Copyright is owned by the Author of the thesis.  Permission is given for 
a copy to be downloaded by an individual for the purpose of research and 
private study only.  The thesis may not be reproduced elsewhere without 
the permission of the Author. 
 



 

 
 

Polyhydroxyalkanoate beads as a particulate vaccine against 

Streptococcus pneumoniae and Neisseria meningitidis 

 

 

A thesis presented in partial fulfilment of the requirements for the degree of 

Doctor of Philosophy 

in 

Microbiology 

at Massey University, Manawatu, New Zealand. 

Majela González Miró 

2017 

 

 

Main Supervisor: Professor Bernd Rehm 

Co-supervisors: Dr Zoe Jordens, Dr Vicente Vérez-Bencomo 

 

 



 

i 
 

Abstract: 

Streptococcus pneumoniae and Neisseria meningitidis are the major causes of 

pneumonia and meningitis, respectively, worldwide. Capsular polysaccharide-protein 

vaccines (conjugate vaccines) provide protection against these diseases but not protection 

against infections caused by serotypes and serogroups not included in these vaccines. 

Proteins have been increasingly considered as antigens for vaccine development due to 

their more structurally conserved composition when compared to capsular 

polysaccharides. Proteins subunit vaccines are safe and protective; however, they have 

limitations such as serotype-dependent immunity, and low immunogenicity of the 

proteins, requiring adjuvant to be included in these formulations or delivery systems that 

enhance the desired immune response. In addition, complex production procedures are 

required, increasing production costs and therefore market prices making these vaccines 

inaccessible for many people affected by these diseases. Recently, bacterial storage 

polymer inclusions have been developed as protein antigen carriers. 

Polyhydroxyalkanoate, in particular 3-polyhydroxybutyrate (PHB) inclusions have been 

successfully bioengineered to display antigens from pathogens like Mycobacterium 

tuberculosis and Hepatitis C virus. These particulate vaccine candidates elicited both a 

Th1 and Th2 immunity patterns combined with a protective immune response against 

Mycobacterium bovis in mice.  

This thesis focuses on the study of polyhydroxybutyrate (PHB) beads properties as 

a carrier/delivery system engineered to display antigens from extracellular bacteria. The 

antigens Pneumococcal adhesin A, Pneumolysin (proteins) and 19F capsular 

polysaccharide (CPS) from Streptococcus pneumoniae, and Neisserial adhesin A, factor 

H binding protein (proteins) and serogroup C CPS from Neisseria meningitidis were 

displayed on the PHB bead surface. These antigenic proteins were produced as fusion 



 

ii 
 

proteins on the PHB bead surface, while the CPS was covalently attached by chemical 

conjugation. Mice vaccinated with these PHB beads produced strong and antigen-specific 

antibody levels. In addition, splenocytes from the same mice generated both IL-17A and 

IFN-ɣ production.  

The antibodies elicited against antigenic pneumococcal proteins were able to 

recognise the same protein in the context of an Streptococcus pneumoniae whole cell 

lysate from more than six different strains, while antibodies produced after vaccination 

with 19F CPS conjugate to PHB showed high opsonophagocytic titers against the 

homologous strain. In the case of Neisseria meningitidis, bactericidal antibodies were 

elicited in mice vaccinated with PHB beads displaying proteinaceous and CPS antigens.  

Overall, this thesis shows that PHB as particulate vaccine candidate holds the 

promise of a broadly protective vaccine that can be produced cost-effectively for 

widespread application to prevent diseases caused by Neisseria meningitidis and 

Streptococcus pneumoniae. 

  



 

iii 
 

 

 

 

 

 

 

With eternal love, gratitude and in memory of my mother 

 

(Mercedes Miró Alonso, 1945-1993) 

 

 

 

 

 

 

 

 

 

 

 



 

iv 
 

Acknowledgements  

It has been a great experience and opportunity to work under the guidance and 

support of my main supervisor Professor Bernd Rehm. After hearing his oral presentation 

in Vaccipharma 2012, about PHA beads and their potential biomedical applications, 

combined with our understanding of the necessity to improve commercial vaccines 

against Neisseria meningitidis and Streptococcus pneumoniae, the idea to explore this 

platform for this purpose was born. This idea became a PhD project giving to me the 

possibility to enter a new field called nanotechnology. For all of these, I would like to 

thank Professor Bernd Rehm. In addition, I would like to thank my co-supervisors Dr Zoe 

Jordens and Dr Vicente Vérez-Bencomo for their support during this journey, respecting 

all the time their experiences, opinions and criticisms.  

