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-decyl aldehyde (luciferase enzyme substrate) onto the back of the machine and ensure the lid is secured and tubes in place.</li>
 <li>Prime luminometer injector with substrate via the prime program, set to inject 100 ucl x 3 into each of 3 empty priming tubes placed in the luminometer.</li>
 <li>Load the 2 tubes into the luminometer and take readings via a program set at injecting 100 ucl of substrate into each tube and read for 20 sec in 1 sec intervals.</li>
 <li>When the stock has reached 1x108 RLU/ml to 2x108 RLU/ml (this takes about 3-4 days of growth), add an equal volume of sterile 30% glycerol to the culture in a 50ml falcon tube and gently mix.</li>
 <li>Aliquot 1.5ml volumes into labeled 2ml screw-cap microtube and store at -80&deg;C.</li>
</ol>
2. Determining stock RLU/CFU correlation and contents of aliquots
<ol>
 <li>Add 15ml of Middlebrook 7H9 culture medium with 10% ADC supplement to a 200ml Erlenmeyer flask with a vented cap.</li>
 <li>Add 15&mu;l of hygromycin and 30&mu;l of 20% Tween.</li>
 <li>Remove a vial of BCG lux from the freezer and defrost at room temperature (RT) in the safety cabinet. Add the contents of the vial to the medium and tighten the cap.</li>
 <li>Incubate with shaking at 200rpm at 37&deg;C for 4 days.</li>
 <li>On each day, make up serial 10-fold dilutions (1:10, 1:100, 1:1000, 1:10,000 and 1:100,000) for CFU determination.</li>
 <li>Set up the same dilutions in parallel and duplicate to determine luminescence (see part 1.2-1.6).</li>
 <li>Prepare two 3-compartment plates of Middlebrook 7H11 agar (1 l of liquid medium containing 0.5% glycerol, 10% OADC supplement, 1 ml of 20% Tween and 1 ml of hygromycin).</li>
 <li>Plate out 100&mu;l of each dilution onto a segment of each plate using separate spreaders, distributing the liquid equally across the individual chamber.</li>
 <li>Seal each plate with parafilm, place in a sterile plastic bag, seal with autoclave tape, and incubate at 37&deg;C for 2 weeks with lids facing down.</li>
 <li>Inspect regularly until CFUs appear (2-3 weeks).</li>
 <li>To count colonies, remove the plates from the incubator and place on a colony counter. Calculate the mean for each dilution from the duplicate plates.</li>
 <li>Calculate the RLU/CFU ratio using equivalent RLU and CFU counts for each dilution. The ratio should be between 3 and 5 RLU/CFU.</li>
</ol>
3. Preparation of BCG lux culture for inoculation into whole blood
<ol>
 <li>Add 15ml of Middlebrook 7H9 culture medium with 10% ADC supplement to a 200ml Erlenmeyer flask with a vented cap.</li>
 <li>Add 15&mu;l of hygromycin and 30&mu;l of 20% Tween.</li>
 <li>Remove a vial of BCG lux from the freezer and defrost at RT in the safety cabinet. Add the contents of the vial to the medium and tighten the cap.</li>
 <li>Incubate with shaking at 200rpm at 37&deg;C for 2 to 4 days.</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>Prime luminometer with substrate (as described above), gently vortex the 2 tubes and load into the luminometer and take readings (as described above).</li>
 <li>Use this reading to dilute down the culture in PBS to give an equivalent of 7x106 RLU (this will give an inoculum of about 2x105 CFU/ml blood and a ratio of BCG to monocytes of about 1:1, assuming a monocyte count of about 2x105 to 4x105 monocytes per ml of blood).</li>
</ol>
4. Preparation of whole blood
<ol>
 <li>Take 3 to 5ml of venous blood in a tube containing preservative-free heparin.</li>
 <li>Transfer the blood to a 50ml falcon tube and dilute with an equal volume of RPMI 1640 containing glutamine and 25mM HEPES (no pen/strep).</li>
 <li>Aliquot 900&mu;l of diluted blood in triplicate into sterile bijou tubes for each time point (6 tubes in total: 3 for 0hrs and 3 for 96hrs. Set up additional tubes for additional timepoints, if necessary.</li>
 <li>Aliquot 900&mu;l of Middlebrooks 7H9 culture medium with 10% ADC supplement + 50&mu;g/ml hygromycin (the same medium as used to culture the BCG lux) in duplicate for the growth controls.</li>
 <li>Add 100&mu;l of the diluted BCG lux to each tube of blood, and to the 2 growth control tubes.</li>
 <li>Mix well and place the 3 bijous for the 96hr time-point and the 2 growth controls on their side in the rocking incubator at 37&deg;C, 20 rev/min (CO2 not required).</li>
</ol>
5. Measuring luminescence (at 0hr and 96hrs)
<ol>
 <li>Centrifuge the 3 bijous at 2000g for 10 min.</li>
 <li>Carefully remove 300&mu;l of supernatant without disturbing the pellet and place in 2ml screw-cap microtubes to freeze at -80&deg;C for subsequent cytokine analysis.</li>
 <li>Add 300&mu;l of PBS to each bijou to replace the volume of supernatant removed.</li>
 <li>Aspirate the contents of each bijou into the corresponding 50ml falcon tube and add 8ml distilled water to each tube. Begin a timer for 10 min. 
 Nb: This is a time-sensitive step, and this incubation must not be any longer than 10 min, beginning from when the cells first come into contact with water. </li>
 <li>Rinse each bijou with 2ml of distilled water and vortex for 5 seconds before tipping into the corresponding falcon tube.</li>
 <li>At the end of the 10 minute incubation, centrifuge the falcon tubes at 2000g for 10 minutes.</li>
 <li>Decant supernatant into Surfanios disinfectant.</li>
 <li>Add a few glass beads to each pellet and vortex.</li>
 <li>Add 1ml of sterile PBS to each tube and vortex.</li>
 <li>In luminometer tubes, make 1:10 dilutions for each sample in duplicate with PBS (900&mu;l PBS + 100&mu;l sample) and at the 96hr time-point. Make the same dilutions for each of the growth controls.</li>
 <li>Gently vortex the tubes and load into the luminometer and take readings (as described above) 
 Nb: Readings between triplicates of the same sample should be within 15% of one another.</li>
</ol>
6. Representative results:
It is important to use bacteria in logarithmic phase of growth for the whole blood lux assays, i.e. grown over 48-72 hours prior to inoculating the samples, as metabolic activity is suboptimal if either straight out of the freezer or in stationary phase. The prior growth should be standardized for a series of experiments to either 48 or 72 hours to avoid variability. Figure 1 shows the growth curve of a representative culture of BCG lux. The doubling time is about 24 hours until stationary phase is reached.
<img alt="Figure 1" src="/files/ftp_upload/3332/3332fig1.jpg" /> 
Figure 1. Representative growth curve of BCG lux over 96 hours in 7H9 medium and correlation of RLU and CFU.
Although RLU values always correlate with CFU, the number of RLU corresponding to a single CFU can vary depending on the stock, which is why it is good practice to use the same frozen stock throughout a series of experiments to guarantee a consistent multiplicity of infection (MOI).
Table 1 shows an example of raw date at time of inoculation (T0) and at 96 hours. The growth ratios are calculated using the formula T96/T0, but other time intervals can of course also be measured. It is, however, advisable not to use the cultures beyond 96 hours, as significant cell death occurs.
<img alt="Table 1" src="/files/ftp_upload/3332/3332table1.jpg" /> 
Table 1. Example of raw data at time of inoculation (T0) and at 96 hours (T96) and calculated growth ratios from 3 adult donors.
Depending on the available antigen-specific memory responses and possibly also neutrophil count, growth ratios vary among individuals, as shown here in Figure 2 in the blood of children with and without HIV-infection. On average, young children have higher growth ratios than adults, Tuberculin skin test (TST) +ve individuals have lower growth ratios than TST-ve individuals, and HIV-infected patients have high growth ratios due to the deficiency of CD4 T cell population, one of the key mediators of cellular immune responses to mycobacteria.
<img alt="Figure 2" src="/files/ftp_upload/3332/3332fig2.jpg" /> 
Figure 2. Inter-donor variability: Growth ratios of T0 versus 96 hours for a set of patients, depending on underlying HIV status.
Reproducibility of growth ratios over a period of time is shown in Figure 3, which summarises results from 64 donors bled twice over a period of 12 months and from a single donor bled repeatedly for control experiments. Potential causes of variability could be changes in mycobacterial sensitization or variability within levels of host cytokines, as observed in many bioassays.
<img alt="Figure 3" src="/files/ftp_upload/3332/3332fig3.jpg" /> 
Figure 3. (a) Single donor on 12 occasions over 12 months. (b) multiple (64) donors on 2 occasions over 12 months.

