1. Immunology and Inflammation
  2. Microbiology and Infectious Disease

Target-agnostic identification of human antibodies to Plasmodium falciparum sexual forms reveals cross-stage recognition of glutamate-rich repeats

  1. Axelle Amen
  2. Randy Yoo
  3. Amanda Fabra-García
  4. Judith Bolscher
  5. William JR Stone
  6. Isabelle Bally
  7. Sebastián Dergan-Dylon
  8. Iga Kucharska
  9. Roos M de Jong
  10. Marloes de Bruijni
  11. Teun Bousema
  12. C Richter King
  13. Randall S MacGill
  14. Robert W Sauerwein
  15. Jean-Philippe Julien  Is a corresponding author
  16. Pascal Poignard  Is a corresponding author
  17. Matthijs M Jore  Is a corresponding author
  1. CNRS, Université Grenoble Alpes, CEA, UMR5075, Institut de Biologie Structurale, France
  2. CHU Grenoble Alpes, France
  3. Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Canada
  4. Department of Biochemistry, University of Toronto, Canada
  5. Department of Medical Microbiology, Radboud University Medical Center, Netherlands
  6. TropIQ Health Sciences, Netherlands
  7. Department of Immunology and Infection, London School of Hygiene and Tropical Medicine, United Kingdom
  8. Center for Vaccine Innovation and Access, PATH, United States
  9. Department of Immunology, University of Toronto, Canada
https://doi.org/10.7554/eLife.97865.3
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Cite this article
  1. Axelle Amen
  2. Randy Yoo
  3. Amanda Fabra-García
  4. Judith Bolscher
  5. William JR Stone
  6. Isabelle Bally
  7. Sebastián Dergan-Dylon
  8. Iga Kucharska
  9. Roos M de Jong
  10. Marloes de Bruijni
  11. Teun Bousema
  12. C Richter King
  13. Randall S MacGill
  14. Robert W Sauerwein
  15. Jean-Philippe Julien
  16. Pascal Poignard
  17. Matthijs M Jore
(2025)
Target-agnostic identification of human antibodies to Plasmodium falciparum sexual forms reveals cross-stage recognition of glutamate-rich repeats
eLife 13:RP97865.
https://doi.org/10.7554/eLife.97865.3
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5 figures, 3 tables and 6 additional files

Figures

Figure 1 with 1 supplement
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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. Figure was created with BioRender.

Figure 1—figure supplement 1
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Memory B cell (MBC) sorting and cell culture supernatant screening.

Gating strategy for agnostic MBCs sorting (A). Gamete extract (B) or gametocyte extract (C) ELISA for cell culture supernatant screening. Wells with signal above or close to positivity threshold (indicated in red) were selected for immunoglobulin variable genes amplification.

Figure 2
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Characterization of the panel of isolated monoclonal antibodies (mAbs).

(A) Percentage positive wild-type gametes and Pfs48/45 knockout (KO) gametes that also lack surface-bound Pfs230 in surface immunofluorescence assay, in a heatmap format (graded color scale: red for high percentage of binding, green for low percentage of binding). The experiment was performed in duplicate and three different mAb concentrations were tested (100 µg/ml, 5 µg/ml, and 1 µg/ml). (B) Transmission-reducing activity (TRA) of the mAb panel in standard membrane feeding assay (SMFA). For mAbs with >80% TRA at 500 µg/ml, experiments were run in duplicates and bars are estimates of the mean and error bars represent the 95% confidence intervals. mAbs with >80% TRA at 500 µg/ml were also tested at 100 µg/ml. Oocyst count data of the SMFA (Standard membrane feeding assay) experiments can be found in Figure 2—source data 1. (C) Reactivity of the mAb 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-Pfs48/45 mAb, RUPA-96 is an anti-Pfs230 mAb, and VRC01 is an anti-HIV mAb (negative control). Pfs48/45 and Pfs230 bands are indicated with a red arrow, antibodies with >80% TRA at 500 µg/ml are indicated with an asterisk (*). (D) Reactivity of the mAb panel at 30 µg/ml against full-length Pfs48/45 in ELISA. (E) B1C5K and B1C5L binding to various Pfs48/45 domains in ELISA, at 10 µg/ml. (F) B2C10L binding to Pfs230CMB domain in ELISA, at 10 µg/ml. Values in (D-F) are means from three technical replicates and error bars represent standard deviation. mAbs were considered positive when the absorbance was higher than the mean absorbance plus three standard deviations of seven negative mAbs, indicated by dashed lines.

