Umbilical cord blood sampling
Cord blood samples were acquired in 10-ml heparin tubes (BD Vacutainer®, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). All relevant guidelines and regulations were followed, and all experimental protocols involving human samples were approved by the Kangwon National University Hospital Ethical committee (IRB No. KNUH-B-2021-06-034). Written informed consent was obtained from all subjects.
Recombinant protein expression and purification
All recombinant proteins were produced using the mammalian human embryonic kidney 293E (HEK293E) cell expression system as described elsewhere . For plasmid preparation, pTT3 and pTT5 containing an appropriate exogenous signal peptide with intended C-terminal tags were generated. A group of reticulocyte receptor proteins and parasite ligand proteins were incorporated with Fc- and His-tags, respectively. The full-length ectodomain was chosen for expression, except for P. vivax reticulocyte binding protein 2b (PvRBP2b, residues 169 to 813), P. vivax GPI-anchored micronemal antigen (PvGAMA, residues 408 to 589), PvDBP (region II, residues 194 to 521), P. vivax merozoite surface protein 1 paralog-19 (PvMSP1P-19, residues 1751 to 1834), and DARC (CD234, residues 1 to 63). The complementary DNA (cDNA) of the intended sequences was codon-optimized for expression in mammalian cells using GeneArt (https://www.thermofisher.com/order/geneartgenes/projectmgmt) and then chemically synthesized by Twist Bioscience (South San Francisco, CA, USA). HEK cells were maintained in suspension in Gibco FreeStyle™ F17 Expression Medium (Life Technologies Corp., Grand Island, NY, USA) at 37 °C under 70% humidity and 8% CO2 in an orbital shaking incubator at 120 revolutions per minute. Twenty-four hours before transfection, fresh F17 medium was used to seed cells to a final density of 6 to 8 × 105 cells/ml. For each transfection, the expression plasmid was mixed with polyethyleneimine MAX transfection reagent (Polysciences, Warrington, PA, USA) before transfection into HEK cells. To produce biotinylated proteins, D-biotin (Sigma-Aldrich, St. Louis, MO, USA) and a plasmid encoding a secreted form of Escherichia coli biotin ligase (BirA) were added during transfection. After 5 days of incubation post-transfection, culture supernatants containing protein were collected for protein purification. His-tagged proteins were purified using Ni–NTA agarose (QIAGEN, Hilden, Germany) and a Poly-Prep® chromatography column (Bio-Rad, Hercules, CA, USA). HiTrap® Protein G HP (Sigma-Aldrich) was used for the purification of Fc-fusion proteins. The purified proteins were subjected to buffer exchange with HEPES-buffered saline (HBS) and concentrated using a 30-kDa Amicon® Ultra-15 Centrifugal Filter (Sigma-Aldrich).
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE) and western blotting
His- and Fc-tagged recombinant proteins were separated by 13% SDS‒PAGE. When needed, a reducing agent was added to the sample to induce reducing conditions. The gels for SDS‒PAGE analysis were stained with Coomassie brilliant blue (Sigma-Aldrich), while others were used for protein blotting onto polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA), after which the membranes were blocked with 5% skim milk for 1 h. The membranes containing His-tagged protein were then incubated with an anti-Pena-His antibody (1:2000) (LI-COR® Biosciences, Lincoln, NE, USA) and then with a secondary IRDye® goat anti-mouse (1:10,000) antibody (LI-COR® Biosciences), while only goat anti-human antibodies (1:5000) were used for Fc-fusion proteins (LI-COR® Biosciences). Data analysis was performed with an Odyssey infrared imaging system and software (LI-COR® Biosciences).
Protein‒protein interaction screening by enzyme-linked immunosorbent assay (ELISA)
The biotinylated proteins were normalized on a streptavidin-coated plate with mouse anti-rat CD4 monoclonal antibody (clone: OX68, Novus Biologicals™, Centennial, CO, USA). After normalization, 100 µl of each biotinylated protein was incubated for 1 h at room temperature (RT) and then washed with phosphate-buffered saline with 0.1% Tween 20 (PBS-T) and PBS. Then, 1 μg of Fc-fusion protein in PBS-T was added, and the mixture was incubated for 1 h with shaking. Following washing steps with PBS-T, the plate was allowed to react with a goat anti-human Fc-specific alkaline phosphatase antibody (Sigma-Aldrich, 1:5000 in PBS-T) for 1 h at RT. After washing with PBS-T, 100 µl of 1-Step™ p-nitrophenyl phosphate disodium salt (PNPP; Thermo Scientific, MA, USA) solution was added. The plate was incubated for 30 min at RT, and then the absorbance was measured at 450 nm.
