Figures and data
General workflow.
IgG+ memory B cells from Donor A were sorted individually regardless of their specificity, at one cell per well. Cells were further cultured in activation medium with CD40L-expressing feeder cells and cytokines to induce antibody secretion. Supernatants were tested for antibody binding to the sexual stage of the parasite through screening using a gamete extract ELISA. Memory B cells from wells displaying reactivity were selected for Ig genes amplification, followed by cloning and production of the corresponding antibody.
Characterization of the panel of isolated monoclonal antibodies.
(A) Percentage of binding to wild type (WT) and Pfs48/45 knock-out (KO) gametes in SIFA, in a heatmap format. The experiment was performed in duplicate and three different monoclonal antibody concentrations were tested (100 µg/ml, 5 µg/ml and 1µg/ml). GMT: gamete. (B) TRA of the mAb panel in SMFA: seven mAbs demonstrated TRA above 80% (red dotted line) when tested at 500 µg/ml in SMFA. For TRAbs, experiments were run in duplicates and bars are estimates of the mean and error bars represent the 95% confidence intervals. TRAbs were also tested at 100 µg/ml. (C) Reactivity of the monoclonal antibody panel against gametocyte extract in Western blot, in non-reducing conditions. Antibodies are classified depending on the antigen recognized: Pfs48/45, Pfs230, or no antigen identified. TB31F is an anti-Pfs 48/45 monoclonal antibody, RUPA-96 is an anti-Pfs230 monoclonal antibody, and VRCO1 is an anti-HIV monoclonal antibody (negative control). (D) Reactivity of the monoclonal antibody panel against full-length Pfs48/45 at 30 µg/ml in ELISA. (E) B1C5K and B1C5L binding to various Pfs48/45 domains in ELISA, at 10 µg/ml. (F) B2C10L binding to Pfs230 CMB domain in ELISA, at 10 µg/ml.
B1E11K binds repeat peptides.
(A) B1E11K binding to recombinant fragments of Pf proteins displayed on a microarray. (B) B1E11K binding to several recombinant proteins in Western blot, in non-reducing conditions. (C) Sequences of the peptides tested for binding. Peptides were N-terminally linked to a biotin moiety using aminohexanoyl (Ahx) spacers. (D) B1E11K binding in ELISA to a panel of peptides.
Binding Characteristics of RESA peptides to B1E11K.
(A) Various peptides based on the EENV repeat region were designed and conjugated to a biotin-AHX-AHX moiety (AHX = ε-aminocaproic acid). EC50 values obtained from ELISA experiments utilizing various EENV repeat peptides with (B) B1E11K mAb or (C) B1E11K Fab. Error bars represent standard deviation. Biolayer interferometry experiments utilizing immobilized (D) RESA 10AA peptide or (E) RESA P2 (16AA) peptide dipped into B1E11K Fab. Representative isothermal titration calorimetry experiments in which B1E11K Fab was injected into (F) RESA 10AA peptide or (G) RESA P2 (16AA) peptide. (H) Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) of a solution of B1E11K Fab incubated with RESA P2 (16AA) peptide in a 6:1 molar ratio. The predicted molecular weight of the B1E11K Fab and RESA P2 peptide are 46.9 kDa and 2.5 kDa respectively. The shaded region indicates the fractions collected used for negative stain electron microscopy. (I) A nsEM map reconstruction which permits the fitting of two B1E11K Fabs (Fab A and Fab B).
ITC thermodynamics and binding affinity of B1E11K Fab to RESA peptides.
Structure of the B1E11K Fab and RESA P2 (16AA) peptide complex.
(A) The overall architecture of the B1E11K:RESA P2 (16AA) peptide complex. (B) The electrostatic potential of the surface of the B1E11K Fabs. (C) Electrostatic interactions occurring with glutamate residues of the RESA P2 (16AA) peptide. Residues that have undergone somatic hypermutation (SHM) are marked with an asterisk. Salt bridges are shown as dashed yellow lines and hydrogen bonds as dashed black lines. (G) Hydrogen bonding interactions through the asparagine residues of the RESA P2 (16AA) peptide are shown as black dashed lines. (E) Variable heavy (VH) and variable kappa (Vκ) residues involved in homotypic interactions are shown as sticks. (F) The first interaction interface and (G) second interface. Residues that have undergone somatic hypermutation (SHM) are marked with an asterisk. Electrostatic interactions are presented as dashed lines and coloured as done previously.
