A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

TEXT RESOURCE AVAILABILITY Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Robert Seder ( rseder@mail.nih.gov ).

Materials Availability

All unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Data and Code Availability

The heavy and light chain gene sequences of anti-PfCSP human monoclonal antibodies isolated in this study, along with the sequences of PCR primers used to amplify the antibody genes, were deposited in GenBank (Accession Numbers MT811859 – MT811914 ). Sequences of synthetic rPfCSP constructs created in this study were deposited in GenBank (Accession Numbers MT891160 – MT891178 ).

EXPERIMENTAL MODELS AND SUBJECT DETAILS Human clinical specimens

Clinical specimens were derived from malaria-naive, healthy adults (18–45 years of age) in the VRC 314 clinical trial ( https://clinicaltrials.gov/ ; NCT02015091 ) after obtaining written informed consent. Briefly, VRC 314 was a multi-institution, phase 1, open-label, dose-escalation trial with controlled human malaria infection (CHMI) that was designed to assess the safety, immunogenicity, and protective efficacy of the Sanaria PfSPZ Vaccine administered by intravenous or intramuscular injection ( Lyke et al., 2017 ). The Sanaria PfSPZ Vaccine is composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites derived from the NF54 strain ( Seder et al., 2013 ).

Mice Female 6- to 8-weeks old

B6(Cg)-Tyrc-2J/J albino mice and female 6- to 12-weeks old C57BL/6 mice were obtained from The Jackson Laboratory. Female 6- to 8-weeks old C57BL/6 mice and female 8-weeks old Swiss Webster mice were obtained from Charles River Laboratories. Female 8–9 month old FRG-huHep were obtained from Yecuris Corp. All mouse research was performed according to National Institutes of Health (NIH) guidelines for use and care of live animals approved by the institutional animal care and use ethics committees of the Vaccine Research Center (Animal Study Protocol VRC-17–702), Johns Hopkins University (Approved protocol permit no. MO18H419), Radboud University Medical Center (ADV103002016452), and Australia National University (A2016/17 & A2019/36).

Cell Lines

Expi293 and 293F cells used were from Thermo Fisher Scientific. The human hepatoma cell line HC-04 (MRA-965, deposited by Jetsumon Sattabongkot) ( Sattabongkot et al., 2006 ) was obtained through the Malaria Research and Reference Reagent Resource Center as part of the Biodefense and Emerging Infections Research Resources Repository. Sporozoites Transgenic P. berghei (strain ANKA 676m1c11, MRA-868) expressing full-length P. falciparum CSP and a green fluorescent protein/luciferase fusion protein (Pb-PfCSP-GFP/Luc-SPZ) were obtained as previously described ( Flores-Garcia et al., 2019 ). GFP-labeled P. berghei SPZ expressing PfCSP (Pb-PfCSP-GFP-SPZ) were generated by crossing the parental Pb-PfCSP-SPZ line ( Espinosa et al., 2017 ) with the previously described P. berghei -ConF parasite line that expresses GFP under the control of an HSP70 promotor ( Amino et al., 2008 ). Briefly, C57BL/6 mice were coinfected with Pb-PfCSP-SPZ and P. berghei -ConF at a ratio of 10:1. Anopheles stephensi mosquitoes were allowed to feed on the mice, and subsequently, sporozoites dissected from these mosquitoes were used to infect naive animals. Parasites expressing the GFP transgene were sorted from the blood of these mice by use of a FACSAria cell sorter and used to infect mice. Subsequently, the GFP + progeny was cloned, and the clones screened for the insertion of the PfCSP knock-in via measurement of anti-PfCSP antibody binding to progeny SPZ. P. falciparum NF54 sporozoites were isolated from an individual near Schiphol Airport (The Netherlands) ( Delemarre-van de Waal and de Waal, 1981 ). METHOD DETAILS Generation of junctional probe S02, a 43-residue structurally stabilized peptide mimicking the junctional epitope (MPESSSNPDCNANPNVDPNEDLIKKCEKINVPTEEIKKEIEEKK), was designed using Rosetta ( Chevalier et al., 2017 ) by modifying the junctional peptide 21 with flanking sequences to stabilize the peptide in a conformation that retains binding by the neutralizing junctional antibody CIS43, but which is unfavorable to binding by the poorly neutralizing junctional antibody CIS42 ( Kisalu et al., 2018 ). Sera from 14 protected subjects in group 6 of VRC 314 who received a total of three doses of radiation-attenuated PfSPZ (9.0 x 10 5 ) intravenously at weeks 0, 8, and 16 were screened for antibody titers against S02 using ELISA. A subject whose sera demonstrated the highest reactivity against S02 at the week 20 timepoint, 4 weeks after the last immunization, was chosen for memory B cell sorting and mAb isolation.

Production of recombinant PfCSP constructs

The amino acid sequence of PfCSP in the 3D7 clone of the NF54 isolate (PlasmoDB ID: PF3D7_0304600.1) was used to generate a codon-optimized synthetic gene for expression in mammalian cells (GenScript). The DNA construct corresponding to the full-length rPfCSP, in which the leader peptide residues 1–20 were replaced with a mammalian secretory signal peptide derived from the modified bovine prolactin (MDSKGSSQKGSRLLLLLVVSNLLLPQGVLA) and the glycosylphosphatidylinositol (GPI) anchor residues 376–397 were excluded, was cloned into a CMV/R-expression vector with a C-terminal AviTag, HRV3C-processing tag, and a 6X histidine tag. This construct, termed PfCSP_SAmut_C5S, encodes the N-terminal domain (with four amino acid mutations that removed processing sites and prevented dimerization upon solubilization to increase yield and facilitate consistent analyses), the central domain consisting of 38 NANP tandem repeats interspersed with 4 NVDP repeats, and the C-terminal domain. Truncated ( Oyen et al., 2018 ) and NVDP repeat mutant rPfCSP synthetic constructs were created in the same expression vector with identical N- and C-termini to full-length PfCSP_SAmut_C5S, expressed through transient transfection in 293F cells using the Freestyle 293F expression system (Thermo Fisher Scientific) at 37°C, 8% CO 2 for 6 days, and purified from culture supernatants through polyhistidine-tag affinity chromatography followed by size-exclusion chromatography on an ÄKTA ™ start (GE Healthcare). Monomer-containing fractions were pooled, concentrated, snap frozen, and stored at −80°C. rPfCSP and S02 probe generation For tetramer probe generation, rPfCSP or S02-DsbC (S02 fused to the disulfide bond C protein) were first biotinylated and then respectively conjugated to the fluorophores FITC (fluorescein isothiocyanate) and BV605 (Brilliant™ Violet 605) (BD Biosciences). Biotinylation was performed using ligase Bir A (Avidity) at 30°C for 4 hours prior to buffer exchange with 1X PBS (pH 7.4) over a 30-kDa Centricon Plus-70 Centrifugal Filter (Millipore) to remove excess free biotin. Biotinylated rPfCSP and S02-DsbC were fluorescently labeled through sequential addition of streptavidin conjugated to FITC (SA-FITC) or BV605 (SA-BV605), respectively, in a 4:1 molar ratio.