I thank members and ex-members of Bernd Rehm team, especially, Shuxiong Chen, 

Patricia Rubio, Jason Lee, Jason Smith, Jinping du, Natalie Burn, Kathryn Grage, Natalie 

Parlane, Andy Hollings…I learned a lot from them. The cooperation and interaction 

between my coworkers and myself, but also the weekly seminar allowed me to increase 

my scientific knowledge and my oral presentation skills.  

I would like to thank my co-workers in The Finlay Vaccine Institute, Havana, Cuba, 

for all their professional and spiritual support, especially, Dr Dagmar García, Laura Marta 

Nodas, Yanet Estrada, Aylin Amador, Mildrey Fariñas, Sandra Madariaga, Dr Caridad 

Zayas, Neissa García, Aniuska Garces, MsC Amarilys Pérez, Leandro Camejo, Yury 

Valdés, Darielys Santana, Danaydis Fonseca, Ubel González, Elizabeth González, Dr 

Reinaldo Acevedo, Dr Reinaldo Oliva, Maria Onelia, Dr Barbara Cedré, Marilé García, 

MS Tamara Hernández, Alex Quintero Pérez, Dr Reinaldo Oliva Hernández, Dr Yanely 

Tirado, Rosmira Nicado. 

https://www.researchgate.net/profile/Jinping_Du


 

v 
 

I would like to thank Barry Bunn and Debra Cresswell for their support with English 

grammar. In addition, I would like to thank Jordan Taylor, Niki Minard and Matthew 

Savoian from the Manawatu Microscopy Imaging Centre for their assistance in TEM 

analysis. Prof Martin Hazelton and Dr Edgar Santos-Fernández are acknowledged for 

their assistance in statistical analysis of data in this thesis. 

I am grateful for the scholarship support, by The MacDiarmid Institute for 

Advanced Materials and Nanotechnology and also, Massey University and The Finlay 

Institute for financial support.  

I cannot forget to mention my friends from Cuba, New Zealand, Argentina, Mexico, 

Hungary, Colombia, Chile, Guatemala, Portugal, Spain, Germany, Austria, Italy, 

Ecuador, USA, France, The Netherlands, Paraguay, UK, Venezuela, Iran, India, Gana, 

Honduras thank you very much to all of them. But I would like to highlight my friend’s 

Dr Edgar Santos-Fernández, Dr Jimena Yapura, Dr Javier Flores and Tony Reid because 

you have helped a lot not only to be a better scientist but to be a better human being. 

Thank you very much. 

Last but not least, I would like to thank my family especially my father (the love of 

my life and my example to follow) and my sister (my friend, my confidante….). Thank 

you forever. I will always love you both.  

  

https://www.massey.ac.nz/massey/learning/colleges/college-of-sciences/about/fundamental-sciences/staff-list.cfm?stref=844930


 

vi 
 

Preface  

This thesis is written according to the regulations of the Handbook for Doctoral Study, 

revised in May 2016 by the Doctoral Research Committee. This thesis complies with the 

format of a thesis based on publication as described in the handbook.  

Chapter 1  

Introduction  

This chapter was written by Majela González Miró as an introductory chapter for this 

thesis only and is not intended for publication  

Chapter 2 

Self-assembled particulate PsaA as vaccine against Streptococcus pneumoniae 

infection 

González-Miró, Majela1,2, Rodríguez-Noda, Laura1, Fariñas-Medina, Mildrey1, García-

Rivera, Dagmar1, Vérez-Bencomo, Vicente1, Rehm, Bernd H.A.2 

Published: Heliyon 11 Apr 2017- Volume 3, Issue 4 Pharmaceutical Science, 

Biochemistry, Immunology 

G.-Miró. M: Conceived and designed the experiments; performed and supervised the 

experiments; analysed and interpreted the data; Wrote the paper. R.-N. L., F-M. M: 

Performed the experiments. G.-R. D.: Analysed and interpreted the data; Contributed 

reagents, materials, analysis tools or data; Wrote the paper. V.-B. V: Contributed 

reagents, materials, analysis tools or data. R. B.H.A.: Conceived and designed the 

experiments; Analysed and interpreted the data; Contributed reagents, materials, analysis 

tools or data; Wrote the paper. 

