概要

Функциональные Всего Пробирная крови для измерения жизнеспособности микобактерий, используя Репортер-Gene меткой БЦЖ или M. Tb (БЦЖ Люкс / М. Tb Люкс)

Published: September 14, 2011
doi:

概要

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

1. Подготовка БЦЖ люкс репортер микобактерий акции Расти БЦЖ люкс (М. Bovis БЦЖ штамма Монреаль трансформированных плазмидой трансфер pSMT1) при встряхивании на 200rpm до середины логарифмической фазы в Миддлбрук 7H9 бульона, содержащего 0,2% глицерина, 0,05% между 80 и 10% АЦП обогащени…

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?…

開示

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    

参考文献

  1. Cheng, S. H., Walker, L. Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children. Clin Exp Immunol. 74, 20-205 (1988).
  2. Cheng, S. H., Walker, K. B. Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom. Infect Immun. 61, 4501-4503 (1993).
  3. Silver, R. F., Li, Q. Expression of virulence of Mycobacterium tuberculosis within human monocytes: virulence correlates with intracellular growth and induction of tumor necrosis factor alpha but not with evasion of lymphocyte-dependent monocyte effector functions. Infect Immun. 66, 1190-1199 (1998).
  4. Hoft, D. F., Worku, S. Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J Infect Dis. 186, 1448-1457 (2002).
  5. Worku, S., Hoft, D. F. Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth. Infect Immun. 71, 1763-1773 (2003).
  6. Kampmann, B., Gaora, P. O. Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria. J Infect Dis. 182, 895-901 (2000).
  7. Cheon, S. H., Kampmann, B. Bactericidal activity in whole blood as a potential surrogate marker of immunity after vaccination against tuberculosis. Clin Diagn Lab Immunol. 9, 901-907 (2002).
  8. Janulionis, E., Sofer, C. Lack of activity of orally administered clofazimine against intracellular Mycobacterium tuberculosis in whole-blood culture. Antimicrob Agents Chemother. 48, 3133-3135 (2004).
  9. Kampmann, B., Tena, G. N. Novel human in vitro system for evaluating antimycobacterial vaccines. Infect Immun. 72, 6401-6407 (2004).
  10. Kampmann, B., Tena-Coki, G. N. Reconstitution of antimycobacterial immune responses in HIV-infected children receiving HAART. Aids. 20, 1011-1018 .
  11. Martineau, A. R., Wilkinson, R. J. A single dose of vitamin D enhances immunity to mycobacteria. Am J Respir Crit Care Med. 176, 208-213 (2007).

Play Video

記事を引用
Newton, S., Martineau, A., Kampmann, B. A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux). J. Vis. Exp. (55), e3332, doi:10.3791/3332 (2011).

View Video