Direct PBMC Mycobacterial Growth Inhibition Assay

Direct PBMC Mycobacterial Growth Inhibition Assay

Table of Contents

1 Purpose...... 2

2 Introduction...... 2

3 Scope...... 2

4 Definitions/abbreviations...... 2

5 Responsibilities...... 3

6 Procedure...... 3

6.1 Reagents...... 3

6.2 Equipment...... 3

6.3 Assay protocol...... 4

6.4 Data processing and reporting...... 9

7 References...... 10

8 Revision History...... 10

1 Purpose

The direct PBMC MGIA is a functional in vitro assay which aims to offer an unbiased measure of the host immune response, taking into account the ability of this response to function in its respective immune environment.

2 Introduction

The development of an effective TB vaccine is hampered by the lack of a validated immune correlate of protection. One alternative to measuring predefined individual parameters (such as antigen-specific IFN-γ ELISpot responses) is the use of MGIAs, which take into account a range of immune mechanisms and their complex interactions. Several MGIAs have been described in the literature [1]; most recently a considerable effort has been applied to developing the direct MGIA, in which whole blood, PBMC or mouse splenocytes are inoculated with mycobacteria at a low MOI and cultured for 96 hours, after which time cells are lysed and the lysate inoculated into Bactec MGITs [2]. It has been demonstrated that the whole blood direct MGIA may be confounded by haemoglobin, supporting the use of PBMC [3]. The use of cryopreserved cells removes the batch effects associated with running the assay in real-time across multiple time-points, and could aid in transferability of the assay to different trial sites. Cells can be stored and analysed in the future, which is not only logistically simpler but negates the need for a Bactec MGIT system at every site [2]. The direct PBMC MGIA has been used to demonstrate an effect of primary BCG vaccination in healthy UK adults [4]. The MGIA also offers a tractable system for exploring the immune mechanisms underlying control of mycobacterial growth.

3 Scope

This protocol outlines the current procedure for performing the direct PBMC MGIA which follows optimisation experiments conducted as part of the EURIPRED consortium.

4 Definitions/abbreviations

CV Coefficient of Variation

BCG Bacillus Calmette-Guerin

CFU Colony Forming Unit

CO2 Carbon Dioxide

ELISpot Enzyme-Linked ImmunoSpot

FBSFoetal Bovine Serum

IFNInterferon

MGIAMycobacterial Growth Inhibition Assay MGITMycobacteria Growth Indicator Tube MOI Multiplicity of Infection

PANTAAntibiotic Mix

PBMCPeripheral Blood Mononuclear Cell PEN/STREP Penicillin/Streptomycin

PHSPooled Human AB Serum

PBSPhosphate-BufferedSaline

TTDTime to Detection

UKUnited Kingdom

5 Responsibilities

All staff performing the direct PBMC MGIA must be trained in tissue culture sterile technique, handling of mycobacteria and preferably receive training specific to this protocol.

6 Procedure

6.1 Reagents

R10 = Sigma RPMI-1610 Medium (Cat. R0883). Add 5ml of L-glutamine (200mM 100X), 5ml of Sodium Pyruvate (Gibco, 11360-039) and 50ml (10%) of Foetal Bovine Serum (FBS) (Biosera Cat. S1810 or alike) (Heat inactivate FBS at 56°C for 30 minutes and cool before use) NB. NO PEN/STREP.
RPMI-MGIA = Sigma RPMI-1640 Medium HEPES modification with 25mM HEPES (Cat. R5886) Add 5ml of L-glutamine (200mM 100X) (Cat. G7513)
Benzonase (25U/µl) (Sigma Cat. E1014)

Mycobacteria stock (Aeras BCG Pasteur stock 051415MF is recommended)

BD BBL MGIT tubes containing 7ml media (BD Ref. 245122)

PANTA/enrichment supplement for MGIT tubes (BD supplement kit Ref. 245124)

Cell culture grade sterile water (Sigma)

6.2 Equipment

BACTEC MGIT 320/960 machine

37°C water bath

Centrifuge and microcentrifuge

37°C incubator with CO2

Cell counter/microscope and related cell counting equipment

48-well plates (Costar 3548, cell culture cluster)

2ml screw-top microtubes (Sarstedt)

Vortex

360° tube rotator (VWR 13916-822)

6.3 Assay protocol

6.3.1 Preparation of Standard Curve (must be completed ahead of time to calculate input inoculum):

Note: The following is adapted from Zelmer et al. “Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing – a protocol for mouse splenocytes.” BioRxiv. 2015 [5]. Please refer to this protocol for more information on preparing a standard curve.