Isolation of PfCSP-specific memory B cells Probe-specific memory

B cells were isolated from cryopreserved peripheral blood mononuclear cells stained with the following panel: Aqua LIVE/DEAD (Thermo Fisher Scientific), rPfCSP-FITC and S02-BV605 tetramer probes, and antibodies against CD3-APC/Cy7 (BioLegend), CD8-V450 (BD Biosciences), CD14-BV785 (BioLegend), CD20-Alexa Fluor 700/PE (BioLegend), IgM-PE/Cy5 (BD Biosciences) and IgG-APC (BD Biosciences). Cells were sorted using a BD FACS Aria II (BD Immunocytometry Systems), and flow cytometry data were analyzed using FlowJo software (Tree Star). PfCSP-reactive (rPfCSP + and/or S02 + ) CD20 + CD3 − CD14 − memory B cells were single-cell sorted into 96-well PCR plates containing lysis buffer (RNase OUT, 5X First-Strand buffer, DTT, IgePAL, and water; SuperScript III First-Strand Synthesis System, Thermo Fisher Scientific). Production of recombinant immunoglobulins Immediately following single cell sorting and lysis of probe-specific memory B cells, all RNA transcripts were reverse transcribed to cDNA (SuperScript III First-Strand Synthesis System; Thermo Fisher Scientific). Amplification of the genes encoding the immunoglobulin variable regions heavy chains, as well as kappa or lambda light chains, was performed using a cocktail of primers followed by sequencing (ACGT) and cloning into the pVRC8400 huIgG1, pVRC8400 huIgK, or SBShuLambda expression vectors (GenScript) containing the relevant constant region. Sequence analysis was performed using The International Immunogenetics Information System (IMGT, http://www.imgt.org/ ). Matched heavy and light chain constructs were co-transfected into Expi293 cells using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) and cultures were incubated at 37°C, 8% CO 2 for 6 days. Supernatants were harvested and purified using rProtein A Sepharose Fast Flow resin (GE Healthcare) and buffer exchanged with 1X PBS (pH 7.4) before being concentrated using Amicon Centrifugal Filters (Millipore). Purified mAb concentrations were determined using a Nanodrop spectrophotometer. The sequences of CIS43, mAb10 ( Kisalu et al., 2018 ), MGU12 ( Tan et al., 2018 ), 1210 ( Imkeller et al., 2018 ), 311, and 317 ( Oyen et al., 2017 ) were retrieved from PDB or GenBank and produced and purified as described above.

Fluorescent antibody labeling

Antibodies were conjugated to Alexa Fluor-405 molecules using a SAIVI Antibody Labeling Kit according to the manufacturers’ directions (ThermoFisher Scientific) but using Alexa Fluor-405 NHS ester (ThermoFisher Scientific), which was mixed with each antibody at an 8:1 molar ratio for 1 hr at room temperature. The reaction was then purified over the SAIVI column, with fractions collected to determine the location of the conjugate. Resulting PfCSP mAb conjugates had degree of labeling (DOL) ratios between 1.4–2.2. For determination of concentration and DOL of Alexa Fluor-405 conjugates: absorbance correction factor = 0.7; extinction coefficient (e) = 34,500. Prior to use, conjugate binding was compared to unlabeled mAbs by rPfCSP ELISA and/or Pb-PfCSP-SPZ flow cytometry to confirm binding was not dramatically altered. ELISA for binding of mAbs to rPfCSP_FL Immulon 4HBX flat bottom microtiter plates (Thermo Fisher Scientific) were coated with 100 µl per well of antigen (1.0 µg/mL) in bicarbonate buffer overnight at 4°C. Coated plates were blocked with 200 µl of PBS + 10% FBS for 2 hrs at room temperature, followed by incubation for 2 hrs at 37°C with 100 µl of PfCSP or control mAbs at varying concentrations (5x10 −7 – 5.0 µg/mL, 10-fold serial dilutions). Plates were incubated with 100 µl/well of 0.1 µg/mL HRP-conjugated goat anti–human IgG (Bethyl Laboratories). Plates were washed six times with PBS-Tween between each step. After a final wash, samples were incubated for 10 min with 1-Step Ultra TMB-ELISA Substrate (Thermo Fisher Scientific). The optical density was read at 450 nm after addition of stopping solution (2N sulfuric acid, 100 µl/well). ELISA for binding of mAbs to NANP peptides MSD Gold microtiter plates (Meso Scale Discovery) were blocked with PBS + 5% BSA (20 µl/well). Blocked plates were coated with 10 µl/well of NANP biotinylated peptides (240 pmol, Genscript) in PBS + 1% BSA for 1 hr at room temperature. The coated plates were incubated for 2 hrs at room temperature with 10 µl of PfCSP or control mAbs at varying concentrations (5x10 −7 – 5.0 µg/mL, 5-fold serial dilutions). Plates were then incubated with 10 µl/well of 1.0 µg/mL Sulfo-tag goat anti–human IgG (Meso Scale Discovery) for 1 hr at room temperature. Plates were washed six times with PBS-Tween between each step. After a final wash, 35 µl of 1X MSD Read T Buffer (Meso Scale Discovery) was added to each well and plates were analyzed on an MSD Sector Image 2400 instrument. Epitope mapping and competition ELISAs Epitope mapping of CIS43 and L9 was performed using PfCSP overlapping peptides (peptides 20–61) that were 15 amino acids in length (GenScript) and overlapped by 11 residues spanning the central repeat region of PfCSP using the MSD U-Plex Assay platform (Meso Scale Discovery) according to the manufacturer’s instructions, with all mAb concentrations at 0.01 µg/mL. Competitive ELISA was also performed using peptides 20–61. Briefly, ELISA plates were coated with 10 µl of rPfCSP (200 ng/mL) for 1 hr at room temperature. After coating, PfCSP-specific monoclonal antibodies (10 ng/mL) preincubated overnight with varying concentrations (0 – 1,000 µg/mL) of selected PfCSP peptides in PBS + 1% BSA were added to the rPfCSP-coated plates, and ELISA was performed on the MSD platform as described above. For the alanine scanning mutagenesis experiments, competitive ELISA was performed as described above using peptide 22 variants where each residue was mutated to an alanine or a serine if the original residue was an alanine (GenScript).

