Human subjects

We have used three sets of samples for conducting our experiments.

Malaria-exposed pregnant women from Malawi (n = 40)

From July 2011 to March 2013, HIV-uninfected pregnant women were enroled at antenatal clinics between 16–28 weeks’ gestation in a randomised trial comparing intermittent preventive treatment in pregnancy (IPTp) and Intermittent Screening and Treatment in Malawi (Pan African Clinical Trials Registry ISRCTN69800930)¹⁵. At enrolment, women’s P. falciparum infection status was established using light microscopy and polymerase chain reaction (PCR). Blood plasma was separated, stored at −80 °C, and shipped frozen to Melbourne. We randomly selected 10 plasma samples each from primigravid women with and without infection and multigravid women with and without infection (Supplementary Table 1). The study was approved by the Malawian National Health Science Research Committee and the Melbourne Health Human Research Ethics Committee. All participants provided informed written consent, including for their samples to be shipped overseas, and to be used in studies of immunity to malaria.

Pregnant women with or without placental infection at delivery from Papua New Guinea (n = 77)

From November 2009 to August 2012, pregnant women in Madang Province, Papua New Guinea were enroled at antenatal clinics between 14–26 weeks’ gestation into a randomised controlled trial of IPTp with sulphadoxine-pyrimethamine (SP) and azithromycin (AZ) or one course of SP and chloroquine (CQ)¹⁶ (ClinicalTrials.gov NCT01136850). Plasma was collected at enrolment, stored at −80 °C, and shipped frozen to Melbourne. Peripheral blood collected at delivery was tested for P. falciparum infection using light microscopy and quantitative PCR and placental biopsies were examined for evidence of placental malaria as described¹⁷. Plasma samples were selected from women with peripheral parasitaemia, but no IEs in the placenta (non-placental malaria, n = 27; all available samples used), and samples from women with active placental infection (IEs in the placenta, n = 50, frequency-matched for primigravidity, IPTp regime receipt, bed net use, rural residency, and age), as previously described¹². Participant characteristics are outlined in Supplementary Table 2. The trial was approved by the PNG Institute of Medical Research Institutional Research Board (IMR IRB 0815), the PNG Medical Research Advisory Council (MRAC 0801), and the Melbourne Health Human Research Ethics Committee (2008.016) and the use of plasma samples was approved by the same bodies (IMR IRB 0922; MRAC 10.50; 2010.017). All participants provided informed written consent including for future use of their samples in studies of immunity to malaria.

Children

Children aged 0.5–12 years of age presenting to Modilon Hospital in Madang, PNG with severe or uncomplicated malaria were recruited from 2006 to 2009¹⁸. Children with severe malaria were part of an observational study¹⁸, which included admission to the Paedatric Ward of the hospital, treatment in accordance with PNG National Treatment Guidelines, and collection of clinical data, including detailed neurological examination. Severe malaria was defined based on World Health Organization (WHO) criteria¹⁹ and approximately 55% were males. Children with uncomplicated malaria were recruited from clinics in the same villages as children with severe malaria and were matched by age (within 12 months of the index case) sex and ethnicity and had no features of severe disease. Approximately 55% of children were male. Plasma separated from venous blood collected at enrolment (acute) and approximately eight weeks later (convalescent) from each group was stored at −80 °C and shipped frozen to Melbourne. Written consent was obtained from the child’s parent or guardian, including for studies of antibody immunity to IE surface antigens. Participant characteristics are outlined in Supplementary Table 3. All available samples were used. The study was approved by the PNG Institute of Medical Research’s Institutional Review Board (IRB No.1103) and the Medical Research Advisory Council of the PNG Department of Health (MRAC 11.12).

