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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

Abstract

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Introduction

Malaria is a vector-borne disease caused in humans upon infection with five different species of the genus Plasmodium. The most prevalent species is P. falciparum, which is also responsible for the most morbidity and mortality1. Malaria clinical presentation varies from asymptomatic or benign infections to complicated/severe disease, the latter occurring mostly in children under the age of five years. Exposure to P. falciparum does not induce sterile immunity, but individuals living in endemic areas slowly develop immunity against the clinical disease. Protection is age/exposure dependent and immunity is normally acquired during the first 5-10 years of life2. Adult women are an important exception, as severe malaria can occur during pregnancy in a clinical presentation known as placental malaria (PM). PM is an important cause of abortion, stillbirth, premature delivery, low birth weight, fetal death, and maternal anemia. Resistance to PM develops over successive pregnancies3. Protection from PM is associated with the acquisition of antibodies against VAR2CSA-type PfEMP14,5, an infected erythrocyte (IE) surface antigen that binds to chondroitin sulphate A (CSA) enabling IE sequestration in the placenta. Antibodies mediate protection performing various functional activities (reviewed in6,7) including opsonization of IEs to induce phagocytosis. Early in vitro studies showed that antibodies can limit P. falciparum growth in the presence of monocytes via phagocytosis8,9. More recent studies have shown that higher levels of phagocytosis-inducing antibodies are associated with better pregnancy outcomes (in the context of HIV co-infection)10,11, indicating the relevance of this effector function in the naturally acquired immune response.

Here we present a protocol to measure this function of antibodies present in human plasma/serum, using in vitro cultured IEs expressing VAR2CSA together with the monocyte line THP-1. The assay has been previously used11,12,13,14,15,16,17,18 and is considered an improved and easier approach compared to earlier microscope-based protocols8, since it allows testing of a larger number of antibody samples in a single run using smaller volumes of antibody and avoiding tedious and biased microscopy counting. Even though the assay has been used by multiple laboratories and its execution is simple enough, it requires careful planning and preparation, therefore, a detailed protocol would allow its application by laboratories and researchers lacking previous experience. We use, as an example, late-stage-synchronized IEs expressing VAR2CSA opsonized with antibodies present in serum collected from women with naturally acquired PM-specific immunity. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination.

Protocol

The human serum samples used for the results presented here were collected in a separate study19. Collection was approved by the Institutional Review Board of Noguchi Memorial Institute for Medical Research, University of Ghana (study 038/10-11), and by the Regional Research Ethics Committees, Capital Region of Denmark (protocol H-4-2013-083).

1. Parasite culture

NOTE: Follow the local regulations for human pathogens handling.

  1. Maintain P. falciparum parasites according to the standard protocol as described before20 in parasite culture medium (see Table of Materials).
  2. Keep the parasites tightly synchronized using sorbitol treatment as previously described21.
  3. To ensure VAR2CSA expression on the surface of the IE, perform repeated rounds of immune-magnetic selection using an anti-VAR2CSA antibody (e.g., PAM1.4 antibody, a cross-reactive human monoclonal VAR2CSA-specific IgG22) coupled to protein G-magnetic beads23.
    1. In brief, incubate late-stage trophozoite-IEs with magnetic beads coupled to an anti-VAR2CSA antibody and positively select using a magnet.
    2. Expand the selected parasites in culture for a few cycles until parasitemia is at least 5%.
    3. Alternatively, select parasites that bind to plastic-immobilized CSA (the receptor for VAR2CSA) as previously described24.
  4. To verify VAR2CSA expression on the selected parasites perform flow cytometry as previously described25.
    1. In brief, label the late-stage trophozoite-IEs with the same antibody used for the magnetic selection (step 1.3) followed by a fluorescently labeled secondary antibody.
    2. Measure the IE surface reactivity by flow cytometry. Successful antibody selection normally results in a parasite population that expresses VAR2CSA in most of the parasites (≈80%).
  5. For the phagocytosis assay, use purified mid- to late-stage trophozoite-IEs. Perform purification using magnetic separation as previously described26.
    NOTE: To accurately determine the stage of the parasites, perform Giemsa staining on blood smears followed by light microscopy observation. Follow standard procedures and morphological guidelines as described before27. Late-stage purification can also be performed using density gradient medium. The IE yield, however, in our experience is lower and, therefore, magnetic purification is preferable.
    1. For magnetic separation, spin the parasite culture (5-10% parasitemia) at 500 x g for 10 min at room temperature. Remove the supernatant and re-suspend the cell pellet in 10 mL of parasite culture medium.
    2. Add the parasite suspension into a CS-column coupled to a magnet (see Table of Materials) and let it slowly pass through the column. Trophozoite-IEs are paramagnetic (due to the presence of hemozoin) and will stay into the column mesh.
    3. Wash the column with 50 mL of parasite culture medium and elute the IEs from the magnetic column using 50 mL of parasite culture medium.
  6. Spin the eluate from 1.5.3 at 500 x g for 10 min at room temperature. Carefully discard the supernatant and re-suspend in 1 mL of parasite culture medium.
  7. Using a hemocytometer, count the number of IEs (easily identified by light microscopy as erythrocytes with dark hemozoin pigment) and total cell number to calculate the final parasitemia (percentage of IEs).
    NOTE: Only use parasites with at least 80% purity (80% IEs).
  8. Based on the number of serum/antibody samples planned for testing, estimate the total amount of IEs needed and scale up the parasite cultures accordingly. A 75 cm2 culture flask with 25 mL of culture at 5% hematocrit and 5-10% parasitemia should yield enough IEs to run a full 96 well plate. For a full plate (42 samples plus 6 controls in duplicate), a minimum of 1.5 mL parasite suspension at 3.3 x 107 IEs/mL are needed.