Biolayer interferometry kinetic binding assay

Antibody binding kinetics were measured using biolayer interferometry on an Octet HTX instrument (FortéBio) using streptavidin-capture biosensors (fortéBio). PfCSP mAb solutions were plated in black tilted-bottom 384-well microplates (fortéBio); assays were performed with agitation at 30°C. mAb serial concentrations used are as follow: 1.25, 0.625, 0.3125, and 0.15625 µg/mL. Loading of biotinylated peptides 21, peptide 22, and peptide 29 (GenScript) was performed for 300s, followed by dipping of biosensors into buffer (PBS + 1% BSA) for 60s to assess baseline assay drift. Association with whole IgG (serially diluted from 16.67 to 1.04 µM) was done for 300s, followed by a dissociation step in buffer for 600s. Background subtraction of nonspecific binding was performed through measurement of association in buffer alone. Data analysis and curve fitting were performed using Octet software, version 7.0. Experimental data were fitted with the binding equations describing a 1:1 analyte-ligand interaction. Global analyses of the complete data sets, assuming binding was reversible (full dissociation), were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all concentrations of a given mAb dilution series. Isothermal titration calorimetry Isothermal titration calorimetry was carried out using a VP-ITC microcalorimeter (Malvern Panalytical). In all titration experiments, the rPfCSP constructs and mAbs were prepared in PBS, pH 7.4. Each antibody solution, prepared at a concentration of ~40 µM (expressed per antigen binding site), was injected in 5 or 7 µl aliquots into the calorimetric cell containing the respective rPfCSP construct at a concentration of ~0.4 µM except for rPfCSP_5/3, which was prepared at 0.8 µM. All titrations were performed at 25°C. The exact concentrations of the reactants in each experiment were determined from the absorbance at 280 nm. The heat evolved upon each injection of antibody was obtained from the integral of the calorimetric signal. The heat associated with binding to the different rPfCSP constructs was obtained by subtracting the heat of dilution from the heat of reaction. The individual heats were plotted against the molar ratio, and the enthalpy change, ∆H , the association constant, K a (the dissociation constant, K d =1/ K a ) and the stoichiometry (valency of antigen binding sites), N , were obtained by nonlinear regression of the data to a model that takes into account the binding to either one or two sets of sites with different binding affinities. Gibbs energy, ∆G , was calculated from the relation ∆G = − RT ln K a , where R is the universal gas constant, (1.987 cal/(K × mol)) and T the absolute temperature in Kelvin. The entropy contribution to Gibbs energy, -T∆S , was calculated from the known relation ∆G = ∆H − T∆S. The results were expressed per mole of antigen binding sites and the stoichiometry, N , denotes the number of antigen binding sites per mole of the respective rPfCSP construct. FACS analysis of mAb binding to sporozoites Freshly isolated Pb-PfCSP-GFP/Luc-SPZ were purified across an Accudenz density gradient (Accurate Chemical) to remove mosquito debris as previously described ( Kennedy et al., 2012 ) and resuspended in PBS containing the protease inhibitor E64 (Sigma-Aldrich) to prevent proteolytic processing of PfCSP. 8,000 SPZ were aliquoted to each well of a 96-well V-bottom plate (50 µl/well) and incubated for 30 min at 4 °C with various concentrations (0.02 – 20 µg/mL) of PfCSP-specific or control mAbs in PBS+E64, washed with 200 µl PBS+E64, and stained for 20 minutes at 4°C with goat anti-human IgG-Alexa Fluor® 647 secondary antibody (Thermo Fisher Scientific) at a 1:1,000 dilution in PBS+E64. After washing with 200 µl PBS+E64 and fixation in 250 µl PBS with 0.5% paraformaldehyde, events were acquired on a modified LSR II (BD Biosciences). In vitro hepatocyte invasion inhibition assay The human hepatoma cell line HC-04 was used to evaluate the in vitro capacity of the PfCSP mAbs to block hepatocyte invasion by PfSPZ NF54. HC-04 cells were seeded at 50,000 cells/well in rat tail collagen pre-treated 96 well plates for 16–24 hrs. Salivary gland PfSPZ were pre-mixed with heat-inactivated human serum and varying concentrations of PfCSP mAbs (10, 1 or 0.1 µg/mL) for 30 minutes on ice and then seeded in triplicate (50,000 sporozoites/well) onto the HC-04 cells. Plates were centrifuged for 10 minutes at 3000 xg and incubated for 3 hrs at 37°C with 5% C0 2 . Cells were washed with PBS (Gibco) and treated with trypsin (0.05% Trypsin-ethylenediamine tetra acetic acid (EDTA); Gibco) to generate a single-cell suspension. Subsequently, cells were fixed and impermeabilized (eBioscience) for 30 min at 4°C. The staining was done with a cell viability dye (1:2,000; Fixable viability dye (FVD) eFluor 780, eBioscience, Thermo Fisher Scientific) and a fluorescently-labelled mouse anti-PfCSP antibody (1:400; 3SP2-FITC) for 30 min at 4°C. After washing with 2% FCS/PBS, cells were fixed with 1% paraformaldehyde (PFA) and analyzed by flow cytometry using Gallios (Beckman Coulter) and FlowJo software (version 10.0.8, Tree Star). IV challenge with Pb-PfCSP-SPZ To measure mAb neutralization of SPZ and reduction of parasite burden in vivo , specified amounts of PfCSP-specific or control mAbs diluted in sterile filtered 1X PBS (pH 7.4; total volume 200 µl/mouse) were injected into the tail veins of female 6- to 8-week old B6(Cg)-Tyrc-2J/J albino mice (The Jackson Laboratory). Mice were then intravenously challenged in the tail vein with 2,000 freshly harvested Pb-PfCSP-GFP/Luc-SPZ ( Flores-Garcia et al., 2019 ) 2 hours after mAb administration. 40–42 hours post-challenge, mice were injected intraperitoneally with 150 µl of D-Luciferin (30 mg/mL), anesthetized with isoflurane and imaged with the IVIS® Spectrum in vivo imaging system (PerkinElmer) 10 minutes after luciferin injection. Liver burden was quantified by analyzing a region of interest (ROI) in the upper abdominal region and determining the total flux or bioluminescent radiance (photons/sec) expressed by Pb-PfCSP-GFP/Luc-SPZ using the manufacturer’s software (Living Image 4.5, PerkinElmer). Mosquito bite challenge with Pb-PfCSP-SPZ Anopheles stephensi female mosquitoes were allowed to feed on female 8-week old Swiss Webster mice (Charles Laboratories) infected with blood-stage Pb-PfCSP-SPZ. Twenty days after infected bloodmeal, the proportion of infected mosquitoes was between 70–80%, as assessed by microscopic observation of 20 salivary glands. Based on this observation, we determined that 6–7 mosquitoes were needed to expose mice to the bites of ~5 infected mosquitoes. 6–8 week old C57BL/6 female mice (Charles Laboratories) were injected IV with PfCSP mAbs (100, 300, or 600 µg mAb/mouse; blinded and in differing orders per experimental replicate) diluted in 1X PBS (pH 7.4) in a total volume of 200 µL. Forty-eight hours after mAb administration, mice were subjected to a small tail vein bleed to ascertain pre-challenge mAb serum titers. Seventy-two hours after mAb administration, test and control mice were anesthetized with 2% Avertin (Alfa Aesar, Ward Hill, MA). Mosquitoes were allowed to feed on mice for 10 minutes. Following feeding, mosquito abdomens were inspected to confirm the blood meal. Mouse parasitemia was assessed daily through Giemsa staining of blood smears starting on day 4 and up to day 10–12 after exposure to infected mosquito bites. IV challenge with PfSPZ Female 8–9 month old FRG-huHep mice with engrafted human hepatocytes from two different donors (HHM19027 and HHM13022) were purchased from Yecuris Corp. Repopulation of human hepatocytes were confirmed by the level of serum albumin and ranged between 4000 to 8000 µg/mL, evenly divided between the different experimental groups. Mice were intravenously injected with the indicated dose of VRC01 or PfCSP mAbs (100 µL per mouse) 24 hours before sporozoite challenge. On the day of sporozoite challenge, Anopheles stephensi mosquitoes infected with Pf NF54 (on day 14–18 post blood meal) were harvested into Dutch modified RPMI 1640 media (ThermoFisher). 