Positive and negative control plasma

Decomplemented (heat-inactivated) plasma samples were pooled from fifteen Malawian pregnant women with high levels of antibody to CS2 IEs (immune plasma or PPS). Pregnant women gave informed consent to participate in a study examining the relationship between malaria antibody and use of IPTp and insecticide treated nets²⁰ Ethical approval was provided by the Human Research Ethics Committee, Walter and Eliza Hall Institute of Medical Research, Ethical Committee of Pirkanmaa Hospital District in Finland, and the College of Medicine Research and Ethics Committee, University of Malawi. Rabbit immunoglobulins (IgGs) developed against human red blood cells (RaH; Cappel) were used as a commercial positive control at a final concentration of 90 µg/ml. Pooled plasma from malaria-naïve individuals residing in Melbourne (malaria naïve plasma or pooled MC) was obtained from volunteer blood donations from Australian Red Cross Lifeblood (agreement 23-11VIC-13; approved by Melbourne Health Human Research Ethics Committee 2017.319). Donor informed consent included acknowledgement that the blood may be used for research. It was used as the negative control, and phosphate buffered saline (PBS) was used as the no-antibody control to differentiate between opsonic and non-opsonic phagocytosis. All plasmas were used at a final dilution of 1:10.

Whole blood samples

Heparinised whole blood was collected following informed consent from volunteers, as approved by the Melbourne Health Human Research Ethics Committee.

Parasite cell lines

Red blood cells (RBCs) infected with the placental binding parasite line CS2 or a CS2 skeleton binding protein 1 knockout²¹ or IT4VAR19 expressing the it4var19 var gene, a PfEMP1 variant that binds to endothelial protein C receptor through a DC8 domain cassette²², were cultured as previously described and maintained in a gaseous mixture (5% CO₂, 1% O₂ and 94% N₂) in an incubator at 37 °C²³. The IT4VAR19 EPCR-binding parasite isolate was regularly phenotyped for its expression of the specific PfEMP1 (or DC8)²⁴ using antiserum against IT4VAR19-IEs developed in rabbits²⁵. The cultures were synchronized by sorbitol treatment²⁶ and regularly selected for knob expression by gelatin flotation²⁷. Cell cultures were tested for mycoplasma negativity (MycoAlert Kit, Lonza, Mount Waverley, Australia) as per manufacturer’s instructions). Mature trophozoite stage IEs were isolated by Percoll® density gradient centrifugation (GE Healthcare USA)²⁸. Purity by microscopy was at least 95%. Purified IEs were labelled with dihydroethidium (DHE) (Sigma Aldrich) 25 μg/ml diluted 1:400 for 30 min in the dark at RT and washed to remove excess DHE. The final concentration of IEs was adjusted to 1.65 × 107/ml.

Whole blood

Human blood was collected from healthy malaria-naïve adult volunteers from Melbourne into lithium heparin tubes (Beckton, Dickinson and Company, USA), diluted 1:1 with prewarmed RPMI-1640 (Gibco) and plated at 25 µl/well in duplicate in 96-well U-bottom plates (Corning).

Antibody-Dependent Phagocytosis by neutrophils and monocytes

Ninety-six well U bottom plates were precoated with 1% bovine serum albumin (BSA) (Sigma Aldrich) in PBS at RT for one hour. The coating solution was discarded, and heat-inactivated human plasma samples were added for opsonisation along with 30 µl of DHE-labelled IEs (concentration 1.65 ×10⁷/ml). The plates were gently tapped to mix the IEs with plasma and incubated in the dark for one hour at RT. The IEs were then washed three times with RPMI-1640 with 25 mM HEPES and centrifuged at 350 x g for 5 min at RT. Opsonised IEs were resuspended in 50 µl of RPMI-1640 and 25 µl/well were added to the diluted human blood prepared in a separate 96-well U bottom plate to reach a final dilution of 1:4 for whole human blood. The neutrophils and monocytes in blood were allowed to phagocytise opsonised IEs at 37 °C in a 5% CO₂ incubator for one hour. Phagocytosis was stopped by centrifugation at 350 x g for five minutes at 4 °C.

The cell pellets were labelled with fluorescent antibodies against leucocyte cell membrane markers. These included 1:100 FITC antihuman CD14 (BioLegend Cat no. 301804), 1:200 PECy7 antihuman CD16 (BD Biosciences cat no. 557744), 1:800 BV421 antihuman CD66b (Beckton Dickinson Cat no. 562940), and 1:100 FITC antihuman CD45 (BD Biosciences cat no.11-0459-42). All antibodies were diluted in cold RPMI-1640, and 50 µl of the antibody cocktail was added per well. Following incubation on ice in the dark for 30 min, the plates were spun at 350 x g for 5 min at 4 °C and the supernatants were discarded. Erythrocytes were lysed by adding 100 µl of 1X FACS lysing solution (BD BioSciences, cat no. 89882) diluted in sterile distilled water at 4 °C for 10 min. The lysis step was repeated once to enrich the leucocyte population and facilitate cell acquisition. Upon lysis, the plates were fixed with cold 2% paraformaldehyde (PFA) in PBS for 10–15 min at RT. The plates were washed (350 x g, five minutes at RT) and resuspended in cold FACS buffer (containing 2% FBS, 2 mM EDTA, and 0.02% sodium azide in PBS) until acquired by a flow cytometer.

