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

Summary

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). “Survival” of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

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/Abbreviations

BCG 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).

Protocol

1. Preparation of BCG lux reporter mycobacteria stock

1. Grow BCG lux (M. bovis BCG Montreal strain transformed with the shuttle plasmid pSMT1) with shaking at 200rpm to mid-logarithmic phase in Middlebrook 7H9 broth containing 0.2% glycerol, 0.05% tween 80 and 10% ADC enrichment.
2. Prepare 1ml of a 1:10 dilution of BCG lux culture in sterile PBS in a luminometer tube (900μl PBS + 100μl BCG lux culture) in duplicate.
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....

Discussion

Bactericidal assays in the context of tuberculosis vaccine research are well established in 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.

The first generation of human bactericidal assays were designed using macrophages alone, infected with M. tuberculosis. CFU served as the main read-out of mycobacterial survival after a minimum time of 3 weeks........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCG lux	Various	n/a
Middlebrook 7H11 agar	BD Biosciences	283810
Middlebrook 7H9 broth	BD Biosciences	271310 (500g)
Middlebrook ADC enrichment	BD Biosciences	212352
Middlebrook OADC supplement	BD Biosciences	212240
Tween 80	Sigma-Aldrich	274364
Glycerol	Sigma-Aldrich	G5150
Hygromycin B	Roche Group	10843555001 (20ml)
N-decyl aldehyde	Sigma-Aldrich	D7384-100G
Ethanol (>99.7%)	VWR international	101077
Sterile PBS	In House	n/a
RPMI-1640 with L-Glutamine, 25mM HEPES	Sigma-Aldrich	R0883
Sterile distilled Water	In House	n/a
Tube luminometer (injectable port mandatory)	Berthold Technologies
Luminometer tubes, 12 x 75mm, pyrex rimless	Corning	99445-12
Erlenmeyer flasks with vented caps, sterile polycarbonate 250ml	Corning	2150329
BD Vacutainer Blood Collection Tubes (preservative-free heparin)	BD Biosciences	367676
50ml Falcon tubes, conical	BD Biosciences	352077
7ml Sterilin specimen polypropylene bijou tubes	VWR international	99445-12
Sterilin Universal containers (30 ml)	Laboratory Analysis LTD	128C
Glass beads 2mm diameter	Sigma-Aldrich	Z143928
10ml pipettes and pipette boy	Any Supplier
1000μl and 200μl pipettes and filter tips	Any Supplier
Class II safety cabinet cabinet
Refrigerator (4°C)
Centrifuge
Rocking shaker platform (1800) to mix tubes
Orbital Shaker incubator set at 37°C for growing up bacterial cultures