100,000 sporozoites (in a total volume of 100 µl) were injected IV in the tail vein of each mouse. Six days following challenge, serum was collected via cardiac bleed and livers of each mice were harvested as previously described ( Foquet et al., 2013 ; Yang et al., 2017 ). Briefly, lobes were pooled and emulsified to obtain single-cell suspensions for subsequent genomic DNA (gDNA) extraction. gDNA were extracted from roughly 25% of the chimeric livers and used to quantify parasite load using oligonucleotides specific for Pf 18S rRNA as previously described ( McCall et al., 2017 ; Yang et al., 2017 ). qPCR was used to quantify the relative amount of human hepatocytes engrafted onto the mouse liver, as previously described ( Alcoser et al., 2011 ). ELISA for quantitation of mAb serum titers ELISA was performed on serum from mice passively transferred human PfCSP mAbs as previously described ( Kisalu et al., 2018 ) using rPfCSP-coated plates (200 ng/mL). A standard curve for each mAb was generated using eight two-fold serial dilutions of mAb starting at 10 ng/mL. Serum samples were applied at various dilutions in dilution/blocking buffer. For datapoints in the linear range of the standard curve, the average of the calculated concentration values was used for each individual sample. Intravital liver imaging of Pb-PfCSP-SPZ Female 6- to 12-weeks old C57BL/6 mice (The Jackson Laboratory) received sequential IV injections of Alexa Fluor-405-labeled mAbs (30 µg, blinded), 1 x 10 5 Pb-PfCSP-GFP-SPZ, and rhodamine-labeled dextran (20 µg/mL, 50µL). Mice were immediately prepared for multiphoton microscopy as previously described ( McNamara et al., 2017 ). Briefly, mice were anaesthetized with a mix of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). The mouse temperature was maintained at 37ºC using a heating mat attached to a feedback probe inserted in the mouse rectum throughout the surgery and imaging procedure. A lateral incision was made over the left lobe of the liver and any vessels cauterized by applying light pressure to the vessel until clotting occurred naturally. The mouse was then placed in a custom-made holder. The liver was exposed and directly adhered to a coverslip that was secured in the holder. Once stable, the preparation was transferred to a Fluoview FVMPE-RS multiphoton microscope system (Olympus) equipped with a XLPLN25XWMP2 objective (25x; NA1.05; water immersion; 2mm working distance). For quantification of parasites, damaged hepatocytes, and shedding of PfCSP, a single 50µm Z-stack (2µm/slice) was acquired using a resonance scanner. Fluorescence of Alexa Fluor-405, GFP and rhodamine were detected using an 860nm wavelength laser. For videos of traversal or shedding, a sequence of between 1,000–2,000 50µm Z-stacks (2µm/slice) was acquired using a resonance scanner. Images were acquired using FV30 software (Olympus) and exported to Imaris (Bitplane) for downstream processing. Sporozoites and shed PfCSP were measured using the measurement function on Imaris. Sporozoites were classified as either sinusoid-bound, traversing hepatocytes, or having infected a hepatocyte based on the following criteria. Any sporozoite located within a sinusoid and not within 40µm (i.e., diameter of a hepatocyte)of a rhodamine + hepatocyte was considered to be vessel bound in the sinusoid. Any sporozoite within 40µm of at least one rhodamine + hepatocyte and still in contact with the traversed hepatocyte was considered to be traversing. Any sporozoite within 40µm of at least one rhodamine + hepatocyte, but not in a sinusoid nor in contact with a rhodamine + hepatocyte, was considered to have established infection. Additionally, any sporozoite with a discontinuation (i.e., fragmentation) of GFP along its length was classified as undergoing “dotty death”, and any sporozoite with a length of Alexa Fluor-405 not colocalized with GFP was considered to be shedding its mAb-bound PfCSP coat and undergoing a CSPR. Parasite death in any of the resonance videos was characterized as bursting if a sudden loss of membrane integrity and release of GFP into the surrounding tissue was observed. NVDP conservation in global field isolates PfCSP sequences and country in which the sequences were isolated were retrieved from GenBank. N- and C-terminal sequences were trimmed in Geneious Prime (Geneious) and the central repeat region sequences was exported into Microsoft Excel. All NPDP tetrapeptides were transformed into 0, NANP repeats into 1, and NVDP repeats into 2; sequences were numerically ordered based on number of NVDP repeats and the geographic region in which they were isolated was indicated. Heat map classifying the panel of PfCSP mAbs Numerical values for each parameter describing each of the seven mAbs in the panel were collated and ranked using conditional formatting (Microsoft Excel), with darker colors denoting improved performance and lighter colors indicating poorer performance. All numerical values that varied logarithmically were log-transformed into linear values to facilitate consistent analysis.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical tests used, exact value of n, what n represents, and precision measures can be found in figure legends. Unless otherwise stated, all mAbs were compared for significance to untreated/isotype control or to each other using the Kruskal-Wallis test with Dunn’s post-hoc test correcting for multiple comparisons. For the PfSPZ challenge and L9 titers in FRG-huHep mice and the comparison of L9 and CIS43 serum titers, the two-tailed Mann-Whitney test was used. For the in vitro hepatocyte invasion assay, two-way ANOVA was used to compare each mAb to the isotype control at each respective concentration and adjusted for multiple comparisons using Bonferroni’s post-hoc test. For the intravital liver imaging data (locations, traversal, CSPR, and dotty death), all mAbs were first compared to each other using an omnibus chi-squared test; if the distribution varied significantly, then each individual mAb was compared to isotype control (or the best performing mAb) and adjusted for multiple comparisons using Bonferroni’s post-hoc test. For measurement of PfCSP mAbs in mouse serum, standard curves were fitted with a hyperbolic parameter curve, and concentration values in the linear range of the standard curve were interpolated. For the ITC stoichiometry data, errors with 95% confidence were estimated from the fits of the data. Unless otherwise indicated, all data were plotted using GraphPad Prism, version 7.0. For the mosquito bite challenge experiments, L9 was tested for superiority against the other mAbs (one-sided tests) and P -values were corrected using the Holm method ( Holm, 1979 ). For the Kaplan–Meier curves, comparisons were tested using the log-rank test; an exact P -value was calculated when the sample size did not exceed 15 in either group ( Hothorn et al., 2008 ). The relationship between dose or circulating serum mAb concentration prior to challenge and protection were modeled using two-parameter logistic (2PL) models with the following functional form: y = 1/1 + (x/ED 50 ) h ) ( Raghunandan et al., 2020 ). The ED 50 (or EC 50 for concentration) is the effective dose that elicits 50% protection and the hill slope, h, determines the steepness of the logistic curve. The 2PL model was fit to the data using logistic regression with a log-transformed dose or concentration covariate. For mAbs eliciting at least 50% protection, a single model was fit assuming a common hill slope and a mixed model was used with a random intercept specified for each experiment. ED 50 , EC 50 , ED 80 , and ED 80 with 95% confidence intervals were estimated from these fitted models using parametric simulation. Protection was compared between L9 and the other mAbs using the dose-adjusted or concentration-adjusted odds ratio from these models. For mAbs eliciting less than 50% protection, separate logistic models with small-sample size corrections (Firth-correction) were fit for each paired adjusted odds ratio comparison ( Kosmidis and Firth, 2009 ). P -values of odds ratios were corrected together within dose or concentration comparisons. Protection analyses were conducted in R programming language (CRAN) ( R Core Team ). Mixed effects logistic regression models were implemented with the lme4 package ( Bates et al., 2015 ), simulations were implemented with the arm package ( Gelman et al., 2020 ), and data manipulation and visualization were implemented with the tidyverse packages ( Wickham et al., 2019 ; Wilke, 2019 ).

