Ethics Statement- For parasite culture, heparinized venous blood was collected from healthy donors after a written consent. Blood was collected following the Institutional ethical guidelines. Ethics committee of All India Institute of Medical Sciences, New Delhi, had approved the study via approval number IECPG-532/26.10.2016.
Materials- RPMI 1640, hypoxanthine, penicillin-streptomycin, Foetal Calf Serum, glutamine, glutaraldehyde, HBS-EP buffer (degassed and ready to use 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) (GE Healthcare, Piscataway, NJ, USA), Matchmaker Gold Systems (Clontech, Palo Alto, CA, USA), and Trypsin (Promega Corporation, Madison, Wisconsin, USA) were commercially obtained. Recombinant histidine-tagged PvTRAgs, bacterial (Desulfovibrio desulfuricans) thioredoxin, and human Basigin were available in the lab from previous studies (6, 9, 11).
Solid phase binding assay - A 96 well microtiter plate was coated with 50 nM of histidine-tagged individual PvTRAg or histidine-tagged bacterial thioredoxin in carbonate buffer (pH 9.6) and incubated overnight at 4oC. The coated ELISA plate was blocked with 5% BSA in PBS for 2 h at 37oC. The different concentration of histidine-tagged recombinant Basigin (0–3.2 µM) was added to these wells and plate was incubated for 2 h at 37oC. After washing, the plate was developed with polyclonal anti-Basigin antibodies as described earlier (8).
Surface Plasmon Resonance (SPR) – This assay was done on Autolab Esprit instrument (Eco Chemie, Utrecht, The Netherlands) at 25ºC, using HBS buffer (10 mM HEPES + 100 mM NaCl, 2 mM EDTA, pH 7.4). The recombinant Basigin (1 µM) was immobilized on CM5 sensor chip using Amine-coupling method. Kinetic analysis was then performed by flowing different concentration of the analyte (PvTRAg, PvTRAg36, PvTRAg33.5, PvTRAg35.2, PvTRAg69.4, PvTRAg34, or PvTRAg38) on the immobilized Basigin as well as reference flow cell, followed by regeneration with 50 mM NaOH at the end of each cycle. The association kinetics was monitored for 400s followed by dissociation for the next 100s. Reference-subtracted sensorgrams were analyzed using in built software in Autolab Esprit Instrument. The binding constant, KD, was calculated as kd/ka using data analysis software, and kinetic rate constants were determined by fitting the corrected response data to a simple 1:1 Hill-Langmuir binding isotherm model.
Plasmodium falciparum culture and growth inhibition assay- Growth inhibition assay was performed as described by Persson et al (12). Briefly, the P. falciparum 3D7 strain was cultured in complete RPMI 1640 medium containing, 0.5 g/L Albumax I 27.2 mg/L hypoxanthine, and 2 g/L sodium bicarbonate, using O+ human erythrocytes (4% hematocrit) under mixed gas (5% O2, 5% CO2 and 90% N2). Parasite culture was synchronized by sorbitol treatment and synchronous culture at late schizont stage with parasitemia of 1% were treated with different concentration (0–20 µM) of the PvTRAg, PvTRAg33.5, PvTRAg35.2, PvTRAg34, PvTRAg36, and PvTRAg69.4, PvTRAg38, or PvTRAg38.7 in a 96-well culture plate in triplicate. Uninfected erythrocytes, infected erythrocytes alone and infected erythrocytes with PBS were taken as controls. Parasites were maintained for 40 h and stained with ethidium bromide. One hundred thousand total events were acquired per sample, using Cell Quest software on a FACS Caliber flow cytometer (Becton Dickenson Biosciences, Palo Alto, CA, USA).