Journal: Nature Communications

Article Title: Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9

doi: 10.1038/s41467-023-38509-2

Figure Lengend Snippet: a Buried surface area (BSA) contributions of individual residues to rsCSP binding in the L9 heavy chain. Sequence alignment to the IGHV3-33 germline gene shown below. b Same as in a, for the L9 light chain. c , d Structural details of NPNV binding. e NPNA 2 epitope structure in the NPNA-specific mAb 2243 (PBD 6O23), highlighting the two key CH-π interactions of germline-encoded aromatic residues (W52 H and Y94 L ) with the repeat prolines. f Same as in e, with X-ray structures of six NPNA-specific mAbs superimposed to highlight structural conservation. These six mAbs are shown in g . g Electrostatic surface potentials from L9 cryo-EM structure +/− peptide (upper left two panels) and X-ray structures of six other NPNA-specific mAbs bound to peptide; electrostatic potentials were calculated in PyMol . The PDB accession codes are in parentheses. K b : Boltzmann constant; T : temperature in kelvin; c e : electron charge in coulombs. Source data are provided as a Source Data file.

Article Snippet: L9 heavy and light chain sequences were synthesized and codon-optimized for mammalian expression and cloned into pHCMV3 by Genscript Inc.

Techniques: Binding Assay, Sequencing, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural basis of epitope selectivity and potent protection from malaria by PfCSP antibody L9

doi: 10.1038/s41467-023-38509-2

Figure Lengend Snippet: a Ribbon diagram of Fab B (cyan) and C (maroon); side chains of interacting residues are shown. b – d Structural details of key homotypic interactions. Dashed lines indicate specific contacts. e Buried surface area (BSA) contributions of individual residues to the homotypic interface in L9 light chain. Sequence alignment with F10 K and germline IGKV1-5 gene is shown below. f Same as in e , for L9 heavy chain, with sequence alignment to F10 H and germline IGHV3-33 gene. Source data are provided as a Source Data file.

Article Snippet: L9 heavy and light chain sequences were synthesized and codon-optimized for mammalian expression and cloned into pHCMV3 by Genscript Inc.

Techniques: Sequencing

Journal: Cell reports

Article Title: The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria

doi: 10.1016/j.celrep.2022.110367

Figure Lengend Snippet: (A) Ig heavy and light chain pairings matched to create normal mAbs (L9, F10, L9MRCA) and chimeric mAbs (L9HF10κ, F10HL9κ, D2HL9κ, D2HF10κ) used in this study. Each heavy and light chain’s divergence from germline is depicted. (B) Binding of various concentrations of indicated mAbs to rPfCSPFL (left), peptide 22 (NANPNVDPNANPNVD, middle), and (NANP)9 (right) measured by ELISA; optical density at 450 nm (OD450) is plotted. The NANP-preferring PfCSP mAb 317 and the anti-gp120 mAb VRC01 were included as positive and negative controls, respectively. (C) Binding response (nm) and apparent avidity (KDapp, nM) of indicated mAbs binding to peptide 22 determined through BLI. (D) Competition ELISA of indicated mAbs binding to rPfCSPFL and various concentrations of peptide 22 (rightmost bar) or variant peptides (other bars) where the indicated amino acid was mutated to alanine or serine. Area under the curve (AUC) is plotted. (E) SEC plots of indicated Fabs incubated with peptide 22 (Fabs alone, black peak; Fabs+pep22, blue peak). All data are representative of two independent experiments. See also Figure S2 and Table S1.

Article Snippet: L9 lineage PfCSP immunoglobulin heavy and light chain variable region sequences , GenScript , GenBank Accession Numbers MZ686952 – MZ686956.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Variant Assay, Incubation

Journal: Cell reports

Article Title: The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria

doi: 10.1016/j.celrep.2022.110367

Figure Lengend Snippet: (A) Left: binding of various concentrations of indicated mAbs to Pb-PfCSP-SPZ determined by flow cytometry; median fluorescence intensity (MFI) of mAb-bound SPZ is plotted. Mean and standard error of the mean (SEM) MFI values from four independent experiments are shown except for L9MRCA, which was from two experiments. Right: area under the curve (AUC) and SEM values calculated from the MFI plots. (B) Percentage (%) of SPZ undergoing the CSPR after incubation with 50 μg/mL of indicated mAb for 15 min. Bar graphs show the mean and SEM from six independent experiments. (C) Percentage of non-motile SPZ after incubation with 25 μg/mL of indicated mAb for 15 min. Bar graphs show the mean and SEM from two independent experiments. (B-C): P-values reflect comparisons between L9κ-containing mAbs and their F10κ-containing counterparts using Fisher’s exact test. (D) Reduction in parasite bioluminescence (total flux, photons/sec) in the livers of mice (n=15/group; data pooled from three independent experiments indicated by squares, circles, and triangles) mediated by 100 μg of indicated mAbs administered 2 hrs before IV challenge with 2,000 Pb-PfCSP-SPZ. P-values reflect comparisons between L9κ-containing mAbs and their F10κ-containing counterparts using the two-tailed Mann–Whitney test. (E) Survival curves of mice (n=7/group) challenged with five infected mosquito bites 24 hrs after passive transfer of 300 μg of indicated mAb.

Article Snippet: L9 lineage PfCSP immunoglobulin heavy and light chain variable region sequences , GenScript , GenBank Accession Numbers MZ686952 – MZ686956.

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Incubation, Two Tailed Test, MANN-WHITNEY, Infection

Journal: Cell reports

Article Title: The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria

doi: 10.1016/j.celrep.2022.110367

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: L9 lineage PfCSP immunoglobulin heavy and light chain variable region sequences , GenScript , GenBank Accession Numbers MZ686952 – MZ686956.

Techniques: Virus, Recombinant, Construct, SYBR Green Assay, Staining, Expressing, Crystallization Assay, Luciferase, Amplification, Software, In Vivo Imaging, Fluorescence, Microscopy