Materials Availability

All unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

EXPERIMENTAL MODELS AND SUBJECT DETAILS Human clinical specimens

Clinical specimens were derived from malaria-naive, healthy adults (18–45 years of age) in the VRC 314 clinical trial ( https://clinicaltrials.gov/ ; NCT02015091 ) after obtaining written informed consent. Briefly, VRC 314 was a multi-institution, phase 1, open-label, dose-escalation trial with controlled human malaria infection (CHMI) that was designed to assess the safety, immunogenicity, and protective efficacy of the Sanaria PfSPZ Vaccine administered by intravenous or intramuscular injection ( Lyke et al., 2017 ). The Sanaria PfSPZ Vaccine is composed of radiation-attenuated, aseptic, purified, cryopreserved Plasmodium falciparum sporozoites derived from the NF54 strain ( Seder et al., 2013 ).

Mice Female 6- to 8-weeks old

B6(Cg)-Tyrc-2J/J albino mice and female 6- to 12-weeks old C57BL/6 mice were obtained from The Jackson Laboratory. Female 6- to 8-weeks old C57BL/6 mice and female 8-weeks old Swiss Webster mice were obtained from Charles River Laboratories. Female 8–9 month old FRG-huHep were obtained from Yecuris Corp. All mouse research was performed according to National Institutes of Health (NIH) guidelines for use and care of live animals approved by the institutional animal care and use ethics committees of the Vaccine Research Center (Animal Study Protocol VRC-17–702), Johns Hopkins University (Approved protocol permit no. MO18H419), Radboud University Medical Center (ADV103002016452), and Australia National University (A2016/17 & A2019/36).

Cell Lines

Expi293 and 293F cells used were from Thermo Fisher Scientific. The human hepatoma cell line HC-04 (MRA-965, deposited by Jetsumon Sattabongkot) ( Sattabongkot et al., 2006 ) was obtained through the Malaria Research and Reference Reagent Resource Center as part of the Biodefense and Emerging Infections Research Resources Repository. Sporozoites Transgenic P. berghei (strain ANKA 676m1c11, MRA-868) expressing full-length P. falciparum CSP and a green fluorescent protein/luciferase fusion protein (Pb-PfCSP-GFP/Luc-SPZ) were obtained as previously described ( Flores-Garcia et al., 2019 ). GFP-labeled P. berghei SPZ expressing PfCSP (Pb-PfCSP-GFP-SPZ) were generated by crossing the parental Pb-PfCSP-SPZ line ( Espinosa et al., 2017 ) with the previously described P. berghei -ConF parasite line that expresses GFP under the control of an HSP70 promotor ( Amino et al., 2008 ). Briefly, C57BL/6 mice were coinfected with Pb-PfCSP-SPZ and P. berghei -ConF at a ratio of 10:1. Anopheles stephensi mosquitoes were allowed to feed on the mice, and subsequently, sporozoites dissected from these mosquitoes were used to infect naive animals. Parasites expressing the GFP transgene were sorted from the blood of these mice by use of a FACSAria cell sorter and used to infect mice. Subsequently, the GFP + progeny was cloned, and the clones screened for the insertion of the PfCSP knock-in via measurement of anti-PfCSP antibody binding to progeny SPZ. P. falciparum NF54 sporozoites were isolated from an individual near Schiphol Airport (The Netherlands) ( Delemarre-van de Waal and de Waal, 1981 ).

METHOD DETAILS Generation of junctional probe S02, a 43-residue structurally stabilized peptide mimicking the junctional epitope (MPESSSNPDCNANPNVDPNEDLIKKCEKINVPTEEIKKEIEEKK), was designed using Rosetta ( Chevalier et al., 2017 ) by modifying the junctional peptide 21 with flanking sequences to stabilize the peptide in a conformation that retains binding by the neutralizing junctional antibody CIS43, but which is unfavorable to binding by the poorly neutralizing junctional antibody CIS42 ( Kisalu et al., 2018 ). Sera from 14 protected subjects in group 6 of VRC 314 who received a total of three doses of radiation-attenuated PfSPZ (9.0 x 10 5 ) intravenously at weeks 0, 8, and 16 were screened for antibody titers against S02 using ELISA. A subject whose sera demonstrated the highest reactivity against S02 at the week 20 timepoint, 4 weeks after the last immunization, was chosen for memory B cell sorting and mAb isolation.