  

http://www.heliyon.com/subject/Pharmaceutical-Science
http://www.heliyon.com/subject/Biochemistry
http://www.heliyon.com/subject/Immunology


 

vii 
 

Chapter 3 

Biologically assembled polyester beads displaying pneumolysin and capsular 

polysaccharide induce protective immunity against Streptococcus pneumoniae 

Majela González-Miró 1,2, Anna-Maria Radecker 2, Laura M Rodríguez-Noda 1, Mildrey 

Fariñas-Medina1, Caridad Zayas-Vignier1, Mabel Heránndez-Cedeño 1, Yohana 

Serrano1, Félix Cardoso 1, Darielys Santana-Mederos1, Dagmar García-Rivera 1, Yury 

Valdés-Balbín 1, Vicente Vérez-Bencomo1, Bernd H.A. Rehm2, 3 

M. G.-Miró, A-M. R., L.M. R.-N., M. F.-M., C. Z.-V., M. H.-C., Y.S., F.C. performed 

the studies and analysed the data. D. G.-R., V. V.-B., M.G.- Miró., Y. V.-B., D. S.-M. 

B.H.A. R. analysed and interpreted the data; contributed reagents, materials, analysis 

tools or data. M.G.- Miró. and B.H.A. R. conceived and designed and supervised the 

experiments. M.G.- Miró. and B.H.A. R. wrote the manuscript. 

Under review by Frontiers in Immunology, Jan 2018 

Chapter 4 

Bioengineered polyester beads co-displaying protein and carbohydrate-based 

antigens enhance protective efficacy against bacterial infection  

Majela González-Miró1,2, Laura M Rodríguez-Noda1, Mildrey Fariñas-Medina1, 

Barbara Cedré-Marrero1, Sandra-Madariaga-Zarza1, Caridad Zayas-Vignier1, Mabel 

Hernández-Cedeño1, Torsten Kleffmann3, Dagmar García-Rivera1, Vicente Vérez-

Bencomo1, Bernd H. A. Rehm2, 4 

M. G.- Miró, L.M. R.-N., M. F.-M., B. C.-M., S.M.-Z., C. Z.-V., M. H.-C., T.K. 

performed the studies and analysed the data. D.G.-R., V.V.-B., M.G.- Miró., B.H.A.R. 

analysed and interpreted the data; contributed reagents, materials, analysis tools or data. 

M.G.- Miró. and B.H.A. R. conceived and designed and supervised the experiments. 

M.G.- Miró. and B.H.A. R. wrote the manuscript. 

Published: Scientific Reports Journal, 30 Jan 2018, Volume 8, Issue 1 

 

 

https://www.nature.com/natbiomedeng/


 

viii 
 

Chapter 5.  

General Discussion, Conclusion and Future work 

This chapter was written by Majela González Miró for this thesis only and is not intended 

for publication. 

 

 

  



 

ix 
 

Table of Contents 

Abstract: ............................................................................................................................. i 

Acknowledgements .......................................................................................................... iv 

Preface .............................................................................................................................. vi 

List of Figures ................................................................................................................ xiv 

List of Tables.................................................................................................................. xvi 

Abbreviations ................................................................................................................ xvii 

Chapter 1. Introduction ..................................................................................................... 1 

