DOI: 10.5061/dryad.8931zcrzv
title: PfRH5-induced human monoclonal antibodies show broadly neutralizing activity in P. falciparum clinical isolates

description: Vaccines to the Plasmodium falciparum reticulocyte binding-like protein
 homolog 5 (PfRH5) target the blood stage of the parasite’s life cycle.
 PfRH5 has the potential to trigger the production of strain-transcendent
 antibodies and has proven its efficacy both in pre-clinical and early
 clinical studies. Vaccine-induced monoclonal antibodies (mAbs) to PfRH5
 showed promising outcomes in cultured P. falciparum strains from distinct
 geographic areas. Here, we assessed the functional impact of
 vaccine-induced mAbs to PfRH5 on the genetically more complex P.
 falciparum clinical isolates. We used mAbs isolated from single-cell
 sorted B-cells of volunteers enrolled in the phase 1a (NCT02181088)
 clinical trial of the viral-vectored PfRH5 vaccine and used ex-vivo growth
 inhibition assays (GIA) to assess their efficacy in P. falciparum clinical
 isolates. Next-generation sequencing (NGS) was used to assess the breadth
 of genetic diversity in P. falciparum clinical isolates and to infer the
 genotype/phenotype relationship involved in antibody susceptibility. We
 showed a dose-dependent inhibition of clinical isolates with three main
 GIA groups, high, medium, and low. Except for one isolate, our data shows
 no significant differences in antibody GIA profile between the P.
 falciparum clinical isolates and the 3D7 reference strain, which harbours
 the vaccine allele. We observed an additive relationship, where the
 combination of GIA-low and GIA-medium antibodies resulted in increased GIA
 activities, having important implications for the contribution of specific
 monoclonal antibodies in polyclonal IgG responses. While our NGS analysis
 showed the occurrence of novel mutations in the pfrh5 gene, these
 mutations were predicted to have little or no functional impact on the
 antigen’s structure or recognition by known mAbs. Our present findings
 complement earlier reports on the strain transcendent potential of mAbs to
 PfRH5 and constitute, to our knowledge, the first report on the
 susceptibility of P. falciparum clinical isolates from natural infections
 to vaccine-induced mAbs to PfRH5.
description: <strong>Monoclonal antibody
 production</strong> The generation of the mAbs
 used here has previously been described. Briefly, the variable heavy and
 light chains of the antibody-producing genes were amplified from
 single-cell-sorted plasmablasts isolated from peripheral blood mononuclear
 cells (PBMCs) of vaccinees from the first-in-human viral-vectored PfRH5
 vaccine trial (NCT02181088). Cognate, paired heavy and light chains were
 cloned into separate human IgG vectors and transformed into E. coli Mix
 & go competent cells. The panel of mAbs used in this study
 included earlier well-characterized human-vaccine-induced antibodies
 (R5.001, R5.004, R5.007, R5.008, R5.011, R5.016 and R5.017) as well as two
 chimeric mAbs (c2AC7 and c9AD4). These mAbs were expressed by transfection
 of Expi293F cells with paired plasmids encoding matched heavy and light
 chains using the ExpiFectamine 293 (Invitrogen) reagents and purified from
 culture supernatants using a protein G column. Purified antibodies were
 subsequently buffer-exchanged and resuspended at 1mg/ml in incomplete
 parasite medium (RPMI 1640 containing 25 mM HEPES, 0.1mg/ml Hypoxanthine,
 and 50 μg/mL Gentamicin) and stored at -20°C until use.
 <strong>Parasite culturing and growth inhibition activity
 assays</strong> P. falciparum clinical isolates
 were cultured at 4% hematocrit in complete parasite medium (RPMI 1640
 containing 25 mM HEPES, 0.1mg/ml Hypoxanthine, 0.5% Albumax II, 2 mg/mL
 sodium bicarbonate and 50 μg/mL Gentamicin and 5% normal human serum).
