Primary Supervisor: Professor Tim Mitchel, Institute of Microbiology and Infection
Secondary supervisor: Dr Hasan Yesilkaya (University of Leicester)
PhD project title: Development of new biological adjuvants and vaccines for prevention of bacterial infections
University of Registration: University of Birmingham
BACKGROUND: Immunization is a major way to prevent infectious diseases in man and other animals. In order to generate protective immunity it is important to generate the appropriate type of immunity (e.g. antibody vs cellular immunity). The structural features of microbial antigens play a fundamental role the way immune system responds. In this project bacterial protein toxins will be developed as adjuvants to develop appropriate immune responses in vaccines for protection against infectious diseases in man and animals. Preliminary data demonstrates that bacterial pore-forming toxins such as pneumolysin (from S.pneumoniae), suilysin (from S.suis) and others act as potent activators of the immune system. We have eliminated the toxicity of these proteins by deletion of key amino acids from the proteins that are crucial to the pore formation process (1). There are also natural non-toxic variants of pneumolysin in some pneumococcal strains and these will be cloned and expressed to allow evaluation of biological activity (2). We have shown that these engineered or natural toxoids stimulate cell signalling events that generate antibody and T-cell responses. We have also shown that the membrane-binding domain of the toxin (C-terminal domain) can be expressed separately and also acts as an adjuvant for proteins that are linked to it (3). Furthermore, we have identified several naturally occurring polymorphisms affecting the haemolytic activity of these pore forming toxins (2). An advantage of use of these genetically engineered and naturally occurring toxoids to modulate immune response is that they can be coupled with other proteins, which then provides impressive immune responses to the carried protein. Vaccines generated using this technology will then be used to assess protective efficacy against infectious challenges with multiple serotypes of the pneumococcus in animal models of infection.
OBJECTIVES: Generate and purify protein fusions of selected pathogen antigens with membrane binding but non-toxic versions of pore forming proteins (including pneumolysin and suilysin). Pneumolysin is chosen as a human pathogen associated and suilysin as a pig-associated pathogen. Further members of the large pore forming toxin family may also be investigated. The ability of the fusion vaccines to generate antibody and T-cell responses will be tested in animal models of infection with multiple strains of human and veterinary pathogens (using Streptococcus pneumoniae and Streptococcus suis as examples). The mechanisms by which the toxoids generate different types of immunity will be evaluated using cell typing (flow cytometry) and analysis of gene expression by single cell transcriptomic analysis of murine T-cells. In addition to assessing toxoids in their potency to induce host response, the student will also evaluate their impact on bacterial colonisation and virulence. The student will engineer S.pneumoniae strain D39 to express the altered versions of the toxin and evaluate the effects on ability to cause colonisation and disease using well established mouse models of pneumococcal pneumonia and colonisation (these experiments will be overseen by Yesilkaya at the University of Leicester) . For infection studies, the student will use our existing unmarked pneumococcal strains in type 2 D39 strain background each expressing different toxoids under the native ply promoter. In the colonisation model, the student will quantify the bacterial load over 4 weeks in nasopharynx, while in pneumonia model, the survival time and bacterial counts in blood at predetermined post infection times will be assessed. The in vivo analysis will be complemented with histopathological analysis of lung tissues and by measuring the levels of selected cytokines and chemokines in infected tissues.
METHODS: Standard molecular biology techniques will be used to construct the non-pore forming versions of the toxins and to express the single domain versions of the toxins. Protein-fusions will be constructed by genetic fusion of the test antigen with a toxoid carrier. The toxoid used will be a version of the toxin termed delta6 in which we have deleted 2 amino acids from the protein and this blocks the ability of the toxin to form pores. This toxoid has no toxicity to cells or animals. Green fluorescent protein (GFP) will be used as one of the fusion partners to allow visualisation of the protein in animal tissues and on cells. Fusion proteins will be expressed in standard bacterial expression systems and purified by standard affinity-tag purification methods. We have a well-established animal model of bacterial colonisation and infection and an established pipeline for the transcriptional analysis of T-cell responses.
- Kirkham L-AS, Kerr AR, Douce GR, Paterson GK, Dilts DA, Liu D-F, et al. Construction and Immunological Characterization of a Novel Nontoxic Protective Pneumolysin Mutant for Use in Future Pneumococcal Vaccines. Infect Immun. 2006;74(1):586-93.
- Kirkham L-AS, Jefferies JMC, Kerr AR, Jing Y, Clarke SC, Smith A, et al. Identification of Invasive Serotype 1 Pneumococcal Isolates That Express Nonhemolytic Pneumolysin. J Clin Microbiol. 2006;44(1):151-9.
- Pope C, Oliver EH, Ma J, Langton Hewer C, Mitchell TJ, Finn A. Genetic conjugation of components in two pneumococcal fusion protein vaccines enhances paediatric mucosal immune responses. Vaccine. 2015;33(14):1711-8.
BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology
Techniques that will be undertaken during the project:
- Molecular biology including gene cloning protein expression and purification
- Immune responses to vaccination. Characterisation of T-cell and antibody responses
- Microbial polymorphism analysis for selection of antigens
- Construction of bacterial gene replacement strains
- Animal models of infection
- Analysis of cellular responses to vaccine/toxoid at single cell level using transcriptomics
Contact: Professor Tim Mitchell, University of Birmingham