The protective and immunomodulatory properties of Bordetella pertussis adenylate cyclase toxin

MacDonald-Fyall, Julia (2002) The protective and immunomodulatory properties of Bordetella pertussis adenylate cyclase toxin. PhD thesis, University of Glasgow.

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Bordetella pertussis is the causative agent of the disease whooping cough in man, and although there is an effective whole-cell vaccine (WCV), recent efforts have focused on the development of the acellular vaccines (ACVs) to avoid the problems of reactogenicity associated with WCVs. The cell-invasive toxin, adenylate cyclase toxin (CyaA), is an important virulence factor of B. pertussis and, when used as a vaccine, is able to protect mice against intranasal challenge with B. pertussis. The project was designed to investigate the mouse immune responses to two forms of the toxin, an enzymically-active, acylated form (CyaA) and an enzymically-inactive, acylated form (CyaA*). These were expressed as recombinant forms in E. coli, purified and their enzymic, haemolytic and cytotoxic properties characterised. The adenylate cyclase (AC) and cytotoxic activities of CyaA* were low compared to CyaA, but the haemolytic activities of the two toxins were similar. This indicated that the toxic activity of CyaA is dependent on AC enzymic activity, but that haemolytic activity is independent of AC activity. Mouse responses to intraperitoneal immunisation with CyaA or CyaA* were examined, initially when each toxin was administered alone and later when administered in combination with protective antigens commonly used in ACV preparations (pertussis toxin (PT), filamentous haemagglutinin (FHA), and pertactin (P.69)). Both forms of the toxin when administered alone produced a strong serum IgG response which was boosted by a second vaccination. Anti-PT, -FHA and -P.69 IgG levels, in response to immunisation with PT, FHA and P.69 mixture in the presence of CyaA* were all raised compared to mice immunised with just the antigen mixture alone when measured at five weeks post-immunisation, however only anti-P.69 levels were raised by coadministration with CyaA. When detoxified PT (dPT) was co-administered alone with CyaA*, the anti-PTd levels decreased but if FHA or P.69 were also included in the antigen mixture, the anti-PTd levels increased in comparison to mice immunised with PTd alone. When either FHA or P.69 antigen was co-administered with CyaA*, both anti-FHA and anti-P.69 levels respectively were raised in comparison to those immunised with FHA or P.69 alone, again when measured at five weeks postimmunisation. Other work had shown that induction of cell-mediated immunity (CMI) was necessary for effective clearance of B. pertussis in the mouse. In this study, nitric oxide production by peritoneal macrophages was measured as a marker of macrophage activation. Macrophages collected from mice vaccinated with PT, FHA and P.69 together with CyaA or CyaA* responded more to stimulation with B. pertussis heat- killed cells or CyaA* than those taken from mice immunised with just the antigen mixture alone. Increased protection following aerosol challenge with B. pertussis was also observed when mice received a vaccination of CyaA* co-administered with PT, FHA and P.69 mixture. Taken together these results indicate that CyaA is a candidate antigen for inclusion in acellular vaccines, even in the detoxified CyaA* form. As well as being a protective antigen in its own right, it may also afford improved CMI responses than the current ACV formulations.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Coote
Keywords: Immunology
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-71965
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 13:37
Last Modified: 17 May 2019 13:37

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