Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay

Obiakor, Harold; Avril, Marion; MacDonald, Nicholas J.; Srinivasan, Prakash; Reiter, Karine; Anderson, Charles; Holmes, Kevin L.; Fried, Michal; Duffy, Patrick E.; Smith, Joseph D.; Narum, David L.; Miller, Louis H.

doi:10.1128/cvi.00638-12

Cited by 24 publications

(19 citation statements)

References 44 publications

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“…After an incubation of 30 min at 37°C, parasite pellets were washed twice with DPBS/FCS medium and resuspended in 200 μL formaldehyde 0.37% (Sigma F-8775). Immuno-fluorescence staining was analysed as described [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

“…Different expression systems have been explored to produce single domain or multidomain constructs [ 22 – 27 ]. Apart from immunisation using DNA constructs [ 28 , 29 ], responses elicited by recombinant proteins were variable, with a wide range of titres, variable proportions of immunised animals producing cytoadherence-inhibiting antibodies [ 22 – 27 , 30 – 36 ] and discordant results in different species of laboratory animals [ 22 , 33 , 34 ]. Systematic immunogenicity studies are scarce for rosette-forming PfEMP1 adhesins.…”

Section: Introductionmentioning

confidence: 99%

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Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

et al. 2015

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Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

et al. 2015

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“…The antibodies inhibited cytoadhesion to CSA under both static and flow conditions ( Figure 5A-B). 26,44,45 Two of the 6 antibodies, namely those directed at DBL3X and DBL5e, seemed to interfere with cooperative binding, indicated by significantly reduced Hill coefficients as determined in parallel assays ( Figure 5C; P , .01). The CSA d 50 values were unaffected by the DBL domain-specific antibodies ( Figure 5D).…”

Section: Antibodies Against Dbl Domains Affect Cooperativitymentioning

confidence: 99%

Cytoadhesion of Plasmodium falciparum–infected erythrocytes to chondroitin-4-sulfate is cooperative and shear enhanced

Rieger

Yoshikawa

Quadt

et al. 2015

Blood

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Key Points• Cytoadhesion of parasitized erythrocytes depends on the intermolecular distance between neighboring chondroitin-4-sulfate molecules.• VAR2CSA is an allosteric adhesin that binds chondroitin-4-sulfate in a cooperative and shear stress-induced manner.Infections with the human malaria parasite Plasmodium falciparum during pregnancy can lead to severe complications for both mother and child, resulting from the cytoadhesion of parasitized erythrocytes in the intervillous space of the placenta. Cytoadherence is conferred by the specific interaction of the parasite-encoded adhesin VAR2CSA with chondroitin-4-sulfate (CSA) present on placental proteoglycans. CSA presented elsewhere in the microvasculature does not afford VAR2CSA-mediated cytoadhesion of parasitized erythrocytes. To address the placenta-specific binding tropism, we investigated the effect of the receptor/ligand arrangement on cytoadhesion, using artificial membranes with different CSA spacing intervals. We found that cytoadhesion is strongly dependent on the CSA distance, with half-maximal adhesion occurring at a CSA distance of 9 6 1 nm at all hydrodynamic conditions. Moreover, binding to CSA was cooperative and shear stress induced. These findings suggest that the CSA density, together with allosteric effects in VAR2CSA, aid in discriminating between different CSA milieus. (Blood. 2015;125(2):383-391)

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“…Plasma IgG reactivity to several VAR2CSA recombinant antigens based on the FCR3 allele were assessed by ELISA as previously described (16). Briefly, Escherichia coli-produced ID1-DBL2X-ID2a (16,55) and DBL3X, DBL4ε, and DBL5ε (16,55) recombinant proteins from the FCR3 strain were coated at 100 ng per well in flat-bottom 96-well plates (Immulon 4; Dynex Technology, Inc., Chantilly, VA). Plates were incubated at 4°C overnight and blocked with 5% (wt/vol) skim milk (Difco, Detroit, MI) in Tris-buffered saline (BioFluids, Camarillo, CA) for 2 h at room temperature (RT).…”

Section: Methodsmentioning

confidence: 99%

Functional Antibodies against Placental Malaria Parasites Are Variant Dependent and Differ by Geographic Region

et al. 2019

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During pregnancy, Plasmodium falciparum-infected erythrocytes (IE) accumulate in the intervillous spaces of the placenta by binding to chondroitin sulfate A (CSA) and elicit inflammatory responses that are associated with poor pregnancy outcomes. Primigravidae lack immunity to IE that sequester in the placenta and thus are susceptible to placental malaria (PM). Women become resistant to PM over successive pregnancies as antibodies to placental IE are acquired. Here, we assayed plasma collected at delivery from Malian and Tanzanian women of different parities for total antibody levels against recombinant VAR2CSA antigens (FCR3 allele), and for surface reactivity and binding inhibition and opsonizing functional activities against IE using two CSA-binding laboratory isolates (FCR3 and NF54). Overall, antibody reactivity to VAR2CSA recombinant proteins and to CSA-binding IE was higher in multigravidae than in primigravidae. However, plasma from Malian gravid women reacted more strongly with FCR3 whereas Tanzanian plasma preferentially reacted with NF54. Further, acquisition of functional antibodies was variant dependent: binding inhibition of P. falciparum strain NF54 (P < 0.001) but not of the strain FCR3 increased significantly with parity, while only opsonizing activity against FCR3 (P < 0.001) increased significantly with parity. In addition, opsonizing and binding inhibition activities of plasma of multigravidae were significantly correlated in assays of FCR3 (r = 0.4, P = 0.01) but not of NF54 isolates; functional activities did not correlate in plasma from primigravidae. These data suggest that IE surface-expressed epitopes involved in each functional activity differ among P. falciparum strains. Consequently, geographic bias in circulating strains may impact antibody functions. Our study has implications for the development of PM vaccines aiming to achieve broad protection against various parasite strains.

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Identification of VAR2CSA Domain-Specific Inhibitory Antibodies of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Using a Novel Flow Cytometry Assay

Cited by 24 publications

References 44 publications

Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

Cytoadhesion of Plasmodium falciparum–infected erythrocytes to chondroitin-4-sulfate is cooperative and shear enhanced

Functional Antibodies against Placental Malaria Parasites Are Variant Dependent and Differ by Geographic Region

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