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).
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.
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.
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.
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).
3. Phagocytosis assay (Figure S1)
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 phagocytosis by THP-1 cells.
The assay specifically measures antibody-mediated phagocytosis and, therefore, interaction with the appropriate Fc-receptors on the surface of the THP-1 cells is required. For this reason, and as mentioned in the protocol, we recommend periodically checking the expression of Fcγ-receptors on the surface of the THP-1 cells by flow cytometry. The cells should be negative for CD16 (Figure 1A) and positive for CD32 and CD64 (Figure 1B,C).
For the assay, purified late-stage IEs were labeled with EtBr and then opsonized with antibodies present in the plasma/serum of malaria-naïve or malaria-exposed individuals. Phagocytosis was measured by flow cytometry, quantifying the percentage of EtBr+ THP-1 cells after 40 min co-incubation with EtBr-labeled and antibody-opsonized IEs. Initially, THP-1 cells were gated using an FSC vs. SSC density plot (Figure 2A). Then, an EtBr+ marker was created, using an FL3 histogram on the THP-1 cells and un-opsonized IEs (Figure 2B). These gates were then used to analyze all the other controls and test samples.
The negative controls (including the THP-1 cells alone, the un-opsonized IE control, and the controls with malaria-naïve and malaria-exposed males) should all generate a single negative peak in the FL3 channel (Figure 3A) with only few events in the EtBr+ marker. Accordingly, the mean phagocytosis values both as absolute EtBr+ THP-1 cells and as relative phagocytosis percentages should be very low (Figure 3B,C, normally less than 2% for all cases). In contrast, the positive controls (including the rabbit anti-human erythrocyte antibody and the malaria-exposed female pool) should generate traces with two peaks (Figure 3A): a negative one (largely overlapping with the one generated by all the negative controls) and a clearly positive and well-separated one located inside the EtBr+ marker. A positive sample, as the presented example (sample from a malaria-exposed multigravid woman/NF20) should generate a similar profile as the positive controls. The mean phagocytosis values, measured as absolute EtBr+ THP-1 cells and as relative phagocytosis, were normally highest for the positive control (58%/100%), followed by the malaria-exposed female pool (29%/53%), and then the single malaria-exposed woman (23%/40%). As observed in Figure 3B,C, where three independent experiments are presented, there was a considerable variability between experiments and we, therefore, recommend running samples intended for comparison in the same experiment. In our hands, at least four full 96 well plates can be handled by a single experienced researcher. The variability between assays was also clearly observed when two identical experiments testing several serum samples from malaria-exposed women were performed simultaneously. The same parasite preparation (after magnetic purification of late-stage IEs) and serum dilutions were used. THP-1 cells were kept in two separate flasks but seeded from the same initial flask and the experiments were performed by two different researchers. Even though the assay seems to generate consistent results when performed separately, with tight linear correlations (r>0.9 for both absolute and relative phagocytosis values) between phagocytosis values measured in the two experiments, the slope coefficient of the adjusted lines deviates from one, indicating the values generated in different experiments were not identical. This deviation was more evident for the absolute values (slope coefficient confidence interval 0.55-0.72) as compared to the relative values (slope coefficient confidence interval 0.68-1) (Figure 4). We, therefore, recommend using relative values, especially if for some reason (e.g., not enough purified IEs, more than 4 full plates, etc.) it is not possible to run all the samples in a single experiment. We also recommend running experiments intended for comparative analysis within the shortest amount of time, to avoid introducing extra variation due to drifting in PfEMP1 expression (as well as other antigens) and due to subtle differences on the THP-1 cells upon extended time in culture.
Figure 1: Fc receptors expressed on the THP-1 cell surface.
(A) Fcγ-receptor III/CD16 (red). (B) Fcγ-receptor II/CD32 (green). C. Fcγ-receptor I/CD64 (orange). Un-labeled cells are shown in blue. Please click here to view a larger version of this figure.
Figure 2: Flow cytometry gating strategy.
(A) THP-1 cells gated on FSC/SSC. (B) Ethidium bromide positive (EtBr+) THP-1 cells in an FL3 histogram. THP-1 cells alone/no IEs added (blue), THP-1 cells incubated with un-opsonized IEs (green), and THP-1 cells incubated with IEs opsonized with a positive control (red) are shown. Please click here to view a larger version of this figure.
Figure 3: Phagocytosis of IT4VAR04-IEs by THP-1 cells.
