https://doi.org/10.5061/dryad.fttdz0930

Description of the data and file structure

# Dataset: Protective antibodies target cryptic epitope unmasked by cleavage of malaria sporozoite protein

[Access this dataset on Dryad]( [https://doi.org/10.5061/dryad.fttdz0930)

#Dataset file formats:

We have submitted our raw dataset on the characterization of the monoclonal antibodies in an Excel file (‘Dryad_dataset_adr0510_Science.xlsx’). Each sheet within the Excel file is labelled with a unique figure number and contains the data relevant to the figure by which it is labelled. Within each sheet, datapoints are entered under the relevant sub-figure number. We have also submitted the isothermal titration calorimetry (ITC) dataset as a compressed zip folder (‘ITC_fig._S8A_raw_data.zip’). Each .itc file within the folder contains data for an individual replicate experiment relevant to the experimental condition by which it is labelled.

# Descriptions

## Methods and analyses:

* Data entries under sub-figure numbers Fig 1A, 1B, 1C, 1D, 1E, 2C, 3D, 3E, 5A, 5B, S1A, S1C, S1D, S1E, S4D, S5A, S6, S9B, S9C, S9D, S10 were derived from flow cytometry measurement of the binding of plasma-derived polyclonal antibodies or recombinant monoclonal antibodies to the surface of whole sporozoites or beads coated with recombinant antigens

* Data entries under sub-figure numbers Fig 2D, 2E, S2C, S8B were derived from quantification of bioluminescence signals from mouse livers as measured by live imaging microscopy

* Data entries under sub-figure numbers Fig 3B and S4A were derived from ELISA measurement of the binding of recombinant monoclonal antibodies to plates coated with sporozoites or recombinant antigens

* Data entries under sub-figure number Fig 5C were derived from meso-scale discovery ELISA measurement of the binding of recombinant monoclonal antibodies to plates coated with R21 vaccine construct

* Data entries under sub-figure number 5D were derived from quantitative real-time PCR (qRT-PCR) analysis of Pf 18S ribosomal RNA levels in the blood samples of mice

* Data entries under sub-figure number S3B were derived from microscopy quantification of fluorescently labelled trails produced when sporozoites move across a solid surface

* Data entries under sub-figure number S3C were derived from quantitative real-time PCR (qRT-PCR) analysis of Pf 18S ribosomal RNA levels in infected primary hepatocyte cultures

* Data entries under sub-figure number S5D were derived from mass spectrometry analyses of Pf-CSP peptides

* Data entries under sub-figure number S8A and S8B were derived from isothermal calorimetry analyses of binding interaction between antibodies and Pf-CSP peptides

* Data entries under sub-figure number S9A were derived from bioinformatic analysis of naturally occurring polymorphisms within the PfCSP sequence of Plasmodium falciparum isolates

Files and variables

File: ITC_fig._S8A_raw_data.zip

Description: We have submitted the isothermal titration calorimetry (ITC) dataset as a compressed zip folder (‘ITC_fig._S8A_raw_data.zip’). Each .itc file within the folder contains data for an individual replicate experiment relevant to the experimental condition by which it is labelled. 

File: Dryad_dataset_adr0510_Science.xlsx

Description: We have submitted our raw dataset on the characterization of the monoclonal antibodies in an Excel file (‘Dryad_dataset_adr0510_Science.xlsx’). Each sheet within the Excel file is labelled with a unique figure number and contains the data relevant to the figure by which it is labelled. Within each sheet, datapoints are entered under the relevant sub-figure number. Please see variables below for more information.

Variables

* Naturally exposed cohort: Individuals living in malaria endemic region (Mali) and exposed to the parasite via mosquito bite

* Vaccinated cohort: Individuals living in non-malaria endemic region (USA) and exposed to the parasite via vaccination with irradiated sporozoites

* Donor reference: Identifier unique to each study participant

* IgG positive sporozoites, (%): Percentage of the sporozoite population on which IgG antibody binding was detected

* PfCSP: Plasmodium falciparum circumsporozoite protein

* PfSPZ: Plasmodium falciparum sporozoite

* Unblocked: Samples incubated with buffer

* rCSP blocked: Samples incubated with recombinant PfCSP

* Age: Age of individual in years

* PfSPZ Negative: Supernatants for which reactivity to Pf sporozoites was not detected

