An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

This protocol requires the recruitment of donors for serum and RBC to be used for parasite culture, and HIV(+) and uninfected donors for PBMC isolation. Institutional Review Boards must approve all studies and all donors must provide informed consent prior to blood draw.

CAUTION: Working with human blood samples and human malaria parasites requires precautionary measures. Always wear a lab coat, gloves, and work in a level 2 Biosafety cabinet. In the event of accidental percutaneous exposure to human malaria, report to Health and Safety for prophylactic treatment. Additional safety consideration should also be put in place for working with HIV(+) blood. Wear a back closing lab coat. Double glove (top glove should be latex). Perform all handling in a level 2 Biosafety cabinet. Do not use any glass or sharp objects. Do not use aspirator. Place all contaminated items in virox solution or bleach for a minimum of 1 hr prior to discarding. Wash all surfaces with virox and UV for 1 hr following use. Note that each institution will have their own specific biosafety regulations that need to be followed. Report all accidental exposures to HIV-infected blood to Health and Safety for evaluation and consideration of possible post-exposure prophylaxis.

Note: Different strains of P. falciparum malaria parasites exist. ITG was used for these experiments, but different strains can be used. Excellent instructions on freezing and thawing Plasmodium falciparum parasites are available at the MR4 website ¹³.

1. Making RPMI-A for Malaria Parasite Culture

1. Make RPMI-0 by mixing 950 ml of ddH₂O, 1 packet of RPMI-1640 powder, 6 g of HEPES, 2 g of sodium bicarbonate, and 1.35 mg of hypoxanthine.
2. Thaw heat inactivated human serum from two different donors. AB donors are best but any can be used if parasites are grown in O type red blood cells (RBC). Invert tubes to mix. If serum contains particulate matter or is thick spin at 2,000 rpm and then filter liquid portion using a 0.45 µm filter unit.
3. Using a 0.2 µm filter unit filter 180 ml of RPMI-0, 20 ml of human serum (10 ml from each donor), and 0.5 ml of 10 mg/ml gentamycin.
   Note: Human serum can clog the filter so more than one filter unit may be required.
4. Label the medium bottle with RPMI-A, the date, and the source of the serum. Refrigerate until required. RPMI-A may turn cloudy when refrigerated. This is normal, but increasing cloudiness is a sign of contamination.
   Note: Parasite growth may vary in different donor serum. It is a good idea to test all human serum batches for good parasite growth before using.

2. Preparing Human Red Blood Cells for Parasite Culture

Note: Blood donors should be type O.

1. Collect 7-10 ml of blood into acid-citrate-dextrose (ACD) tubes. Write the donor’s ID and date of collection on the label.
2. Store blood at 4 ^oC until needed. Use blood for parasite cultures within 1 month.
3. Wipe the top of the tube with 70% ethanol. Carefully remove stopper and discard. Transfer blood into a 15 ml tube. Spin for 3 min at 1,000 x g. Remove plasma by aspiration.
4. Suspend RBC with equal volume of warm RPMI-0. Spin for 5 min at 1,000 x g. Remove the buffy coat by aspiration, resuspend in 5 ml of RPMI-0 and repeat the wash 2 more times.
5. Remove the supernatant and add enough RPMI-A to produce a mixture that is 50% RBC by volume.
6. Store at 4 ^oC until needed.