<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>

No conflicts of interest declared.

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 ...

a-functional-whole-blood-assay-to-measure-viability-mycobacteria

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JoVE Journal

Immunology and Infection

13.8K visualizações.  Imperial College London. Descrevemos uma abordagem alternativa para a enumeração de micobactérias In vitro, Que usa repórter gene-tagged micobactérias em vez de unidades formadoras de colônia (UFC). "Survival" de organismos, bem como anfitrião-resposta marcadores são medidos simultaneamente, proporcionando um sistema de baixo custo, versátil e funcional para os estudos de host / patógeno interações no contexto da tuberculose.

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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for protection against children&#39;s severe TB meningitis and some forms of disseminated TB, but fails to protect against pulmonary TB, which is the most prevalent form of the disease. Promising strategies to improve BCG currently rely either on its transformation with genes encoding immunodominant M. tuberculosis (Mtb)-specific antigens and/or complementation with genes encoding co-factors that would stimulate antigen presenting cells. Major limitations to these approaches include low efficiency, low stability, and the uncertain level of safety of expression vectors. In this study, we present an alternative approach to vaccine improvement, which consists of BCG complementation with exogenous proteins of interest on the surface of bacteria, rather than transformation with plasmids encoding corresponding genes. First, proteins of interest are expressed in fusion with monomeric avidin in standard E. coli expression systems and then used to decorate the surface of biotinylated BCG. Animal experiments using BCG surface decorated with surrogate ovalbumin antigen demonstrate that the modified bacterium is fully immunogenic and capable of inducing specific T cell responses. Altogether, the data presented here strongly support a novel and efficient method for reshaping the current BCG vaccine that replaces the laborious conventional approach of complementation with exogenous nucleic acids.

Expression of Exogenous Antigens in the Mycobacterium bovis BCG Vaccine via Non-genetic Surface Decoration with the Avidin-biotin System

A novel technique for rapid antigen display on a bacterial surface is presented, which involves surface biotinylation followed by exposure to proteins of interest in fusion with monomeric avidin. Loading BCG with selected antigens successfully improves its immunogenicity, suggesting that surface decoration can replace traditional genetic approaches.

Tuberculosis (TB) is a serious infectious disease and the only available vaccine M. bovis bacillus Calmette-Gu&#233;rin (BCG) is safe and effective for ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...