Production of recombinant PfCSP constructs

The amino acid sequence of PfCSP in the 3D7 clone of the NF54 isolate (PlasmoDB ID: PF3D7_0304600.1) was used to generate a codon-optimized synthetic gene for expression in mammalian cells (GenScript). The DNA construct corresponding to the full-length rPfCSP, in which the leader peptide residues 1–20 were replaced with a mammalian secretory signal peptide derived from the modified bovine prolactin (MDSKGSSQKGSRLLLLLVVSNLLLPQGVLA) and the glycosylphosphatidylinositol (GPI) anchor residues 376–397 were excluded, was cloned into a CMV/R-expression vector with a C-terminal AviTag, HRV3C-processing tag, and a 6X histidine tag. This construct, termed PfCSP_SAmut_C5S, encodes the N-terminal domain (with four amino acid mutations that removed processing sites and prevented dimerization upon solubilization to increase yield and facilitate consistent analyses), the central domain consisting of 38 NANP tandem repeats interspersed with 4 NVDP repeats, and the C-terminal domain. Truncated ( Oyen et al., 2018 ) and NVDP repeat mutant rPfCSP synthetic constructs were created in the same expression vector with identical N- and C-termini to full-length PfCSP_SAmut_C5S, expressed through transient transfection in 293F cells using the Freestyle 293F expression system (Thermo Fisher Scientific) at 37°C, 8% CO 2 for 6 days, and purified from culture supernatants through polyhistidine-tag affinity chromatography followed by size-exclusion chromatography on an ÄKTA ™ start (GE Healthcare). Monomer-containing fractions were pooled, concentrated, snap frozen, and stored at −80°C. rPfCSP and S02 probe generation For tetramer probe generation, rPfCSP or S02-DsbC (S02 fused to the disulfide bond C protein) were first biotinylated and then respectively conjugated to the fluorophores FITC (fluorescein isothiocyanate) and BV605 (Brilliant™ Violet 605) (BD Biosciences). Biotinylation was performed using ligase Bir A (Avidity) at 30°C for 4 hours prior to buffer exchange with 1X PBS (pH 7.4) over a 30-kDa Centricon Plus-70 Centrifugal Filter (Millipore) to remove excess free biotin. Biotinylated rPfCSP and S02-DsbC were fluorescently labeled through sequential addition of streptavidin conjugated to FITC (SA-FITC) or BV605 (SA-BV605), respectively, in a 4:1 molar ratio.

Isolation of PfCSP-specific memory B cells Probe-specific memory

B cells were isolated from cryopreserved peripheral blood mononuclear cells stained with the following panel: Aqua LIVE/DEAD (Thermo Fisher Scientific), rPfCSP-FITC and S02-BV605 tetramer probes, and antibodies against CD3-APC/Cy7 (BioLegend), CD8-V450 (BD Biosciences), CD14-BV785 (BioLegend), CD20-Alexa Fluor 700/PE (BioLegend), IgM-PE/Cy5 (BD Biosciences) and IgG-APC (BD Biosciences). Cells were sorted using a BD FACS Aria II (BD Immunocytometry Systems), and flow cytometry data were analyzed using FlowJo software (Tree Star). PfCSP-reactive (rPfCSP + and/or S02 + ) CD20 + CD3 − CD14 − memory B cells were single-cell sorted into 96-well PCR plates containing lysis buffer (RNase OUT, 5X First-Strand buffer, DTT, IgePAL, and water; SuperScript III First-Strand Synthesis System, Thermo Fisher Scientific). Production of recombinant immunoglobulins Immediately following single cell sorting and lysis of probe-specific memory B cells, all RNA transcripts were reverse transcribed to cDNA (SuperScript III First-Strand Synthesis System; Thermo Fisher Scientific). Amplification of the genes encoding the immunoglobulin variable regions heavy chains, as well as kappa or lambda light chains, was performed using a cocktail of primers followed by sequencing (ACGT) and cloning into the pVRC8400 huIgG1, pVRC8400 huIgK, or SBShuLambda expression vectors (GenScript) containing the relevant constant region. Sequence analysis was performed using The International Immunogenetics Information System (IMGT, http://www.imgt.org/ ). Matched heavy and light chain constructs were co-transfected into Expi293 cells using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) and cultures were incubated at 37°C, 8% CO 2 for 6 days. Supernatants were harvested and purified using rProtein A Sepharose Fast Flow resin (GE Healthcare) and buffer exchanged with 1X PBS (pH 7.4) before being concentrated using Amicon Centrifugal Filters (Millipore). Purified mAb concentrations were determined using a Nanodrop spectrophotometer. The sequences of CIS43, mAb10 ( Kisalu et al., 2018 ), MGU12 ( Tan et al., 2018 ), 1210 ( Imkeller et al., 2018 ), 311, and 317 ( Oyen et al., 2017 ) were retrieved from PDB or GenBank and produced and purified as described above.

Fluorescent antibody labeling

Antibodies were conjugated to Alexa Fluor-405 molecules using a SAIVI Antibody Labeling Kit according to the manufacturers’ directions (ThermoFisher Scientific) but using Alexa Fluor-405 NHS ester (ThermoFisher Scientific), which was mixed with each antibody at an 8:1 molar ratio for 1 hr at room temperature. The reaction was then purified over the SAIVI column, with fractions collected to determine the location of the conjugate. Resulting PfCSP mAb conjugates had degree of labeling (DOL) ratios between 1.4–2.2. For determination of concentration and DOL of Alexa Fluor-405 conjugates: absorbance correction factor = 0.7; extinction coefficient (e) = 34,500. Prior to use, conjugate binding was compared to unlabeled mAbs by rPfCSP ELISA and/or Pb-PfCSP-SPZ flow cytometry to confirm binding was not dramatically altered. ELISA for binding of mAbs to rPfCSP_FL Immulon 4HBX flat bottom microtiter plates (Thermo Fisher Scientific) were coated with 100 µl per well of antigen (1.0 µg/mL) in bicarbonate buffer overnight at 4°C. Coated plates were blocked with 200 µl of PBS + 10% FBS for 2 hrs at room temperature, followed by incubation for 2 hrs at 37°C with 100 µl of PfCSP or control mAbs at varying concentrations (5x10 −7 – 5.0 µg/mL, 10-fold serial dilutions). Plates were incubated with 100 µl/well of 0.1 µg/mL HRP-conjugated goat anti–human IgG (Bethyl Laboratories). Plates were washed six times with PBS-Tween between each step. After a final wash, samples were incubated for 10 min with 1-Step Ultra TMB-ELISA Substrate (Thermo Fisher Scientific). The optical density was read at 450 nm after addition of stopping solution (2N sulfuric acid, 100 µl/well). ELISA for binding of mAbs to NANP peptides MSD Gold microtiter plates (Meso Scale Discovery) were blocked with PBS + 5% BSA (20 µl/well). Blocked plates were coated with 10 µl/well of NANP biotinylated peptides (240 pmol, Genscript) in PBS + 1% BSA for 1 hr at room temperature. The coated plates were incubated for 2 hrs at room temperature with 10 µl of PfCSP or control mAbs at varying concentrations (5x10 −7 – 5.0 µg/mL, 5-fold serial dilutions). Plates were then incubated with 10 µl/well of 1.0 µg/mL Sulfo-tag goat anti–human IgG (Meso Scale Discovery) for 1 hr at room temperature. Plates were washed six times with PBS-Tween between each step. After a final wash, 35 µl of 1X MSD Read T Buffer (Meso Scale Discovery) was added to each well and plates were analyzed on an MSD Sector Image 2400 instrument. Epitope mapping and competition ELISAs Epitope mapping of CIS43 and L9 was performed using PfCSP overlapping peptides (peptides 20–61) that were 15 amino acids in length (GenScript) and overlapped by 11 residues spanning the central repeat region of PfCSP using the MSD U-Plex Assay platform (Meso Scale Discovery) according to the manufacturer’s instructions, with all mAb concentrations at 0.01 µg/mL. Competitive ELISA was also performed using peptides 20–61. Briefly, ELISA plates were coated with 10 µl of rPfCSP (200 ng/mL) for 1 hr at room temperature. After coating, PfCSP-specific monoclonal antibodies (10 ng/mL) preincubated overnight with varying concentrations (0 – 1,000 µg/mL) of selected PfCSP peptides in PBS + 1% BSA were added to the rPfCSP-coated plates, and ELISA was performed on the MSD platform as described above. For the alanine scanning mutagenesis experiments, competitive ELISA was performed as described above using peptide 22 variants where each residue was mutated to an alanine or a serine if the original residue was an alanine (GenScript).

