Name: measuring naturally acquired phagocytosis-inducing antibodies to plasmodium falciparum parasites by a flow cytometry-based assay
Uploaded: 2020-08-06T16:20:00
Description: Watch this Scientific Journal Video about Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay at JoVE.com

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.

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.
<ol>
	<li>Mai...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets+of+antibodies+against+erythrocytes+in+malaria+immunity.">Targets of antibodies against erythrocytes in malaria immunity.</a> J Clin Invest. 122 (9), 3227-3238 (2012).</li><li>Quintana, M. P., Angeletti, D., Moll, K., Chen, Q., Wahlgren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+inducing+antibodies+to+Plasmodium+falciparum+upon+immunization+with+a+recombinant+PfEMP1+NTS+DBL1&#945;+domain.">Phagocytosis inducing antibodies to Plasmodium falciparum upon immunization with a recombinant PfEMP1 NTS DBL1&#945; domain.</a> Malaria Journal. 1, 1-9 (2016).</li><li>Chan, J. A., et al. <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+point+in+protein+trafficking+by+Plasmodium+falciparum+determines+the+expression+of+major+antigens+on+the+surface+of+infected+erythrocytes+targeted+by+human+antibodies.">A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.</a> Cellular and Molecular Life Sciences. 73, 4141-4158 (2016).</li><li>Quintana, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+in+children+with+malaria+to+PfEMP1,+RIFIN+and+SURFIN+expressed+at+the+Plasmodium+falciparum+parasitized+red+blood+cell+surface.">Antibodies in children with malaria to PfEMP1, RIFIN and SURFIN expressed at the Plasmodium falciparum parasitized red blood cell surface.</a> Scientific Reports. 8 (1), 3262 (2018).</li><li>Hommel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+antibody+functional+activity+and+strain-specificity+of+vaccine+candidates+for+malaria+in+pregnancy+using+in+vitro+phagocytosis+assays.">Evaluating antibody functional activity and strain-specificity of vaccine candidates for malaria in pregnancy using in vitro phagocytosis assays.</a> Parasites &amp; Vectors. 11 (69), 1-7 (2018).</li><li>Ampomah, P., Stevenson, L., Ofori, M. 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Part I. Learner's Guide.</a> World Health Organization. , (1991).</li><li>Tsuchiya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> International Journal of Cancer. Journal International Du Cancer. 26 (2), 171-176 (1980).</li><li>Fleit, H. B., Kobasiuk, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+monocyte-like+cell+line+THP-1+expresses+FcyRl+and+Fc'yRIl.">The human monocyte-like cell line THP-1 expresses FcyRl and Fc'yRIl.</a> Journal of Leukocyte Biology. 49, 556-565 (1991).</li><li>Auwerx, J., Staels, B., Van Vaeck, F., Ceuppens, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+IgG+Fc+receptor+expression+induced+by+phorbol+12-myristate+13-acetate+treatment+of+THP-1+monocytic+leukemia+cells.">Changes in IgG Fc receptor expression induced by phorbol 12-myristate 13-acetate treatment of THP-1 monocytic leukemia cells.</a> Leukemia research. 16 (3), 317-327 (1992).</li><li>Ata&#237;de, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+an+improved+phagocytosis+assay+to+evaluate+the+effect+of+HIV+on+specific+antibodies+to+pregnancy-associated+malaria.">Using an improved phagocytosis assay to evaluate the effect of HIV on specific antibodies to pregnancy-associated malaria.</a> PloS One. 5 (5), 10807 (2010).</li></ol>

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...

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.

