Name: a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)
Uploaded: 2011-09-14T13:00:00
Description: Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

This work was funded by personal fellowships from the Wellcome Trust to Beate Kampmann (056608, GRO77273).

1. Preparation of BCG lux reporter mycobacteria stock
<ol>
 <li>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.</li>
 <li>Prepare 1ml of a 1:10 dilution of BCG lux culture in sterile PBS in a luminometer tube (900&mu;l PBS + 100&mu;l BCG lux culture) in duplicate.</li>
 <li>Load tube containing N-decy...

<ol>
	<li>Cheng, S. H., Walker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+increased+anti-mycobacterial+activity+in+peripheral+blood+monocytes+after+BCG+vaccination+in+British+school+children.">Demonstration of increased anti-mycobacterial activity in peripheral blood monocytes after BCG vaccination in British school children.</a> Clin Exp Immunol. 74, 20-205 (1988).</li><li>Cheng, S. H., Walker, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+antimycobacterial+activity+before+and+after+Mycobacterium+bovis+BCG+vaccination+in+Chingleput,+India,+and+London,+United+Kingdom.">Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom.</a> Infect Immun. 61, 4501-4503 (1993).</li><li>Silver, R. F., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Infect Immun. 66, 1190-1199 (1998).</li><li>Hoft, D. F., Worku, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+relationships+between+immune-mediated+inhibition+of+mycobacterial+growth+and+other+potential+surrogate+markers+of+protective+Mycobacterium+tuberculosis+immunity.">Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity.</a> J Infect Dis. 186, 1448-1457 (2002).</li><li>Worku, S., Hoft, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+control+and+antigen-specific+T+cells+on+intracellular+mycobacterial+growth.">Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth.</a> Infect Immun. 71, 1763-1773 (2003).</li><li>Kampmann, B., Gaora, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+human+antimycobacterial+immunity+using+recombinant+reporter+mycobacteria.">Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria.</a> J Infect Dis. 182, 895-901 (2000).</li><li>Cheon, S. H., Kampmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bactericidal+activity+in+whole+blood+as+a+potential+surrogate+marker+of+immunity+after+vaccination+against+tuberculosis.">Bactericidal activity in whole blood as a potential surrogate marker of immunity after vaccination against tuberculosis.</a> Clin Diagn Lab Immunol. 9, 901-907 (2002).</li><li>Janulionis, E., Sofer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+activity+of+orally+administered+clofazimine+against+intracellular+Mycobacterium+tuberculosis+in+whole-blood+culture.">Lack of activity of orally administered clofazimine against intracellular Mycobacterium tuberculosis in whole-blood culture.</a> Antimicrob Agents Chemother. 48, 3133-3135 (2004).</li><li>Kampmann, B., Tena, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+human+in+vitro+system+for+evaluating+antimycobacterial+vaccines.">Novel human in vitro system for evaluating antimycobacterial vaccines.</a> Infect Immun. 72, 6401-6407 (2004).</li><li>Kampmann, B., Tena-Coki, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+antimycobacterial+immune+responses+in+HIV-infected+children+receiving+HAART.">Reconstitution of antimycobacterial immune responses in HIV-infected children receiving HAART.</a> Aids. 20, 1011-1018 .</li><li>Martineau, A. R., Wilkinson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+dose+of+vitamin+D+enhances+immunity+to+mycobacteria.">A single dose of vitamin D enhances immunity to mycobacteria.</a> Am J Respir Crit Care Med. 176, 208-213 (2007).</li></ol>

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....

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Company</td>
			<td>Cat No</td>
		</tr>
		<tr>
			<td>BCG lux</td>
			<td>Various</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>Middlebrook 7H11 agar</td>
			<td>Becton Dickinson</td>
			<td>283810</td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 broth </td>
			<td>BD Biosciences</td>
			<td>271310 (500g)</td>
		</tr>
		<tr>
			<td>Middlebrook ADC enrichment</td>
			<td>BD Biosciences</td>
			<td>212352</td>
		</tr>
		<tr>
			<td>Middlebrook OADC supplement</td>
			<td>Becton Dickinson</td>
			<td>212240</td>
		</tr>
		<tr>
			<td>Tween 80</td>
			<td>Sigma Aldrich</td>
			<td>274364</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5150</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Roche</td>
			<td>10843555001 (20ml)</td>
		</tr>
		<tr>
			<td>N-decyl aldehyde</td>
			<td>Sigma</td>
			<td>D7384-100G</td>
		</tr>
		<tr>
			<td>Ethanol (&gt;99.7%) </td>
			<td>VWR</td>
			<td>101077</td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>In House</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td>RPMI-1640 with L-Glutamine, 25mM HEPES</td>
			<td>Sigma</td>
			<td>R0883</td>
		</tr>
		<tr>
			<td>Sterile distilled Water</td>
			<td>In House</td>
			<td>n/a</td>
		</tr>
		<tr>
			<td colspan="3">Equipment</td>
		</tr>
		<tr>
			<td>Tube luminometer (injectable port mandatory)</td>
			<td>Berthold AutoLumat LB953 or single-tube luminometer Sirius</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminometer tubes, 12 x 75mm, pyrex rimless</td>
			<td>Corning from VWR</td>
			<td>99445-12</td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks with vented caps, sterile polycarbonate 250ml </td>
			<td>Corning via Fisher Scientific </td>
			<td>2150329</td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tubes (preservative-free heparin)</td>
			<td>BD</td>
			<td>367676</td>
		</tr>
		<tr>
			<td>50ml Falcon tubes, conical</td>
			<td>BD</td>
			<td>352077</td>
		</tr>
		<tr>
			<td>7ml Sterilin specimen polypropylene bijou tubes</td>
			<td>VWR</td>
			<td>99445-12</td>
		</tr>
		<tr>
			<td>Sterilin Universal containers (30 ml)</td>
			<td>Laboratory Analysis LTD</td>
			<td>128C</td>
		</tr>
		<tr>
			<td>Glass beads 2mm diameter </td>
			<td>SigmaAldrich</td>
			<td>Z143928</td>
		</tr>
		<tr>
			<td>10ml pipettes and pipette boy</td>
			<td>any</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1000&mu;l and 200&mu;l pipettes and filter tips</td>
			<td>any</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="3">General laboratory equipment:</td>
		</tr>
		<tr>	
			<td>Class II safety cabinet cabinet</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>	
			<td>Refrigerator (4&deg;C)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>	
			<td>Centrifuge</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>	
			<td>Rocking shaker platform (1800) to mix tubes</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>	
			<td>Orbital Shaker incubator set at 37&deg;C for growing up bacterial cultures</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged bcg or m.tb (bcg lux/m.tb lux)

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).

Watch this Scientific Journal Video about A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux at JoVE.com

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

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; 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.

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

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

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