1.1 Streptococcus pneumoniae ................................................................................. 1 

1.1.1 Pneumococcal diseases and epidemiology ............................................................ 1 

1.1.2 Host immune defences against Streptococcus pneumoniae .................................. 2 

1.1.3 Relevant pneumococcal virulence factors ............................................................. 3 

1.1.4 Pneumococcal vaccines ......................................................................................... 5 

1.2 Neisseria meningitidis ........................................................................................ 6 

1.2.1 Meningococcal diseases and epidemiology .......................................................... 7 

1.2.2 Host immune defences against Neisseria meningitidis ......................................... 8 

1.2.3 Relevant meningococcal virulence factors ............................................................ 9 

1.2.4 Meningococcal vaccines ..................................................................................... 11 

1.3 Adjuvants and delivery systems ....................................................................... 12 

1.4 Polyhydroxyalkanoate beads as potential particulate vaccines ........................ 13 

1.5 General Hypothesis .......................................................................................... 18 

1.6 Aims and scope of the thesis ............................................................................ 18 

1.7 References ........................................................................................................ 19 

Preface to the next Chapter ............................................................................................. 33 

Chapter 2. Self-assembled particulate PsaA as vaccine against Streptococcus pneumoniae 

infection ...................................................................................................................... 34 

2.1 Abstract ............................................................................................................ 35 



 

x 
 

2.2 Introduction ...................................................................................................... 36 

2.3 Materials and methods ...................................................................................... 37 

2.3.1 Bacterial strains, oligonucleotides, plasmids and cultivation conditions ............ 37 

2.3.2 Construction of plasmids mediating production of PHB beads displaying PsaA39 

2.3.3 Construction of the plasmid encoding N-terminally His-tagged PsaA for 

production of soluble PsaA ................................................................................. 39 

2.3.4 Production, isolation and purification of PHB beads .......................................... 39 

2.3.5 Production, isolation and purification of recombinant soluble protein ............... 39 

2.3.6 Confirmation of the PhaC in vivo activity using transmission electron microscopy 

(TEM) .................................................................................................................. 40 

2.3.7 Protein analysis ................................................................................................... 40 

2.3.8 Measurement of the PHA bead size distribution and zeta potential ................... 40 

2.3.9 Analysis of immunological properties of PHB beads ......................................... 41 

2.3.10 Statistical analysis ............................................................................................... 43 

2.4 Results .............................................................................................................. 43 

2.4.1 Construction of plasmids mediating production of the PsaA-PhaC fusion protein 

and His6-PsaA ..................................................................................................... 43 

2.4.2 Production and characterization of PsaA displaying PHA beads ....................... 44 

2.4.3 Humoral immune response .................................................................................. 50 

2.5 Discussion ........................................................................................................ 54 

2.6 Acknowledgements .......................................................................................... 57 

2.7 References ........................................................................................................ 58 

2.8 Supplementary material .................................................................................... 63 

Preface to the next Chapter ............................................................................................. 64 

Chapter 3. Biologically assembled polyester beads displaying pneumolysin and capsular 

polysaccharide induce protective immunity against Streptococcus pneumoniae ...... 65 

3.1 Abstract ............................................................................................................ 66 

3.2 Introduction ...................................................................................................... 67 



 

xi 
 

3.3 Materials and methods ...................................................................................... 68 

3.3.1 Strains and cultivation conditions ....................................................................... 68 

3.3.2 Construction of plasmids for production of soluble Ply and Ply displayed on PHB 

beads .................................................................................................................... 69 

3.3.3 PHB bead production .......................................................................................... 69 

3.3.4 Production of soluble Ply .................................................................................... 69 

3.3.5 Conjugation of CPS to PHB beads ...................................................................... 70 

3.3.6 Transmission electron microscopy (TEM) .......................................................... 70 

3.3.7 Proteins analysis by SDS-PAGE and immunoblot ............................................. 70 

3.3.8 Immune response evaluation ............................................................................... 71 

3.3.9 Statistical analysis ............................................................................................... 75 

3.4 Results .............................................................................................................. 75 

3.4.1 Bioengineering E. coli for production of Ply-PHB beads and soluble His6-Ply 75 

3.4.2 Immunological properties of the various PHB beads and soluble proteins ........ 79 

3.5 Discussion ........................................................................................................ 87 

3.6 Acknowledgements .......................................................................................... 91 

3.7 References ........................................................................................................ 93 

3.8 Supplementary material .................................................................................. 100 

Preface to the next Chapter ........................................................................................... 101 

Chapter 4. Bioengineered polyester beads co-displaying protein and carbohydrate-based 

antigens enhance protective efficacy against bacterial infection ............................. 102 