Note: All work must be performed under sterile conditions

1) Prepare MGIT PANTA enrichment: reconstitute one bottle of lyophilized MGIT PANTA by adding contents of one bottle MGIT growth supplement. Mix by turning over until completely dissolved.

2) Add 800 μl of MGIT PANTA enrichment to each BACTEC MGIT tube. Prepare 2 tubes per dilution. Tightly recap the tube(s).

Note: Keep tubes sealed whenever possible as they are oxygen-enriched.

Note: Do not store MGIT tubes after the addition of PANTA enrichment. The tubes must be used the same day or discarded.

3) Thaw one vial of the mycobacteria to be tested at room temperature.

Note: The homogeneity of the mycobacterial stock has an impact on the reproducibility of the results in this assay. If the mycobacterial stock appears clumpy, it can be sonicated in a sonication water bath for 1 min. Place vial on ice for 1 min and repeat.

4) Prepare seven 10‐fold serial dilutions of the stock: add 1.08ml PBS-Tween 80 (or other appropriate diluent as desired) to each of 7 sterile 2ml tubes.

5) Add 120μl undiluted stock to the first tube. Mix thoroughly by pipetting up and down.

6) From the first tube (1:10 dilution), remove 120μl and transfer it to the next tube, mix. Repeat until all 7 dilutions are prepared.

Note: The number and range of dilutions can be adjusted according to the stock concentration, if known.

7) Inoculate 2 MGIT tubes for each dilution by adding 500 μl of the appropriate dilution to each tube. Tightly seal tubes as soon as possible. Turn over to mix.

8) Place MGIT tubes into the BACTEC MGIT instrument. Note time to detection (TTD) as tubes come positive.

Note: Record tube numbers (given on barcode) or position within the drawer for each sample

– this will make it easier to identify samples from the MGIT report.

9) Divide two 7H10 or 7H11 agar plates into 3-4 sectors each. Spot 3x20 μl from each dilution onto a section. Leave plates to dry. Seal plates in sealable bags or with Parafilm to prevent drying out. Place in incubator at 37°C. Count colonies as soon as they are visible (after 3-4 weeks for most standard BCG strains; colonies of BCG Pasteur from Aeras are countable after approx. 10-12 days). Counts from spots should be approx. between 4 and 30 colonies.

10) Calculate a standard curve by plotting TTD against input CFU as determined by plating of equivalent volumes (step 9) and use regression analysis to obtain the equation that can be used to convert any TTD to volume or CFU. Log10 CFU can be fitted with a linear regression, whilst using CFU requires the fitting of a semi-log line. The regression analysis will provide an R2 value (a measure of how well the line fits the data), and the equation describing the line. Solve the equation for X: Y = A*X+B -> X = (Y-B)/A where A = slope and B = intersect). By inserting the TTD (=Y), the corresponding number of CFU (or log10 CFU) can now be calculated. See Zelmer et al. “Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing – a protocol for mouse splenocytes.” BioRxiv. 2015 [5] for more information.

Figure 1: Schematic of Direct MGIA

6.3.2 MGIA Day 0 (Assay set-up)

1) Thaw cryopreserved cells according to standard protocol:

Thaw in water bath at 37°C until a small amount of frozen material remains

Pipette up and down and gradually add to 10ml R10 (NO PEN/STREP) using a Pasteur pipette
Rinse out the cryovial contents with 1ml of fresh medium and add the rest of the cells

Centrifuge at 1500rpm for 5 minutes

Pour off supernatant and resuspend cells at approx. 2-3x10^6 cells per ml of R10 (NO PEN/STREP), plus 2µl (50U) benzonase per ml
Rest for 2 hours in 37°C incubator with CO2

2) Count cells and resuspend at 10x106 cells per ml of RPMI-MGIA

3) Place 300μl of cell mix into labelled wells of a 48-well plate

Note: Duplicate cultures may be performed if sufficient cells are available, but have been demonstrated to be consistent (CV <5%) such that a single culture is acceptable.