Reticulocytes were enriched from umbilical cord blood using a 19% Nycodenz solution (Axis-Shield, Oslo, Norway) in high-KCl buffer with gradient centrifugation as mentioned previously . Briefly, fresh cord blood was washed twice with incomplete Roswell Park Memorial Institute (RPMI) 1640 medium, and white blood cells were removed by a non-woven fabric (NWF filter (ZhiXing Bio S&T Co. Ltd., Bengbu, China). After centrifugation, the packed cells were resuspended in high-KCl buffer (115 mM KCl, pH 7.4) and then incubated at 4 °C for 3 h with rotation. Each of 5 ml of the red blood cell (RBC)-high-KCl buffer mixture was overlaid on Nycodenz solution (19%, 3 ml) in a 15-ml tube. After centrifugation at 3000×g for 30 min without braking, the enriched reticulocytes in the interface layer were collected and washed three times with incomplete RPMI 1640 medium. The purity of reticulocytes was evaluated using thin blood smears with new methylene blue stain using light microscopy and thiazole orange (TO) (Becton Dikinson, San Jose, CA, USA) staining with flow cytometric analysis. A total of 100,000 events were obtained from each sample using a fluorescence-activated cell sorting (FACS) Accuri™ C6 Flow Cytometer (Becton Dikinson, Mansfield, MA, USA).
Flow cytometric analysis of CD36 expression on erythrocytes
Samples for flow cytometric experiments were prepared as described previously with slight modifications . The enriched reticulocytes were washed three times with PBS containing 1% bovine serum albumin (BSA), and then 1 million RBCs were incubated with 5 μg of PE/Cyanine7 anti-human CD36 (BioLegend, San Diego, CA, USA) or the same amount of PE/Cyanine7 Mouse IgG2 α,κ isotype control antibody as a control (BioLegend) for 30 min at 4 °C in the dark. After washing three times with PBS-1% BSA, the cells were incubated with TO for 30 min at RT. A total of 100,000 events were obtained per sample using a FACS Accuri™ C6 Flow Cytometer.
Flow cytometry-based erythrocyte binding assay
The reticulocyte binding assay was performed as mentioned previously . Briefly, 1 × 106 cells were incubated with a gradient concentration of purified His-tagged recombinant PvMTRAP for 3 h at 25 °C. The PvRBP2b-His and glutathione S-transferase (GST)-His proteins were used in parallel as positive and negative controls, respectively. After incubation, the samples were washed with 200 µl of PBS-1% BSA twice and then incubated with 5 µg of APC anti-His Tag monoclonal antibody (BioLegend, San Diego, CA, USA) in 100 µl staining volume for 1 h at 4 °C in the dark. The samples were washed three times with PBS-1% BSA and incubated with TO for 30 min at 25 °C. A total of 100,000 events were counted per sample using a FACS Accuri™ C6 Flow Cytometer. All flow cytometric data were analyzed by FlowJo software (TreeStar, Ashland, OR, USA).
Octet® bio-layer interferometry analysis
The interactions between His-tagged P. vivax ligands and Fc-tagged reticulocyte receptors were assessed using protein G (ProG) biosensors in the Octet RED96 system (ForteBio Inc., Menlo Park, CA, USA). The Fc-tagged reticulocyte receptors were immobilized on the ProG biosensor, and then a twofold serial dilution of parasite ligands in HBS was loaded into the biosensor to generate the kinetic parameters. The data were analyzed with Octet System Data Analysis Software ver. 7.0. (Sartorius, Göttingen, Germany).
The data were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2016 (Microsoft, Redmond, WA, USA). For comparison, paired Student’s t-tests were utilized, and a P-value of < 0.05 was considered to indicate significance.