2. THP-1 cells

NOTE: The THP-1 cell line is used in this assay. This monocyte cell line is derived from a patient with monocytic leukemia28 and can be purchased from ATCC. Maintain the cell line according to the provider’s instructions in THP-1 culture medium (see Table of Materials).

  1. For regular maintenance, start THP-1 cell culture at 2-4 x 105 cells/mL and subculture when cell concentration reaches 8 x 105 cells/mL.
    NOTE: Do not allow the cell concentration to exceed 1 x 106 cells/mL and keep track of passage number. Avoid the use of cells that are beyond passage 25. The average doubling time of the THP-1 cell line varies between 19-50 h. Determine the doubling time of the cell batch before starting the phagocytosis experiments. This will help to roughly estimate the necessary amount of culture needed for a determined number of serum samples to be tested (e.g., for a full 96 well plate, 10 mL of culture at 5 x 105 cells/mL are needed).
  2. Periodically check the THP-1 cells for the surface expression of the Fcγ-receptors I (CD64), II (CD32) and III (CD16) as previously described15.
    1. In brief, stain the THP-1 cells (separately) with anti-CD64, anti-CD32 and anti-CD16 fluorescently labeled antibodies (Table of Materials) for 30 min at room temperature (1:100 dilution prepared in 2% FBS in PBS).
    2. Wash three times with 2% FBS in PBS and measure surface staining by flow cytometry.
      NOTE: The THP-1 cells are negative for CD16 and positive for CD32 and CD64 as previously reported29,30 (Figure 1).
  3. For the phagocytosis assay, set up a THP-1 cell culture flask with 2.5 x 105 cells/mL the day before the experiment to yield around 5 x 105 cells/mL on the day of assay.
    NOTE: Ensure 4-6 x 105 cells/mL are present on the day of the assay.

