Article

Recognition of mycobacterial antigens delivered by genetically detoxified Bordetella pertussis adenylate cyclase by T cells from cattle with bovine tuberculosis.

TB Research Group, Veterinary Laboratory Agency, Weybridge, Woodham Lane, New Haw, Addlestone KT15 3NB, UK.
Infection and Immunity (Impact Factor: 4.16). 12/2004; 72(11):6255-61. DOI: 10.1128/IAI.72.11.6255-6261.2004
Source: PubMed

ABSTRACT The exponential increase in the incidence of tuberculosis in cattle over the last two decades in the British national herd constitutes a significant economic problem. Therefore, research efforts are under way to develop cattle tuberculosis vaccines and specific diagnostic reagents to allow the distinction of vaccinated from infected animals. Mycobacterial antigens like ESAT-6 and CFP10 allow this distinction. This study investigates whether fusion protein of ESAT-6 or CFP10 with genetically detoxified Bordetella pertussis adenylate cyclase (CyaA) are recognized by Mycobacterium bovis-infected cattle more effectively than conventional recombinant proteins are, thus enhancing sensitivity or reducing the amount of antigens required. By measuring the frequencies of gamma interferon (IFN-gamma)-producing cells, we were able to show that the presentation of CFP10 as a CyaA fusion protein enhanced the molecular efficiency of its recognition 20-fold, while the recognition of ESAT-6 was not improved by CyaA delivery. Furthermore, in the whole-blood IFN-gamma test currently used in the field, the delivery of CFP10 and ESAT-6 by fusion to CyaA increased the amount of IFN-gamma produced and hence the proportion of infected animals responding to CFP10. The improved T-cell recognition of CyaA336/CFP10 was found to be dependent upon interaction with CD11b. In addition, presentation of CyaA336/CFP10 to CD4+ T cells was chloroquine sensitive, and CFP10 delivery by CyaA resulted in its accelerated presentation to T cells. In conclusion, the use of CyaA fusion proteins with ESAT-6 and CFP10 has the potential to improve the sensitivity of immunodiagnostic tests detecting bovine tuberculosis in cattle.

0 Bookmarks
 · 
51 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The adenylate cyclase toxin-hemolysin (ACT, AC-Hly or CyaA) is a key virulence factor of Bordetella pertussis. It targets bactericidal activities of phagocytes, such as oxidative burst and complement- or antibody-mediated opsonophagocytic killing of bacteria. Through cAMP signaling, CyaA also skews TLR-triggered maturation of dendritic cells, inhibiting proinflammatory IL-12 and TNF-α secretion and enhancing IL-10 production and Treg expansion, likely hampering induction of adaptive immune responses to Bordetella infections. Non-enzymatic CyaA toxoid is a potent protective antigen and adjuvant that boosts immunogenicity of co-administered B. pertussis antigens and improves potency of acellular pertussis (aP) vaccines in mice. This makes CyaA a prime antigen candidate for inclusion into a next generation of aP vaccines. Moreover, recombinant CyaA toxoids were recently shown to be safe in humans in frame of Phase I clinical evaluation of a CyaA-based immunotherapeutic vaccine that induces Th1-polarized CD8(+) cytotoxic T-lymphocyte responses targeting cervical tumors.
    Expert Review of Vaccines 08/2014; 13(10):1-13. DOI:10.1586/14760584.2014.944900 · 4.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium tuberculosis is a significant threat to global health. Mycobacterium bovis BCG vaccine provides only partial protection, and the skin test reagent used to aid diagnosis of both active and latent tuberculosis, purified protein derivative (PPD), lacks specificity and sensitivity. The use of genetically detoxified Bordetella pertussis adenylate cyclase toxin (CyaA) as a delivery system for two immunodominant proteins of M. tuberculosis that are of greater specificity than PPD, early-secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10), was therefore investigated. CyaA toxoids incorporating these antigens were able to restimulate T cells from more than 91% tuberculosis patients and healthy sensitized donors. Delivery of antigen by CyaA decreased by 10-fold the amount of ESAT-6 and CFP-10 required to restimulate T cells, and in low responders, the overall frequency of gamma interferon-producing cells detected by enzyme-linked immunospot assay was increased (P < 0.01 for both antigens). Delivery of ESAT-6 and CFP-10 by CyaA enabled the detection of both CD4(+) and CD8(+) T cells: these responses could be blocked by inhibition of major histocompatibility complex class II or class I, respectively. Covalent linkage of antigen to the CyaA vector was required for enhancement to occur, as a mixture of mock CyaA toxoid plus recombinant ESAT-6 did not lead to enhancement. In a simplified whole-blood model to detect tuberculosis infection, the frequency of positive responses to CFP-10 was increased by CyaA delivery, a potentially important attribute that could facilitate the identification of latent infection.
    Infection and Immunity 05/2005; 73(5):2991-8. DOI:10.1128/IAI.73.5.2991-2998.2005 · 4.16 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: CyaA is crucial for colonization by Bordetella pertussis, the etiologic agent of whooping cough. Here we report crystal structures of the adenylyl cyclase domain (ACD) of CyaA with the C-terminal domain of calmodulin. Four discrete regions of CyaA bind calcium-loaded calmodulin with a large buried contact surface. Of those, a tryptophan residue (W242) at an alpha-helix of CyaA makes extensive contacts with the calcium-induced, hydrophobic pocket of calmodulin. Mutagenic analyses show that all four regions of CyaA contribute to calmodulin binding and the calmodulin-induced conformational change of CyaA is crucial for catalytic activation. A crystal structure of CyaA-calmodulin with adefovir diphosphate, the metabolite of an approved antiviral drug, reveals the location of catalytic site of CyaA and how adefovir diphosphate tightly binds CyaA. The ACD of CyaA shares a similar structure and mechanism of activation with anthrax edema factor (EF). However, the interactions of CyaA with calmodulin completely diverge from those of EF. This provides molecular details of how two structurally homologous bacterial toxins evolved divergently to bind calmodulin, an evolutionarily conserved calcium sensor.
    The EMBO Journal 10/2005; 24(18):3190-201. DOI:10.1038/sj.emboj.7600800 · 10.75 Impact Factor

Full-text (2 Sources)

Download
2 Downloads
Available from
Jun 10, 2014