Biolayer interferometry kinetic binding assay

Antibody binding kinetics were measured using biolayer interferometry on an Octet HTX instrument (FortéBio) using streptavidin-capture biosensors (fortéBio). PfCSP mAb solutions were plated in black tilted-bottom 384-well microplates (fortéBio); assays were performed with agitation at 30°C. mAb serial concentrations used are as follow: 1.25, 0.625, 0.3125, and 0.15625 µg/mL. Loading of biotinylated peptides 21, peptide 22, and peptide 29 (GenScript) was performed for 300s, followed by dipping of biosensors into buffer (PBS + 1% BSA) for 60s to assess baseline assay drift. Association with whole IgG (serially diluted from 16.67 to 1.04 µM) was done for 300s, followed by a dissociation step in buffer for 600s. Background subtraction of nonspecific binding was performed through measurement of association in buffer alone. Data analysis and curve fitting were performed using Octet software, version 7.0. Experimental data were fitted with the binding equations describing a 1:1 analyte-ligand interaction. Global analyses of the complete data sets, assuming binding was reversible (full dissociation), were carried out using nonlinear least-squares fitting allowing a single set of binding parameters to be obtained simultaneously for all concentrations of a given mAb dilution series. Isothermal titration calorimetry Isothermal titration calorimetry was carried out using a VP-ITC microcalorimeter (Malvern Panalytical). In all titration experiments, the rPfCSP constructs and mAbs were prepared in PBS, pH 7.4. Each antibody solution, prepared at a concentration of ~40 µM (expressed per antigen binding site), was injected in 5 or 7 µl aliquots into the calorimetric cell containing the respective rPfCSP construct at a concentration of ~0.4 µM except for rPfCSP_5/3, which was prepared at 0.8 µM. All titrations were performed at 25°C. The exact concentrations of the reactants in each experiment were determined from the absorbance at 280 nm. The heat evolved upon each injection of antibody was obtained from the integral of the calorimetric signal. The heat associated with binding to the different rPfCSP constructs was obtained by subtracting the heat of dilution from the heat of reaction. The individual heats were plotted against the molar ratio, and the enthalpy change, ∆H , the association constant, K a (the dissociation constant, K d =1/ K a ) and the stoichiometry (valency of antigen binding sites), N , were obtained by nonlinear regression of the data to a model that takes into account the binding to either one or two sets of sites with different binding affinities. Gibbs energy, ∆G , was calculated from the relation ∆G = − RT ln K a , where R is the universal gas constant, (1.987 cal/(K × mol)) and T the absolute temperature in Kelvin. The entropy contribution to Gibbs energy, -T∆S , was calculated from the known relation ∆G = ∆H − T∆S. The results were expressed per mole of antigen binding sites and the stoichiometry, N , denotes the number of antigen binding sites per mole of the respective rPfCSP construct. FACS analysis of mAb binding to sporozoites Freshly isolated Pb-PfCSP-GFP/Luc-SPZ were purified across an Accudenz density gradient (Accurate Chemical) to remove mosquito debris as previously described ( Kennedy et al., 2012 ) and resuspended in PBS containing the protease inhibitor E64 (Sigma-Aldrich) to prevent proteolytic processing of PfCSP. 8,000 SPZ were aliquoted to each well of a 96-well V-bottom plate (50 µl/well) and incubated for 30 min at 4 °C with various concentrations (0.02 – 20 µg/mL) of PfCSP-specific or control mAbs in PBS+E64, washed with 200 µl PBS+E64, and stained for 20 minutes at 4°C with goat anti-human IgG-Alexa Fluor® 647 secondary antibody (Thermo Fisher Scientific) at a 1:1,000 dilution in PBS+E64. After washing with 200 µl PBS+E64 and fixation in 250 µl PBS with 0.5% paraformaldehyde, events were acquired on a modified LSR II (BD Biosciences). In vitro hepatocyte invasion inhibition assay The human hepatoma cell line HC-04 was used to evaluate the in vitro capacity of the PfCSP mAbs to block hepatocyte invasion by PfSPZ NF54. HC-04 cells were seeded at 50,000 cells/well in rat tail collagen pre-treated 96 well plates for 16–24 hrs. Salivary gland PfSPZ were pre-mixed with heat-inactivated human serum and varying concentrations of PfCSP mAbs (10, 1 or 0.1 µg/mL) for 30 minutes on ice and then seeded in triplicate (50,000 sporozoites/well) onto the HC-04 cells. Plates were centrifuged for 10 minutes at 3000 xg and incubated for 3 hrs at 37°C with 5% C0 2 . Cells were washed with PBS (Gibco) and treated with trypsin (0.05% Trypsin-ethylenediamine tetra acetic acid (EDTA); Gibco) to generate a single-cell suspension. Subsequently, cells were fixed and impermeabilized (eBioscience) for 30 min at 4°C. The staining was done with a cell viability dye (1:2,000; Fixable viability dye (FVD) eFluor 780, eBioscience, Thermo Fisher Scientific) and a fluorescently-labelled mouse anti-PfCSP antibody (1:400; 3SP2-FITC) for 30 min at 4°C. After washing with 2% FCS/PBS, cells were fixed with 1% paraformaldehyde (PFA) and analyzed by flow cytometry using Gallios (Beckman Coulter) and FlowJo software (version 10.0.8, Tree Star). IV challenge with Pb-PfCSP-SPZ To measure mAb neutralization of SPZ and reduction of parasite burden in vivo , specified amounts of PfCSP-specific or control mAbs diluted in sterile filtered 1X PBS (pH 7.4; total volume 200 µl/mouse) were injected into the tail veins of female 6- to 8-week old B6(Cg)-Tyrc-2J/J albino mice (The Jackson Laboratory). Mice were then intravenously challenged in the tail vein with 2,000 freshly harvested Pb-PfCSP-GFP/Luc-SPZ ( Flores-Garcia et al., 2019 ) 2 hours after mAb administration. 40–42 hours post-challenge, mice were injected intraperitoneally with 150 µl of D-Luciferin (30 mg/mL), anesthetized with isoflurane and imaged with the IVIS® Spectrum in vivo imaging system (PerkinElmer) 10 minutes after luciferin injection. Liver burden was quantified by analyzing a region of interest (ROI) in the upper abdominal region and determining the total flux or bioluminescent radiance (photons/sec) expressed by Pb-PfCSP-GFP/Luc-SPZ using the manufacturer’s software (Living Image 4.5, PerkinElmer). Mosquito bite challenge with Pb-PfCSP-SPZ Anopheles stephensi female mosquitoes were allowed to feed on female 8-week old Swiss Webster mice (Charles Laboratories) infected with blood-stage Pb-PfCSP-SPZ. Twenty days after infected bloodmeal, the proportion of infected mosquitoes was between 70–80%, as assessed by microscopic observation of 20 salivary glands. Based on this observation, we determined that 6–7 mosquitoes were needed to expose mice to the bites of ~5 infected mosquitoes. 