* PfSPZ Positive/rPfCSP Positive: Supernatants for which reactivity to Pf sporozoites was detected, but reactivity to recombinant CSP was not detected

* PfSPZ Positive/PfCSP Negative: Supernatants for which reactivity to both Pf sporozoites and recombinant CSP was not detected

* Number of wells: Number of wells for which the respective supernatant reactivity profile was recorded

* Median fluorescence intensity, MFI: Median signal intensity of the respective fluorophore (or fluorophore-labelled antibody) as measured by flow cytometry, taken as an index of mAb binding

* Pf-WT: P. falciparum wild-type sporozoites

* Pb-WT: *P. berghei *wild-type sporozoites

* Pb-PfCSP: Transgenic P. berghei sporozoites expressing P. falciparum CSP

* Liver burden: Readout of signal intensity from bioluminescent parasites (total flux, photons/second), taken as an index of parasite load in liver

* Max burden: Condition for which the highest liver burden is expected as animals were inoculated with sporozoites but did not receive mAb prophylaxis

* Naïve: Condition for which the lowest liver burden is expected as animals were not inoculated with sporozoites

* Neutralization: Percentage by which the indicated treatment reduces liver parasite load versus the naive condition

* Optical Density, OD: Chemiluminescent signal intensity measured during enzyme linked immunosorbent assay (ELISA), taken as an index of mAb binding

* Titration curve: Plot of the measured antibody binding signal versus the antibody concentration tested

* Area under curve, AUC: Total area enclosed by the titration curves of antibody binding, taken as an index of the magnitude of mAb binding

* Fluorescence-activated cell sorting (FACS) plots: Scatter plots showing the distribution of each event acquired versus the intensity of the relevant binding signal measured by flow cytometry

* Sporozoite motility: Locomotion of sporozoites as measured by microscopy

* Untreated: Condition which did not receive mAb treatment

* Isotype: A negative control condition in which the primary antibody used is of the same class and type of the test antibody but lacks specificity to the antigen of interest. Included to estimate background signal due to non-specific binding.

* Relative peak height: The height of a specific peak on a plot of mass-to-charge ratios (m/z) wherein the ions present in a sample are plotted against their intensities. Plotted data is derived from mass spectrometry analyses and relative peak height is interpreted as the relative abundance of the respective species.

* Percent converted (%): Calculated portion (%) of the sample that has undergone conversion of its N-terminal glutamine residue to a pyroglutamate residue

* Isothermal calorimetry, ITC: Quantitative method to determine the heat change associated with the binding interaction between the antigen (peptide) and relevant antibody of interest, used to estimate affinity

* Affinity: Strength of the binding interaction between the peptide and relevant antibody

* Fragment antigen binding, Fab: Generated antibody fragment that contains only the antigen binding site. The Fab consists of only one constant and one variable domain from each of the heavy and light chains, and lacks the fragment crystallizable region (Fc) portion of the antibody

* Dissociation constant, Kd: Measurement of the binding affinity between the peptide and relevant antibody

* Variant: Parasite isolate with a specific amino acid residue identity at the indicated position

* Mutation: A change in the amino acid residue sequence versus the consensus sequence of the Plasmodium falciparum 3D7 strain

* Substitution: Occurrence of one or more amino acid residue being replaced by another residue at the indicated position

* Fold change: Calculated ratio of the signal intensity (measured by flow cytometry) for binding to the peptide with the wild-type amino acid sequence versus the binding to the sequence containing the indicated mutation

## Sharing/Access information:

* Mass spectrometry raw data may be accessed at https://www.ebi.ac.uk/pride/login by logging in with these credentials: Username: reviewer_pxd052186@ebi.ac.uk; Password: LJKcpuX0

* Data related to structural analyses can be found at the Protein Data Bank (https://www.rcsb.org/) with accession numbers: 9C79 for MAD21-101, 9C7D for MAD22-38 and 9C7F for MAD24-01

Code/software

Microsoft Excel or a free-access version such as from OpenOffice is required to read the data in the Excel file ‘Dryad_dataset_adr0510_Science.xlsx’. The isothermal titration calorimetry data files with the ‘.itc’ file extension are readable text files and can be read with TextEdit or Notepad software.