Optimisation of a functional mycobacterial growth-inhibition assay to improve its suitability for infant TB vaccine studies

Department of Pediatrics, St Mary's Campus, Imperial College London, Norfolk Place, London W2 1PG, UK. Electronic address: .
Journal of immunological methods (Impact Factor: 1.82). 05/2013; 394(1-2). DOI: 10.1016/j.jim.2013.05.006
Source: PubMed


The development of vaccines against tuberculosis continues to be hindered by the lack of correlates of protection. Immunity to Mycobacterium tuberculosis (M.tb) infection relies predominantly on cell mediated response, which are routinely measured using a read-out of host cytokine profiles, However, to date none of the cytokine profiles have been found to predict protection. A number of functional in vitro approaches have been developed that measure growth of mycobacteria pre and post vaccination as a potential functional surrogate marker for vaccine take. The use of a reporter-gene tagged BCG-lux assay measuring the viability of mycobacteria in whole blood samples has previously been described by our group to assess vaccine immunogenicity. Since only very small blood samples are usually available in paediatric studies, we now report a modification of the BCG-lux assay to reduce the volume required and make it more field-friendly. Our results show that a 2-fold reduction in blood volume made no significant difference to bacterial growth ratios, used as the main read-out. These results confirm the suitability of the BCG-lux assay for functional studies of vaccine immunogenicity and immunopathogenesis in young children and could play a role in late-phase TB vaccine trials of novel candidates.

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    • "This has been facilitated by the development of genetically modified luminescent mycobacteria that allow rapid and accurate mycobacterial quantification, overcoming their slow growth on solid media and inaccuracies caused by mycobacteria clumping [21,22]. A whole-blood model of the extent to which blood cells support or restrict the growth of luminescent mycobacteria has been used effectively for investigating aspects of human antimycobacterial cellular immunity [23]. These include correlation with cytokine production and tuberculin skin test status [24], evaluation of vaccine candidates [14,25] and of nutritional augmentation of antimycobacterial immunity [26]. "
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    ABSTRACT: Tuberculosis infection, disease and mortality are all less common at high than low altitude and ascent to high altitude was historically recommended for treatment. The immunological and mycobacterial mechanisms underlying the association between altitude and tuberculosis are unclear. We studied the effects of altitude on mycobacteria and antimycobacterial immunity. Antimycobacterial immunity was assayed in 15 healthy adults residing at low altitude before and after they ascended to 3400 meters; and in 47 long-term high-altitude residents. Antimycobacterial immunity was assessed as the extent to which participants' whole blood supported or restricted growth of genetically modified luminescent Bacille Calmette-Guérin (BCG) mycobacteria during 96 hours incubation. We developed a simplified whole blood assay that could be used by a technician in a low-technology setting. We used this to compare mycobacterial growth in participants' whole blood versus positive-control culture broth and versus negative-control plasma. Measurements of mycobacterial luminescence predicted the number of mycobacterial colonies cultured six weeks later. At low altitude, mycobacteria grew in blood at similar rates to positive-control culture broth whereas ascent to high altitude was associated with restriction (p≤0.002) of mycobacterial growth to be 4-times less than in culture broth. At low altitude, mycobacteria grew in blood 25-times more than negative-control plasma whereas ascent to high altitude was associated with restriction (p≤0.01) of mycobacterial growth to be only 6-times more than in plasma. There was no evidence of differences in antimycobacterial immunity at high altitude between people who had recently ascended to high altitude versus long-term high-altitude residents. An assay of luminescent mycobacterial growth in whole blood was adapted and found to be feasible in low-resource settings. This demonstrated that ascent to or residence at high altitude was associated with decreased mycobacterial growth in whole blood relative to controls, consistent with altitude-related augmentation of antimycobacterial cellular immunity.
    PLoS ONE 09/2013; 8(9):e74220. DOI:10.1371/journal.pone.0074220 · 3.23 Impact Factor
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    ABSTRACT: Tuberculosis (TB) is a premier example of a disease complex with pathogens primarily affecting humans (i.e., Mycobacterium tuberculosis) or livestock and wildlife (i.e., Mycobacterium bovis) and with a long history of inclusive collaborations between physicians and veterinarians. Advances in the study of bovine TB have been applied to human TB, and vice versa. For instance, landmark discoveries on the use of Koch's tuberculin and interferon-γ release assays for diagnostic purposes, as well as Calmette and Guérin's attenuated M. bovis strain as a vaccine, were first evaluated in cattle for control of bovine TB prior to wide-scale use in humans. Likewise, recent discoveries on the role of effector/memory T cell subsets and polyfunctional T cells in the immune response to human TB, particularly as related to vaccine efficacy, have paved the way for similar studies in cattle. Over the past 15 years, substantial funding for development of human TB vaccines has led to the emergence of multiple promising candidates now in human clinical trials. Several of these vaccines are being tested for immunogenicity and efficacy in cattle. Also, the development of population-based vaccination strategies for control of M. bovis infection in wildlife reservoirs will undoubtedly have an impact on our understanding of herd immunity with relevance to the control of both bovine and human TB in regions of the world with high prevalence of TB. Thus, the one-health approach to research on TB is mutually beneficial for our understanding and control of TB in humans, livestock, and wildlife. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
    ILAR journal / National Research Council, Institute of Laboratory Animal Resources 05/2015; 56(1):26-43. DOI:10.1093/ilar/ilv001 · 2.39 Impact Factor
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    ABSTRACT: The testing of vaccines for tuberculosis is costly and time-consuming, and dependent on preclinical animal challenge models and clinical trials. We have recently developed a mycobacterial growth inhibition assay (MGIA) to test vaccine efficacy ex vivo. This assay measures the summative effect of the host immune response and may serve as a novel tool to facilitate vaccine testing. It has generated much interest recently, and to facilitate technology transfer and reproducibility between laboratories, we here describe a detailed protocol for an ex vivo MGIA in mouse splenocytes.

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