Note: Do not use wells on the outside of the 48-well plate for cultures. These should contain 600μl of RPMI medium only.

4) Add 120µl (to give a final concentration of 20%) of non-heat inactivated autologous serum or plasma matched to the volunteer and time-point.

Note: Filtered pooled human AB serum may be used if autologous serum is not available, but will not capture the influence of serum factors such as antibodies on control of mycobacterial growth.

Note: If plasma is viscous, it may be warmed in a 37°C incubator and pulse-vortexed. Note: Ensure the serum is mixed well before adding.

5) Thaw mycobacteria stock at room temperature and prepare to the correct concentration in RPMI-MGIA. The appropriate inoculum volume will depend on the particular stock, but should correspond to 100 CFU, calculated using the standard curve generated in section

6.3.1. The stock should be made up to a concentration of 100 CFU per 180µl of media.

Note: If stock is highly concentrated, the stock should be diluted in several steps (eg. serial 1:10 dilutions) to avoid pipetting very small volumes.

6) Add 180µl (100 CFU) of the BCG Pasteur final preparation to each sample well.

7) Incubate the 48-well plates in a CO2 incubator at 37°C for 96 hrs (4 days).

8) Supplement one MGIT tube with 800µl PANTA/enrichment to produce supplemented Middlebrook 7H9. Decant the contents into a fresh falcon tube for use in step 10.

9) Supplement 2 further MGIT tubes with 800µl PANTA/enrichment. These are the direct-to- MGIT controls.

10) Add an equal volume of BCG Pasteur final preparation as step 6 to each of the 2 direct- to-MGIT controls. Using the extra supplemented Middlebrook 7H9 produced in step 8, make up the added volume to 500µl (so if 180µl of BCG preparation is added, add an additional 320µl of supplemented Middlebrook 7H9). Invert to mix and place on the Bactec MGIT machine.

6.3.3 MGIA Day 4 (Assay processing)

Before harvesting, supplement 1 MGIT tube per culture well with 800μl PANTA enrichment and label.

1) Pipette the cultures in the well up and down three times, collect the liquid and transfer to a 2ml screw-cap tube.

2) Centrifuge tubes at 12,000rpm (15,300g) for 10 minutes.

3) Add 500μl of sterile tissue culture grade endotoxin free water to each well, and incubate at room temperature for at least 5 minutes.

4) Remove 500μl of supernatant from the 2ml tubes, ensuring the pellet remains intact. Supernatant can be discarded unless required for later cytokine analysis.

5) Pipette the water in the wells up and down 5 times to detach monocytes that have attached to the bottom of the well and completely remove the water from the well, transferring it to the corresponding tube containing the cell/BCG pellet.

6) Pulse vortex for 1 second just before adding, and add the 600μl of sample from the 2ml tube to the corresponding MGIT tube. Use some media from the MGIT tube to wash out the 2ml tube and add to MGIT tube.

7) Invert all MGIT tubes to mix and place on the Bactec MGIT machine until positivity is reached.

6.3.4 Alternative protocol

If cell number is limiting, an alternative protocol may be used whereby 1x106 PBMC are added to 2ml screw-cap tubes, inoculated and rotated for the 96 hour period.

Note: This protocol may be associated with reduced cell viability, reduced IFN-γ production and increased variability.

1) Thaw cryopreserved cells as detailed in section 6.3.2.

2) Count cells and resuspend at 1x106 PBMC per 300µl of RPMI-MGIA containing 10% pooled human AB serum (filtered and heat-inactivated).

3) Label 2 x 2ml screw-cap tubes per sample, and add 300µl of the corresponding cell mixture to each.