Figure 2—source data 1

Raw standard membrane feeding assay (SMFA) data.

https://cdn.elifesciences.org/articles/97865/elife-97865-fig2-data1-v1.xlsx
Figure 2—source data 2

Original western blots.

https://cdn.elifesciences.org/articles/97865/elife-97865-fig2-data2-v1.zip
Figure 3 with 4 supplements
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B1E11K binds repeat peptides.

(A) B1E11K binding to recombinant fragments of Plasmodium falciparum (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.

Figure 3—source data 1

Raw microarray data.

https://cdn.elifesciences.org/articles/97865/elife-97865-fig3-data1-v1.xlsx
Figure 3—source data 2

Original western blot.

https://cdn.elifesciences.org/articles/97865/elife-97865-fig3-data2-v1.zip
Figure 3—figure supplement 1
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Further characterization of B1E11K.

(A) B1E11K binding to a panel of human self-proteins, single-stranded DNA (ssDNA) and lipopolysaccharide (LPS) in ELISA. 4E10 is a polyreactive anti-HIV monoclonal antibody (mAb) (positive control). (B) Immunoprecipitation of B1E11K mAb against gametocyte extract. A Native PAGE 3–12% Bis-Tris gel was used for protein separation followed by silver staining. VRC01, an anti-HIV mAb was used as a negative control. *: BSA.

Figure 3—figure supplement 2
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Sequences of the recombinant protein fragments in the microarray.
Figure 3—figure supplement 3
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Glutamic acid-rich repeats in RESA (A), RESA3 (B), LSA3 (C), and Pfs230 (D).

Sequences from UniProt database. 'EENVEE' repeats are highlighted in pink and 'EEVGEE' in green.

Figure 3—figure supplement 4
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Glutamic acid-rich repeats in Pf11.1.

Sequence from UniProt database. 'EELVEE' are highlighted in light blue, 'EEVVEE' in dark blue, and other repeats following the 'EEXXEE' pattern in yellow.

Figure 4 with 1 supplement
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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 (nsEM). (I) An nsEM map reconstruction which permits the fitting of two B1E11K Fabs (Fab A and Fab B).

Figure 4—figure supplement 1
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BE11K ELISA binding curves to RESA peptides.

Binding to RESA peptides in ELISA: for B1E11K mAb (A), three independent experiments; for B1E11K Fab (B), two independent experiments. Curves were used to calculate EC50s shown in Figure 4.

Figure 5 with 4 supplements
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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. Fab residues involved in electrostatic interactions with (C) residues 1–8 and (D) 9–16 of the RESA P2 peptide are shown as sticks. (E) 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. (F) Hydrogen bonding interactions through the asparagine residues of the RESA P2 (16AA) peptide are shown as black dashed lines. (G) Variable heavy (VH) and variable kappa (Vκ) residues involved in homotypic interactions are shown as sticks. (H) The first interaction interface and (I) second interface. Residues that have undergone SHM are marked with an asterisk. Electrostatic interactions are presented as dashed lines and colored as done previously.

Figure 5—figure supplement 1
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Composite omit maps of residues involved in inter-chain interactions.

Composite omit maps of (A) RESA P2 (16AA) peptide (red) which contain the repetitive elements. Composite omit maps of the residues of (B) B1E11K Fab A (heavy chain in green and kappa chain in light green) and (C) B1E11K Fab B (heavy chain in teal and kappa chain in light blue) that interact with the RESA P2 peptide. Composite omit map of residues in (D) B1E11K Fab A and (E) B1E11K Fab B involved in homotypic interaction interface (same coloring scheme).

Figure 5—figure supplement 2
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Buried surface area plots of B1E11K Fabs and RESA P2 (16AA) peptide interactions.

Bar graphs of the buried surface area of each residue in the (A) RESA P2 (16AA) peptide and both heavy and kappa chains of (B) B1E11K Fab A and (C) B1E11K Fab B. Kabat numbered CDRs are marked with bars.

Figure 5—figure supplement 3
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Buried surface area plots of B1E11K Fabs of residues buried at the homotypic interaction interface.

Bar graphs of the buried surface area of each residue of both the heavy and kappa chain of (A) B1E11K Fab A and (B) B1E11K Fab B. Kabat numbered CDRs are marked with bars.

Figure 5—figure supplement 4
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IgBLAST of B1E11K heavy chain and light chain.

Amino acid sequence alignments of the B1E11K heavy chain and light chain with the (A) IGHV3-7 and (B) IGKV3-20 loci. Residues that have undergone somatic hypermutation and partake in electrostatic interactions with RESA are highlighted in yellow. Residues that have undergone somatic hypermutation and take part in homotypic interactions (electrostatic) are highlighted in cyan. Residues that partake in both types of interactions are highlighted in green.