 Human erythrocytes of blood group O+ from a single donor were used for all
 cultures and growth inhibition activity (GIA) assays. For ex-vivo GIAs,
 parasites were maintained at 0.5-1% parasitemia at 2% hematocrit and
 co-incubated at ring stage with different concentrations of human mAbs to
 PfRH5 (150, 50, 25, and 10μg/ml), while a mouse IgG was used as isotype
 control in all assay. All assays were set in duplicates in flat bottom
 half-area 96-well plates (Corning), with additional smear wells to monitor
 parasite growth; and incubated at 37º C in an atmosphere of 5%
 O<sub>2</sub>, 5% CO<sub>2</sub> and balanced with
 N<sub>2</sub>. Cultures were harvested after a re-invasion
 rate of at least 95% was reached in the control wells. Parasite cultures
 were transferred into U-bottom 96 well plates and washed with 1X PBS-3%
 BSA (100μl/well) at 1500 rpm for 5 minutes. The red blood cell pellets
 were subsequently stained with 1/2000 Sybr Green in PBS for 20 minutes at
 room temperature in a shaking plate and washed twice with 1X PBS-3% BSA at
 1500 rpm for 5 minutes. The pellets were finally resuspended into 200 ul
 1X PBS and data were acquired using a Cytoflex cytometer (Beckman
 Coulter), where 100,000 events were recorded per well. Flow cytometry data
 outputs were analyzed to determine parasitemia in each well using the Flow
 Jo_v10.8.1 software. Finally, the FlowJo outputs were exported as Excel
 files and the percent GIA for each well was calculated from the invasion
 rate of each well relative to that of the IgG isotype control (Mouse IgG1
 kappa mAb, abcam ab81032) at any given concentration. For each
 concentration, parasitemia from the duplicate test wells were averaged and
 invasion inhibition, relative to isotype control of the matched
 concentration, was calculated as follows: %GIA = 100- [(Average Percent
 invasion in anti-RH5 mAb wells)/ (Average parasitemia in IgG1 isotype
 control wells) * 100]. <strong>PCR amplification
 and next-generation sequencing</strong>   DNA was
 extracted from ring-infected red blood cell pellets using the QIAmp DNA
 Blood Mini Kit (Qiagen) following the manufacturer’s instructions. The
 PfRH5 gene was amplified as previously described39 using the high-fidelity
 platinum taq (Invitrogen) and the amplicon size was resolved in a 1%
 agarose gel electrophoresis. Successfully amplified products were
 subsequently subjected to Next-generation Sequencing using the Illumina
 Novaseq 6000 platform using the following procedure. Briefly, PCR
 amplicons were bead-purified and quantified by Qubit and Bioanalyzer
 before library preparation using unique dual indexes (UDIs), which
 associate each sample with a unique dual index for easy identification
 after sequencing. Indexed samples were subsequently bead-purified to
 select for that fragmented DNA of 200-300 bp sizes, which were then
 quantified with qPCR using Illumina’s KAPA Library Quantification Kit and
 normalized to a concentration of 4nM. These normalized samples were pooled
 into 8 sets of 12 samples each and bead-purified once more to further
 select DNA with the desired range of sizes, which were run on another KAPA
 qPCR to assess concentration. The concentration of these 8 sub-pools was
 normalized to 4nM and combined in equal parts to form one final pool,
 which was submitted to the Yale Center for Genome Analysis (YCGA) for High
 Throughput Next Generation Sequencing with a Novaseq with targeted
 coverage of 500,000 reads per sample.
 <strong>Variant Calling</strong>
 For each sample demultiplexed forward and reverse sequencing
 reads were obtained from the sequencing platform and imported into the
 Geneious prime software, where sequencing reads were paired using the
 Illumina, paired end-setting and subsequently trimmed using the BBDuk
 plug-in, with a minimum quality score of Q30 and a minimum length of 75
 base pairs set, as we were expecting reads around 100 base pairs. Trimmed
 sequences were mapped to the 3D7 reference PfRH5 sequence (PF3D7 0424100)
 already annotated with all known synonymous and non-synonymous mutations.
 Sequence mapping was set for two iterations and coverage criteria were set
 at 1000 reads. The criteria for single nucleotide polymorphism (SNP)
 calling was set to a minimum frequency of 0.02 (2%) and 1000 read
 coverage. At least 3 individuals performed sequence data analysis and SNP
 calling analysis for each sample using defined guidelines to ensure data
 quality.
description: # PfRH5-induced human monoclonal antibodies show broadly neutralizing
 activity in *P. falciparum* clinical isolates
 [https://doi.org/10.5061/dryad.8931zcrzv](https://doi.org/10.5061/dryad.8931zcrzv) The data contains growth inhibition assay data (reported as percent inhibition relative to isotype control) for all antibodies used in the study. The data is organized by tab by antibody and a codebook is included preceding the following tabs for each analysis type. There are three main analyses for which data is included: 1) RH5 Antibody GIA, which gives the percent GIA for each anti-RH5 monoclonal antibody (c2AC7, c9AD4, R5.016, R5.017, R5.004, R5.008, R5.011, R5.007, R5.001); 2) RH5 Antibody Comb GIA, which gives the percent GIA for individual and combinations of anti-RH5 monoclonal antibodies (R5.011 + R5.001; R5.011 + R5.007; R5.011 + R5.008); and 3) BSG Antibody GIA, which gives the percent GIA for anti-BSG monoclonal antibody (MEM-M6_6) ## Description of the data and file structure GIA_KDG22.xlsx describes the growth inhibition assay data (as percent inhibition relative to isotype control) for all antibodies used in the study, and for clinical samples and 3D7 and FVO controls. Genotype_KDG22.xlsx describes the result of next-generation sequencing and SNP calling for clinical isolates. Taken together, Genotype_KDG22.xlsx and GIA_KDG22.xlsx permit genotype-phenotype association analyses. Missing data indicates that a particular antibody was not used for the given clinical isolate, the experiment was not performed. NA indicates not applicable. ## Sharing/Access information This work is licensed under a CC0 1.0 Universal (CC0 1.0) Public Domain Dedication license. Data was derived from the following sources: * Date of data collection: (2022-07-14 to 2022-08-10) * Geographic location of data collection: Kédougou, Sénégal ## Code/Software Flow data were processed using Flow Jo_v10.8.1 to calculate parasitemia. Parasitemia data, invasion percentages, and inhibition percentages were analyzed using and Excel and GraphPad Prism software. No instrument or software-specific information applicable.