3. Maintaining Parasite Cultures

1. Pre-warm RPMI-A to 37 ^oC.
2. Place thawed parasites (see MR4 protocol for thawing procedure) into a T25 flask with 5 ml of RPMI-A and 75 μl of washed human RBC for a hematocrit of ~3%. Note: Hematocrit measures the volume of RBC compared to total volume. To calculate hematocrit measure the volume of packed RBC to the total volume of medium plus RBC. Assume that the thawed parasites will contribute 75 μl of RBC. By adding 150 μl of RBC stock (which is equivalent to adding 75 μl of packed RBC) to the flask there is a total of 150 μl of RBC into 5 ml of medium, which equals to a hematocrit of 3% (150 μl/5,000 μl x 100% = 3%).
3. Gas the flask for 30 sec with parasite gas mixture (1% O₂, 3% CO₂, balance N₂). Seal and place the flask wide side down in a 37 ^oC incubator as to allow for the greatest surface area for gas exchange.
4. To change the medium and check for parasitemia (needs to be done daily), carefully move the flask as to not disturb the RBC layer. Using a sterile unplugged Pasteur pipette draw off the culture medium without disturbing the blood layer.
5. To check parasitemia, remove a 10 μl sample from the blood layer, place it on a glass slide and using a second glass slide create a thin blood film. Allow slide to dry.
6. Place 4 ml of fresh RPMI-A into the flask, gently mix, gas for 30 sec, and place in incubator at 37 °C.
7. Stain the slide using the Hema3 staining kit according to manufacturer’s protocol.
   1. For optimal staining, dip slides in the fixing solution for 10 sec, solution I for 10 sec, and solution II for 30 sec. Rinse in water and leave slides to dry.
8. Calculate parasitemia by counting the number of PfRBC (stained dark purple) versus the total number of RBC (stained pink). Count a total of 300 cells to ensure accuracy.
9. When parasites have reached a parasitemia of 5%, expand the culture into a T75 flask, using 40 ml of RPMI-A, and 500 μl of RBC.

4. Parasite Synchronization

Note: The day before the experiment, synchronize the parasite culture by treating with alanine. Only ring stage parasites and uninfected RBC will survive this treatment. Alanine synchronization will give you a pure trophozoite culture the next day that can be used in the co-culture experiments. Make sure to start with a parasite culture that contains a majority of ring stage parasites.

1. Prepare alanine solution by mixing 8.01 g of alanine (300 mM) and 0.365 g of Tris (10 mM) into 300 ml of ddH₂O. Bring pH to 7.4. Filter sterilize using a 0.2 μm filter unit.
2. Pre-warm alanine solution to 37 ^oC.
3. Spin down the parasite culture (5 min x 1,000 x g), and remove medium.
4. Resuspend pellet in 19 volumes of alanine solution (1 ml packed RBC to 19 ml alanine solution). Incubate for 15 min at RT.
5. Spin 5 min x 1,000 x g. Aspirate supernatant. Wash in RPMI-0 once. Aspirate supernatant and resuspend in RPMI-A and adjust hematocrit to ~3%. Gas flask and return to 37 ^oC.
   Note: For co-culture experiments a minimum parasitemia of 5% trophozoites is needed, with 10% parasitemia considered optimal.

5. Parasite and RBC Preparation for Co-culture Experiments

1. Use a ratio of 3 PfRBC per PBMC in co-culture experiments to induce an inflammatory response. To calculate total PfRBC, quantify parasitemia by making a thin blood smear as described in 3.5, and hematocrit by counting the number of RBC per mL of parasite culture using a hemocytometer.
   Number of PfRBC = % parasitemia x total RBC/ml x ml of culture. For example: 10 ml of culture at 10 x 10⁶ RBC/ml and 10% parasitemia is equal to 0.1 x 10⁶/ml x 10 ml = 10 x 10⁶ PfRBC.
2. Spin the parasite culture for 5 min at 1,000 x g (RT). Aspirate medium, and resuspend at 6 x 10⁶ PfRBC per ml in RPMI-S+ (500 ml RPMI-1640 supplemented with L-glutamine and HEPES, 10% heat inactivated FBS, 1.5 ml gentamicin, 5 ml of 100 mM sodium pyruvate, 5 ml of 10 mM MEM non-essential amino acids, 5 ml of 5mM β-mercaptoethanol).
3. Take control blood (uninfected RBC from the same donor used for maintaining the parasite culture), calculate hematocrit and resuspend in RPMI-S+ at the same number of RBC per ml as the parasite culture.