Tables

Table 1
Isothermal titration calorimetry (ITC) thermodynamics and binding affinity of B1E11K Fab to RESA peptides.
RESA 10AA peptide (n=2)RESA P2 peptide (n=3)
N1.0±0.22.1±0.1
KD (nM)78±1273±21
ΔG (kcal/mole)–9.7±0.3–9.8±0.3
ΔH (kcal/mole)–18.3±0.1–20.5±0.1
–TΔS (kcal/mole)8.6±0.210.7±0.3

  1. Error reported as standard deviation.

Table 2
Crystallography statistics.
CrystalB1E11K:RESA P2 (16AA) peptide
BeamlineAPS-23-ID-B
Wavelength (Å)1.0332
Space groupC 2 2 21
Cell dimensions
a, b, c (Å)78.7, 186.3, 131.5
α, β, γ (°)90, 90, 90
Resolution (Å)*40.0–2.56 (2.65–2.56)
No. molecules in ASU1
No. of observations236,070 (23,834)
No. unique observations31,520 (3067)
Multiplicity7.5 (7.8)
Rmerge (%)†14.5 (222.8)
Rpim (%)‡5.7 (85.5)
<I/σI>10.8 (1.0)
CC1/2 (%)99.7 (36.5)
Completeness (%)99.8 (98.5)
Refinement statistics
Reflections used in refinement31,512
Reflections used in R-free1575
Non-hydrogen atoms6781
Macromolecule6627
Water130
Heteroatom24
Rwork§/Rfree (%)21.5/24.5
Rms deviations from ideality 
Bond lengths (Å)0.002
Bond angle (°)0.48
Ramachandran plot 
Favored regions (%)96.0
Allowed regions (%)3.8
Ramachandran outliers (%)0.2
B-factors (Å2) 
Wilson B-factor65.5
Average B-factors89.8
Average macromolecule90.3
Average heteroatom83.1
Average water molecule61.0
  1. *

    Values in parentheses refer to the highest resolution bin.

  2. †

    Rmerge = Σhkl Σi | Ihkl, i -<Ihkl > | / Σhkl <Ihkl >.

  3. ‡

    Rpim = Σhkl [1/(N – 1)]1/2 Σi | Ihkl, i -<Ihkl > | / Σhkl <Ihkl>.

  4. §

    Rwork = (Σ | |Fo | − |Fc | |) / (Σ | |Fo |) – for all data except as indicated in footnote ¶.

