PfRH5-induced human monoclonal antibodies show broadly neutralizing activity in P. falciparum clinical isolates
Data files
Apr 17, 2024 version files 101.87 KB
Abstract
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.
README: PfRH5-induced human monoclonal antibodies show broadly neutralizing activity in P. falciparum clinical isolates
https://doi.org/10.5061/dryad.8931zcrzv
The data contains growth inhibition assay data (reported as percent inhibition relative to isotope 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 isotope 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.
Methods
Monoclonal antibody production
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.
Parasite culturing and growth inhibition activity assays
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% O2, 5% CO2 and balanced with N2. 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].
PCR amplification and next-generation sequencing
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.
Variant Calling
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.