6. Isolation of Human Peripheral Blood Mononuclear Cells (PBMC)

1. Collect venous blood in sodium heparin tubes (green top) from both a chronic HIV(+) donor and an HIV(-) control. Do not use EDTA as an anti-coagulant as EDTA’s calcium chelating action affects cell function. On average expect between 5-10 x 10⁶ PBMC per 10 ml of blood. For a typical experiment collect 30 ml of blood from each donor. The blood volume will need to be adjusted according to the experimental requirements.
2. As quickly as possible and definitely within an hour of blood being collected, remove the blood from the tubes and place into a plastic 50 ml tube. Monocytes in particular will be activated and stick to glass, so it is important that if glass blood collection tubes are used cells are removed as quickly as possible.
3. Spin the blood at 1,000 x g for 15 min and collect plasma (this can be used for other studies if required – if not this step can be skipped). Dilute the blood in equal volume of cold DPBS.
4. Place 15 ml of RT Ficoll into a 50 ml tube. Slowly, using a sterile plastic transfer pipette, layer the blood/DPBS solution over the Ficoll without any mixing. A maximum of 25 ml of blood/DPBS solution can be layered over each 15 ml of Ficoll.
5. Spin the gradients for 30 min at RT at 600 x g. Make sure the ‘no brake’ setting is on.
6. Once the cells have spun, look for the interface between the two phases. This is where the PBMC are. Using a sterile plastic transfer pipette collect the PBMC from the interface (see Figure 1). Minimize contamination by RBC.
7. Place collected cells in 50 ml tubes, with no more than 20 ml per tube. Top tube up to 50 ml with sterile cold DPBS.
8. Spin the cells for 10 min at 4 ^oC at 300 x g.
9. Discard supernatant, resuspend pellet in 5 ml sterile cold DPBS and pool all cells from the same donor into one tube. Top up to 50 ml with sterile DPBS, and repeat the wash steps 2 more times.
10. Resuspend cells in 10 ml RPMI-S+ medium.
11. Remove a 20 μl aliquot and mix with 20 μl of trypan blue. Pipette 10 μl in a haemocytometer and count the living cells (cells that have not taken up the blue dye – all blue cells are dead). Use a haemocytometer to calculate cell number in a solution as follows.
    Note: A haemocytometer has a grid of specified dimensions so that the area covered by the lines is known.
    1. Make sure the haemocytometer is clean. Place the coverslip over the counting area. Load 10 μl of cell solution by placing the pipette tip into one of the V-shaped wells. The area under the coverslip will fill by capillary action.
    2. Place the loaded haemocytometer under a microscope. The full grid of the haemocytometer contains 9 squares of 1 mm² each. Count all cells within each large square. If too many cells are present, dilute the solution further and recount.
    3. Calculate cell concentration as follows: Total cells/ml = (total cells counted/# of squares) x dilution factor x 10,000 cells/ml, e.g., (500 cells/5 squares) x dilution factor of 20 x 10,000 cells/ml = 20 x 10⁶ cells total.
12. Adjust medium to a final concentration of 10 x 10⁶ per ml (RPMI-S+ medium).

7. Malaria/HIV Co-infection Culture

1. Plate 100 μl of isolated PBMCs and 400 μl of RPMI-S+ medium to a 24-well plate (1 x 10⁶ PBMC per well). Ensure that wells are set up in triplicate: 3 wells for uninfected RBC, 3 wells with PfRBC, 3 wells with medium, and 3 wells with PMA/Ionomycin. This is both for the HIV(+) and HIV(-) sample.
2. For the PfRBC wells, place 3 x 10⁶ PfRBC in each of the wells (500 μl of parasite culture prepared in 5.2).
3. For uninfected RBC wells, place 500 μl of the uninfected RBC culture prepared in 5.3 in each of the wells.
4. For medium wells, place 500 μl of RPMI-S+ medium in each of the wells.
5. For PMA/Ionomycin wells, prepare PMA/Ionomycin solution (2.5 pg/ml, 250 pg/ml respectively). Place 500 μl of this solution in each well.
   Note: As potent stimulators of T cell cytokine secretion, PMA and Ionomycin are used as a positive control to ensure cells are functional.
6. Place plate at 37 ^oC in a 5% CO₂ incubator.
7. Optional: use excess cells for phenotypic analysis using flow cytometry or store in an RNA stabilization solution for future mRNA expression analysis.

8. Detection of Malaria Immune Responses

Note: Perform co-culture experiments for as long as 4 days. No medium change is required during this time. The optimal time point will depend on the cell type of interest and the question asked. An incubation of 12-48 hr is optimal for monocytic responses to PfRBCs, while lymphocyte responses were best observed at 72-96 hr. The time points will need to be optimized based on the experimental question. Shorter periods (2-4 hr) may be used if interaction between intact PfRBC and PBMCs is of interest.