(A) Representative flow cytometry histograms of one of the experiments presented in B (identified by larger symbols). (B) Percentage of EtBr+ THP-1 cells (means and standard deviations of three independent experiments). (C) Same data as in B, after normalization against the corresponding positive control. Color coding is the same in all panels: THP-alone (black), un-opsonized/no antibody control (blue), malaria-naïve control (cyan), malaria-exposed male pool (green), malaria-exposed female pool (orange), a malaria-exposed female donor (pink), and positive control/rabbit anti-human erythrocytes (red). Mean and standard deviations are shown. Please click here to view a larger version of this figure.
Figure 4: Phagocytosis of IT4VAR04 IEs by THP-1 cells upon opsonization with serum from 10 malaria-exposed women.
The plots present linear regression analysis for two identical experiments performed on the same day, but by different researchers. (A) Data presented as absolute values and as (B)relative phagocytosis values. Analysis performed using statistical analysis software. Please click here to view a larger version of this figure.
Figure S1: Phagocytosis assay flow chart. Flow chart depicting the main steps of the assay. Please click here to download this figure.
Figure S2: 96 well plate experiment layout. (A) Layout for IEs EtBr labeling. (B) Layout for opsonization; 6 wells are always reserved for controls. (C) Layout for THP-cells plating. (D)Layout for phagocytosis. Please click here to download this figure.
Figure S3: Color coding as in Figure 3. (A) Flow cytometry histogram overlay of one experiment acquired immediately and (B) after storage at 4 °C for 12 h. (C) Percentage of EtBr+ THP-1 cells measured before and after storage. NF## represent different malaria-exposed female donors. Please click here to download this figure.
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 a type of PfEMP1 involved in placental sequestration of IEs, and a key determinant in the pathogenesis of PM.
The assay can be used for antibodies induced by immunization and/or targeting other PfEMP1 variants or any other parasite antigen present on the IE surface, provided the antibody tested interacts with the human Fcγ-receptors expressed by the THP-1 cells (CD32 and CD64). The assay is simple, high-throughput (allowing the analysis of large sample sets) and can be performed in one day. Phagocytosis is measured by flow cytometry, quantifying the percentage of EtBr+ THP-1 cells after 40 min co-incubation with EtBr-labeled and antibody-opsonized IEs.
Even though the assay gives consistent results over experimental replicates, there is variability between the absolute values measured and, therefore, we recommend calculating relative values using a positive control that must always be included. We also recommend running all samples to be tested in a single experiment, to avoid inter-assay variation as discussed above. When testing serum samples collected from individuals exposed to P. falciparum infection, we recommend always including a set of samples from naïve individuals to be used as a control group. This control group can be used to set up a threshold to determine which of your test samples are to be considered positive.
We have previously used this approach to compare the phagocytosis-inducing capacity of sera collected from children with different malaria clinical presentations (severe vs. mild)17.
The authors have nothing to disclose.
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.
96 well cell culture plates, round bottom with lid | Corning | 3799 | Any similar plate can be used, make sure it is compatible with the flow cytometer instrument you intend to use |
AlbuMAX-II | Gibco | 11021-037 | |
AlbuMAX-II (5%) | – | – | 5% AlbuMAX-II (Gibco, 11021-037), 0.2g/L hypoxanthine (Sigma, H9377) in RPMI1640 (Sigma, R5886) |
Anti-Red Blood Cells antibody | Abcam | ab34858 | Prepare 2��l aliquots and freeze a -20°C. Use one aliquot per experiment. |
DPBS | Sigma | P8622 | |
Dynabeads Protein G | Invitrogen | 10003D | |
Ethidium bromide solution | Sigma | E1510 | Prepare a stock solution at 0.1mg/mL in RPMI1640 (R5886). Store protected from light |
FC500 flow cytometer | Beckman Coulter | Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used. | |
Fetal Bovine Serum (FBS) | Gibco | 10099-141 | Heat inactivate before use. |
FITC mouse anti-human CD16 | BD Biosciences | 555406 or 556618 | |
FITC mouse anti-human CD32 | BD Biosciences | 552883 | |
FITC mouse anti-human CD64 | BD Biosciences | 555527 | |
FlowLogic software | Inivai technologies | Any flow cytometry analysis can be used, for example FlowJo or Winlist | |
Gentamicin (10mg/mL) | Sigma | G1272 | |
Hypoxanthine | Sigma | H9377 | |
L-glutamine (200mM) | Sigma | G7513 | |
Lysing solution | – | – | 15mM NH4Cl, 10mM NaHCO3, 1mM EDTA |
MACS CS-column and accesories | Miltenyi Biotec | 130-041-305 | |
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) | Sigma | P0781 | |
RPMI-1640 medium | Sigma | R5886 | |
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 magnet | Miltenyi Biotec |