Функциональные Всего Пробирная крови для измерения жизнеспособности микобактерий, используя Репортер-Gene меткой БЦЖ или M. Tb (БЦЖ Люкс / М. Tb Люкс)
Summary
Мы опишем альтернативный подход к перечислению микобактерий<em> В пробирке</em>, Который использует репортер-гена меткой микобактерий вместо колониеобразующих единиц (КОЕ). "Выживание" организмов, а в качестве принимающей ответ-маркеров измеряются одновременно, обеспечивая недорогой, универсальный и функциональная система для исследования хост / патогена взаимодействия в контексте туберкулеза.
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
Discussion
Бактерицидное анализов в контексте исследований проблемы туберкулеза, вакцина хорошо известны на животных моделях, где они применяются для отбора потенциально перспективных вакцин-кандидатов. Сокращение бактериальной нагрузки в различных функциях органов в качестве основной для ч?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Эта работа финансировалась личным стипендии из Wellcome Trust, чтобы Беате Kampmann (056608, GRO77273).
Materials
| Reagent | Company | Cat No |
| BCG lux | Various | n/a |
| Middlebrook 7H11 agar | Becton Dickinson | 283810 |
| Middlebrook 7H9 broth | BD Biosciences | 271310 (500g) |
| Middlebrook ADC enrichment | BD Biosciences | 212352 |
| Middlebrook OADC supplement | Becton Dickinson | 212240 |
| Tween 80 | Sigma Aldrich | 274364 |
| Glycerol | Sigma Aldrich | G5150 |
| Hygromycin B | Roche | 10843555001 (20ml) |
| N-decyl aldehyde | Sigma | D7384-100G |
| Ethanol (>99.7%) | VWR | 101077 |
| Sterile PBS | In House | n/a |
| RPMI-1640 with L-Glutamine, 25mM HEPES | Sigma | R0883 |
| Sterile distilled Water | In House | n/a |
| Equipment | ||
| Tube luminometer (injectable port mandatory) | Berthold AutoLumat LB953 or single-tube luminometer Sirius | |
| Luminometer tubes, 12 x 75mm, pyrex rimless | Corning from VWR | 99445-12 |
| Erlenmeyer flasks with vented caps, sterile polycarbonate 250ml | Corning via Fisher Scientific | 2150329 |
| BD Vacutainer Blood Collection Tubes (preservative-free heparin) | BD | 367676 |
| 50ml Falcon tubes, conical | BD | 352077 |
| 7ml Sterilin specimen polypropylene bijou tubes | VWR | 99445-12 |
| Sterilin Universal containers (30 ml) | Laboratory Analysis LTD | 128C |
| Glass beads 2mm diameter | SigmaAldrich | Z143928 |
| 10ml pipettes and pipette boy | any | |
| 1000μl and 200μl pipettes and filter tips | any | |
| General laboratory equipment: | ||
| 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 | ||
References
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