4.1 Abstract .......................................................................................................... 103 

4.2 Introduction .................................................................................................... 104 

4.3 Materials and Methods ................................................................................... 106 

4.3.1 Ethics statement ................................................................................................. 106 

4.3.2 Construction of plasmids mediating production of PHB beads ........................ 107 

4.3.3 Construction of plasmids for production of soluble His-tagged proteins ......... 107 

4.3.4 Production, isolation and purification of PHB beads and soluble proteins ....... 107 



 

xii 
 

4.3.5 Conjugation of MenC polysaccharide (CPS) to carrier proteins ....................... 108 

4.3.6 Transmission electron microscopy analysis (TEM) .......................................... 108 

4.3.7 Measurement of PHB bead size distribution and surface charge ...................... 108 

4.3.8 NMR spectroscopy. ........................................................................................... 108 

4.3.9 Protein analysis ................................................................................................. 109 

4.3.10 Protein and carbohydrate quantification............................................................ 109 

4.3.11 Immunization schedule for proteinaceous antigens .......................................... 110 

4.3.12 Immunization schedule for conjugated vaccine prototypes .............................. 110 

4.3.13 Assessment of anti-NadA and anti-fHbp antibody titers in mice ...................... 111 

4.3.14 Assessment of anti-MenC antibody titers in mice ............................................ 111 

4.3.15 Analysis of the Ig subclass profile in sera ......................................................... 111 

4.3.16 Analysis of the cytokine production .................................................................. 112 

4.3.17 Serum bactericidal assay ................................................................................... 112 

4.3.18 Statistical analysis ............................................................................................. 113 

4.4 Results ............................................................................................................ 114 

4.4.1 Bioengineering of Escherichia coli for production of antigen-displaying PHB 

inclusions and soluble antigens. ........................................................................ 114 

4.4.2 Immunological properties of antigen displaying PHB beads versus soluble 

antigens. ............................................................................................................. 119 

4.4.3 Chemical conjugation of capsular polysaccharides to PHB beads. .................. 120 

4.4.4 Immunological properties of antigen-coated PHB beads displaying CPS. ....... 123 

4.5 Discussion ...................................................................................................... 129 

4.6 Acknowledgements ........................................................................................ 135 

4.7 References ...................................................................................................... 136 

4.8 Supplementary material .................................................................................. 144 

Chapter 5. General Discussion, Conclusion, and Future work ..................................... 156 

5.1 General Discussion ......................................................................................... 156 

5.2 General Conclusion ........................................................................................ 160 



 

xiii 
 

5.3 Future work .................................................................................................... 161 

5.3.1 Gene design ....................................................................................................... 161 

5.3.2 New targets ........................................................................................................ 161 

5.3.3 Antigen Multivalency vaccine .......................................................................... 162 

5.3.4 Mucosal Immunity ............................................................................................ 162 

5.3.5 Immunological studies ...................................................................................... 162 

5.4 References ...................................................................................................... 164 

Appendix ....................................................................................................................... 169 

 

 

  



 

xiv 
 

List of Figures 

Chapter 1 

Figure 1. Electron microscopy image of Pseudomonas aeruginosa containing PHA 

granules. .......................................................................................................................... 14 

Figure 2. The biosynthetic pathway of PHB production. ............................................... 15 

Figure 3. PHB granules Self-assembly model. .............................................................. 16 

Chapter 2 

Figure 1. Schematic presentation of the construction of plasmid pET-14b-psaA-phaC 

encoding the PsaA-PhaC fusion protein for the formation of PHB beads in recombinant 

ClearColi. ........................................................................................................................ 44 

Figure 2. TEM analysis of recombinant ClearColi cells (pMCS69) harbouring various 

plasmids and respective isolated PHB beads. ................................................................. 45 

Figure 3. SDS-PAGE and immunoblot analysis of proteins attached to PHB beads and 

purified His6-PsaA. ......................................................................................................... 46 

Figure 4. Immunological assessment of PsaA display on the PHB bead surface. ......... 48 

Figure 5. Correlation between zeta potential and pH of various PHB beads. ................ 50 

Figure 6. Anti-PsaA IgG antibody response. ................................................................. 51 