  5. 5% of data were used for the Rfree calculation.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Plasmodium falciparum)NF54Radboud University Medical Center; Ponnudurai et al., 1989
Strain, strain background (Anopheles stephensi)Nijmegen Sind-Kasur strainRadboud University Medical Center, Ponnudurai et al., 1989
Genetic reagent (Plasmodium falciparum)Pfs48/45 knockoutRadboud University Medical Center; Dijk et al., 2001Pfs48/45 knockout in Pf NF54 background
Genetic reagent (Homo sapiens)Fibroblasts expressing CD40L 'L cells'Laboratory for Immunological Research, Schering-Plough; Garrone et al., 1995
Cell line (Homo sapiens)HEK293FThermo Fisher ScientificRRID:CVCL_6642
Cell line (Homo sapiens)Freestyle 293FThermo Fisher ScientificRRID:CVCL_D615
Biological sample (Homo sapiens)PBMCsRadboud University Medical Center; Stone et al., 2018
AntibodyAnti-human CD3 VioBlue (human monoclonal)Miltenyi#130-114-519Single B cell sorting (1:50),
Recombinant human IgG1
AntibodyAnti-human CD19 PE-Vio 770 (human monoclonal)Miltenyi#130-113-647Single B cell sorting (1:10),
Recombinant human IgG1
AntibodyAnti-human CD20 PE-Vio 770 (human monoclonal)Miltenyi#130-111-340Single B cell sorting (1:50),
Recombinant human IgG1
AntibodyAnti-human CD27 APC (human monoclonal)Miltenyi#130-113-636Single B cell sorting (1:10), Recombinant human IgG1
AntibodyAnti-human IgM PE (mouse monoclonal)Miltenyi#130-093-075Single B cell sorting (1:50),
Mouse IgG1
AntibodyAnti-human IgD PE (human monoclonal)Miltenyi#130-110-643Single B cell sorting (1:50),
Recombinant human IgG1
AntibodyAnti-human IgA PE (mouse monoclonal)Miltenyi#130-113-476Single B cell sorting (1:50),
Mouse IgG1k
AntibodyAnti-human IgG AP (goat polyclonal)Thermo Fisher Scientific#A18814ELISA (1:2000)
AntibodyAlexa Fluor 488 Goat Anti-Mouse IgG (goat polyclonal)Invitrogen#A11001SIFA (1:200)
AntibodyAnti-human IgG-HRP (goat polyclonal)Pierce#31412Western blot (1:5000), ELISA (1:60,000)
AntibodyAnti-Human IgG-TXRD (goat polyclonal)Southern Biotech#2040-07Microarray, (1:2000)
Recombinant DNA reagentVariable domains of heavy and light chains cloned into gamma1 HC, kappa LC, and lambda LC expression vectorsThis paper; Tiller et al., 2008Inserts are provided in Supplementary file 2
Recombinant DNA reagentpCDNA3.4_B1E11K (Fab)This paperInserts are provided in Supplementary file 2
PeptidePfs230 (P1)This paperBiotin-AHX-AHX-EEVG-EEVG-EEVG-EEVG-GG
PeptidePfs230 (P2)=RESA P2 (16AA)This paperBiotin-AHX-AHX-EENV-EENV-EENV-EENV-GG
PeptidePf11.1 (P3)This paperBiotin-AHX-AHX-EELV-EEVIP-EELV-EEFIP-GG
PeptidePf11.1 (VIP)This paperBiotin-AHX-AHX-EELV-EEVIP-EELV-EE
PeptidePf11.1 (VVP)This paperBiotin-AHX-AHX-EELV-EEVVP-EELV-EE
PeptideRESA 8AAThis paperBiotin-AHX-AHX-EENV-EENV
PeptideRESA 10AAThis paperBiotin-AHX-AHX-EENV-EENV-EE
PeptideRESA 12AAThis paperBiotin-AHX-AHX-EENV-EENV-EENV-
PeptideRESA 14AAThis paperBiotin-AHX-AHX-EENV-EENV-EENV-EE
Chemical compound, drugAqua LIVE/DEAD stainThermo Fisher Scientific#L34957
Chemical compound, drug293FectinThermo Fisher Scientific#12347500Tranfection reagent for mAb expression
Chemical compound, drugFectoproPolyplus#101000014Tranfection reagent for Fab expression
Chemical compound, drugssDNASigma#D8899-5MGPolyreactivity testing
Chemical compound, drugDisialoganglioside GD1αSigma#G2392-1MGPolyreactivity testing
Chemical compound, drugLipopolysaccharideSigma#L2630-10MGPolyreactivity testing
Chemical compound, drugTransferrinSigma#T3309-100MGPolyreactivity testing
Chemical compound, drugApotransferrinSigma#T1147-100MGPolyreactivity testing
Chemical compound, drugHemocyaninSigma#H7017-20MGPolyreactivity testing
Chemical compound, drugInsulinSigma#I2643-25MGPolyreactivity testing
Chemical compound, drugCardiolipinSigma#C0563-10MGPolyreactivity testing
Chemical compound, drugHistoneSigma#H9250-100MGPolyreactivity testing
Chemical compound, drugTosyl-activated beadsInvitrogen#14203For immunoprecipitation
Chemical compound, drugSAX biosensorsSartorius#18-5117For BLI experiments
Commercial assay, kitmRNA TurboCapture kitQIAGENCat# 72271

Additional files

Supplementary file 1

Genetic characteristics of isolated antibodies.

https://cdn.elifesciences.org/articles/97865/elife-97865-supp1-v1.docx
Supplementary file 2

Sequences of isolated antibodies.

https://cdn.elifesciences.org/articles/97865/elife-97865-supp2-v1.xlsx
Supplementary file 3

PDB validation report.

https://cdn.elifesciences.org/articles/97865/elife-97865-supp3-v1.pdf
Supplementary file 4

Interactions between B1E11K Fabs and RESA P2 (16AA) peptide.

https://cdn.elifesciences.org/articles/97865/elife-97865-supp4-v1.docx
Supplementary file 5

Homotypic interactions between B1E11K Fab A and Fab B.

https://cdn.elifesciences.org/articles/97865/elife-97865-supp5-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/97865/elife-97865-mdarchecklist1-v1.pdf

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  1. Axelle Amen
  2. Randy Yoo
  3. Amanda Fabra-García
  4. Judith Bolscher
  5. William JR Stone
  6. Isabelle Bally
  7. Sebastián Dergan-Dylon
  8. Iga Kucharska
  9. Roos M de Jong
  10. Marloes de Bruijni
  11. Teun Bousema
  12. C Richter King
  13. Randall S MacGill
  14. Robert W Sauerwein
  15. Jean-Philippe Julien
  16. Pascal Poignard
  17. Matthijs M Jore
(2025)
Target-agnostic identification of human antibodies to Plasmodium falciparum sexual forms reveals cross-stage recognition of glutamate-rich repeats
eLife 13:RP97865.
https://doi.org/10.7554/eLife.97865.3