<table>
	<tbody>
		<tr>
			<td>96 well cell culture plates, round bottom with lid</td>
			<td>Corning</td>
			<td>3799</td>
			<td>Any similar plate can be used, make sure it is compatible with the flow cytometer instrument you intend to use</td>
		</tr>
		<tr>
			<td>AlbuMAX-II</td>
			<td>Gibco</td>
			<td>11021-037</td>
			<td></td>
		</tr>
		<tr>
			<td>AlbuMAX-II (5%)</td>
			<td>-</td>
			<td>-</td>
			<td>5% AlbuMAX-II (Gibco, 11021-037), 0.2g/L hypoxanthine (Sigma, H9377) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Anti-Red Blood Cells antibody</td>
			<td>Abcam</td>
			<td>ab34858</td>
			<td>Prepare 2&#65533;&#65533;l aliquots and freeze a -20&#176;C. Use one aliquot per experiment.</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma</td>
			<td>P8622</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Protein G</td>
			<td>Invitrogen</td>
			<td>10003D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution</td>
			<td>Sigma</td>
			<td>E1510</td>
			<td>Prepare a stock solution at 0.1mg/mL in RPMI1640 (R5886). Store protected from light</td>
		</tr>
		<tr>
			<td>FC500 flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Any flow cytometer supporting 96 well plate format and having the appropriate lasers/filters to measure EtBr fluorescence can be used.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td>Heat inactivate before use.</td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD16</td>
			<td>BD Biosciences</td>
			<td>555406 or 556618</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD32</td>
			<td>BD Biosciences</td>
			<td>552883</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC mouse anti-human CD64</td>
			<td>BD Biosciences</td>
			<td>555527</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowLogic software</td>
			<td>Inivai technologies</td>
			<td></td>
			<td>Any flow cytometry analysis can be used, for example FlowJo or Winlist</td>
		</tr>
		<tr>
			<td>Gentamicin (10mg/mL)</td>
			<td>Sigma</td>
			<td>G1272</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxanthine</td>
			<td>Sigma</td>
			<td>H9377</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine (200mM)</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing solution</td>
			<td>-</td>
			<td>-</td>
			<td>15mM NH4Cl, 10mM NaHCO3, 1mM EDTA</td>
		</tr>
		<tr>
			<td>MACS CS-column and accesories</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-305</td>
			<td></td>
		</tr>
		<tr>
			<td>Parasite culture medium</td>
			<td>-</td>
			<td>-</td>
			<td>2mM L-glutamine (Sigma, G7513), 50&#181;g/mL Gentamicin (Sigma, G1272), 0.5% AlbuMAX-II (AlbuMAX-II 5%) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (10000U and 10mg/mL)</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>Sigma</td>
			<td>R5886</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 culture medium</td>
			<td>-</td>
			<td>-</td>
			<td>10%FBS (Gibco, 10099-141), 2mM L-glutamine (Sigma, G7513), 100U/mL Penicillin, 0.1mg/mL Streptomycin (Sigma, P0781) in RPMI1640 (Sigma, R5886)</td>
		</tr>
		<tr>
			<td>Vario MACS magnet</td>
			<td>Miltenyi Biotec</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring naturally acquired phagocytosis-inducing antibodies to plasmodium falciparum parasites by a flow cytometry-based assay

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.

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...

Watch this Scientific Journal Video about Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay at JoVE.com

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

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).