4) Thaw mycobacteria stock at room temperature and prepare to the correct concentration in RPMI-MGIA containing 10% pooled human AB serum (filtered and heat-inactivated). The

appropriate inoculum volume will depend on the particular stock, but should correspond to a direct-to-MGIT control TTD of 8.5 days (approximately 100 CFU), calculated using the standard curve generated in section 6.3.1. The stock should be made up at a volume of 300µl per well.

5) Supplement 2 MGIT tubes with 800µl PANTA/enrichment and 200µl of PANTA/Enrichment supplemented Middlebrook 7h9.

6) Add 300µl of the mycobacteria master mix to each of the 2 positive control tubes, invert to mix and place on the MGIT machine.

7) Add 300µl of the mycobacteria master mix to each sample 2ml tube.

8) Place tubes on a 360° tube rotator in a 37°C incubator for 96 hours.

9) On day 4, add 800µl PANTA/enrichment to the same number of MGIT tubes as you have 2ml sample tubes and label accordingly.

10) Add the 600µl sample to its corresponding MGIT tube. Use some media from the MGIT tube to wash out the 2ml tube and add to MGIT tube. Invert to mix and place on MGIT machine until positivity is reached.

6.4 Data processing and reporting

1) Record time to detection (TTD) for controls and samples, and convert to CFU values using the stock standard curve prepared in section 6.3.1.

Note: The TTD will indicate total number of bacteria per MGIT tube (ie. the number of bacteria per 600µl culture) rather than CFU/ml, so will need to be converted accordingly.

2) If desirable, log10 CFU can be converted to CFU and vice versa. If more than one time- point or experiment is to be compared, it is advisable to normalise data to the direct-to-MGIT controls to account for differences in input inocula and BCG stocks between assay runs. In

this case, the number of total CFU per sample is divided by the number of total CFU in the direct-to-MGIT control to give a read-out of fold-change in bacterial number, or ∆log10 CFU.

7 References

[1] Tanner, R., O'Shea, M. K., Fletcher, H. A., and McShane, H., 2016, "In vitro mycobacterial growth inhibition assays: A tool for the assessment of protective immunity and evaluation of tuberculosis vaccine efficacy," Vaccine.

[2] Brennan, M. J., Tanner, R., Morris, S., Scriba, T. J., Achkar, J. M., Zelmer, A., Hokey, D. A., Izzo, A., Sharpe, S., Williams, A., Penn-Nicholson, A., Erasmus, M., Stylianou, E., Hoft,

D. F., McShane, H., and Fletcher, H. A., 2017, "The Cross-Species Mycobacterial Growth

Inhibition Assay (MGIA) Project, 2010-2014," Clin Vaccine Immunol, 24(9).

[3] Tanner, R., O'Shea, M. K., White, A. D., Müller, J., Harrington-Kandt, R., Matsumiya, M., Dennis, M. J., Parizotto, E. A., Harris, S., Stylianou, E., Naranbhai, V., Bettencourt, P., Drakesmith, H., Sharpe, S., Fletcher, H. A., and McShane, H., 2017, "The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays," Sci Rep, 7, p. 43478.

[4] Fletcher, H. A., Tanner, R., Wallis, R. S., Meyer, J., Manjaly, Z. R., Harris, S., Satti, I.,

Silver, R. F., Hoft, D., Kampmann, B., Walker, K. B., Dockrell, H. M., Fruth, U., Barker, L., Brennan, M. J., and McShane, H., 2013, "Inhibition of mycobacterial growth in vitro following primary but not secondary vaccination with Mycobacterium bovis BCG," Clin Vaccine Immunol, 20(11), pp. 1683-1689.

[5] Zelmer, A., Tanner, R., Stylianou, E., Morris, S., Izzo, A., Williams, A., Sharpe, S., Pepponi, I., Walker, B., Hokey, D., McShane, H., Brennan, M., and Fletcher, H., 2015, "Ex vivo mycobacterial growth inhibition assay (MGIA) for tuberculosis vaccine testing - a protocol for mouse splenocytes,"BioRxiv.

8 Revision History

Version
Number / Date / What Changed / Why it Changed / Who Changed it
1.0 / 27/11/17 / First Written / Rachel Tanner