6–8 week old C57BL/6 female mice (Charles Laboratories) were injected IV with PfCSP mAbs (100, 300, or 600 µg mAb/mouse; blinded and in differing orders per experimental replicate) diluted in 1X PBS (pH 7.4) in a total volume of 200 µL. Forty-eight hours after mAb administration, mice were subjected to a small tail vein bleed to ascertain pre-challenge mAb serum titers. Seventy-two hours after mAb administration, test and control mice were anesthetized with 2% Avertin (Alfa Aesar, Ward Hill, MA). Mosquitoes were allowed to feed on mice for 10 minutes. Following feeding, mosquito abdomens were inspected to confirm the blood meal. Mouse parasitemia was assessed daily through Giemsa staining of blood smears starting on day 4 and up to day 10–12 after exposure to infected mosquito bites. IV challenge with PfSPZ Female 8–9 month old FRG-huHep mice with engrafted human hepatocytes from two different donors (HHM19027 and HHM13022) were purchased from Yecuris Corp. Repopulation of human hepatocytes were confirmed by the level of serum albumin and ranged between 4000 to 8000 µg/mL, evenly divided between the different experimental groups. Mice were intravenously injected with the indicated dose of VRC01 or PfCSP mAbs (100 µL per mouse) 24 hours before sporozoite challenge. On the day of sporozoite challenge, Anopheles stephensi mosquitoes infected with Pf NF54 (on day 14–18 post blood meal) were harvested into Dutch modified RPMI 1640 media (ThermoFisher). 100,000 sporozoites (in a total volume of 100 µl) were injected IV in the tail vein of each mouse. Six days following challenge, serum was collected via cardiac bleed and livers of each mice were harvested as previously described ( Foquet et al., 2013 ; Yang et al., 2017 ). Briefly, lobes were pooled and emulsified to obtain single-cell suspensions for subsequent genomic DNA (gDNA) extraction. gDNA were extracted from roughly 25% of the chimeric livers and used to quantify parasite load using oligonucleotides specific for Pf 18S rRNA as previously described ( McCall et al., 2017 ; Yang et al., 2017 ). qPCR was used to quantify the relative amount of human hepatocytes engrafted onto the mouse liver, as previously described ( Alcoser et al., 2011 ). ELISA for quantitation of mAb serum titers ELISA was performed on serum from mice passively transferred human PfCSP mAbs as previously described ( Kisalu et al., 2018 ) using rPfCSP-coated plates (200 ng/mL). A standard curve for each mAb was generated using eight two-fold serial dilutions of mAb starting at 10 ng/mL. Serum samples were applied at various dilutions in dilution/blocking buffer. For datapoints in the linear range of the standard curve, the average of the calculated concentration values was used for each individual sample. Intravital liver imaging of Pb-PfCSP-SPZ Female 6- to 12-weeks old C57BL/6 mice (The Jackson Laboratory) received sequential IV injections of Alexa Fluor-405-labeled mAbs (30 µg, blinded), 1 x 10 5 Pb-PfCSP-GFP-SPZ, and rhodamine-labeled dextran (20 µg/mL, 50µL). Mice were immediately prepared for multiphoton microscopy as previously described ( McNamara et al., 2017 ). Briefly, mice were anaesthetized with a mix of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). The mouse temperature was maintained at 37ºC using a heating mat attached to a feedback probe inserted in the mouse rectum throughout the surgery and imaging procedure. A lateral incision was made over the left lobe of the liver and any vessels cauterized by applying light pressure to the vessel until clotting occurred naturally. The mouse was then placed in a custom-made holder. The liver was exposed and directly adhered to a coverslip that was secured in the holder. Once stable, the preparation was transferred to a Fluoview FVMPE-RS multiphoton microscope system (Olympus) equipped with a XLPLN25XWMP2 objective (25x; NA1.05; water immersion; 2mm working distance). For quantification of parasites, damaged hepatocytes, and shedding of PfCSP, a single 50µm Z-stack (2µm/slice) was acquired using a resonance scanner. Fluorescence of Alexa Fluor-405, GFP and rhodamine were detected using an 860nm wavelength laser. For videos of traversal or shedding, a sequence of between 1,000–2,000 50µm Z-stacks (2µm/slice) was acquired using a resonance scanner. Images were acquired using FV30 software (Olympus) and exported to Imaris (Bitplane) for downstream processing. Sporozoites and shed PfCSP were measured using the measurement function on Imaris. Sporozoites were classified as either sinusoid-bound, traversing hepatocytes, or having infected a hepatocyte based on the following criteria. Any sporozoite located within a sinusoid and not within 40µm (i.e., diameter of a hepatocyte)of a rhodamine + hepatocyte was considered to be vessel bound in the sinusoid. Any sporozoite within 40µm of at least one rhodamine + hepatocyte and still in contact with the traversed hepatocyte was considered to be traversing. Any sporozoite within 40µm of at least one rhodamine + hepatocyte, but not in a sinusoid nor in contact with a rhodamine + hepatocyte, was considered to have established infection. Additionally, any sporozoite with a discontinuation (i.e., fragmentation) of GFP along its length was classified as undergoing “dotty death”, and any sporozoite with a length of Alexa Fluor-405 not colocalized with GFP was considered to be shedding its mAb-bound PfCSP coat and undergoing a CSPR. Parasite death in any of the resonance videos was characterized as bursting if a sudden loss of membrane integrity and release of GFP into the surrounding tissue was observed. NVDP conservation in global field isolates PfCSP sequences and country in which the sequences were isolated were retrieved from GenBank. N- and C-terminal sequences were trimmed in Geneious Prime (Geneious) and the central repeat region sequences was exported into Microsoft Excel. All NPDP tetrapeptides were transformed into 0, NANP repeats into 1, and NVDP repeats into 2; sequences were numerically ordered based on number of NVDP repeats and the geographic region in which they were isolated was indicated. Heat map classifying the panel of PfCSP mAbs Numerical values for each parameter describing each of the seven mAbs in the panel were collated and ranked using conditional formatting (Microsoft Excel), with darker colors denoting improved performance and lighter colors indicating poorer performance. All numerical values that varied logarithmically were log-transformed into linear values to facilitate consistent analysis.

Supplementary Material 2