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

This protocol allows to measure the ability of antibodies to opsonize and induce the phagocytosis of plasmodium falciparum infected erythrocytes. Antibodies present in the serum of individuals naturally exposed to parasite infection, as well as those induced by immunization with parasite antigens, can be tested. Demonstrating the procedure with me will be Maiken Visti, a technician from my laboratory.To set up a THP-1 cell culture for a phagocytosis assay, the day before the experiment seed the cells in a 25-centimeter squared culture flask and a concentration of 2.5 times 10 to the fifth cells per milliliter in THP one cell culture medium. On the day of the experiment, at least one hour before the experiment block to round-bottom 96-well plates with 150 microliters of sterile 2%FBS in PBS per well. For mid-to late-stage trophozoite-infected erythrocytes harvest and purification, prepare around 1.2 milliliters of packed P falciparum parasite culture in 10 milliliters of parasite culture medium, with at least 5%parasitemia and add to a magnetic column.Wash the column with 50 milliliters of parasite culture medium. To elute the infected the erythrocytes, remove the column from the magnet and flush it with 50 milliliters of parasite culture medium. Then, collect the infected erythrocytes by centrifugation and resuspend the pellet in one milliliter of fresh parasite culture medium for counting.Adjust the purified infected erythrocyte suspension by 3.3 times 10 to the seventh cells per milliliter in parasite culture medium containing ethidium bromide, to a final concentration of 2.5 micrograms per milliliter. Next, remove blocking solution from one 96-well plate by flicking the plate into a waste container. Then remove any excess over a paper towel and add 30 microliters of the ethidium bromide label infected erythrocyte suspension to all but one well in the upper half of the plate.Add 30 microliters of parasite culture medium to the empty well, and incubate the plate for 10 minutes at room temperature, protected from light. At the end of the incubation, add 170 microliters of parasite culture medium to each well and sediment the infected erythrocytes at the bottom of the plate by centrifugation. Remove the supernatant by flicking the plate into a waste container, and wash the ethidium bromide-labeled infected erythrocytes two more times, with 200 microliters of parasite culture medium per well, as just demonstrated.After the second wash, use a multi-channel pipette to carefully remove the entire volume of supernatant from each well, without disturbing the pellets. And resuspend the ethidium bromide-labeled infected erythrocytes in 30 microliters of antibody solution, including the appropriate controls and test samples previously prepared in parasite culture medium. Then, place the plate at 37 degrees Celsius for 45 minutes, protected from light.While the infected erythrocytes are being opsonized, collect the THP-1 cells by centrifugation, and resuspend the pellet in 12 milliliters of fresh, pre-warmed THP-1 cell culture medium. After a second centrifugation, resuspend the pellet in one milliliter of THP-1 cell culture medium for counting, and adjust the cell concentration to five times 10 to the fifth cells per milliliter in THP-1 cell culture medium. Remove the blocking solution from the second 96-well plate and add 100 microliters of THP-1 cells to each well.Then, place the plate in the cell culture incubator. At the end of the opsonization incubation, add 170 microliters of parasite culture medium to each well of the opsonization plate, and sediment the infected erythrocytes to the bottom of the plate by centrifugation. Wash the opsonized infected erythrocytes two times with 200 microliters of parasite culture medium per well, using a multi-channel pipette to carefully remove the supernatant after the second wash.Resuspend the opsonized infected erythrocytes in 100 microliters of pre-warmed THP-1 cell culture medium per well, and transfer 50 microliters of each opsonized infected erythrocyte suspension to a corresponding well in the phagocytosis plate. When all of the cells have been added, place the plate in the cell culture incubator for no more than 40 minutes, protected from light. At the end of the incubation, stop the phagocytosis by centrifugation at four degrees Celsius.Remove the supernatant and resuspend the pellets with 150 microliters of room-temperature ammonium chloride lysing solution per well. After exactly three minutes, add 100 microliters of ice cold at 2%FBS in PBS to stop the lysis, and sediment the intact cells by centrifugation. Next, wash the cells three times with 200 microliters of fresh ice cold 2%FBS in PBS per well, per wash.After the final wash, resuspend the cell pellet in 200 microliters of fresh ice cold 2%FBS in PBS per well. For flow cytometry acquisition and analysis, immediately load the cells onto a flow cytometer and use the linear forward versus linear side scatter plot for the wells without infected erythrocytes to gate the THP-1 cells. Acquire 10, 000 events in this gate, and use the THP-1 cells with the infected erythrocytes opsonized with a positive control to set up a histogram plot to measure the ethidium bromide fluorescence intensity.For analysis, open the linear forward versus linear side scatter plot for the wells without infected erythrocytes, and gate the THP-1 cells. Then, set up a positive gate in an FL-3 histogram and copy these gates onto all of the other sample wells to determine the percentage of ethidium bromide-positive THP-1 cells. For each sample tested, phagocytosis can be reported as the absolute value or as the relative phagocytosis calculated as a percentage using the positive control as the maximum.The THP-1 cells should be periodically checked for FC gamma receptor surface expression by flow cytometry. The cells should be negative for CD16, and positive for CD32 and CD64. In this opsonization experiment, the THP-1 cells were gated first according to their forward and side scatter profiles, to allow quantification of the percentage of ethidium bromide-labeled cells, indicative of cells that have phagocytized at least one antibody opsonized infected erythrocyte.The negative control samples should all generate a single negative peak in the FL3 channel, with few events observed in the ethidium bromide marker. Accordingly, the mean phagocytosis values, both as absolute ethidium bromide-positive THP-1 cells and as relative phagocytosis percentages, should be very low. In contrast, the positive control should generate traces with two peaks, a negative peak and a clearly positive and well-separated peak located within the ethidium bromide marker.The mean phagocytosis values are normally highest for the positive control, followed by the malaria-exposed female pool and the single malaria-exposed woman controls. Although this methodology generates consistent results, deviations in the slope coefficient of the adjusted lines have been observed. Therefore, the use of relative values is recommended, especially if it's not possible to run all of the samples in a single experiment.Be sure to carefully plan the experiment in advance, preparing both the THP-1 cells and the parasites accordingly, using only trophocytes with a purity greater than 80%In addition, be sure to always include the appropriate controls, to allow the quality of the experiment to be assessed and facilitate data analysis.

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