A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG <em>lux</em>/M.Tb <em>lux</em>)

Abstract

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model. Note: Definitions/AbbreviationsBCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. CFU = Colony Forming Unit (a measure of mycobacterial viability). Video LinkThe video component of this article can be found at http://www.jove.com/details.php?id=3332 3. Load tube containing N-decyl aldehyde (luciferase enzyme substrate) onto the back of the machine and ensure the lid is secured and tubes in place. 4. Prime luminometer injector with substrate via the prime program, set to inject 100 ucl x 3 into each of 3 emp...

Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex

“…In this study we set out to evaluate the use of G. mellonella as an infection model for M. bovis BCG lux , a luminescent derivative of the vaccine strain that has the advantages of faster measurement of mycobacterial viability (luminescence requires co-factors only found in living metabolically active cells) compared to the slow growing M. tuberculosis with a doubling time of approximately 24 h [21,22,24]. Additionally, M. bovis BCG lux requires containment level (CL) 2, rather than CL3 laboratories.…”

“…Luminescence measurement has been shown to be a measure of mycobacterial metabolic activity, related to viability [21,22,24,33]. We also isolated and enumerated mycobacterial CFU from infected G. mellonella at the same respective time-points as luminescence measurements up to 168 h to determine the correlation between RLU and CFU in the G. mellonella model.…”

“…M. bovis BCG lux luminescence was measured after addition of decanal (Sigma) substrate (1% v/v in ethanol), as relative light units (RLU)/ml in a luminometer (Berthold Technologies) and correlated with CFU/ml counts. A ratio of 3 RLU correlated to 1 CFU in liquid broth culture as previously described [22]. …”

“…Prior to infection experiments, a vial of mycobacteria was defrosted and grown in liquid medium (as described above) to mid-log phase for 72 h with M. bovis BCG lux growth monitored by luminescent measurements on a daily basis [22]. Cultures were centrifuged at 3880 g for 10 minutes, supernatant decanted and the cell pellet was resuspended in phosphate buffered saline (PBS) (Sigma-Aldrich) containing 0.05% (v\v) Tween 80 (PBS-Tween 80) and washed a further twice.…”

“…Due to safety and practical considerations when using M. tuberculosis as an experimental organism, we have carried out this study using the non-pathogenic, luminescent reporter vaccine strain, M. bovis BCG lux [21], which has been well characterized and validated in a wide range of in vitro and in vivo models and clinical studies (e.g. liquid broth, human whole blood and cells, mouse) and has the advantage of faster measurement of mycobacterial viability [22–32]. Over a time-course we have assessed the survival of G. mellonella larvae in response to infection with different inocula of M. bovis BCG lux , and monitored the survival of M. bovis BCG lux by measuring bioluminescence and correlating with CFU.…”