Figure 7. Isotype IgG profile evaluated by direct ELISA using ELISA plates coated with 

0.5 µg of soluble His6-PsaA. .......................................................................................... 52 

Figure 8. Recognition of PsaA in various serotypes of S. pneumoniae by sera from mice 

immunized with PsaA displayed on PHB beads or soluble PsaA. .................................. 53 

Supplementary Figure 1. SDS-PAGE of whole cell lysates of the various S. pneumoniae 

serotypes. ......................................................................................................................... 63 

Chapter 3 

Figure 1. Schematic representation of genes encoding proteins relevant to this study. . 75 

Figure 2. TEM images of E. coli with PHB inclusions and the corresponding purified 

PHB beads. ...................................................................................................................... 77 

Figure 3. SDS-PAGE and immunoblot analysis of proteins attached to PHB beads as well 

as purified His6-Ply. ....................................................................................................... 78 

Figure 4. Analysis of induction of anti-Ply antibodies by various vaccine formulations.

 ......................................................................................................................................... 80 

Figure 5. IgG subclass profile as assessed by ELISA. ................................................... 81 

Figure 6. Cytokine profiles induced by various vaccine formulations. ......................... 82 



 

xv 
 

Figure 7. Cross-reactivity of anti-Ply antibodies with Ply from various serotypes of S. 

pneumoniae. .................................................................................................................... 84 

Figure 8. Schematic representation of conjugation reaction between activated serotype 

19F polysaccharide and PhaC on PHB beads or soluble TT. ......................................... 85 

Figure 9. Analysis of anti-19F CPS antibody titers. ...................................................... 86 

Figure. 10. The opsonophagocytic activity of sera against S. pneumoniae serotype 19F.

 ......................................................................................................................................... 87 

Supplementary Figure 1. SDS-PAGE of whole cell lysates of the various S. pneumoniae 

serotypes. ....................................................................................................................... 100 

Chapter 4 

Figure 1. Biological production and characterization of antigen coated PHB beads ... 115 

Figure 2. PHB bead production and their immunogenicity. ........................................ 117 

Figure 2. PHB bead production and their immunogenicity (continued). ..................... 118 

Figure 3. Schematic representation of the chemical conjugation of the CPS (MenC) to 

soluble and insoluble antigens displayed on PHB beads and characterization of their 

immunological properties.............................................................................................. 121 

Figure 4. Structural models of PhaC depicting lysine residues proposed as sites 

conjugated to the activated polysaccharide. .................................................................. 123 

Figure 5. Immunogenicity studies of the various antigens conjugated to MenC. ........ 125 

Figure 6. Bactericidal activity of various sera.............................................................. 128 

Supplementary Figure 1. FHbp confirmation of molecular identity and bead display by 

ELISA using a commercial monoclonal anti-fHbp antibody (JAR4, NIBCS, UK). .... 144 

Supplementary Figure 2. 1H NMR monodimensional spectra of CPS. ..................... 145 

Supplementary Figure 3. MenC confirmation of molecular identity by ELISA using a 

commercial anti-CPS (MenC) monoclonal antibody (NIBS, UK). .............................. 145 

Supplementary Figure 4. Assessment of IgG subclass binding to MenC evaluated by 

ELISA. .......................................................................................................................... 146 

Supplementary Figure 5. Assessment of IgG subclass binding to NadA protein 

evaluated by ELISA. ..................................................................................................... 147 

 

  



 

xvi 
 

List of Tables 

Chapter 1 

Table 1. Pneumococcal protein vaccines under study. ..................................................... 6 

Chapter 2 

Table 1. Description of bacterial strains, plasmids and oligonucleotides used in this 

study ................................................................................................................................ 38 

Table 2. Tryptic peptide fingerprinting analysis (MALDI-TOF/MS). ........................... 47 

Table 3. PHB bead yield and composition ..................................................................... 49 

Chapter 3 

Table 1. Characteristics of plasmids and oligonucleotides used in this study................ 76 

Table 2. Tryptic peptide fingerprinting analysis (Triple TOF) ...................................... 79 

Table 3. PHB beads yields and the antigen/ mg of wet beads ratio ............................... 79 

