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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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+Plasmodium+falciparum+erythrocytic+stages+in+culture.">Synchronization of Plasmodium falciparum erythrocytic stages in culture.</a> Journal of Parasitology. 65 (3), 418-420 (1979).</li><li>Barfod, L., 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=Human+pregnancy-associated+malaria-specific+B+cells+target+polymorphic,+conformational+epitopes+in+VAR2CSA.">Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA.</a> Molecular Microbiology. 63 (2), 335-347 (2007).</li><li>Staalsoe, T., 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=In+vitro+selection+of+Plasmodium+falciparum+3D7+for+expression+of+variant+surface+antigens+associated+with+severe+malaria+in+African+children.">In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children.</a> Parasite Immunology. 25 (8-9), 421-427 (2003).</li><li>Chan, S., 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=Regulation+of+PfEMP1-VAR2CSA+translation+by+a+Plasmodium+translation-enhancing+factor.">Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor.</a> Nature Microbiology. 2, 17068 (2017).</li><li>Barfod, L., 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=Evasion+of+immunity+to+Plasmodium+falciparum+malaria+by+IgM+masking+of+protective+IgG+epitopes+in+infected+erythrocyte+surface-exposed+PfEMP1.">Evasion of immunity to Plasmodium falciparum malaria by IgM masking of protective IgG epitopes in infected erythrocyte surface-exposed PfEMP1.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (30), 12485-12490 (2011).</li><li>Moll, K., Kaneko, A., Scherf, A., 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=.">.</a> Methods in Malaria Research. , (2013).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+malaria+microscopy.+Part+I.+Learner's+Guide.">Basic malaria microscopy. 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 ...

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Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...