”, which extends from Senegal to Ethiopia (Fig. 1). In this area, A, C, W135 and X are the major disease causing meningococcal serogroups. The development of capsular polysaccharide-conjugated vaccines has allowed the prevention of disease caused by these serogroups1,2.  On the other hand, there is a great meningococcal serogroup B incidence in Europe and the USA. Its capsular polysaccharide is poorly immunogenic due to its antigenic structure, which mimics the cell surface glycoproteins of human neurological tissue. For this reason, developing a group B vaccine has supposed a true challenge1. Reverse vaccinology has allowed identification of new vaccine candidates. The first meningococcal serogroup B vaccine based on this technique (Bexsero®) was approved in Europe and Australia in 20131,3. In this work, another approach to a vaccine development against Neisseria meningitidis serogroup B is proposed. Using NOMVs present several advantages than using classic detergent-extracted OMVs10:  • LOS and lipoproteins are not depleted from the first vesicles. • Surface exposure of membrane proteins is similar to that on the intact bacterium. • Retaining lipoproteins allows some proteins to be present in the vaccine at increased levels.  Expression of key epitopes and the level at which they are expressed are verified by colony blotting and Western blotting, respectively10. 1. Caesar, N. M. et al. Microbial Pathogenesis Neisseria meningitidis serogroup B vaccine development. Microb. Pathog. 57, 33–40 (2013). 2. Caugant, D. A. Genetics and evolution of Neisseria meningitidis: importance for the epidemiology of meningococcal disease. Infect. Genet. Evol. 8, 558–65 (2008). 3. O’Ryan, M. et al. A multi-component meningococcal serogroup B vaccine (4CMenB): the clinical development program. Drugs 74, 15–30 (2014). 4. Harrison, L. H. et al. Global epidemiology of meningococcal disease. Vaccine 27 Suppl 2, 51–63 (2009). 5. Lewis, L. A. et al. Meningococcal disease and the complement system. Landes Bioscience 5, 1–29 (2014). 6. Serruto, D. et al. Molecular mechanisms of complement evasion: learning from staphylococci and meningococci. Nat. Rev. Microbiol. 8, 393-9 (2010). 7. McNeil, L. K. et al. Role of factor H binding protein in Neisseria meningitidis virulence and its potential as a vaccine candidate to broadly protect against meningococcal disease. Microbiol. Mol. Biol. Rev. 77, 234–52 (2013). 8. Lewis, L. A. et al. The meningococcal vaccine candidate neisserial surface protein a (NspA) binds to factor H and enhances meningococcal resistance to complement. PLoS Pathog. 6, 1–20 (2010). 9. Lewis, L. A. et al. Factor H-Dependent Alternative Pathway Inhibition Mediated by Porin B Contributes to Virulence of Neisseria meningitidis. MBio 4, 1–9 (2013). 10. Zollinger, W. D. et al. Design and evaluation in mice of a broadly protective meningococcal group B native outer membrane vesicle vaccine. Vaccine 28, 5057–67 (2010). 

Novel NOMV-based vaccine against Neisseria meningitidis serogroup B

http://ddd.uab.cat/pub/tfg/2014/119348/TFG_marcguaschrodriguez.pdf

Novel NOMV-based vaccine against Neisseria meningitidis serogroup B

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