Chapter 4 

Supplementary Table 1. Strains, Plasmids and primers ............................................. 148 

Supplementary Table 2. Identification of fusion proteins by peptide fingerprinting 

analysis (MALDI-TOF/MS). ........................................................................................ 149 

Supplementary Table 3. Correlation between Zeta potential and pH of various PHB 

beads. ............................................................................................................................. 149 

Supplementary Table 4. Amount of neisserial antigen attached to PHB beads and 

immunization doses. ...................................................................................................... 150 

Supplementary Table 5. Carbohydrate/protein ratios and carbohydrate yield after 

conjugation and purification. ........................................................................................ 150 

Supplementary Table 6. Conjugation site analysis results by liquid chromatography-

coupled tandem mass spectrometry (LC-MS/MS). ....................................................... 151 

Supplementary Table 7. IgG/IgM ratio after first (1D) and third (3D) blood collection 

assayed against MenC ................................................................................................... 155 

Supplementary Table 8. Size distribution of PHB beads in vaccine formulations (µm) 

as measured by dynamic laser scattering. ..................................................................... 155 

 

  



 

xvii 
 

Abbreviations 

APCs: antigen presenting cells 

APS: activate polysaccharide 

BCA: Bicinchoninic acid assay 

CLSM: Confocal Laser Scanning Microscope 

CPS: capsular polysaccharide 

CFU: colony-forming unit 

CON A: Concanavalin A 

DC: dendritic cell 

DF: Dilution Factor 

DIC: Differential interference contrast 

DMEM: Dulbecco's Modified Eagle's Medium 

DT: Diphtheria toxoid 

ELISA: enzyme-linked immunosorbent assay  

FCS: fetal calf serum  

fHbp: factor H binding protein 

GNA2091: genome Neisseria antigen 2091 

GNA2091-fHbp-PhaC: genome Neisseria antigen 2091 fuse to factor H binding protein 

and PhaC 

HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 

Ig: Immunoglobulin 

IgG: Immunoglobulin G 

IgG1, IgG2a, IgG2b and IgG3: Immunoglobulin G 1,2a,2b,3 

IgM: Immunoglobulin M 

IL17A: cytokine 17A 

INF-γ: Interferon gamma 

LB: Luria-Bertani broth (Lennox) 

M. bovis: Mycobacterium bovis 

N. meningitidis: Neisseria meningitidis 

S. pneumoniae: Streptococcus pneumoniae 

E. coli: Escherichia coli 

MALDI-TOF-MS/MS: matrix assisted laser desorption ionization-time of flight mass 

spectrometry  

http://www.sigmaaldrich.com/life-science/cell-culture/classical-media-salts/dmem.html


 

xviii 
 

MW: molecular weight 

NaBH3CN: Sodium Cyanoborohydride  

NadA: Neisseria adhesin A 

NadA-PhaC: NadA-PhaC fusion protein 

NIBSC: National Institute for Biological Standards and Control 

OVA: Ovalbumin  

OPA: opsonophagocytic assay 

PBS: Phosphate Buffered Saline 

PCR: polymerase chain reactions 

PHA: Polyhydroxyalkanoate 

PhA: β- ketothiolase 

PhaC: Polyhydroxyalkanoate synthase 

PhB: Acetoacetyl-CoA reductase 

PHB: Polyhydroxybutyrate 

Ply: Pneumolysin 

Ply-PhaC: Pneumolysin fused to PhaC 

PsaA: Pneumococcal surface adhesin A 

PsA-PhaC: Pneumococcal Surface protein A fused to PhaC 

PspA: Pneumococcal Surface protein A 

PspC: Pneumococcal Surface protein C 

rpm: revolutions per minute 

SBA: serum bactericidal activity 

SD: standard deviation 

SDS-PAGE: sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) 

SEM: standard error of the mean 

TEM: Transmission electron microscopy. 

Th17: Lymphocyte T helper 17 

Triple TOF: mass spectrometry by Triple TOF 

TT: Tetanus toxoid 

TLR: Toll-like receptor 

0C: degrees Celsius 

1H NMR: Proton nuclear magnetic resonance 

UNICEF: United Nations Children's Fund 

WHO: World Health Organization