3. Phagocytosis assay (Figure S1)

  1. Before starting the assay, block (>1 h) two round-bottom 96 well plates (one for the opsonization and another for the phagocytosis) using 150 μL per well of sterile 2% FBS in PBS.
    NOTE: Label plate 1 as opsonization plate and use it both for ethidium bromide (EtBr) staining and opsonization. Label plate 2 as phagocytosis plate and use both for THP-1 cell plating and for the phagocytosis step.
  2. Parasite staining and opsonization
    1. Take the IE suspension prepared in 1.6 and adjust cell count to 3.3 x 107 IEs/mL by adding parasite culture medium and EtBr to achieve a final concentration of 2.5 μg/mL (1:40 dilution, from a 0.1 mg/mL stock).
      NOTE: EtBr stains the parasite DNA, allowing detection by flow cytometry.
      CAUTION: EtBr is a mutagen and skin, eye, and respiratory irritant. Use appropriate protection and dispose waste according to local regulations.
    2. Remove the blocking solution from one of the 96 well plates (opsonization plate), flicking the plate and removing liquid excess over a piece of towel paper.
    3. Add 30 μL per well of the IE suspension prepared above in the upper half of the plate. Leave one well empty and add 30 µL of parasite culture medium (without IEs for the THP-1 alone control). (Figure S2A). Incubate for 10 min at room temperature and protected from light.
    4. Add 170 µL per well of parasite culture medium. Spin the plate at 500 x g for 3 min at room temperature and remove the supernatant by flicking the plate over an appropriate waste container.
    5. Wash the EtBr-labeled IEs two more times using 200 µL per well of parasite culture medium spinning the plate at 500 x g for 3 min at room temperature. The supernatant from the first wash can be removed by flicking the plate. Carefully remove the supernatant from the second wash using a multichannel pipette to make sure the entire volume of washing medium is removed. Do not disturb the pellet.
    6. Re-suspend the EtBr-labeled IEs in 30 μL of antibody/plasma/serum solution prepared at the desired concentrations in parasite culture medium.
    7. Always include the following controls (Figure S2B): a control without IEs/THP-1 cells control (parasite culture medium); a control without any antibody or plasma/serum (un-opsonized control); a positive control using the commercially available rabbit anti-human erythrocyte antibody at 1:100 dilution (see Table of Materials); two negative plasma/serum controls at 1:5 dilution (a malaria-naïve pool and a pool from malaria-exposed males); and a positive serum control at 1:5 dilution (a pool from malaria-exposed women, who have previously been pregnant (preferably multigravidae)).
      NOTE: A 1:100 dilution for the positive control seems to work consistently across different batches of purchased antibody (data not shown). However, it is recommended to rule out variations between batches by testing several dilutions every time a new batch of antibody is used.
    8. Incubate for 45 min in the dark at 37 °C.
  3. THP-1 cells preparation and phagocytosis
    1. While the IEs are being opsonized (3.2.8), begin preparing the THP-1 cells.
    2. Remove the THP-1 cells from the culture flask, spin them down (500 x g for 5 min at room temperature), decant the supernatant and re-suspend the pellet in THP-1 cell culture medium.
    3. Spin again (500 x g for 5 min at room temperature), decant the supernatant and re-suspend the cell pellet in 1 mL of THP-1 cell culture medium.
    4. Determine cell count in the solution prepared above and add more medium to obtain a final concentration of 5 x 105 cells/mL.
    5. Remove the blocking solution from the remaining 96 well plate (phagocytosis plate) by flicking the plate and removing liquid excess over a piece of towel paper.
    6. Add 100 μL per well of the THP-1 cell suspension prepared in 3.3.4 and put back in the cell culture incubator (Figure S2C).
    7. Once the antibody/plasma/serum incubation time has finished, add 170 µL per well of parasite culture medium. Spin the plate at 500 x g for 3 min at room temperature and remove the supernatant by flicking the plate over an appropriate waste container.
    8. Wash the opsonized IEs two more times using 200 µL per well of parasite culture medium, spinning the plate at 500 x g for 3 min at room temperature. The supernatant from the first wash can be removed by flicking the plate. Carefully remove the supernatant from the second wash, using a multichannel pipette to make sure the entire volume of washing medium is removed. Do not disturb the pellet.
    9. Finally re-suspend the opsonized IEs in 100 µL of pre-warmed THP-1 cell culture medium. Transfer 50 µL of opsonized IE suspension to each well in the phagocytosis plate. Since there is a total of 100 µL of IEs, each antibody/serum dilution can be run in duplicates in the phagocytosis plate (Figure S2D)
      NOTE: The amount of IEs and THP-1 cells used in the assay correspond to a 10:1 ratio.
    10. Incubate for 40 min in the dark at 37 °C, 5% CO2.
      NOTE: Do not allow the phagocytosis to proceed for more than 40 min.
    11. Stop the phagocytosis by centrifugation at 4 °C (500 x g, 5 min) and discard the supernatant by flicking the plate. Add 150 µL of room-temperature ammonium chloride lysing solution (Table of Materials) and mix by pipetting, incubate for exactly 3 min.
      NOTE: This step will lyse the erythrocytes that have not been phagocytosed by the THP-1 cells.
    12. Stop the lysis by adding 100 µL of ice-cold 2% FBS in PBS. Spin the plate at 4 °C (500 x g for 3 min) and remove the supernatant by flicking the plate over an appropriate waste container.
    13. Wash three times using 200 µL per well of ice-cold 2% FBS in PBS spinning the plate at 4°C (500 x g for 3 min). After the final wash, re-suspend in 200 µL of ice-cold 2% FBS in PBS and immediately analyze by flow cytometry.
      NOTE: Previous publications have used cell fixation in 2% paraformaldehyde prior to flow cytometry31, but the results presented here were acquired immediately after assay completion. Postponing flow cytometry data acquisition by storing the plate at 4°C is not recommended, since the percentage of EtBr+ THP-1 cells decays rapidly (Figure S3).
  4. Flow cytometry acquisition and analysis
    ​NOTE: Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.
    1. For acquisition, gate on THP-1 cells using a linear forward-scatter (FSC) vs. linear side-scatter (SSC) plot using the wells were no IEs were added (Figure 2A) and acquire 10,000 events on this gate.
    2. Measure fluorescence intensity for EtBr (FL3-Log) on the THP-1 gate using a histogram plot.
    3. For gating, first gate on THP-1 cells in a FSC vs. SSC density plot, using the wells were no IEs were added (Figure 2A). Then set up a positive gate in an FL3 (EtBr) histogram, using the THP-1 cells (no IEs added) and the un-opsonized control (Figure 2B).
    4. Copy these gates in all the other samples tested in the same plate and determine the percentage of EtBr-positive THP-1 cells (THP-1 cells that have phagocytized at least one IE). For each of the samples tested, phagocytosis can be reported as the absolute values (percentage of EtBr+ THP-1 cells) or as relative phagocytosis calculated as percentage using the positive control as maximum.