1. Spin plate at 300 x g for 3 min to pellet cells. Collect 700 μl of culture supernatant from each well.
2. Spin supernatant at 1,000 x g for 5 min to clear of any debris.
3. Aliquot cleared supernatant as needed, label, and freeze at below -20 °C until analysis of secreted factors is to be performed.
4. Analyze cytokine/chemokine responses by ELISA or by bead array (follow the manufacturer’s suggested protocols).
5. Collect cells remaining in the plate into an RNA stabilizing solution and utilize for mRNA expression analysis by quantitative real-time PCR ^14-16.

9. Intracellular Flow Cytometry for Cell-specific Ccytokine Responses Using PBMC Co-cultured with P. falciparum Infected RBC

Note: As mentioned above the length of co-culture will depend on the cell type of interest. If interested in monocytic responses to PfRBC a shorter incubation period is needed. Times will be longer for innate lymphocyte responses γδ T cells, NK cells, NKT cells), and longer still for CD4 and CD8 T cells. Optimization will be required.

1. 6-8 hr prior to analysis add 1 μl of 1,000x brefeldin A to all wells. Return the plate to 37 ^oC incubator. After 6-8 hr incubation, place plate on ice for 15 min.
2. Remove cells from the wells using vigorous pipetting and place them into labeled microcentifuge tubes. Scraping may be required to remove monocytes.
3. Spin cells for 5 min at 1,000 x g. Remove supernatants. Resuspend cells in 500 μl flow cytometry buffer (1x DPBS, 2% heat inactivated FBS, 0.02% sodium azide).
4. Divide cells up so that each sample has: one tube for full antibody staining (240 μl), and one tube for fluorescent minus one (FMO) control antibody staining (240 μl). Pool a small amount of each sample (20 μl) for the unstained flow cytometry control (this will be used in setting up the flow cytometer).
5. Spin cells for 5 min at 500 x g. Remove supernatants.
6. Incubate cells with unconjugated anti-human CD16/CD32 (5 μg/ml) in 50 μl flow cytometry buffer for 15 min at 4 ^oC to block Fc receptors.
7. Add 1 ml of flow cytometry buffer to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.
8. Resuspend cells in 50 μl of flow cytometry buffer with a pre-titrated concentration of fluorophore-conjugated antibodies to desired cell surface markers. Pre-mix enough antibody solution for all samples to be stained. Incubate cells at 4 ^oC for 20 min. Protect from light.Note: Amount of each antibody will have to be optimized. We routinely stain for CD56, CD3, γδ, CD4, CD8, CD14. Titrated volumes for these antibodies are shown in Table 1.
9. Add 1 ml of flow cytometry buffer to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.
10. Add 100 μl of cytofix/cytoperm solution to each tube. Incubate cells for 20 min at 4 ^oC. Protect from light.
11. Add 1 ml of perm/wash buffer (provided as 10x concentrate – dilute to 1x using ddH₂O) to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.
12. Add 100 μl of perm/wash buffer to FMO control antibody staining tubes and mix to resuspend cells.
13. Add 100 μl of perm/wash buffer with a pre-titrated concentration of fluorophore-conjugated antibodies to desired intracellular markers (i.e., cytokines/chemokines) to full antibody staining tubes.
    Note: Amount of each antibody will have to be optimized. We routinely stain with anti-TNF and anti-IFNγ antibodies. Titrated volumes for these antibodies are shown in Table 1.
14. Incubate all tubes for 30 min at 4 ^oC. Protect from light.
15. Add 1 ml of Perm/Wash buffer to each tube. Spin cells for 5 min at 500 x g. Remove supernatants.
16. Resuspend cells in 300 μl flow cytometry buffer with 1% paraformaldehyde. Let sit for a minimum of 15 min to neutralize HIV.
17. Prepare single antibody stained samples using compensation beads, to be used for compensation set up on the flow cytometer. The type of compensation beads will depend on the antibodies used (i.e., anti-mouse Ig, anti-rat/hamster Ig). Vortex beads. Add one drop of beads per antibody. Add equal amounts of antibody as used for staining. Add 200 μl of flow cytometry buffer. These are now ready for use. There is no need to wash the beads.
18. Acquire samples on a flow cytometer as soon as possible and within 24 hr for best results. Tandem dyes are susceptible to dissociation with storage so we recommend immediate acquisition if possible.