Phagocytosis was measured by gating on neutrophils and monocytes using forward scatter (FSC) and side scatter (SSC) parameters. All samples were run in duplicate, and duplicates were averaged. Assays were repeated with three different whole blood donors. For each donor, the percentages of neutrophils or monocytes positive for DHE in the duplicates were averaged. The mean percentage phagocytosis of the three donors for each cell type was used.

Flow cytometry

We utilised the differences in light scatter properties of RBCs and leucocytes under blue SSC-A (488 nm) and violet light SSC-A (405 nm) to separate the cell types. Leucocytes were identified using fluorescently labelled antibodies against CD45 (leucocyte common antigen), and specific populations were identified using the fluorescent antibodies described above.

Image stream analysis

DHE-labelled IEs opsonised with immune plasma, malaria naive plasma, or unopsonised IEs were incubated with prediluted whole blood. Following phagocytosis, the RBCs were lysed, and the leucocytes were labelled with fluorescent antibodies against cell surface markers for neutrophils and monocytes as described above. Two thousand five hundred leucocytes in focus were acquired using an Amnis ImageStream flow cytometer (Amnis) based on CD45 positivity. CD45 was used as a standard leucocyte membrane marker to demonstrate whether DHE-labelled parasites were located within leucocytes.

Complement and complement inhibition

To inhibit the complement pathway, we used compstatin, a potent C3 inhibitor that selectively inhibits C3 activity of complement cascade²⁹ and a lipocalin protein, OmCI (Ornithodoros moubata Complement Inhibitory protein, 1 µM), that specifically binds to C5, inhibiting the downstream complement cascade³⁰.

Whole blood was prediluted at a volume ratio of 1:1 in RPMI-1640 and compstatin was used at a range of concentrations from 200 μM-1.6 μM. Whole blood was incubated at RT for 15 min with compstatin 200 μM or 1 μM OmCI before being used in ADP assays.

Stimulation of whole blood for phagocytosis

Two stimulants were used, TNFα and C5a. For TNFα, we used ten-fold dilutions from 10 ng/ml to 0.1 ng/ml, while for C5a, we used concentrations ranging from 1–500 nM at semi-log intervals. After selecting an optimal concentration of C5a and TNFα for whole blood priming, we stimulated prediluted whole blood with either C5a, TNFα, or both C5a and TNFα together to investigate the effect of whole blood priming on ADP of IEs. The stimulations were carried out at RT.

ELISA for antibody to VAR2CSA

Antibody to full-length VAR2CSA (gift from Morten Nielsen and Ali Salanti, University of Copenhagen) was measured by ELISA as described¹² with the following modifications. Plates were coated with VAR2CSA at a final concentration of 0.5μg/mL in PBS. Plasma samples were diluted 1/200. The detection antibody was goat anti-human IgG HRP (Millipore AP112P). Antibody levels were expressed as arbitrary units, calculated from raw absorbance readings derived against a standard curve made using serial doubling dilutions of PPS starting at a dilution of 1 in 250.

Statistics and reproducibility

The data obtained from flow cytometry were analysed using FlowJo and exported to Microsoft Excel. The analyses were conducted, and graphs were generated in GraphPad Prism.

All phagocytosis assays were run in duplicate, and the percentages of neutrophils or monocytes positive for DHE in the duplicates were averaged. Assays were repeated with two or three different whole blood donors. For comparisons of clinical samples, three whole blood donors were used and the mean percentage phagocytosis for each cell type was obtained. Phagocytosis levels by groups were compared using the Mann-Whitney U test. For paired samples, the Wilcoxon matched pairs rank test was used. Spearman’s correlation was used to compare phagocytosis between donors.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.