Results

Here we present in detail a protocol that has previously been described31 and used11,12,13,14,15,16,17,18 to measure the capacity of antibodies targeting the surface of P. falciparum IEs to induce opsonization and phagocytos...

Discussion

The protocol presented here has been previously described and used12,15,17,31 to measure the capacity of antibodies targeting the surface of P. falciparum IEs to induce opsonization and phagocytosis by THP-1 cells. The results presented here focus on naturally acquired VAR2CSA-specific antibodies in the plasma/serum of women living in a P. falciparum endemic region. VAR2CSA is...

Disclosures

The authors have nothing to disclose.

Acknowledgements

Maiken Visti is thanked for excellent technical assistance. This work was partly funded by a grant (MAVARECA II; 17-02-KU) from the Ministry of Foreign Affairs of Denmark and administered by Danida Fellowship Centre. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Materials

NameCompanyCatalog NumberComments
96 well cell culture plates, round bottom with lidCorning3799Any similar plate can be used, make sure it is compatible with the flow cytometer instrument you intend to use
AlbuMAX-IIGibco11021-037
AlbuMAX-II (5%)--5% AlbuMAX-II (Gibco, 11021-037), 0.2g/L hypoxanthine (Sigma, H9377) in RPMI1640 (Sigma, R5886)
Anti-Red Blood Cells antibodyAbcamab34858Prepare 2μl aliquots and freeze a -20°C. Use one aliquot per experiment.
DPBSSigmaP8622
Dynabeads Protein GInvitrogen10003D
Ethidium bromide solutionSigmaE1510Prepare a stock solution at 0.1mg/mL in RPMI1640 (R5886). Store protected from light
FC500 flow cytometerBeckman CoulterAny flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.
Fetal Bovine Serum (FBS)Gibco10099-141Heat inactivate before use.
FITC mouse anti-human CD16BD Biosciences555406 or 556618
FITC mouse anti-human CD32BD Biosciences552883
FITC mouse anti-human CD64BD Biosciences555527
FlowLogic softwareInivai technologiesAny flow cytometry analysis can be used, for example FlowJo or Winlist
Gentamicin (10mg/mL)SigmaG1272
HypoxanthineSigmaH9377
L-glutamine (200mM)SigmaG7513
Lysing solution--15mM NH4Cl, 10mM NaHCO3, 1mM EDTA
MACS CS-column and accesoriesMiltenyi Biotec130-041-305
Parasite culture medium--2mM L-glutamine (Sigma, G7513), 50µg/mL Gentamicin (Sigma, G1272), 0.5% AlbuMAX-II (AlbuMAX-II 5%) in RPMI1640 (Sigma, R5886)
Penicillin/Streptomycin (10000U and 10mg/mL)SigmaP0781
RPMI-1640 mediumSigmaR5886
THP-1 culture medium--10%FBS (Gibco, 10099-141), 2mM L-glutamine (Sigma, G7513), 100U/mL Penicillin, 0.1mg/mL Streptomycin (Sigma, P0781) in RPMI1640 (Sigma, R5886)
Vario MACS magnetMiltenyi Biotec

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